==================================BSR20================================== 20. Processing enzymes of peptide hormones (oxytocin, vasopressin, neurophysin, enkephalin, endorphin, ACTH, insulin, glucagon. endoproteolytic enzymes, exo, carboxypeptidase B-like enzymes, amidating enzymes. 1 UI - 87057677 AU - Eskridge EM ; Shields D TI - The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes. AB - To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing. MH - Acetyltransferases/*GENETICS ; Animal ; Bacillus Subtilis/ENZYMOLOGY/ *GENETICS ; Cytoplasm/ENZYMOLOGY ; DNA Restriction Enzymes ; DNA/ METABOLISM ; Fishes ; Genes, Bacterial ; Genes, Structural ; Intracellular Membranes/*METABOLISM ; Islands of Langerhans/METABOLISM ; Microsomes/*METABOLISM ; Proinsulin/*GENETICS ; Protein Precursors/ *GENETICS ; *Protein Processing, Post-Translational ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic ; *Translation, Genetic SO - J Cell Biol 1986 Dec;103(6 Pt 1):2263-72 2 UI - 87006401 AU - Neal GW ; Solomon SS ; Duckworth WC TI - Internalization and degradation of glucagon by human mononuclear cells. AB - The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism. MH - Binding Sites ; Cell Fractionation ; Ethylmaleimide/PHARMACODYNAMICS ; Glucagon/*METABOLISM ; Human ; Insulin/METABOLISM ; Kinetics ; Lymphocytes/*METABOLISM/ULTRASTRUCTURE ; Receptors, Gastrointestinal Hormone/METABOLISM/PHYSIOLOGY ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. ; Time Factors SO - Horm Metab Res 1986 Aug;18(8):540-5 3 UI - 87004333 AU - Levy JR ; Murray E ; Manolagas S ; Olefsky JM TI - Demonstration of insulin receptors and modulation of alkaline phosphatase activity by insulin in rat osteoblastic cells. AB - Osteoporosis is a known complication of diabetes mellitus, suggesting a role for insulin in bone homeostasis. We studied insulin receptors and insulin action in the osteoblast-like rat osteogenic sarcoma cell line ROS 17/2.8. These cells share many common features with the osteoblast, such as 1,25-dihydroxyvitamin D3 receptors, PTH receptors, and 1,25-dihydroxyvitamin D3-induced modulation of alkaline phosphatase activity and osteocalcin. Competition binding studies revealed high affinity insulin receptors, with an ED50 for insulin of 1 nM. The receptors were highly specific for insulin, with 60% inhibition of insulin binding by an antireceptor antibody, no competition by epidermal growth factor, and an ED50 of 300 nM for proinsulin. Steady state maximal insulin binding was obtained by 40 min at 37 C, and insulin degradation, as measured by trichloroacetic acid solubility, was 1%/h at 37 C. ROS cells readily internalized insulin, and under steady state binding conditions at 37 C, 56% of the cell-associated radioactivity consisted of intracellular material. Chloroquine (100 microM) inhibited intracellular processing of insulin, leading to a 300% increase in cell-associated insulin by 2 h (37 C). Photoaffinity labeling of the insulin receptor with the photosensitive analog of insulin, B2 (2-nitro-4-azidophenyl-acetyl)des-pheB1-insulin, followed by solubilization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed specific bands of 125K and 430K mol wt under reducing and nonreducing conditions, respectively. Thus, the structure of insulin receptors in ROS cells appears comparable to that of insulin receptors of known target tissues. Insulin action was also examined. Insulin did not stimulate [2-3H]deoxyglucose uptake or [1-14C]leucine incorporation into protein. In contrast, physiological concentrations of insulin inhibited alkaline phosphatase activity in nonconfluent cells. After exposure to insulin for 24 h, alkaline phosphatase activity was decreased compared to basal by 39.5% and 50% with 5 and 50 ng/ml insulin, respectively. In conclusion, ROS cells bind insulin to specific receptors that are similar to insulin receptors on other target tissues; receptors internalize insulin, which is then processed through a chloroquine-sensitive pathway; insulin does not affect membrane substrate transport; and insulin does inhibit the