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 mapped the in vivo phosphorylation sites for the matrix (M) protein of the Orsay and San Juan strains of vesicular stomatitis virus, Indiana serotype, using limited proteolysis and phosphoamino acid analysis. M protein was solubilized from 32P-labeled virions by using detergent and high-salt conditions, then treated with either trypsin or Staphylococcus aureus V8 protease, and analyzed by polyacrylamide gel electrophoresis and autoradiography to determine which fragments contained phosphate residues. The M protein fragment extending from amino acid 20 to the carboxy terminus contained approximately 70% of the control 32P label, while the fragment extending from amino acid 35 to the carboxy terminus had only trace amounts of label. These data indicate that the major phosphorylation site was between amino acids 20 and 34 in the Orsay strain M protein. Phosphoamino acid analysis of M protein by thin-layer electrophoresis showed the presence of phosphothreonine and phosphoserine and that phosphothreonine continued to be released after prolonged vapor-phase acid hydrolysis. These data identify Thr-31 as the primary in vivo phosphate acceptor for M protein of the Orsay strain of vesicular stomatitis virus. The San Juan strain M protein has serine at position 32, which may also be an important phosphate acceptor. In addition, phosphorylation at Ser-2, -3, or -17 occurs to a greater extent in the San Juan strain M protein than in the Orsay strain M protein. The subcellular distribution of phosphorylated M protein was investigated to determine a probable intracellular site(s) of phosphorylation. Phosphorylated M protein was associated primarily with cellular membranes, suggesting phosphorylation by a membrane-associated kinase. Virion M protein was phosphorylated to a greater extent than membrane-bound M protein, indicating that M protein phosphorylation occurs at a late stage in virus assembly. Phosphorylation of wild-type and temperature-sensitive mutant M protein was studied in vivo at the nonpermissive temperature. The data show that phosphorylated M protein was detected only in wild-type virus-infected cells and virions, suggesting that association with nucleocapsids may be required for M protein phosphorylation or that misfolding of mutant M protein at the nonpermissive temperature prevents phosphorylation.
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PMID:Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein. 132 2

Tumor cells and urine-voided cells from patients with invasive bladder carcinoma as well as from healthy patients were examined cytologically, ultrastructurally and immunocytochemically. The ultrastructure of tumor cells showed an abundant, dilated, rough endoplasmic reticulum in the form of membrane-bound vacuoles full of granular to fibrillar material located perinuclearly and/or paranuclearly. Some cells exhibited enlarged modified lysosomes containing sparce flocculent and particulate precipitate. Papanicolaou staining of these cells showed two basophilic cytoplasmic textures, one green glossy-patchy, perinuclearly and/or paranuclearly, well segregated from the other texture of peripheral hematoxylinophilic foamy cytoplasm, comparable to the cytologic features of cell cultures originating in invasive bladder carcinoma. PAS diastase showed double distribution and texture of the perinuclear glycosaminoglycans, a glossy accumulated mass and large granules. Glycosaminoglycan sacs similar to those of cell cultures were also present in tumor-dispersed cells. There was a nonspecific binding of antisera against lysozyme, human chorionic gonadotropin and alpha 1-trypsin in normal and tumor cells. Tumor cells and tissues were positive for alpha 1-chymotrypsin distributed perinuclearly and in large spheres. Normal cells lacked the above characteristics. The results indicate that it is feasible to use the aforementioned characteristics in conjunction with the existing bladder-cytologic criteria for malignancy as markers in urothelial cancer with regard to prognosis of superficial tumors with high malignant potential.
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PMID:A cytologic, ultrastructural and immunocytochemical comparison of tumor cells and cell cultures originating in invasive bladder carcinoma. 137 23

