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)

Contrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin-trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin-trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to 'see' sensitive parasite proteases sufficiently to mount a response against the parasite.
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PMID:Schistosoma mansoni host-parasite relationship: interaction of contrapsin with adult worms. 780 Apr 17

Purified budded virions of Autographa californica nuclear polyhedrosis virus (AcNPV) contain abundant amounts of free ubiquitin, which has an altered electrophoretic mobility on SDS gels as compared with standard ubiquitin. Phase extraction of virion proteins with Triton X-114 indicated that the modified form of ubiquitin behaved as an integral membrane protein. The membrane-bound form of ubiquitin was labeled with both phosphate and palmitate, and its electrophoretic mobility was altered by treatment with phospholipase A2 and a phosphatidylcholine-specific phospholipase D. Mild trypsin digestion indicated that the acyl group was not linked to the C-terminus of the protein. Acylated ubiquitin could not be radiolabeled with a membrane-impermeable Bolton-Hunter reagent unless virus was pretreated with detergent. Together, these experiments suggest that ubiquitin is attached to the inner face of the viral membrane by a novel type of phospholipid anchor.
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PMID:Ubiquitin is attached to membranes of baculovirus particles by a novel type of phospholipid anchor. 783 50

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.
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PMID:Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. 796 84

Synaptotagmin, an integral membrane protein localized to secretory vesicles, has been implicated in the docking and fusion steps in calcium-regulated exocytosis. The large cytoplasmic domain contains two C2 motifs, each similar to the Ca2+ and phospholipid binding domain of protein kinase C. To study the membrane binding and aggregating properties of these C2 domains, three recombinant fragments of rat synaptotagmin I were expressed in Escherichia coli and purified. A recombinant protein containing both C2 domains (p65 1-5) was found to bind to and aggregate bovine chromaffin granules in a calcium-dependent manner, with half-maximal binding and aggregation occurring at approximately p Ca2+ = 4.2. However, recombinant proteins containing either the first (p65 1-3) or second (p65 3-5) C2 domain alone were not able to bind to the granules, indicating that both C2 domains are required for binding to chromaffin granules. p65 1-5 also bound to and aggregated liposomes made from chromaffin granule lipid extracts, as well as granules treated extensively with trypsin, suggesting that p65 1-5 binding to granules is mediated by the lipids in the granule membrane and not the granule membrane proteins. Although p65 1-3 and p65 3-5 did not bind to granules or lipids extracted from granules, both did bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (10%-40%PS). Half-maximal binding of p65 1-3 to vesicles occurred at approximately p Ca2+ = 5.2, while p65 3-5 appeared to bind independently of calcium over the range of pCa2+ = 5.5-2.8. p65 1-5 exhibited binding to PS/PC vesicles with characteristics of both the smaller proteins, displaying some binding in EGTA and increased binding in calcium. Larger amounts of p65 1-5 bound to PS/PC vesicles than of either of the smaller fragments. These results suggest that the two C2 domains of synaptotagmin act synergistically to promote binding to biological membranes and to affect calcium sensitivity and membrane binding capacity.
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PMID:Synergistic membrane interactions of the two C2 domains of synaptotagmin. 798 52

Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.
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PMID:Structural integrity of the membrane domains in extensively trypsinized Na,K-ATPase from shark rectal glands. 802 9

(Na,K)-ATPase is an integral membrane protein responsible for maintaining the Na+ and K+ ion concentration gradients across the plasma membranes of cells. All active (Na,K)-ATPase preparations consist of two subunits, designated alpha and beta. The alpha-subunit is the catalytic subunit and contains the cardiac glycoside binding site. In contrast, the physiological function of the beta-subunit remains unclear although it appears to be involved in the processes of folding, membrane insertion, and stabilization of the alpha-subunit. Previous work has determined the amino acid sequence and disulfide bond arrangements for the beta-subunit from both lamb and dog kidney. In this report, we describe the isolation and structural characterization of the glycan moieties of the beta-subunit from both lamb and dog kidney (Na,K)-ATPase. The three glycosylation sites of these beta-subunits were fractionated using reverse phase chromatography after cleavage of the polypeptide chain with trypsin and thermolysin. Glycopeptides derived from each glycosylation site were analyzed by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry results indicated that the predominant glycoforms at the three glycosylation sites of these beta-subunits were a combination of the tetraantennary glycan form and the unusual glycan form of tetraantennary with a limited number of repeating N-acetyllactosamine units. These results further define the covalent structure for the beta-subunit from both lamb and dog kidney (Na,K)-ATPase and suggest that the beta-subunit may be derived from an adhesion molecule.
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PMID:Structures of the complex glycans found on the beta-subunit of (Na,K)-ATPase. 839 Sep 82

The serum level of placental leucine aminopeptidase (P-LAP) increases during pregnancy. P-LAP degrades several peptide hormones such as oxytocin and vasopresin, suggesting a role in maintaining homeostasis during pregnancy. In the study reported here, we have isolated a cDNA clone with 4084 base pairs encoding P-LAP from a human placental cDNA library. The amino acid sequence deduced from the cDNA contained all of the sequences of the peptide fragments obtained by digestion of the purified protein with trypsin. The predicted P-LAP contains the HEXXH consensus sequence of zinc metallopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase N and aminopeptidase A. The deduced sequence also contains a hydrophobic region near the N terminus, suggesting that the enzyme is a type II integral membrane protein. Northern blot analysis revealed that P-LAP was expressed in several tissues, some of which expressed two forms of mRNAs. These results suggest that the enzyme is synthesized as an integral membrane protein and is released into blood under some physiological conditions.
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PMID:Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. 855 Jun 19

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.
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PMID:Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein. 862 62

ORF 1a of lactate dehydrogenase-elevating virus, strain P (LDV-P), encodes a protein of 2206 amino acids. Eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the C-terminal half of the molecule (amino acids 980-1852) that flank the serine protease domain. cDNAs encoding ORF 1a protein segments encompassing transmembrane segments 5 to 11 and its amphipathic C-terminal end as well as the N-terminal 80 amino acids of the downstream ORF 1b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. The synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. When synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. Furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, pH 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase K. No N-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. The results indicate that the C-terminal half of the ORF 1a protein represents a non-glycosylated integral membrane protein. Potential modes of synthesis and function of the protein are discussed. In addition, the results showed that the synthesis of the ORF 1a protein was generally terminated at its termination codon, but that read-through into the ORF 1b gene occurred with low frequency.
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PMID:Membrane association of the C-terminal half of the open reading frame 1a protein of lactate dehydrogenase-elevating virus. 877 92

gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.
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PMID:The propeptide binding site of the bovine gamma-glutamyl carboxylase. 911 24


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