==================================BSR06================================== 6. Amino acid pools in bacteria; Amino acid concentrations in bacteria; Intracellular concentrations; Intracellular free amino acids; Gram- positives; Streptococci. 1 UI - 87089099 AU - Gregory RL TI - Streptococcus mutans ribosomal preparations: purification and properties. AB - Ribosomal preparations were obtained from Streptococcus mutans. Sucrose density gradient analyses showed the ribosomes to be 70S and dissociated subunits to be 56S and 34S. The ribosomal preparation contained 57.4% RNA and 42.6% protein and gave an absorption maximum at 260 nm and a minimum at 235 nm and ribosomal particles were approximately 150-180 X 190-220 A as determined by electron microscopy. Immunodiffusion analysis of pooled antiserum raised by injecting the ribosomal preparation into rabbits disclosed precipitin lines with glucosyltransferase and lipoteichoic acid preparations from S. mutans. Gas chromatography showed rhamnose and glucose to be present in the ribosomal preparation indicating the presence of nonribosomal carbohydrate materials. The ribosomes were able to synthesize precipitable polypeptides when exogenous mRNA and tRNA were added and anti-ribosomal antibodies reduced this activity. Protease treatment rendered the ribosomal preparation less immunogenic in rats and less antigenic when the ribosomal preparation was used to coat erythrocytes for passive haemagglutination assays, while RNase treatment of the ribosomal preparation had no effect, suggesting that a protein(s) is the principal immunogenic moiety of the ribosomal antigen. Polyacrylamide gel electrophoresis of the ribosomal preparation revealed 27 protein bands of which five were found to react with hyperimmune rabbit antisera to the S. mutans ribosomal preparation by Western blot analysis. Washing the ribosomal preparation with 1 M NH4Cl did not remove any of the five immunogenic ribosomal protein antigens indicating that these were innate ribosomal proteins. MH - Amino Acids/ANALYSIS ; Cell Fractionation/METHODS ; Cross Reactions ; Gel Diffusion Tests ; Immune Sera ; Microscopy, Electron ; Ribosomal Proteins/ ANALYSIS ; Ribosomes/*ULTRASTRUCTURE ; Streptococcus mutans/ *ULTRASTRUCTURE ; Support, U.S. Gov't, P.H.S. SO - Microbios 1986;48(194):43-60 2 UI - 87075647 AU - Williams RA ; Andrews P TI - Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms. AB - The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo. MH - Amino Acids/ANALYSIS ; Animal ; Chromatography, Gel ; Electrophoresis, Polyacrylamide Gel ; Fructosediphosphates/*PHYSIOLOGY ; Hexosediphosphates/*PHYSIOLOGY ; Lactate Dehydrogenase/*ISOLATION & PURIFICATION ; Ligands ; Molecular Weight ; Rabbits ; Spectrophotometry ; Streptococcus/*ENZYMOLOGY ; Support, Non-U.S. Gov't ; Temperature ; Ultracentrifugation SO - Biochem J 1986 Jun 15;236(3):721-7 3 UI - 87072186 AU - Usui Y TI - Biochemical properties of fibrinogen binding protein (clumping factor) of the staphylococcal cell surface. AB - The staphylococcal fibrinogen binding protein of a strain of coagulase-negative Staphylococcus aureus was purified 229 fold in terms of the haemagglutination unit compared to the starting material by affinity chromatography on fibrinogen-Sepharose. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed one major band and minor bands with relative molecular masses of 62,000, 61,000, and 59,000, respectively. The isoelectric point was between 10.2 and 10.8 determined by isoelectric focusing in polyacrylamide gel. By the agar diffusion test one band was obtained against anti-whole cell rabbit serum. Amino acid analysis of fibrinogen binding protein showed glycine, glutamic acid, lysine, alanine, aspartic acid, and arginine as the major components. Protein A or teichoic acid extracted from the homologous strain did not show fibrinogen binding activity assayed by haemagglutination and anti-fibrinogen binding activity of anti-whole cell Fab. The amount of the fibrinogen binding protein to absorb the activity of anti-whole cell Fab was decreased to 1/60 of that of the starting material. Sheep red blood cells coated with the fibrinogen binding protein agglutinated in 0.001% (w/v) fibrinogen solution. MH - Amino Acids/ANALYSIS ; Chromatography, Affinity ; Coagulase/*ANALYSIS/ ISOLATION & PURIFICATION ; Electrophoresis, Polyacrylamide Gel ; Fibrinogen/METABOLISM ; Gel Diffusion Tests ; Hemagglutination ; Human ; Isoelectric Point ; Molecular Weight ; Staphylococcus aureus/*ANALYSIS SO - Zentralbl Bakteriol Mikrobiol Hyg [A] 1986 Sep;262(3):287-97 4 UI - 87057322 AU - Ferone R ; Hanlon MH ; Singer SC ; Hunt DF TI - alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli. AB - Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups. We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli. Folates were extracted from E. coli grown with [7-14C] p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al. (Foo, S. K., Cichowicz, D. J., and Shane, B. (1980) Anal. Biochem. 