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)

Murine leukemia viruses, such as Rauscher leukemia virus (RLV), contain a proteolytic factor which becomes activated after detergent treatment of the virus. This factor specifically cleaves P70, the gag precursor polyprotein which is enriched for in preparations of immature virus core subparticles. The factor has been partially purified on Sephadex G-75 columns. It has a molecular weight of 10,000-12,000 daltons but does not coincide in elution position with the major peaks of the viral polypeptides p10 or p12. Under optimal conditions, that is 2% NP-40 (v/v), 10 mM DTT, (pH 7.2) and incubation for 16 hr at 22 degrees C, cleavage of labeled P70 occurs and increasing amounts of the four gag polypeptides p30, p15, p12 and p10 are obtained. The P70 cleavage activity is blocked by TLCK, TAME, CBZ-lysine and other lysyl-containing protease inhibitors. Further, the CBZ-lysine inhibition is reversible, while an inhibition by phenyl-methylsulfonyl fluoride (PMSF) is irreversible. These inhibition studies suggest that a similarity exists between the P70 proteolytic factor and some serine proteases, such as trypsin. The cleavage pattern of P70-rich immature cores treated with trypsin or chymotrypsin is different from that obtained with the P70 proteolytic factor. Thus murine leukemia virions apparently contain a unique, highly specific protease which is present in small amounts and cleaves P70.
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PMID:Properties of a P70 proteolytic factor of murine leukemia viruses. 7 13

This report presents evidence which supports a relationship between intracytoplasmic A particles (CAP) and mouse mammary tumour virus (MMTV). Three MMTV-specific antigenic determinants in CAP (MMTV p27, p14 and p10) uere detected by immunodiffusion. No structural proteins of comparable mol. wt. were found in CAP; however, exposure of CAP to trypsin resulted in the cleavage of the CAP structural proteins to MMTV-like polypeptides. This process was accompanied by the preservation of MMTV-specific antigen determinants. Disulphide bonds were necessary for the structural maintenance of CAP. Reducing agents destroyed the organized structure of CAP, whereupon processing of CAP proteins to MMTV-like polypeptides by trypsin was prevented. CAP p82, possessed only MMTV p27 antigenic determinants, while CAP polypeptides p20--18 possessed p10 antigenic determinants. Following processing of CAP structural proteins by trypsin, MMTV-specific p27 antigenic determinants were shifted from CAP p82 to CAP p27; MMTV-p10 antigenic determinants were found with CAP p15--10. These results suggest a model wherein CAP structural proteins are modified by protease during maturation, resulting in the shift of their proteins to sizes consistent with those which have been currently identified as the major internal components of the virion and that this phenomenon is largely predicated on the folding of CAP proteins into the morphologically intact A particle.
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PMID:Evidence for a precursor-product relationship between intracytoplasmic A particles and mouse mammary tumour virus cores. 8 Dec 68

Intracytoplasmic A particles were analyzed by immunodiffusion and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) before and after enzymatic cleavage with trypsin. A common antigen in A particles was detected by antisera prepared against purified intracytoplasmic A particles, purified mouse mammary tumor virus (MMTV), and a purified MMTV core polypeptide (p28). Despite this correlation, no SDS--polyacrylamide band migrating at p28 was observed in purified intracytoplasmic A particles. However, after incubation with trypsin, A particles subjected to SDS--PAGE produced only two polypeptide bands. They were observed at p28 and p15-10. Ouchterlony analysis of the trypsin-cleaved A particles revealed no alteration in the antigenicity of the particles. These results suggested that some structural components of intracytoplasmic A particles are polypeptide precursors of MMTV core proteins.
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PMID:Mouse mammary tumor virus polypeptide precursors in intracytoplasmic A particles. 16 80

A simple method for obtaining high-resolution banding patterns on elongated chromosomes is devised as follows: Peripheral lymphocytes cultured for 3 days with phytohemagglutinin were exposed to 10 micrograms/ml ethidium bromide for 2 hours and with 0.02 micrograms/ml colcemid for 1 hour prior to harvesting. After preparation by steam-dry method, high-resolution G-bands were obtained by Giemsa staining following exposure to hydrogen peroxide and trypsin treatment. 19 cases of the Prader-Willi syndrome, 3 cases of the aniridia-Wilms' tumor syndrome, 2 cases of the Langer-Giedion syndrome, 2 cases of retinoblastoma and 6 cases with multiple congenital anomalies whose diagnosis was not made by routine chromosome analysis were examined by this method. 18 of the 19 cases of the Prader-Willi syndrome had a deletion of 15q11.2. 2 of the 3 cases of the aniridia-Wilms' tumor syndrome had a deletion of 11p13 and the other had a balanced insertion with a breakpoint at 11p13. The 2 cases of the Langer-Giedion syndrome had a deletion of 8q23.3-24.13. One of retinoblastoma had a deletion of 13q14 and the other had a balanced reciprocal X/13 translocation with breakpoints at Xp11.21 and 13q12.3. The last 6 cases had subtle chromosomal aberrations: 46, XX, del (5) (q15q22), 46, XX, del (13) (q32.3), 46, XX, inv dup (6) (p25p21.3), 46, XX, -5, +der (5), t (3; 5) (p23; p13) mat, 46, XX, -5, +der (5), t (5; 9) (p13.3p21) mat, and 46, XX, -4, +der (4), t (4; 16) (p15.2; p13.1) pat. The simple method for high-resolution banding of chromosomes is useful for diagnosis of many kinds of birth defects.
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PMID:[A simple method for high-resolution banding of chromosomes and its application to diagnosis of birth defects]. 303 Sep 12