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses we have identified immunoreactive prolactin (PRL) proteins with molecular weights of 24 and 16 kD in the female rat brain. Because PRL target tissues have been shown to contain enzymes which, in vitro, cleave PRL into a 16-kD PRL fragment, studies were performed to characterize PRL proteolysis in the female rat brain. In vitro proteolysis of PRL was examined by incubating [125]I-PRL with 25,000 g subcellular fractions followed by SDS-PAGE under reducing conditions. At acidic pHs, incubation of PRL with 25,000 g hypothalamic fractions consistently resulted in the generation of a 16-kD fragment. The generation of the 16-kD fragment was time and tissue concentration dependent. Enzyme inhibitor analysis indicated that PRL proteolysis could be blocked by aspartate and serine protease inhibitors, but not sulfhydryl, metalloenzyme or trypsin protease inhibitors. Subcellular localization of hypothalamic PRL proteolytic activity by equilibrium density centrifugation revealed a bimodal distribution of proteolytic activity with modal densities of 1.12 and 1.24 g/ml. Homogenization of the tissue in a hypo-osmotic medium disrupted the high density peak resulting in a single low-density peak at the top of the gradient. These data indicate that subcellular fractions of the rat brain contain enzymes which can cleave PRL into a 16-kD fragment under acidic conditions. The majority of the enzymatic activity is localized in membrane-bound particles with a density similar to subcellular particles which contain PRL.
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PMID:Proteolytic modification of prolactin by the female rat brain. 147 17

We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S. typhimurium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9 polymerization. Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex. These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes.
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PMID:Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck. 152 43

Human monocyte serine esterase 1 (HMSE1) was purified from U937 cell extract. Since the N terminus of the enzyme was blocked, cleavage with trypsin was used to obtain several peptides accessible to amino acid sequencing. Based on partial amino acid sequence information, an oligonucleotide probe was synthesized and used to screen a U937 cDNA library. One clone was isolated and sequenced by us which contains an open reading frame of 503 amino acids that lacks about 50 amino acids at the N terminus relative to the protein. Computer analysis revealed an active site characteristic of known carboxylesterases with a catalytic active serine. Northern blot hybridization analysis revealed that the expression of HMSE1 is restricted to cells of the monocyte/macrophage system. In contrast to the moderate expression of HMSE1 in monocytes, alveolar macrophages showed very high amounts of the transcript. With the sequence features detected by computer analysis a structure model of HMSE1 as a dimeric, membrane-bound ectoenzyme was developed.
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PMID:Heterogenous expression and putative structure of human monocyte/macrophage serine esterase 1. 156 41

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

Surface proteins of the mammalian stage of the parasitic protozoan, Trypanosoma brucei, were biotinylated with sulfosuccinimidyl 6-(biotinamido) hexanoate. Since the predominant protein labeled by this reagent is the membrane form of the variant surface glycoprotein (mfVSG), a procedure was developed to convert mfVSG to its soluble form by the endogenous glycosylphosphatidylinositol-specific phospholipase C while retaining other biotinylated surface proteins in a membrane-bound state. From these membranes, three novel glycoproteins of 60, 65, and 75 kDa could be isolated by a combination of Triton X-114 phase separation and precipitations by streptavidin and concanavalin A coupled to solid supports. These polypeptides were detected in trypanosomes expressing different mfVSGs and are thus considered to be invariant. In a variant clone in which the mfVSG is trypsin-sensitive, the invariant surface glycoproteins of 65 and 75 kDa, designated ISG65 and ISG75, respectively, were proteolytically degraded with similar kinetics as the mfVSG. Neither ISG65 nor ISG75 could be detected in procyclic trypanosomes, the stage of the parasite characteristic for the insect midgut. Gene cloning reported in the accompanying paper (Ziegelbauer, K., Multhaup, G., and Overath, P. (1992) J. Biol. Chem. 267, 10797-10803) suggests that ISG65 and ISG75 are transmembrane proteins.
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PMID:Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei. 158 55

The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid beta precursor protein (beta APP). It is known that beta APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present beta APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in beta APP have an evolutionarily close relationship with the inter-alpha-trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4+ lymphocytes.
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PMID:Evolutionary origin of a Kunitz-type trypsin inhibitor domain inserted in the amyloid beta precursor protein of Alzheimer's disease. 159 45

Purified cytoplasmic and membrane-bound lactate dehydrogenases (LDH) from white muscle of skate were characterized, Km for pyruvate and NADH for purified LDH were 150 +/- 16 and 29 +/- 7 microM, and for membrane-bound LDH were 185 +/- 22 and 7.5 +/- 1.5 microM, respectively. The membrane-bound enzyme was not inhibited by high pyruvate concentration (up to 20 mM) in contrast to purified LDH. Part of membrane-bound LDH was released by incubation in solutions with a high level of KCl (up to 1 M) or at alkaline pH. The inactivation rate during trypsin digestion for solubilized LDH was 2-3-fold higher than that for the membrane-bound enzyme.
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PMID:Free and membrane-bound lactate dehydrogenase from white driving muscles of skate. 161 Mar 89

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.
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PMID:Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study. 161 60


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