Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In contrast to other studies, our results demonstrate that low concentration of trypsin degrades a high proportion of proteolipid from CNS myelin. The Wolfgram protein and BP are vulnerable and completely lost on trypsinolysis, perhaps accounting for some of the peptides retained by the myelin. In PNS myelin, the major PO protein, a hydrophobic glycoprotein, is readily degraded to a stable 18,000--19,000 molecular weight unit, referred to as TPO protein, still retaining the carbohydrate unit which probably exists as a nonasaccharide grouping. Production of the TPO glycoprotein results from cleavage of a lysinyl-methionine or arginyl-methionine linkage probably found approximately 80--100 residues from the NH2-terminal isoleucine of the PO molecule. This linkage must be especially accessible to trypsin since the TPO protein is also generated in high yield when isolated PO protein is treated with trypsin in solution for 0.5 hours. Further incubation for 24 hours fully degrades the TPO protein to over 20 tryptic peptides, shown by peptide mapping, unlike the situation in myelin where the TPO unit is stable and resists further proteolysis. The TPO unit is also produced when PO protein is treated with BrCN. The PO protein contains 3 methionine residues but presumably the methionine residue in the trypsin-sensitive region is crucial; cleavage leads to the same TPO unit minus NH2-terminal methionine. Another methionine residue also exists in the TPO protein but it may be resistant to BrCN cleavage or else occupy a near-end position. Other proteins were also identified on PAGE of trypsinized PNS myelin: albumin, P2 protein, and PO protein. Albumin and P2 protein were identified in the acidic extract by reaction with specific antibody. The PO protein was isolated; it moved similarly to standard protein on SDS-PAGE and gave the appropriate amino acid analysis. However, it cannot be determined at this time whether a portion of these proteins remains because they are partially inaccessible to trypsin, or else are slightly attacked and thus represent early stages of trypsinolysis. The P2 protein of trypsinized myelin appears to migrate slightly faster than standard P2 protein on PAGE. Further work should clarify this point. Amino acid analysis and sequence data show that the PO protein is particularly hydrophobic, very likely existing in PNS myelin as an amphipathic molecule which penetrates the bilayer but which has a hydrophilic portion exposed. It is this hydrophilic region that contains much lysine, particularly the crucial lysinyl-methionine linkage, that is so trypsin-sensitive. Determination of the amino acid sequence of terminal portions of the isolated PO and TPO proteins serves to firmly establish the PO protein as a unique entity probably exclusive to PNS myelin. It can be concluded that the study of trypsin activity toward PNS myelin has made possible a new understanding of how proteins are positioned in the membrane, and provided valuable insight into the PO protein.
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PMID:The action of trypsin on central and peripheral nerve myelin. 69 76

2',3' cyclic nucleotide-3'-phosphodiesterase (CNP) is phosphorylated in the peripheral nervous system after immunoprecipitation of myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Only radiolabeled phosphoserine was detected after partial acid hydrolysis of immunoprecipitated CNP. Two major phosphopeptides were resolved by two dimensional electrophoresis-chromatography after digestion with trypsin of CNP phosphorylated in the nerve slices. Phosphorylation of CNP was not stimulated a) by forskolin in the nerve slices and b) after incubation of purified nerve myelin with cAMP. However, CNP phosphorylation was increased after incubation of PNS myelin with catalytic unit of protein kinase A. Phosphorylation of the central nervous system myelin CNP was dramatically stimulated by cAMP. These results suggest that PKA may be absent from peripheral nerve myelin or CNP may not be accessible to this enzyme in the PNS. Incubation of nerve slices with phorbol 12 myristate-13-acetate caused a marked increase in the phosphorylation of CNP. These results provide strong evidence that CNP is phosphorylated in the PNS and its phosphorylation in vivo is in all probability regulated by protein kinase C.
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PMID:2',3'cyclic nucleotide-3'-phosphodiesterase in peripheral nerve myelin is phosphorylated by a phorbol ester-sensitive protein kinase. 216 8