107, 109-115). The pAB(Glu)n derived from E. coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu. E. coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments. Molecular weight determinations on the methyl ester derivatives of E. coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8. Chiral HPLC of hydrolyzed and dansylated E. coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer. pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E. coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu. Thus, in E. coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate. MH - p-Aminobenzoic Acid/*METABOLISM ; Aminobenzoic Acids/*METABOLISM ; Carbon Radioisotopes ; Carboxypeptidases ; Chromatography, High Pressure Liquid ; Escherichia Coli/*GROWTH & DEVELOPMENT ; Folic Acid/*ANALOGS & DERIVATIVES ; Glutamates/*ANALYSIS ; Pteroylpolyglutamic Acids/BLOOD/ *ISOLATION & PURIFICATION ; Spectrum Analysis, Mass ; Stereoisomers SO - J Biol Chem 1986 Dec 15;261(35):16356-62 5 UI - 87056972 AU - Fiedler F ; Draxl R TI - Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum. AB - The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls. MH - Amino Acids/ANALYSIS ; Amino Sugars/ANALYSIS ; Animal ; Antigens, Bacterial/*ANALYSIS/IMMUNOLOGY ; Antigens, Surface/ANALYSIS/IMMUNOLOGY ; Bacterial Proteins/IMMUNOLOGY ; Cell Wall/ANALYSIS ; Gram-Positive Bacteria/*ANALYSIS/IMMUNOLOGY/ULTRASTRUCTURE ; Membrane Proteins/ IMMUNOLOGY ; Peptidoglycan/*ANALYSIS/IMMUNOLOGY ; Polysaccharides, Bacterial/*ANALYSIS/IMMUNOLOGY ; Salmonidae/MICROBIOLOGY SO - J Bacteriol 1986 Nov;168(2):799-804 6 UI - 87033657 AU - Poole LB ; Claiborne A TI - Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis. AB - The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide. MH - Amino Acids/ANALYSIS ; Dithionite/PHARMACODYNAMICS ; FAD/ANALYSIS/ *METABOLISM ; Kinetics ; NAD/*METABOLISM ; Oxidation-Reduction ; Peroxidases/ISOLATION & PURIFICATION/*METABOLISM ; Spectrometry, Fluorescence ; Spectrophotometry ; Spectrophotometry, Atomic Absorption ; Streptococcus Faecalis/*ENZYMOLOGY ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 Nov 5;261(31):14525-33 7 UI - 87033639 AU - Claiborne A TI - Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase. AB - An FAD-containing L-alpha-glycerophosphate oxidase has been purified to homogeneity from Streptococcus faecium. The purified protein exists as a dimer (subunit Mr = 65,000); each subunit contains 1 mol of FAD. The enzyme contains no iron, as determined by atomic absorption spectroscopy. The alpha-glycerophosphate oxidase reacts reversibly with sulfite to form a covalent N(5) adduct; it preferentially binds the anionic form of the native oxidized FAD, and it also stabilizes the p-quinonoid form of 8-mercapto-FAD. The enzyme shows an unusually high reactivity with ferricyanide in the absence of oxygen; however, there is no evidence for any superoxide ion (O2-.) generation under standard assay conditions. Dithionite titrations of the enzyme reveal an unusual pH dependence for the stabilization of the flavin semiquinone; only at pH 8.5 does significant anionic semiquinone accumulate. L-alpha-Glycerophosphate rapidly reduces the enzyme-bound FAD; in addition, a small amount of catalytically insignificant red semiquinone appears under these conditions. The 5-deaza-FAD-reconstituted enzyme is also reduced by substrate, strongly suggesting that a radical mechanism is not involved in the oxidation of alpha-glycerophosphate. Furthermore, nitroethane anion reduces the native enzyme; this observation suggests that an electron transfer mechanism involving a substrate carbanion is possible with this enzyme. MH - Amino Acids/ANALYSIS ; Anaerobiosis ; Dithionite/PHARMACODYNAMICS ; FAD/ ANALYSIS ; Glycerolphosphate Dehydrogenase/ISOLATION & PURIFICATION/ *METABOLISM ; Kinetics ; Macromolecular Systems ; Molecular Weight ; Spectrophotometry ; Streptococcus/*ENZYMOLOGY ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 Nov 5;261(31):14398-407 8 UI - 87033438 AU - Keagy PM TI - Computerized semiautomated microbiological assay of folacin. AB - A semiautomated microbiological folacin assay system is described. A microcomputer controls sample dilutions, medium addition, turbidity determination, and data acquisition. Assay capacity is 600 tubes per day, approximately twice that of comparable manual assays. Using the automated equipment, more samples can be compared within one assay, eliminating many sources of between-assay variation in large studies. Additional advantages of this system are reduced human errors, flexibility of assay design, and multifunctional component equipment. Folacin results from chicken liver, spinach, and breakfast cereal samples show equivalent precision for manual and automated assays. MH - Animal ; Autoanalysis ; Cereals/ANALYSIS ; Chickens ; Folic Acid/ *ANALYSIS/PHARMACODYNAMICS ; *Food Analysis ; Lactobacillus Casei/DRUG EFFECTS/GROWTH & DEVELOPMENT ; Liver/ANALYSIS ; Meat/ANALYSIS ; Microcomputers ; Nephelometry and Turbidimetry/METHODS ; Vegetables/ ANALYSIS SO - J Assoc Off Anal Chem 1986 Sep-Oct;69(5):773-6 9 UI - 87031506 AU - K:ery V ; Both V ; Sev:c:ik J ; Zelinka J TI - The number and role of histidine residues in the active site of guanyloribonuclease Sa. AB - The number and role of histidine residues in the active site of extracellular guanyloribonuclease Sa produced by Streptomyces aureofaciens (RNAase Sa) were studied via chemical modification by ethoxyformic anhydride by means of circular dichroism measurements. It was shown that only one of two histidines of RNAase Sa is situated in the active site of the enzyme. Ethoxyformylation of RNAase Sa in the presence of Guo-3'-P, Guo-5'-P and dGuo-5-P, all of them being competitive inhibitors of the enzyme, supported the assumption that an essential histidine residue is bound to the phosphate group in the position 3' of the ribose ring. The circular dichroism measurements of native and modified RNAase Sa and of its complex with Guo-3'-P showed that the modification of the essential histidine residue resulted in alteration of binding of RNAase Sa to Guo-3'-P; histidine thus may play a key role in the formation of such a complex. MH - Binding Sites/DRUG EFFECTS ; Binding, Competitive/DRUG EFFECTS ; Circular