activity of an enzyme that is important in bone metabolism. ROS cells represent a model for studying insulin effects on bone. MH - Affinity Labels ; Alkaline Phosphatase/*METABOLISM ; Aminoisobutyric Acids/METABOLISM ; Animal ; Binding, Competitive ; Cell Line ; Chloroquine/PHARMACODYNAMICS ; Deoxyglucose/METABOLISM ; Insulin/ANALOGS & DERIVATIVES/METABOLISM/*PHARMACODYNAMICS ; Kinetics ; Osteoblasts/DRUG EFFECTS/*METABOLISM ; Photochemistry ; Proinsulin/METABOLISM ; Rats ; Receptors, Insulin/*METABOLISM ; Sarcoma, Osteogenic ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - Endocrinology 1986 Oct;119(4):1786-92 4 UI - 87004297 AU - Singer EA ; Mitra SP ; Carraway RE TI - Plasma protein(s) yields met-enkephalin-related peptides in near-micromolar concentrations when treated with pepsin. AB - Treatment of animal and human plasmas with pepsin yielded large quantities of immunoreactive methionine5-enkephalin (i-met-ENK). The concentrations measured after pepsin treatment were 0.1-0.5 microM, about 1000 times the normal circulating level of i-met-ENK (0.03-0.3 nM). The reaction was shown to be time and pH dependent and to involve the action of pepsin on a protein(s) of about 65,000 mol wt. Pepsin-generated i-met-ENK from rat plasma gave three major peaks during reverse phase HPLC, one of which (approximately 25% of the total) coeluted with methionine5-enkephalin sulfoxide and also completed in a radioreceptor assay for opiate-related substances. In addition, this material produced met-ENK-like effects on vascular permeability in rat skin and inhibited electrically induced contractions of the isolated guinea pig ileum in a naloxone-sensitive manner. The plasma substrate(s) that yielded i-met-ENK was distinguished from adrenal proenkephalins, since partially purified plasma substrate(s) did not liberate i-met-ENK upon digestion with trypsin and carboxypeptidase B. Although it is possible that these peptides differ from met-ENK in amino acid sequence, the results presented here suggest that met-ENK-related substances might be formed physiologically by the action of a pepsin-related processing enzyme(s) on plasma substrate(s). Such a mechanism would be analogous to that used in the renin-angiotensin system. MH - Adult ; Animal ; Biological Assay ; Blood Proteins/*METABOLISM ; Capillary Permeability/DRUG EFFECTS ; Cats ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Dogs ; Enkephalin, Methionine/ *BLOOD ; Guinea Pigs ; Human ; Hydrogen-Ion Concentration ; Kinetics ; Muscle Contraction/DRUG EFFECTS ; Pepsin/*PHARMACODYNAMICS ; Radioimmunoassay ; Radioligand Assay ; Rats ; Support, U.S. Gov't, P.H.S. ; Swine SO - Endocrinology 1986 Oct;119(4):1527-33 5 UI - 86285423 AU - Fischer-Colbrie R ; Diez-Guerra J ; Emson PC ; Winkler H TI - Bovine chromaffin granules: immunological studies with antisera against neuropeptide Y, [Met]enkephalin and bombesin. AB - Antisera against neuropeptide Y, [Met]enkephalin and bombesin were used for characterizing the immunoreactive material in subcellular fractions of bovine adrenal medulla. Neuropeptide Y was identified by high performance liquid chromatography and by immunoblotting. Subcellular fractionation established that neuropeptide Y is present in chromaffin granules. During stimulation of the adrenal it is released concomitantly with catecholamines. The soluble proteins of chromaffin granules contain 1.9 micrograms neuropeptide Y/mg protein which gives 429 copies of neuropeptide Y for a single granule. In two-dimensional immunoblots two peptides of the same molecular size, but with differing pI (6.4 and 7.3) react with the antiserum against neuropeptide Y. There was no evidence for the presence of a larger neuropeptide Y precursor in chromaffin granules. On the other hand, larger enkephalin-containing peptides could be detected by immunoblotting. The subcellular distribution of these enkephalin precursors differed. The larger peptides (23.3 and 18.2 kD) were more concentrated in lighter granules when compared to the smaller precursors (12.6 and 8.6 kD) which is consistent with proteolytic