The sensory neurons of the olfactory epithelium form an anatomically uniform population but are differentially excited by odorants. We have discovered an unexpected biochemical heterogeneity within this population that extends to its axonal projection onto the olfactory bulb. This heterogeneity is recognized by a newly generated monoclonal antibody, designated RB-8, that differentially stains the primary olfactory projection in rats and divides it into 2 nonoverlapping zones. With light-microscopic immunohistochemistry, RB-8 densely labels the fascicles of the olfactory nerve from the ventral and lateral parts of the olfactory epithelium, where there is also some epithelial staining. This area, which we designate RB-8-positive, comprises about two-thirds of the epithelial sheet. RB-8 labeling of the other third of the epithelium, which includes the dorsal recess and medial tips of the dorsal turbinals, is not detectable, and the fascicles from these RB-8-negative areas are only weakly stained. These RB-8-negative areas form a contiguous zone on flattened maps of the epithelial sheet. In the olfactory bulb, RB-8 staining of the glomeruli in the ventrolateral part is correspondingly dense, while that in the dorsomedial glomeruli is undetectable or very light. In the labeled glomeruli, the RB-8 staining is precisely coextensive with anti-olfactory marker protein staining, which serves as a marker for the olfactory axons and terminals. In addition, knife-cut lesions of the olfactory nerve totally eliminate the RB-8 staining in the glomeruli where the destruction of the olfactory terminals is complete. There is also a good correlation between the staining patterns in the bulb and epithelium and what is known from tract-tracing studies of the arrangement of the axonal projection of the epithelium onto the bulb. This evidence strongly suggests that, in the olfactory nerve and glomeruli, RB-8 stains the olfactory axons and their terminals. A survey of the CNS and peripheral tissues demonstrates that staining with RB-8 is nervous system-specific; not all components of the CNS and PNS are stained. The antigen recognized by RB-8 was characterized in immunoblots and by use of a direct radioimmunoassay (RIA) which assessed binding of 125I-RB-8. With this assay, the RB-8 binding sites in whole brain are shown to be membrane-associated, saturable, immunologically specific for RB-8, and trypsin-sensitive. In SDS-PAGE immunoblots of membrane proteins, the antigen in rat forebrain and in the olfactory nerve is a protein of 125 kDa Mr, which comigrates in mixtures of membranes from the 2 sources.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The primary olfactory projection has two chemically distinct zones. 377 38

P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P0 was labeled in rat sciatic nerve slices with [3H]palmitic acid and subsequently treated with various reagents. The protein-bound palmitate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydroxylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM NaBH4 with the concomitant production of [3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [14C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [14C]carboxyamidomethylated P0 was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmembrane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.
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PMID:Identification of the palmitoylation site in rat myelin P0 glycoprotein. 750 74

The distribution of anionic microdomains has been described in cerebral vessels and more recently in capillaries of peripheral nerve. Evidence is accumulating that these sites play a role in the barrier function of vascular endothelia in the PNS and CNS. The chemical nature of anionic sites has been at least partly determined for cerebral vessels but not in peripheral nerve. This study reports our preliminary investigations to determine the nature of endothelial anionic sites in sciatic nerve. The effects of digestion of ultra-thin sections of nerve with a battery of proteolytic and glycolytic enzymes (papain, trypsin, proteinase K, hyaluronidase, heparinase, heparitinase and neuraminidase) on the distribution of anionic sites was determined using the label, cationic colloidal gold. Papain, a proteolytic enzyme of broad specificity, succeeded in removing the majority of cationic colloidal gold-binding sites on the luminal surface of vascular endothelia. In contrast trypsin and proteinase K were less effective, reflecting their narrower specificity. Hyaluronidase, heparinase and heparitinase did not significantly affect cationic colloidal gold-labelling. However, a considerable reduction in cationic colloidal gold-binding occurred following neuraminidase digestion. These results suggest that, as in cerebral vessels, sialic acid-containing glycoproteins are largely responsible for the negatively charged domains on the luminal membrane of endothelial cells in peripheral nerve.
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PMID:Molecular characterization of anionic sites on the luminal front of endoneurial capillaries in sciatic nerve. 817 16