Dichroism ; Comparative Study ; Diethyl Pyrocarbonate/PHARMACODYNAMICS ; Dose-Response Relationship, Drug ; Endoribonucleases/*PHYSIOLOGY ; Guanyloribonuclease/ANALYSIS/*PHYSIOLOGY ; Histidine/*ANALYSIS/PHYSIOLOGY ; Streptomyces Aureofaciens/ENZYMOLOGY ; Structure-Activity Relationship ; Time Factors SO - Gen Physiol Biophys 1986 Aug;5(4):405-14 10 UI - 87007985 AU - Omura S ; Imamura N ; Kawakita K ; Mori Y ; Yamazaki Y ; Masuma R ; Takahashi Y ; Tanaka H ; Huang LY ; Woodruff HB TI - Ahpatinins, new acid protease inhibitors containing 4-amino-3-hydroxy-5-phenylpentanoic acid. AB - A soil isolate, Streptomyces sp. WK-142, was found to produce new acid protease inhibitors, ahpatinins A, B, D, E, F and G active against pepsin and renin. Ahpatinin C was found to be identical with pepstatin A. The structure determinations were based on mass spectral data. Four of the compounds contain the unusual amino acid, 4-amino-3-hydroxy-5-phenylpentanoic acid as a building component. MH - Amino Acids/*ANALYSIS ; Chemistry ; Oligopeptides/*ISOLATION & PURIFICATION ; Pepsin/ANTAGONISTS & INHIBITORS ; Pepstatins/*ISOLATION & PURIFICATION/PHARMACODYNAMICS ; *Protease Inhibitors ; Renin/ANTAGONISTS & INHIBITORS ; Spectrum Analysis, Mass ; Streptomyces/CLASSIFICATION/ METABOLISM SO - J Antibiot (Tokyo) 1986 Aug;39(8):1079-85 11 UI - 86226157 AU - Loridan C ; Alouf JE TI - Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin. AB - RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration. MH - Amino Acids/ANALYSIS ; Bacterial Toxins/*ISOLATION & PURIFICATION ; Chromatography ; Chromatography, Gel ; Electrophoresis, Polyacrylamide Gel ; Hemolysis ; Isoelectric Focusing ; *RNA, Fungal ; Saccharomyces Cerevisiae ; Streptococcus Pyogenes/ANALYSIS ; Streptolysins/*ISOLATION & PURIFICATION ; Support, Non-U.S. Gov't SO - J Gen Microbiol 1986 Feb;132 ( Pt 2):307-15 12 UI - 86304377 AU - van der Ley P ; Struyv:e M ; Tommassen J TI - Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies. AB - Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues. MH - Alleles ; Amino Acids/*ANALYSIS ; *Antibodies, Monoclonal ; Bacterial Outer Membrane Proteins/*ANALYSIS/GENETICS ; Base Sequence ; Electrophoresis, Polyacrylamide Gel ; Escherichia Coli/*ANALYSIS/GENETICS ; Mutation ; Support, Non-U.S. Gov't SO - J Biol Chem 1986 Sep 15;261(26):12222-5 13 UI - 86304299 AU - Bradley FC ; Lindstedt S ; Lipscomb JD ; Que L Jr ; Roe AL ; Rundgren M TI - 4-Hydroxyphenylpyruvate dioxygenase is an iron-tyrosinate protein. AB - A resonance Raman investigation into the blue chromophore of 4-hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron-tyrosinate proteins. MH - Electron Spin Resonance/METHODS ; Iron/*ANALYSIS ; Oxygenases/*METABOLISM ; Pseudomonas/ENZYMOLOGY ; Spectrum Analysis, Raman/METHODS ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tyrosine/*ANALYSIS ; 4-Hydroxyphenylpyruvate Dioxygenase/*METABOLISM SO - J Biol Chem 1986 Sep 5;261(25):11693-6 14 UI - 86304230 AU - Iijima S ; Oh MJ ; Saiki T ; Beppu T TI - Characterization of an essential histidine residue in thermophilic malate dehydrogenase. AB - Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed. MH - Diethyl Pyrocarbonate/PHARMACODYNAMICS ; Heat ; Histidine/*ANALYSIS ; Hydrogen-Ion Concentration ; Malate Dehydrogenase/ANTAGONISTS & INHIBITORS/*METABOLISM ; NAD/METABOLISM ; Oxidation-Reduction ; Photochemistry ; Thermus/*ENZYMOLOGY SO - J Biochem (Tokyo) 1986 Jun;99(6):1667-72 15 UI - 86304219 AU - Takeuchi M ; Asano N ; Kameda Y ; Matsui K TI - Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase. AB - 3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis. MH - Amino Acids/ANALYSIS ; Binding Sites/DRUG EFFECTS ; Diethyl Pyrocarbonate/ ANTAGONISTS & INHIBITORS/*PHARMACODYNAMICS ; Enzyme Reactivators ; Flavobacterium/ENZYMOLOGY ; Formates/*PHARMACODYNAMICS ; Histidine/ *ANALYSIS ; Kinetics ; Lyases/*ANTAGONISTS & INHIBITORS ; Spectrophotometry, Ultraviolet SO - J Biochem (Tokyo) 1986 Jun;99(6):1571-7 16 UI - 86276911 AU - Babu JP ; Beachey EH ; Simpson WA TI - Inhibition of the interaction of Streptococcus sanguis with hexadecane droplets by 55- and 60-kilodalton hydrophobic proteins of human saliva. AB - The effect of salivary secretions on the hydrophobicity of Streptococcus sanguis was investigated. Pretreatment of the bacteria with paraffin-stimulated whole saliva resulted in a 79% inhibition of adhesion to hexadecane droplets. Column chromatography on Sepharose 4B and sodium dodecyl sulfate gel electrophoretic analysis indicated that the inhibitory activity of saliva resided in a fraction containing material of approximately 60,000 molecular weight. The active components, which we have termed the hydrophobic components (HC), bind to octyl-Sepharose beads. Pretreatment of S. sanguis with HC resulted in a dose-dependent inhibition of the streptococcus-hexadecane interaction that reached a maximum of 85%. Furthermore, HC effectively blocked the ability of S. sanguis to adhere to hydroxyapatite beads coated with either whole saliva or HC. Sodium dodecyl sulfate-polyacrylamide gel analysis indicated that HC eluted from octyl-Sepharose consisted primarily of two proteins (60 kDa and 55 kilodaltons) which could be resolved by high-pressure liquid chromatography. Both of these proteins were able to inhibit the binding of S. sanguis to hexadecane in a dose-dependent manner; however, the 60-kilodalton molecule was slightly more effective in this assay. Amino acid analysis of these proteins showed that both proteins contained a high percentage of nonpolar amino acids. These findings suggest that certain components of saliva