Gazdar-murine sarcoma virus (Gz-MSV) particles, obtained from tissue culture fluids of chronically infected HTG-2 hamster cells are immature in morphology and contain uncleaved Pr65gag as the predominant protein (greater than 95% Coomassie blue stain) (A. Pinter and E. deHarven, 1979, Virology 99, 103-110; Y. Yoshinaka and R. B. Luftig, 1982, Virology 118, 380-388). When Gz-MSV particles are disrupted in 1% sodium dodecyl sulfate (SDS) and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) in the absence of reducing agents, such as beta-mercaptoethanol (beta-MSH) almost half of the Pr65gag Coomassie blue-stained band is detected as a band at a Mr of 130K. Electrophoretic blotting studies with monospecific antisera against MuLV p30, p15, p12, and p10 showed that the 130K band cross-reacted with all four antigens suggesting that it was a dimer of Pr65gag. Two-dimensional (2D) SDS-PAGE where the first dimension was run under nonreducing conditions and the second with beta-MSH, supported the contention that the 130K band was a dimeric complex of Pr65gag. One also saw minor amounts of a 260K and higher polymeric forms of Pr65gag on the SDS gels, suggesting that polymeric forms may exist as well. When 32P-labeled Gz-MSV particles obtained by in vivo labeling of infected HTG-2 cells with [32P]PPi were electrophoresed on SDS-PAGE, only 10% of the 32P label was detected at the 130K position. In contrast, 30% of the Coomassie blue-stained Pr65gag material was found at 130K on the 2D gels. This suggests that unphosphorylated Pr65gag is more likely to participate in dimer formation than phosphorylated Pr65gag. Pr65gag of Moloney murine leukemia virus (M-MuLV), which is present as a minor (5% of stain) protein band on SDS-PAGE also showed 130K dimers. Further, in beta-MSH-deficient SDS preparations of Gz-MSV, electrophoresed after trypsin treatment, a 32K band that stained with p15, but not p10, p12, nor p30, antisera was observed. If beta-MSH was added, this band was no longer present. Thus Pr65gag dimerization in immature MuLV particles appears to at least involve the p15 region of the polyprotein. Since p15 is an extremely hydrophobic protein, formation of Pr65gag dimers may occur when virion precursor proteins are brought to the cell membrane during virus assembly.
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PMID:Murine retrovirus Pr65gag forms a 130K dimer in the absence of disulfide reducing agents. 608 46

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.
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PMID:Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors. 615 35

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.
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PMID:Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s. 619 30

The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.
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PMID:Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein. 629 63

Controlled proteolysis of MuLV gp70s results in the generation of several fragments which correspond to distinct structural domains of the molecules. The orientation of these regions in gp70 was determined by analysis of the immunoreactivities of proteolytic products generated from the MuLV PrENV polyprotein toward monoclonal alpha p15(E) and alpha gp70 antibodies, and by fragmentation analysis of gp70s specifically labeled with [35S]cysteine and [35S]methionine. These studies confirmed our previous assignment of a p15(E)-disulfide-linked 33K fragment to the carboxy terminus of Akv gp70 (Pinter, Honnen, Tung, O'Donnell, and Hammerling, Virology 116, 345-351, 1982). Using similar fragmentation procedures, the sizes and structural features of gp70 domains of Akv and MCF 247 MuLV gp70s were compared. Trypsinization of MCF-247 gp70 resulted in the production of a carboxy terminal fragment which resembled that of the ecotropic gp70 in that (1) it was disulfide linked to p15(E) but not to the amino terminal fragments, (2) reacted with monoclonal antibody 35/56, (3) contained cysteines but no methionines, and (4) carried only endo H-resistant oligosaccharide chains. Amino terminal MCF gp70 fragments were obtained with apparent molecular weights of 42K and 30K, considerably smaller than the corresponding Akv fragments of 49K and 35K. These MCF fragments were much more stable to degradation by trypsin than the Akv amino terminal components, indicating the loss or inaccessibility of several trypsin sites in the MCF amino terminal domain. These results demonstrated the Akv and MCF 247 gp70s contained highly conserved carboxy terminal domains but unique amino terminal sequences. Common features for both gp70s were the presence of an endo H-sensitive oligosaccharide chain near the amino terminus, and the presence of internal disulfide bonds in the amino terminal domains which resulted in an increased mobility for these fragments when analyzed under nonreducing conditions. Thus, while the amino terminal domains of the two gp70s are structurally different, certain aspects of glycosylation specificity and secondary conformation are conserved, suggesting that these structural features may be important for common biological properties of these molecules.
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PMID:Comparison of structural domains of gp70s of ecotropic Akv and dualtropic MCF-247 MuLVs. 631 Aug 85

The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected cells exhibited fewer microfilament cables than untransfected neighboring cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a cellular protein alters the properties of the tumor cell to provide a selective advantage for tumor growth in the animal.
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PMID:Molecular cloning and sequencing of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli with sequence similarities to retroviral Gag proteins. 819 43


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