processing of these peptides during granule maturation. An antiserum against bombesin reacts in immunoblots with the chromogranin B family. This study further illustrates that chromaffin granules contain a complex mixture of neuropeptide-immunoreactive material. The combination of immunoblotting with subcellular fractionation appears as a useful tool to characterize these peptides and their precursors. MH - Adrenal Medulla/ANALYSIS/ULTRASTRUCTURE ; Animal ; Antigen-Antibody Complex ; Bombesin/*ANALYSIS ; Cattle ; Cell Fractionation/METHODS ; Centrifugation, Density Gradient ; Chromaffin Granules/ANALYSIS/ *ULTRASTRUCTURE ; Chromaffin System/*ULTRASTRUCTURE ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Enkephalin, Methionine/*ANALYSIS ; Nerve Tissue Proteins/*ANALYSIS ; Support, Non-U.S. Gov't SO - Neuroscience 1986 May;18(1):167-74 6 UI - 86285331 AU - Pruss RM ; Mezey E ; Forman DS ; Eiden LE ; Hotchkiss AJ ; DiMaggio DA ; O'Donohue TL TI - Enkephalin and neuropeptide Y: two colocalized neuropeptides are independently regulated in primary cultures of bovine chromaffin cells. AB - We have found that Neuropeptide Y is colocalized with enkephalin in bovine adrenal chromaffin cells. The two peptides can be found in the same granules in those cells where they coexist. These cells correspond to the adrenergic subpopulation of chromaffin cells since they contain the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase. Despite their coexistence, production of the two peptides is independently regulated. Enkephalin levels are doubled after nicotinic depolarization (which increases enkephalin synthesis) or after treatment with reserpine (which increases enkephalin precursor processing). Neither of these treatments, acting by different mechanisms, has any effect on the levels of Neuropeptide Y. MH - Adrenal Glands/CYTOLOGY ; Animal ; Cattle ; Cells, Cultured ; Chromaffin Granules/METABOLISM ; Chromaffin System/CYTOLOGY/*PHYSIOLOGY ; Enkephalins/*PHYSIOLOGY ; Histocytochemistry ; Immunochemistry ; Microscopy, Electron ; Microscopy, Fluorescence ; Nerve Tissue Proteins/ *PHYSIOLOGY ; Support, U.S. Gov't, P.H.S. SO - Neuropeptides 1986 May-Jun;7(4):315-27 7 UI - 86250701 AU - Andrews PC ; Hawke DH ; Lee TD ; Legesse K ; Noe BD ; Shively JE TI - Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide. AB - The principal products derived from in vivo processing of anglerfish preproglucagon II were isolated and their structures determined. The structures were confirmed by a combination of automated Edman degradation, amino acid analysis, and fast atom bombardment mass spectrometry. The peptide corresponding to anglerfish preproglucagon II-(22-49) (numbering from the amino terminus of preproglucagon) was isolated intact and defines the site of signal cleavage to be between Gln-21 and Met-22. Glucagon from the anglerfish preproglucagon gene II was found to correspond to preproglucagon II-(52-80) (numbering from the amino terminus). Three forms of a glucagon-like peptide derived from preproglucagon II were also isolated. The structure of the longest form was consistent with the sequence of preproglucagon II-(89-122) deduced from the cDNA, His-Ala-Asp-Gly-Thr-Tyr-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Asp-Gln-Ala- Ala-Lys-Asp-Phe-Val-Ser-Trp-Leu-Lys-Ala-Gly-Arg-Gly-Arg-Arg-Glu. The carboxyl-terminal portion deduced from the cDNA remains intact in this form. A second form, preproglucagon II-(89-119) appears to result from proteolytic processing of the major form at the two adjacent arginine residues occurring at the carboxyl terminus. This second form has a glycine residue at its carboxyl terminus and is processed to the third form (preproglucagon II-(89-118)) which contains a carboxyl-terminal arginineamide. Radiolabeling studies in primary tissue culture support the observation that glucagon (preproglucagon II-(52-80], preproglucagon II-(89-122), and preproglucagon II-(89-119) are products of proglucagon processing in vivo. MH - Amino Acid Sequence ; Amino Acids/ANALYSIS ; Animal ; Base Sequence ; Chromatography, High