influence the interaction of S. sanguis with hydrophobic surfaces. MH - Adhesiveness ; Alkanes ; Amino Acids/ANALYSIS ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Human ; Molecular Weight ; Pepsin/PHARMACODYNAMICS ; Salivary Proteins/ANALYSIS/ ISOLATION & PURIFICATION/*PHYSIOLOGY ; Streptococcus sanguis/*PHYSIOLOGY ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Trypsin/ PHARMACODYNAMICS SO - Infect Immun 1986 Aug;53(2):278-84 17 UI - 86270003 AU - Jacobs AA ; van den Berg PA ; Bak HJ ; de Graaf FK TI - Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli. AB - Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins. MH - Amino Acid Sequence ; Amino Acids/ANALYSIS ; Animal ; Bacterial Proteins/ METABOLISM ; Binding Sites ; Chlorobenzoates/PHARMACODYNAMICS ; Chymotrypsin/METABOLISM ; Erythrocytes/MICROBIOLOGY ; Escherichia Coli/ *METABOLISM ; Horses ; Lysine/*ANALYSIS ; Macromolecular Systems ; Peptide Fragments/ANALYSIS ; Support, Non-U.S. Gov't SO - Biochim Biophys Acta 1986 Jul 25;872(1-2):92-7 18 UI - 86251239 AU - McDonald WA ; Watts J ; Bowmer MI TI - Factors affecting Staphylococcus epidermidis growth in peritoneal dialysis solutions. AB - Staphylococcus epidermidis is the most frequent cause of peritonitis complicating continuous ambulatory peritoneal dialysis. We studied factors that might influence the growth of S. epidermidis in commercially available peritoneal dialysis solution (PDS). Test strains were inoculated into PDS and incubated overnight at 37 degrees C. Samples were removed at appropriate intervals, bacterial counts were performed, and growth curves were constructed. We studied the effects of various osmolarities, the neutralization and acidification of fresh and spent PDS, and the effect of intraperitoneal dwell time on the ability PDS to support growth of S. epidermidis. In fresh PDS, numbers of bacteria remained constant after 24 h. No significant differences in growth were observed among PDS with 0.5, 1.5, 2.5, and 4.25% glucose. Neutralizing acidic fresh PDS had no effect on bacterial growth. However, growth did occur in spent PDS. PDS which was recovered after only 2 h in the peritoneal cavity supported growth to the same extent as did PDS recovered after 4 to 6 h. Mean log10 changes after 24 h of incubation were as follows: for fresh PDS, -1.3; after 2 h dwell time, 2.9; after 4 h dwell time, 1.9; and after 6 h dwell time, 1.3. Acidification of spent PDS to less than pH 6.35 produced less rapid growth; mean log10 increases after 24 h of incubation were 1.9 for pH 7.75, 1.6 for pH 6.35, 0.6 for pH 5.75, and 0.7 for pH 4.95. Fresh PDS of all available osmolarities neither supported the growth of S. epidermidis nor was bactericidal. Spent PDS supported bacterial growth, and this growth was partly independent of the neutralization which occurred during the dialysis. MH - Amino Acids/ANALYSIS ; *Drug Contamination ; Human ; Hydrogen-Ion Concentration ; Osmolar Concentration ; *Peritoneal Dialysis, Continuous Ambulatory ; Peritonitis/*MICROBIOLOGY ; *Solutions ; Staphylococcus epidermidis/*GROWTH & DEVELOPMENT SO - J Clin Microbiol 1986 Jul;24(1):104-7 19 UI - 86223927 AU - Ishijima S ; Izui K ; Katsuki H TI - Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue. AB - The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog. MH - Amino Acid Sequence ; *Carboxy-Lyases/ISOLATION & PURIFICATION ; Catalysis ; Chromatography, High Pressure Liquid ; Cysteine/*ANALYSIS ; Electrophoresis, Polyacrylamide Gel ; Escherichia Coli/*ENZYMOLOGY ; Hydroxylamines ; Lactates ; Maleimides ; Peptide Fragments/ANALYSIS ; *Phosphoenolpyruvate Carboxylase/ISOLATION & PURIFICATION ; Support, Non-U.S. Gov't ; Trypsin SO - J Biochem (Tokyo) 1986 May;99(5):1299-310 20 UI - 86221157 AU - Ezepchuk YuV ; Noskov AN TI - NH2-terminal localization of that part of the staphylococcal enterotoxins polypeptide chain responsible for binding with membrane receptor and mitogenic effect. AB - It is established that the part of the SEA and SEC2 polypeptide chain responsible for the binding of these toxin proteins with the membrane receptor on the surface of rabbit thymocytes and mitogenic effect is localised in the NH2-terminal region of the molecule. The SEC2 splits in two fragments T1 (17 kdalton) and T2 (12.5 kdalton) under limited proteolysis by trypsin in the presence of 2-ME. The amino acid terminal residues of SEA, SEC2 and their proteolytic fragments are also studied. MH - Amino Acids/ANALYSIS ; Animal ; Enterotoxins/*METABOLISM ; *Mitogens ; Peptide Fragments/METABOLISM/PHARMACODYNAMICS ; Peptides/*METABOLISM ; Rabbits ; Receptors, Immunologic/*METABOLISM ; Staphylococcus/*METABOLISM ; T Lymphocytes/METABOLISM SO - Int J Biochem 1986;18(5):485-8 21 UI - 86214077 AU - Lewis RN ; George R ; McElhaney RN TI - Structure-function investigations of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B: studies of reactive amino acid residues using group-specific reagents. AB - The purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B was treated with a variety of reagents which specifically modify various amino acid residues on the enzyme. In all cases reaction of this enzyme with any of the reagents tested results in at least a partial inactivation of its activity. The modification of one reactive lysine by dinitrofluorobenzene, of one reactive arginine by phenylglyoxal, or of two tyrosine residues by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or fluorosulfonylbenzoyl adenosine results in a complete inactivation of the enzyme. Partial inactivation of enzymatic activity with N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dicyclohexylcarbodiimide, and