Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have shown that encephalitogenic, myelin basic protein (MBP)-specific CD4+ T cells can cause astrocyte and oligodendrocyte detachment in vitro. Similar processes may damage the central nervous system (CNS) in vivo by causing disorganization and destruction of brain tissue structure. The finding that 'bystander' allogeneic fibrosarcoma cells were detached by MBP-specific CD4+ T cells only when syngeneic astrocytes were present, suggested that a soluble cell-detaching factor (CDF) is released during the specific astrocyte-CD4+ effector interaction. In this study, CDF activity was detected in the supernatants of MBP-reactive CD4+ T cells incubated with concanavalin A or astrocytes. Lymphocyte-induced astrocyte lysis, but not detachment, was inhibited by the protein synthesis inhibitors, cycloheximide and puromycin, indicating that de novo protein synthesis is required for this type of lysis, but not for detachment. Astrocyte detachment was not inhibited, but rather augmented, by the trypsin inhibitors, soybean trypsin inhibitor (SBTI) and alpha-1-antitrypsin (alpha 1), suggesting that the CDF activity is not due to tryptic serine proteases, although it may be protease susceptible. The heparanase inhibitor, heparin, inhibited CD4+ T cell-mediated astrocyte detachment at low doses, but augmented detachment at higher doses, indicating that detaching activity is not due to heparanases. The actin microfilament disrupting agent, cytochalasin B (CB), inhibited astrocyte detachment induced by MBP-specific CD4+ T cells. CB pretreatment of the target astrocytes, but not of the effector CD4+ T cells, inhibited astrocyte detachment, suggesting that the integrity of the target's, but not the effector's, cytoskeleton is required for astrocyte detachment. The results herein suggest that during astrocyte interaction with MBP-specific CD4+ T cells, soluble factors are released that trigger an intrinsic astrocyte detachment mechanism.
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PMID:Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). II. Characteristics of astrocyte detachment mediated by myelin basic protein (MBP) specific CD4+ T lymphocytes. 138 28

The structural characteristics of myelin basic protein (MBP) involved in protein-protein and protein-lipid interactions were investigated. Rabbit MBP could bind calmodulin (CaM) in the presence of Ca2+ to form a complex that remained undissociated in 8 M urea. However, no tight complex formation was observed when the divalent cation was absent. These results suggest that MBP may contain a hydrophobic domain similar to those in the other well-characterized CaM-binding proteins. The stoichiometry of calmodulin binding to MBP was approximately 1:1. Prior limited proteolysis of MBP with trypsin abolished the formation of the MBP-CaM complex, indicating that the entire MBP polypeptide may be involved in the recognition of the hydrophobic clefts in CaM. MBP also formed tight complexes with gangliosides, but the presence of Ca2+ was not required. Binding of gangliosides to MBP-CaM complex released CaM from the complex. The ganglioside-binding sites in MBP were determined after trisecting the protein at two glutamic acid residues with Staphylococcus aureus V8 protease. Subsequent binding studies revealed that a 9.5-kDa polypeptide, which may correspond to the NH2-terminal domain (residues 1-83) of MBP, had higher affinity for the binding of lucifer yellow CH-labeled GM1 than did the other two polypeptides, of apparent molecular mass (Mr) 5,500 and 4,500, respectively. Among the various proteins in purified guinea pig brain myelin, synaptosomes, and synaptosomal membranes, MBP was found to have the highest affinity in binding lucifer yellow CH-GM1.
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PMID:Myelin basic protein: interaction with calmodulin and gangliosides. 169 93

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.
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PMID:Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. 170 Sep 79

Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S-16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein.
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PMID:Expression of the myelin-associated glycoprotein in cultures of immortalized Schwann cells. 170 58

Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. Protein sequence analysis of bFGF isolated from tissue sources initially established that it is composed of 146 amino acids (apparent Mr 18,000). More recently larger apparent molecular weight forms have been identified and partially characterized. In addition, these high molecular weight forms (apparent Mr 22,000 and 25,000) have been shown to localize preferentially to nuclear fractions of transfected cells. In this report we demonstrate that the higher molecular weight, amino terminally extended forms of bFGF contain methylated arginine residues. The demonstration is based on 1) amino acid sequence analysis of a protein known to contain methylated arginine (myelin basic protein) and a comparison with amino acid sequence analysis of trypsin-derived fragments of the high molecular weight bFGF purified from guinea pig brain and 2) the ability to label in vivo the high molecular weight forms of bFGF with S-adenosyl-L-(methyl-3H)-methionine, the substrate of arginine-protein methylase I. These results are suggestive of a role of arginine methylation in directing nuclear localization of certain forms of bFGF.
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PMID:Direct evidence for methylation of arginine residues in high molecular weight forms of basic fibroblast growth factor. 171 85

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.
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PMID:Calcium-stimulated proteolysis in myelin: evidence for a Ca2+-activated neutral proteinase associated with purified myelin of rat CNS. 240 35

A reversed-phase high-performance liquid chromatography (HPLC) system was developed to obtain individual tryptic peptides of myelin basic protein (BP). Because of the similar charge and hydrophobicity of some of the tryptic peptides of the whole protein, several of these were not clearly separated by a single HPLC system. Therefore, the BP was first cleaved specifically between residues 97 and 98 with thrombin, and the two resulting fragments were separated by ion-exchange chromatography. When the thrombic fragments were digested with trypsin separately and subjected to HPLC, all of the peptides were satisfactorily separated. Elution times of all of the tryptic peptides of human BP were established. Differences among homologous peptides, derived from different mammalian BPs, were readily detected from their elution patterns inasmuch as a change in a single amino acid residue was usually sufficient to cause a shift in the retention time of the peptide. An amino acid difference detected by a peak shift could be confirmed by amino acid analysis. The technique has been used to isolate short peptides of rabbit, monkey, porcine, bovine, and human BP for sequence analysis.
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PMID:Isolation of tryptic peptides of myelin basic protein by reversed-phase high-performance liquid chromatography. 241 47

The substrate specificity of protein kinase C was studied and compared with that of cyclic AMP-dependent protein kinase (protein kinase A) by using bovine brain myelin basic protein as a model substrate. This basic protein was phosphorylated at multiple sites by both of these protein kinases. In this analysis, the basic protein was thoroughly phosphorylated in vitro with [gamma-32P]ATP and each protein kinase, and then digested with trypsin. The resulting radioactive phosphopeptides were isolated by gel filtration followed by high performance liquid chromatography on a reverse-phase column. Subsequent amino acid analysis and/or sequential Edman degradation of the purified phosphopeptides, together with the known primary sequence of this protein, revealed that Ser-46 and Ser-151 were specifically phosphorylated by protein kinase C, whereas Thr-34 and Ser-115 were phosphorylated preferentially by protein kinase A. Both kinases reacted with Ser-8, Ser-11, Ser-55, Ser-110, Ser-132, and Ser-161 at various reaction velocities. Contrary to protein kinase A, protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine. The seryl residues that are phosphorylated commonly by these two protein kinases have basic amino acids at both the amino- and carboxyl-terminal sides. These results provide some clues to understanding the rationale that these kinases may show different but sometimes similar functions depending on the structure of target phosphate acceptor proteins.
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PMID:Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. 241 24

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.
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PMID:Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid. 242 99

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.
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PMID:Preparation and characterization of unilamellar myelin vesicles. 243 Sep 66


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