Pressure Liquid ; DNA/ANALYSIS ; Fishes ; Glucagon/ ANALYSIS/GENETICS/ISOLATION & PURIFICATION/*METABOLISM ; Human ; Protein Precursors/ANALYSIS/GENETICS/ISOLATION & PURIFICATION/*METABOLISM ; Rats ; Species Specificity ; Spectrum Analysis, Mass ; Support, U.S. Gov't, P.H.S. SO - J Biol Chem 1986 Jun 25;261(18):8128-33 8 UI - 86313716 AU - White JD ; Gall CM ; McKelvy JF TI - Proenkephalin is processed in a projection-specific manner in the rat central nervous system. AB - The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields. MH - Animal ; Brain/*METABOLISM ; Corpus Striatum/METABOLISM ; Enkephalins/ BIOSYNTHESIS/*METABOLISM ; Hippocampus/METABOLISM ; Hypothalamus/ METABOLISM ; Male ; Protein Precursors/*METABOLISM ; Rats ; Rats, Inbred Strains ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S. SO - Proc Natl Acad Sci USA 1986 Sep;83(18):7099-103 9 UI - 86313658 AU - Thim L ; Hansen MT ; Norris K ; Hoegh I ; Boel E ; Forstrom J ; Ammerer G ; Fiil NP TI - Secretion and processing of insulin precursors in yeast. AB - A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing. MH - Amino Acid Sequence ; Chromatography, High Pressure Liquid ; Glucagon/ BIOSYNTHESIS ; Human ; Insulin/BIOSYNTHESIS ; Proinsulin/ANALYSIS/ ISOLATION & PURIFICATION/*METABOLISM ; Saccharomyces Cerevisiae/ *METABOLISM SO - Proc Natl Acad Sci USA 1986 Sep;83(18):6766-70 10 UI - 86295693 AU - Demmer W ; Brand K TI - A putative opioid-peptide processing activity in enriched Golgi fraction from rat brain. AB - A Golgi enriched fraction from rat brain was prepared. The preparation has no carboxypeptidase activity and is not contaminated with cytosol, mitochondria and lysosomes as judged by marker enzyme activities for these constituents. Associated with the Golgi membranes a putative opioid peptide processing activity was demonstrated, which acts on Dynorphin 1-13, alpha- and beta-Neoendorphin. The enzyme cleaves the bond between the paired basic residues, releasing Leucine-enkephalin-Arg6. The activity has a pH-optimum around 9 and is inhibited by serine-protease inhibitors. Intracellular location and substrate specificity suggest that this endopeptidase activity may be involved in proenkephalin processing. MH - Amino Acid Sequence ; Animal ; Brain/*ULTRASTRUCTURE ; Chromatography, High Pressure Liquid ; Endorphins/*METABOLISM ; Golgi Apparatus/ *ENZYMOLOGY ; Hydrogen-Ion Concentration ; Intracellular Membranes/ ENZYMOLOGY ; Peptide Peptidohydrolases/*METABOLISM ; Protease Inhibitors/ PHARMACODYNAMICS ; Rats ; Rats, Inbred Strains ; Substrate Specificity ; Support, Non-U.S. Gov't SO - Biochem Biophys Res Commun 1986 Jul 16;138(1):356-62 11 UI - 86233291 AU - Lawrence JC Jr ; Hiken JF ; Inkster M ; Scott CW ; Mumby MC TI - Insulin stimulates the generation of an adipocyte phosphoprotein that is isolated with a monoclonal antibody against the regulatory subunit of bovine heart cAMP-dependent protein kinase. AB - Incubating 32P-labeled fat cells with insulin increased by as much as 80-fold the amount of 32Pi in a soluble species of apparent Mr 62,000. This species, designated isp62, was specifically immunoprecipitated from cellular extracts with a monoclonal antibody against the type II regulatory subunit (RII) of cAMP-dependent protein kinase. Fat-cell RII, purified from extracts with cAMP-Sepharose or labeled with 8-azido [32P]cAMP, had an apparent Mr 51,000. Peptide mapping indicated that isp62 and adipocyte RII were different proteins. When cells were metabolically labeled with [35S]methionine, insulin stimulated the appearance of 35S-labeled isp62, indicating that the hormonal effect involves generation of the protein. The insulin-induced increase in isp62 could be observed within 1 min, occurred with physiological concentrations of the hormone, and was rapidly reversible. The increase in isp62 was unaffected by cycloheximide, indicating that insulin stimulates the posttranslational processing of a precursor, rather than de novo synthesis of the protein. MH - Adipose Tissue/ENZYMOLOGY/*METABOLISM ; Animal ; Antibodies, Monoclonal/ DIAGNOSTIC USE ; Antigenic Determinants ; Cattle ; Cross Reactions ; Insulin/*PHARMACODYNAMICS ; Molecular Weight ; Phosphoproteins/IMMUNOLOGY/ *METABOLISM ; Phosphorylation ; Protein Kinases/IMMUNOLOGY/METABOLISM ; Protein Processing, Post-Translational ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Proc Natl Acad Sci USA 1986 Jun;83(11):3649-53 12 UI - 86132678 AU - Kapcala LP TI - Discordant changes between immunoreactive ACTH and beta-endorphin in rat brain and pituitary during early development. AB - Discordant changes in brain concentrations of immunoreactive (IR-) adrenocorticotropic hormone (ACTH) or beta-endorphin, peptides derived from pro-opiomelanocortin (POMC), have been reported during the latter part of development and with subsequent aging. These changes were believed to be due to age-related alterations in the regulation of metabolism of POMC-related peptides. However, concentrations of IR-ACTH and IR-beta-endorphin have not been simultaneously studied during early development when many changes in brain development and behavior are occurring. To determine whether concentrations of IR-ACTH and IR-beta-endorphin change during early development and whether changes are discordant, concentrations of IR-ACTH and IR-beta-endorphin were measured in several brain regions and pituitary in rats at 10, 29 and 80 days after birth. Whereas IR-ACTH in extrahypothalamic brain increased at day 29, and decreased at day 80, it did not change in hypothalamus and pituitary. Between day 10 and 29, IR-beta-endorphin rose in all brain regions, but subsequent changes in different tissues were variable at day 80. Because concentration changes can be mediated by alterations in one or more regulatory mechanisms, chromatographic profiles of hypothalamus and amygdala and molar ratios of all tissues were subsequently studied to give further insight into the mechanisms of the discordant changes. Molecular profiles of hypothalamic IR-ACTH and amygdalar IR-beta-endorphin exhibited lesser proportions of large molecular forms and greater proportions of ACTH and beta-endorphin during development. Molar ratios of IR-ACTH/IR-beta-endorphin in all tissues and ratios of ACTH1-39/beta-endorphin in hypothalamus and amygdala changed during development. Conclusions: (1) changes in IR-ACTH and IR-beta-endorphin occur in rat pituitary and several brain regions at different ages during early development and are frequently discordant; (2) molecular profiles suggested that the activity of processing enzymes for POMC and its derivatives vary in hypothalamus and amygdala with respect to type of derivative, brain region, and developmental age; and (3) changes in some molecular profiles and changes in molar ratios of IR-ACTH/IR-beta-endorphin and ratios of ACTH1-39/beta-endorphin suggest that changes in processing and possibly changes in neurosecretion and degradation contribute to concentration changes independent of possible alterations in biosynthesis of POMC. MH - Adrenocorticotropic Hormone/*ANALYSIS ; Age Factors ; Animal ; Brain/ GROWTH & DEVELOPMENT ; *Brain Chemistry ; Chromatography, Gel ; Comparative Study ; Endorphins/*ANALYSIS ; Female ; Male ; Pituitary Gland/*ANALYSIS/GROWTH & DEVELOPMENT ; Radioimmunoassay ; Rats SO - Brain Res 1986 Feb 5;364(2):350-9 13 UI - 86165392 AU - Gansler TS ; Smith RM ; Jarett L TI - Cell type-specific variability of bacitracin's effects on insulin binding and intracellular accumulation. AB - Bacitracin is known to inhibit proteolytic degradation of insulin and several other peptide hormones. Previous work with isolated rat adipocytes showed that bacitracin blocked insulin degradation by the plasma membrane and, even in the absence of detectable insulin degradation, bacitracin increased insulin binding by decreasing the rate of insulin dissociation. The present study examined the effects of bacitracin on insulin binding and degradation and on levels of intracellular insulin in a variety of cell types. Bacitracin inhibited insulin degradation in all cell types. Maximal inhibition varied from 70% (H4IIEC3 