Woodward's reagent K suggests an indirect involvement of sulfhydryl and carboxylic acid groups in the maintenance of enzymatic activity, although inhibition by these reagents may also be the result of nonspecific effects such as subunit crosslinking. These studies also show that all of the subunits of the ATPase can be labeled by aqueous-phase reagents directed at amino groups and phenolic groups, and provide evidence for a specific affinity labeling of the alpha subunit of the enzyme by a nucleotide analog directed at phenolic and/or sulfhydryl groups. MH - Acholeplasma laidlawii/*ENZYMOLOGY ; Adenosine/ANALOGS & DERIVATIVES/ PHARMACODYNAMICS ; Adenosine Triphosphatase, Magnesium/ANTAGONISTS & INHIBITORS/*METABOLISM ; Adenosine Triphosphate/PHARMACODYNAMICS ; Amino Acids/*ANALYSIS ; Chemistry ; Dinitrofluorobenzene/PHARMACODYNAMICS ; Guanidines/ANALYSIS ; Indicators and Reagents ; Kinetics ; NBD Chloride/ PHARMACODYNAMICS ; Phenols/ANALYSIS ; Phenylglyoxal/PHARMACODYNAMICS ; Structure-Activity Relationship ; Sulfhydryl Compounds/ANALYSIS ; Support, Non-U.S. Gov't SO - Arch Biochem Biophys 1986 May 15;247(1):201-10 22 UI - 86202959 AU - Okahashi N ; Koga T ; Hamada S TI - Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans. AB - A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests. MH - Amino Acids/ANALYSIS ; Antigens, Bacterial/ANALYSIS/IMMUNOLOGY/*ISOLATION & PURIFICATION ; Bacterial Proteins/ANALYSIS/IMMUNOLOGY/*ISOLATION & PURIFICATION ; Chromatography, Gel ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel ; Gel Diffusion Tests ; Glucose/ANALYSIS ; Heat ; Immunoelectrophoresis ; Isoelectric Point ; Molecular Weight ; Precipitation ; Streptococcus mutans/*IMMUNOLOGY ; Support, Non-U.S. Gov't ; Ultrafiltration SO - Microbiol Immunol 1986;30(1):35-47 23 UI - 86196552 AU - Lin JY ; Chen KC ; Hale J ; Totten PA ; Holmes KK TI - Two-dimensional thin-layer chromatography for the specific detection of hippurate hydrolysis by microorganisms. AB - Glycine, one of the end products of hippurate hydrolysis by microorganisms, was detected by a rapid, specific technique utilizing two-dimensional thin-layer chromatography. A loopful of growth of each organism from its suitable agar medium was washed, suspended, and incubated with 0.1% sodium hippurate for 30 min at 37 degrees C. The supernatant of the incubated suspension from each organism was then dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. Glycine, a product of hippurate hydrolysis, was detected under UV light. This technique does not require prolonged incubation and was found to be more specific and reliable than the standard ninhydrin reaction. In addition, it is inexpensive and can be easily conducted in a clinical microbiological reference laboratory. By this method, 100% (22/22) of Campylobacter jejuni and 0% (0/9) of Campylobacter coli reference strains were positive. In addition, 100% (13/13) of group B streptococci, 100% (24/24) of group D streptococci, and 90% (18/20) of Gardenerella vaginalis clinical isolates were positive for hippurate hydrolysis. This method is useful for the identification to the species level of Campylobacter organisms and the biotyping of Gardnerella organisms and for the detection of hippurate hydrolysis by unknown microorganisms. MH - Bacteria/*METABOLISM ; Campylobacter/CLASSIFICATION/METABOLISM ; Campylobacter fetus/CLASSIFICATION/METABOLISM ; Chromatography, Thin Layer ; Glycine/ANALYSIS/METABOLISM ; Haemophilus vaginalis/ CLASSIFICATION/METABOLISM ; Hippurates/*METABOLISM ; Hydrolysis ; Streptococcus/CLASSIFICATION/METABOLISM ; Streptococcus Agalactiae/ CLASSIFICATION/METABOLISM ; Support, U.S. Gov't, P.H.S. SO - J Clin Microbiol 1986 Jan;23(1):118-23 24 UI - 86192497 AU - Moks T ; Abrahms:en L ; Nilsson B ; Hellman U ; Sj:oquist J ; Uhl:en M TI - Staphylococcal protein A consists of five IgG-binding domains. AB - A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-A--immunoglobulin-G complexes are discussed. MH - Amino Acids/ANALYSIS ; Cloning, Molecular ; Escherichia Coli/GENETICS/ METABOLISM ; Genes, Bacterial ; Peptide Fragments/GENETICS ; Plasmids ; Receptors, Immunologic/*GENETICS ; Signal Peptides/ANALYSIS ; Staphylococcal Protein A/*GENETICS ; Staphylococcus/GENETICS ; Support, Non-U.S. Gov't ; Transformation, Bacterial ; Translation, Genetic SO - Eur J Biochem 1986 May 2;156(3):637-43 25 UI - 86171003 AU - Brown CM ; Smith AM ; Picciano MF TI - Forms of human milk folacin and variation patterns. AB - The pattern of folacin in 180 human milk samples collected from 16 women was studied before and after pteroylglutamic hydroxylase (conjugase) treatment. The differential response of Lactobacillus casei versus Streptococcus faecalis distinguishes N-5-methyltetrahydrofolate from other forms. Growth of Pediococcus cerevisiae differentiates the reduced monoglutamates, other than the N-5-methylated derivative, from oxidized folacins. Mean folacin activity with L. casei was 85.3 ng/ml. Prior to conjugase treatment of samples, mean activity was 46.9 ng/ml with this organism. The responses of S. faecalis and P. cerevisiae were 48% and 7.6%, respectively, of the total L. casei active folacins. The total L. casei active folacins increased from 76.5 ng/ml at 6 weeks of lactation to 97.1 ng/ml at 12 weeks of lactation. Increases were also observed with time of day: from morning (65.7 ng/ml), to afternoon (80.0 ng/ml), and to evening (114.7 ng/ml). Hindmilk had greater total L. casei activity (100.0 ng/ml) than foremilk (73.8 ng/ml). Although folacins active for S. faecalis and P. cerevisiae were not affected by duration of lactation, increases in folacin activity with time of day and from fore- to hindmilk were observed using both of these organisms. Values for the folacin content of human milk are therefore dependent on sampling procedures. Pteroylpolyglutamates of greater than or equal to 3 glutamic acid residues and N-5-methyltetrahydrofolate are the predominant forms of human milk folacin based on differential growth responses of L. casei, S. faecalis, and P. cerevisiae to these forms. MH - *Bacteriological Technics ; Female ; Folic Acid/*ANALYSIS ; Human ; Lactobacillus Casei/GROWTH & DEVELOPMENT ; Milk, Human/*ANALYSIS ; Pediococcus/GROWTH & DEVELOPMENT ; Streptococcus Faecalis/GROWTH & DEVELOPMENT ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Time Factors SO - J Pediatr Gastroenterol Nutr 1986 Mar-Apr;5(2):278-82 26 UI - 86159700 AU - Blomster-Hautamaa DA ; Kreiswirth BN ; Novick RP ; Schlievert PM TI - Resolution of highly purified toxic-shock syndrome toxin 1 into two distinct proteins by isoelectric focusing. AB - Highly purified toxic-shock syndrome toxin 1 (TSST-1) was prepared by differential precipitation with ethanol and resolubilization in water followed by successive electrofocusing in pH gradients of 3-10, 6-8, and 6.5-7.5. TSST-1, thus isolated, migrated as two distinct protein bands with isoelectric points of 7.08 (TSST-1a) and 7.22 (TSST-1b). When tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both toxins migrated as homogeneous bands with molecular weights of 22 000. The gel bands were visualized by silver staining. The two toxins have nearly identical amino acid compositions and are immunologically identical as shown by Ouchterlony reactivity against TSST-1 hyperimmune serum. TSST-1a and TSST-1b have the same biological activities as TSST-1: the capacity to induce fever, enhancement of host susceptibility to lethal endotoxin shock, nonspecific T lymphocyte mitogenicity, and suppression of immunoglobulin M synthesis against sheep erythrocytes. These two proteins have been isolated from several different TSS-associated Staphylococcus aureus strains. The data suggest that the differences in isoelectric point result either from the presence of a cofactor or from alternative conformations. Since only two bands appear, microheterogeneity as a result of deamination or acetylation is unlikely. MH - Amino Acids/ANALYSIS ; Animal ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins/*ISOLATION & PURIFICATION/TOXICITY ; Immune Sera ; Isoelectric Focusing/METHODS ; Molecular Weight ; Rabbits ; Staphylococcus aureus ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Biochemistry 1986 Jan 14;25(1):54-9 27 UI - 86159672 AU - Jackson KW ; Malke H ; Gerlach D ; Ferretti JJ ; Tang J TI - Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues. AB - The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W., & Ferretti, J.J. (1984) MGG, Mol. Gen. Genet. 196, 360-365] while the molecular weight of the native streptokinase is 47 000. The structural and activity differences of the cloned streptokinase (cSK) as expressed by S. sanguis and the native streptokinase (nSK) were investigated. From a partially purified cSK, two active fractions were obtained by reversed-phase HPLC. The minor fraction cSKL was nearly as active as SK in plasminogen activation. The major fraction cSKs had only about one-fourth of the specific activity. The structures of cSKL and cSKs were studied and compared to the known amino acid sequence of SK [Jackson, K. W., & Tang, J. (1982) Biochemistry 21, 6620-6625]. From the NH2- and COOH-terminal sequences and amino acid composition of the cyanogen bromide (CNBr) fragments, it could be deduced that cSKL and cSKs are without 31 and 32 residues, respectively, from the COOH-terminal end of SK. Since the cloned gene contained the full SK structure, the missing structures must have been due to posttranslational proteolysis. An SK fragment similar in size to cSK was observed from a chymotryptic digest of SK. MH - Amino Acids/ANALYSIS ; Carboxypeptidases/METABOLISM ; *Cloning, Molecular ; Comparative Study ; Cyanogen Bromide ; *Genes, Bacterial ; *Genes, Structural ; Kinetics ; Molecular Weight ; Peptide Fragments/ANALYSIS ; Plasminogen ; Recombinant Proteins/*GENETICS/METABOLISM ; Streptococcus sanguis/ENZYMOLOGY/*GENETICS ; Streptokinase/*GENETICS/METABOLISM ; Support, U.S. Gov't, P.H.S. SO - Biochemistry 1986 Jan 14;25(1):108-14 28 UI - 86158757 AU - Little C ; Emanuel EL ; Gagnon J ; Waley SG TI - Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus. AB - Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II. MH - Bacillus Cereus/*ENZYMOLOGY ; Beta-Lactamases/*METABOLISM ; Binding Sites ; Cephalosporinase/ANTAGONISTS & INHIBITORS/*METABOLISM ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Ethyldimethylaminopropyl Carbodiimide ; Glutamates/*ANALYSIS ; Models, Biological ; Peptide Fragments/ANALYSIS ; Support, Non-U.S. Gov't SO - Biochem J 1986 Jan 15;233(2):465-9 29 UI - 86142515 AU - Johnston KH ; Zabriskie JB TI - Purification and partial characterization of the nephritis strain-associated protein from Streptococcus pyogenes, group A. AB - We report the isolation and purification of the nephritis strain-associated protein (NSAP) first described by Villareal et al. (8). Amino acid analysis, and determination of the first 21 amino-terminal amino acids indicated that this 46 kD protein is a streptokinase. Biochemical analysis confirmed that NSAP could act as a plasminogen activator; immunological investigations indicated that NSAP is antigenically different from streptokinase from group C streptococcus, and possibly represents a unique streptokinase. It is