hepatoma cells) to 95% (rat adipocytes); concentrations giving half-maximal inhibition varied from 25 microM (3T3-A31 fibroblasts) to 250 microM (H4IIEC3). Dose-response curves showed three distinctive effects on insulin binding: dose-dependent stimulation (rat adipocytes), a biphasic curve with slight stimulation at low doses and inhibition at concentrations greater than 50 microM (human fibroblasts, H4IIEC3, and 3T3-L1 adipocytes), or dose-dependent inhibition of binding (3T3-L1 preadipocytes and 3T3-A31 fibroblasts). The intracellular accumulation of insulin rat adipocytes was not affected by bacitracin but was decreased in all other cell types. These data illustrated type-specific variability in the effects of bacitracin on insulin processing resulting from cellular heterogeneity either in processing insulin or in response to bacitracin, or both, and suggest that insulin binding studies performed in the presence of bacitracin can be biased. MH - Adipose Tissue/CYTOLOGY/DRUG EFFECTS/METABOLISM ; Animal ; Bacitracin/ *PHARMACODYNAMICS ; Cell Line ; Dose-Response Relationship, Drug ; Fibroblasts/DRUG EFFECTS/METABOLISM ; Human ; Insulin/*METABOLISM ; Liver Neoplasms, Experimental/METABOLISM ; Male ; Mice ; Mice, Inbred BALB C ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Temperature SO - Diabetes 1986 Apr;35(4):392-7 14 UI - 86159816 AU - Juul SM ; Neffe J ; Evans JL ; Jones RH ; S:onksen PH ; Brandenburg D TI - Demonstration that the insulin receptor undergoes an early structural modification following insulin binding. AB - Processing of the insulin receptor by hepatocytes was studied using a 125I-labelled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350 000 molecular weight) and binding (alpha) subunit (135 000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140 000 (non-reduced) which on reduction generated species of 100 000 and 68 000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event. MH - Animal ; Cell Membrane/METABOLISM ; In Vitro ; Insulin/*ANALOGS & DERIVATIVES/METABOLISM ; Kinetics ; Liver/METABOLISM ; Macromolecular Systems ; Molecular Weight ; Rats ; Receptors, Insulin/ISOLATION & PURIFICATION/*METABOLISM ; Support, Non-U.S. Gov't SO - Biochim Biophys Acta 1986 Apr 14;856(2):320-4 15 UI - 86136565 AU - Birch NP ; Davies AD ; Christie DL TI - Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal medullary chromaffin granule membranes by immunoblotting. AB - An antiserum which recognizes high molecular mass enkephalin-containing proteins was used to compare proenkephalin intermediates in both the soluble and membrane components of bovine adrenal chromaffin granules by immunoblotting. While a range of molecular mass forms were identified in the soluble lysate the major form in the membranes corresponded to a 27-kDa enkephalin-containing protein. Enzymic digestion of bands of 27-kDa material and quantitation of the enkephalin released showed that 22% of this material was membrane-associated. High concentrations of chaotropic agents were required to extract this material from the membranes. Association of hormone and neuropeptide precursors with membrane components may be important for targeting of precursors to secretory granules or correct processing. MH - Adrenal Medulla/*ULTRASTRUCTURE ; Animal ; Carboxypeptidases/METABOLISM ; Cattle ; Chromaffin Granules/*ANALYSIS ; Chromaffin System/*ANALYSIS ; Collodion ; Comparative Study ; Electrophoresis, Polyacrylamide Gel ; Enkephalins/*ANALYSIS/METABOLISM ; Immunologic Technics ; Intracellular Membranes/ANALYSIS ; Molecular Weight ; Protein Precursors/*ANALYSIS/ METABOLISM ; Support, Non-U.S. Gov't ; Trypsin/METABOLISM SO - FEBS Lett 1986 Mar 3;197(1-2):173-8 16 UI - 86130616 AU - Clamagirand C ; Camier M ; Boussetta H ; Fahy C ; Morel A ; Nicolas P ; Cohen P TI - An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues. AB - The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo. MH - Amino Acid Sequence ; Animal ; Catalysis ; Cattle ; Chromatography, High Pressure Liquid ; Cytoplasmic Granules/*ENZYMOLOGY ; Neurophysins/ *METABOLISM ; Oxytocin/*METABOLISM ; Peptide Fragments/METABOLISM ; Peptide Peptidohydrolases/*ISOLATION & PURIFICATION/METABOLISM ; Pituitary Gland, Posterior/*ENZYMOLOGY ; Protein Precursors/*METABOLISM ; Substrate Specificity ; Support, Non-U.S. Gov't SO - Biochem Biophys Res Commun 1986 Feb 13;134(3):1190-6 17 UI - 86108875 AU - Maret GE ; Fauch:ere JL TI - The prohormone processing activity is enriched in a low-density subpopulation of chromaffin granules. AB - Bovine adrenomedullary chromaffin granules can be separated into two subpopulations by differential centrifugation. The subpopulation which sediments at the interface of two sucrose layers, 1.6 and 1.8 M respectively, is found to be enriched about 10-times in prohormone processing activity, as measured by in vitro degradation of synthetic peptide substrates. The enhanced proteolytic activity is not due to lysosomal contaminations which are very low and only slightly increased in the more active fraction. The low density of the enriched subpopulation suggests that we are dealing with immature granules. The physiological implications of this finding are discussed. Furthermore, the enriched fraction can be used as the starting material for the isolation of proenkephalin processing enzymes. MH - Adrenal Medulla/*METABOLISM/ULTRASTRUCTURE ; Animal ; Cattle ; Cell Fractionation/METHODS ; Chromaffin Granules/*METABOLISM ; Chromaffin System/*METABOLISM ; Enkephalins/*METABOLISM ; Lysosomes/ENZYMOLOGY ; Oligopeptides/METABOLISM ; Peptide Peptidohydrolases/METABOLISM ; Protein Precursors/*METABOLISM ; Support, Non-U.S. Gov't SO - FEBS Lett 1986 Jan 20;195(1-2):258-60 18 UI - 86081510 AU - Lebouille JL ; Burbach JP ; De Kloet ER ; Rommerts FF TI - gamma-Endorphin-generating endopeptidase: distribution in body tissues and cellular localization in rat testis. AB - The Leu17-Phe18 bond of beta-endorphin is cleaved by a specific endopeptidase that generates the biologically active peptide gamma-endorphin. gamma-Endorphin-generating endopeptidase (gamma EGE) activity was determined by a radiometric assay, using as substrate a radioactively labeled, N- and C-terminally protected pentapeptide: Ac-Val-Thr-Leu-Lys( [14C]CH3)2-NHCH3, a derivative of beta-endorphin-(15-19). Here we report the tissue distribution of gamma EGE activity and its cellular localization in the testis. gamma EGE activity was present in the cytosolic fraction of most tissues. Highest specific activity occurred in the testis, ovary, and the uterus (10-16 nmol X mg protein-1 X h-1). In testis highest specific gamma EGE activity was found in the tubules (42 nmol X mg protein-1 X h-1) and lowest in Leydig cells (8 nmol X mg protein-1 X h-1). Further fractionation of the tubules showed that the germinal cell fraction had a higher specific activity (24 nmol X mg protein-1 X h-1) than the Sertoli cell fraction (8 nmol X mg protein-1 X h-1). In testis depleted of the germinal cells by prenatal irradiation of the rat or hypophysectomy, specific activity of gamma EGE activity decreased 50-fold and 4-fold, respectively. In testis depleted of Leydig cells by treatment of rats with ethane dimethyl sulfonate, specific gamma EGE activity did not decrease. Adrenalectomy had no effect on the enzyme activity. The results suggest that the germinal cells are sites of processing of beta-endorphin into alpha- and gamma-endorphins. It is concluded that 1) gamma EGE activity is widely distributed in tissues; 2) highest gamma EGE activity is located in reproductive tissues; and 3) in the testis gamma EGE activity is mainly associated in the germinal cells. MH - Adrenalectomy ; Animal ; Comparative Study ; Cytosol/ENZYMOLOGY ; Endorphins/*METABOLISM ; Female ; Hypophysectomy ; Leydig Cells/ ENZYMOLOGY ; Male ; Methanesulfonates/PHARMACODYNAMICS ; Oligopeptides/ METABOLISM ; Ovary/ENZYMOLOGY ; Peptide Peptidohydrolases/*METABOLISM ; Rats ; Rats, Inbred Strains ; Sertoli Cells/ENZYMOLOGY ; Support, Non-U.S. Gov't ; Testis/DRUG EFFECTS/*ENZYMOLOGY/RADIATION EFFECTS ; Tissue Distribution ; Uterus/ENZYMOLOGY SO - Endocrinology 1986 Jan;118(1):372-6