this uniqueness that may contribute to the role of NSAP in the pathogenesis of acute poststreptococcal glomerulonephritis. MH - Amino Acid Sequence ; Amino Acids/ANALYSIS ; Antibodies, Monoclonal/ DIAGNOSTIC USE ; Bacterial Proteins/IMMUNOLOGY/*ISOLATION & PURIFICATION ; Chromatography ; Glomerulonephritis/*MICROBIOLOGY ; Immunosorbent Technics ; Molecular Weight ; Plasminogen Activators/ANALYSIS ; Streptococcus Pyogenes/*ANALYSIS/PATHOGENICITY ; Streptokinase/ANALYSIS/ IMMUNOLOGY ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - J Exp Med 1986 Mar 1;163(3):697-712 30 UI - 86140147 AU - Davis DR ; Griffey RH ; Yamaizumi Z ; Nishimura S ; Poulter CD TI - 15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. AB - The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum. MH - Amino Acyl T RNA/*ANALYSIS ; Escherichia Coli/*ANALYSIS ; Hydrogen Bonding ; Nitrogen Isotopes ; Nuclear Magnetic Resonance ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Uridine/*ANALOGS & DERIVATIVES/ANALYSIS SO - J Biol Chem 1986 Mar 15;261(8):3584-7 31 UI - 86136554 AU - Stan-Lotter H ; Clarke DM ; Bragg PD TI - Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence. AB - The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene. MH - Amino Acid Sequence ; Base Sequence ; Comparative Study ; Cysteine/ *ANALYSIS ; *DNA, Bacterial ; DNA, Recombinant/METABOLISM ; Escherichia Coli/*ENZYMOLOGY ; H(+)-Transporting ATPase/ANALYSIS/BIOSYNTHESIS/ *GENETICS ; Isoelectric Focusing ; Peptide Fragments/ANALYSIS ; Support, Non-U.S. Gov't ; Trypsin SO - FEBS Lett 1986 Mar 3;197(1-2):121-4 32 UI - 86130645 AU - Camble R ; Petter NN ; Trueman P ; Newton CR ; Carr FJ ; Hockney RC ; Moore VE ; Greene AR ; Holland D ; Edge MD TI - Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes. AB - A gene was chemically synthesised and expressed in Escherichia coli to produce [Ala30,32,33]IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells. Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions. No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33. MH - Amino Acids/*ANALYSIS ; DNA, Recombinant ; Electrophoresis, Polyacrylamide Gel ; Escherichia Coli/GENETICS ; Genes, Structural ; Human ; Interferon Type II/GENETICS/*PHYSIOLOGY ; Plasmids SO - Biochem Biophys Res Commun 1986 Feb 13;134(3):1404-11 33 UI - 86129467 AU - Verburg JG ; Yoshida M ; Allison WS TI - The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan. AB - When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation. MH - Amino Acid Sequence ; Bacteria/*ENZYMOLOGY ; Binding Sites/DRUG EFFECTS ; Chromatography, High Pressure Liquid ; Dithionite/*PHARMACODYNAMICS ; H(+)-Transporting ATPase/*METABOLISM ; Heat ; NBD Chloride/*METABOLISM ; Oxadiazoles/*METABOLISM ; Oxidation-Reduction/DRUG EFFECTS ; Sulfites/ *PHARMACODYNAMICS ; Support, U.S. Gov't, P.H.S. ; Tyrosine/*ANALYSIS SO - Arch Biochem Biophys 1986 Feb 15;245(1):8-13 34 UI - 86129357 AU - Chatterji D ; Wu CW ; Wu FY TI - Spatial relationship between the intrinsic metal in the beta subunit and cysteine-132 in the sigma subunit of Escherichia coli RNA polymerase: a resonance energy transfer study. AB - Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled sigma subunit and Co in the beta subunit of Co-Zn RNA polymerase (RPase). sigma subunit with or without PM labeling was cleaved with 2-nitro-5-thiocyanobenzoic acid, and the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One molecule of the fluorescent probe (PM) was found to be attached to the cysteine-132 residue of the sigma subunit. When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled sigma overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm. Based on F:orster's equation, the R0 values for the donor-acceptor pair were calculated to be 21.5 and 22 A in the absence and presence of template analog (dA-dT)60, respectively. Using these R0 values and the observed energy transfer efficiencies, the distance between the cysteine-132 of the sigma subunit and Co located at the initiation site of the beta subunit was calculated to be 22 A with or without the template present, indicating that no major conformational change of the enzyme was induced upon template binding. However, a small but significant change in the above distance was observed upon the addition of ATP to RPase in the presence (dA-dT)60 but not in the absence of (dA-dT)60 template. The biological implications of these observations are discussed. MH - Binding Sites ; Cysteine/*ANALYSIS ; Electrophoresis, Polyacrylamide Gel ; Energy Transfer ; Escherichia Coli/*ENZYMOLOGY ; Maleimides ; Mathematics ; RNA Polymerases/*ANALYSIS ; Spectrometry, Fluorescence ; Substrate Specificity ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Templates ; Zinc/*ANALYSIS SO - Arch Biochem Biophys 1986 Jan;244(1):218-25 35 UI - 86118497 AU - Silverman SH ; Turnell DC ; Youngs DJ ; Keighley MR TI - What is the role of histidine in studies of faecal mutagenicity? AB - Mutagenicity testing can be used to assay faeces for genotoxic substances and the results are reported to correlate with population risk for colorectal cancer (Ehrich et al., 1979). It has been suggested that histidine in faeces may cause false positive results (Venitt and Bosworth, 1983). To determine the relationship between histidine and false positive mutagenicity assays aliquots of non-mutagenic faecal extract and saline were supplemented with histidine and subjected to the Ames Salmonella/mammalian microsome mutagenicity assay (Ames et al., 1975). Using high-pressure liquid chromatography the analytical recovery of histidine from water and faecal extract supplemented with histidine was equivalent (r = 0.998, p less than 0.001). Histidine was measured in faecal extracts (1 in 10 dilutions) from 35 volunteers, 10 patients with inflammatory bowel disease and 4 with rectal cancer. These extracts were also assayed for mutagens using the Salmonella/mammalian microsome mutagenicity assay. None of the faecal extracts gave mutagenicity ratios above 2. Faecal extracts from volunteers were free of detectable histidine. Although 9 of those from inflammatory bowel disease patients contained histidine (mean +/- SEM 255 +/- 34 mumoles l-1) as did 1 extract from a rectal cancer patient (50 mumoles l-1), none contained sufficient histidine to give a false positive Salmonella/mammalian microsome mutagenicity assay result (800 mumoles l-1 in test solution). Our results do not implicate histidine as a cause of error in faecal mutagenicity testing by the Salmonella/mammalian microsome mutagenicity assay. MH - Chromatography, High Pressure Liquid ; Colitis, Ulcerative/METABOLISM ; Colonic Neoplasms/ANALYSIS ; Crohn Disease/METABOLISM ; Feces/*ANALYSIS ; Histidine/*ANALYSIS/METABOLISM ; Human ; Mutagenicity Tests/STANDARDS ; Mutagens/*ANALYSIS/METABOLISM ; Salmonella Typhimurium/DRUG EFFECTS SO - Mutat Res 1986 Feb;173(2):99-104 36 UI - 86110556 AU - Reeves MW ; Arko RJ ; Chandler FW ; Bridges NB TI - Affinity purification of staphylococcal toxic shock syndrome toxin 1 and its pathologic effects in rabbits. AB - Toxic shock syndrome toxin 1 (TSST-1) was purified to apparent homogeneity by chromatofocusing and affinity chromatography. The amino acid composition of the toxin was very similar to that reported for TSST-1 by other investigators. The amino-terminal amino acid was serine. A partial specific volume of 0.73 ml/g was calculated for the toxin from the amino acid data, and a molecular weight of 19,200 +/- 1,300 was determined by hydrodynamic methods. New Zealand white rabbits of both sexes were equally susceptible to the lethal effects of the toxin; however, older rabbits (greater than 12 months) were far more susceptible than young adults or weanlings. The 50% lethal dose of TSST-1 in older rabbits was 50 to 60 micrograms/kg when injected subcutaneously and 20 to 30 micrograms/kg when injected intravenously. Enhancement of lethal endotoxin shock by TSST-1 could not be demonstrated when both toxins were injected subcutaneously; however, lethal shock did occur when endotoxin (10 micrograms/kg) was injected intravenously after TSST-1 had been injected by either the subcutaneous (50 to 60 micrograms/kg) or the intravenous (20 to 30 micrograms/kg) route. Endotoxin alone was not lethal at a dose of 500 micrograms/kg of body weight when injected subcutaneously. When injected intravenously, endotoxin at a dose of 500 micrograms/kg was not lethal in weanling males or in females in any age group; however, young (6 to 7 months) and adult (greater than 12 months) males were killed by endotoxin doses as low as 45 to 50 micrograms/kg. Histopathologic studies of rabbits by both sexes which died as a result of TSST-1 alone or in combination with endotoxin showed extensive damage to organs rich in lymphoid and mononuclear phagocytic cells such as the thymus, mesenteric lymph nodes, liver, and spleen. Severe congestion of these organs as well as erythrophagocytosis and lymphoid depletion in the spleen and mesenteric lymph nodes were noted. Congestion and hemorrhage were also found in the heart, lungs, trachea, and thymus. The systemic pathology produced by TSST-1 was strikingly similar to that seen in humans who had died of toxic shock syndrome and in rabbits with subcutaneous chamber inoculated with toxic shock case strains of Staphylococcus aureus. Rabbits that were not killed by the toxin suffered a very rapid and severe leukopenia followed by leukocytosis with a left shift. Lymphopenia was also noted as was a mild but persistent anemia. With the exception of the early leukopenia, very similar hematologic findings have been noted in humans with toxic shock syndrome. MH - Amino Acids/ANALYSIS ; Animal ; Blood Cell Count ; Endotoxins/TOXICITY ; Enterotoxins/ANALYSIS/ISOLATION & PURIFICATION/*TOXICITY ; Female ; Human ; Lymphoid Tissue/PATHOLOGY ; Male ; Molecular Weight ; Rabbits ; Shock, Septic/BLOOD/PATHOLOGY ; Staphylococcus aureus SO - Infect Immun 1986 Feb;51(2):431-9 37 UI - 86082334 AU - Searle MS ; Hammond SJ ; Birdsall B ; Roberts GC ; Feeney J ; King RW ; Griffiths DV TI - Identification of the 1H resonances of valine and leucine residues in dihydrofolate reductase by using a combination of selective deuteration and two-dimensional correlation spectroscopy. AB - Lactobacillus casei dihydrofolate reductase (Mr 18 500) contains 16 valine and 14 leucine residues. By comparing the 2D COSY NMR spectra of normal and [gamma-2H6]valine enzyme we have been able to identify all 60 methyl resonances from these residues, and to connect the pairs arising from the same residue. This pairing of the methyl resonances was aided by the examination of the 2D RELAY spectrum which also allowed the C alpha H resonances (and hence the complete spin systems) of 14 of the valine residues to be identified. The combination of selective deuteration with 2D NMR techniques is shown to be a powerful general method for resolving 1H resonances in the complex spectra of proteins and for assigning them to amino-acid type. MH - Deuterium ; Lactobacillus Casei/ENZYMOLOGY ; Leucine/*ANALYSIS ; Nuclear Magnetic Resonance/METHODS ; Protein Binding ; Support, Non-U.S. Gov't ; Tetrahydrofolate Dehydrogenase/*ANALYSIS ; Trimethoprim/ANALYSIS ; Valine/ *ANALYSIS SO - FEBS Lett 1986 Jan 1;194(1):165-70