Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.
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PMID:Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G. 186 Oct 80

The isolation of cytosol aminopeptidase (CAP) from intestinal mucosa has involved difficult procedures because of low yield, instability of the enzyme and many other factors related to purification. The authors resolved these difficulties in the purification of the enzyme from intestinal mucosa of rats using hydrophobic chromatography on phenyl-TOYOPEARL 650S and ion exchange HPLC on Mono Q 5/5. In this procedure, a 60-fold purification was achieved and the purified enzyme revealed a single band with 56kDa molecular weight by SDS polyacrylamide gel electrophoresis. The purified enzyme preferentially hydrolyzed the Leucyl-containing peptides of Leu-Gly and Leu-Gly-Gly. However, it showed no activity on synthetic substrates of Leu-beta-naphthylamide, Gly-p-nitroanilide, Phe-p-nitroanilide or Met-beta-naphthylamide. The Km and Vmax for Leu-Gly were 0.91 mmol/l and 16.4 mol/min/mg protein, while for Leu-Gly-Gly they were 0.77 mmol/l and 20.6 mol/min/mg protein. The purified enzyme was heat-labile and quickly became less active in highly concentrated ammonium sulfate. The optimum pH was 6.5-10. The enzyme activity was stimulated by Mn2+ and Mg2+, while it was inhibited by EDTA, 1,10-phenanthroline, 2-mercaptoethanol and p-hydroxymercuribenzoate. The results suggested the participation of metal ions and the SH group in the enzyme activity. Furthermore the cytosol aminopeptidase was distinct from the brush-border membrane aminopeptidase in molecular weight, immunoreaction to cytosol aminopeptidase antiserum and the specificities to the substrates.
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PMID:[Purification and characterization of cytosol aminopeptidase from rat intestinal mucosa]. 188 Jan 98

Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and
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PMID:Purification and properties of a neurotensin-degrading endopeptidase from pig brain. 190 21

The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus. 190 14

A novel endogenous inhibitor of the proteasome (high molecular weight multicatalytic protease) has been isolated and characterized from human erythrocytes. After purification by ion-exchange and sizing chromatography, the inhibitor displayed a native molecular mass of approximately 200 kDa and contained a single subunit of 50 kDa with an isoelectric point of 6.9. Although the inhibitor noncompetitively blocks proteolysis of [methyl-14C]-alpha-casein (Ki = 7.1 x 10(-8) M) and inhibits hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC, it did not affect hydrolysis of other peptide substrates, such as MeOSuc-Phe-Leu-Phe-MNA and Z-Ala-Arg-Arg-MNA. To further characterize the 50-kDa inhibitor, a monoclonal antibody (MI-8) was generated that showed specific binding upon Western blot analysis of both native PAGE and SDS-PAGE. Immunoprecipitation with MI-8 specifically removed inhibitor activity against the proteasome. The 50-kDa inhibitor is distinct from a previously described 40-kDa inhibitor of the proteasome (Murakami, K., & Etlinger, J.D. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7588-7592) on the basis of lack of cross-reactivity with MI-8 and dissimilar peptide digest patterns. It is suggested that these endogenous inhibitors may have a role in ATP/ubiquitin-dependent proteolysis and/or other cellular functions involving this protease.
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PMID:Isolation and characterization of a novel endogenous inhibitor of the proteasome. 191 59

Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.
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PMID:Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2. 191 1

Translation of tobacco vein mottling virus (TVMV) RNA in a wheat germ system resulted in two products that were not observed in a rabbit reticulocyte system. One of these was the N-terminal protein, based on its being the most abundant product and its migration on SDS-PAGE at about 34 kDa. The second product was similar or identical to helper component (HC) isolated from TVMV-infected plants, based on co-migration with HC on SDS-PAGE and immunoprecipitation with anti-HC antibodies. The N-terminus of this product was determined by radiochemical Edman degradation to be Ser-257 of the polyprotein. This assignment was supported by peptide mapping with a tryptophan-specific reagent. A similar cleavage was observed when tobacco etch virus was translated in a wheat germ system. Comparison with homologous regions in five other potyviruses indicated conservation of amino acid residues on both sides of the proposed cleavage site. Conversion of Phe-256 to Met, Pro, Arg, His, or Trp by site-directed mutagenesis of a TVMV RNA transcription template inhibited cleavage in the wheat germ system. These results suggest that in vitro cleavage occurs between Phe-256 and Ser-257 and that this cleavage is the same as the in vivo cleavage which liberates the N-terminus of HC.
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PMID:In vitro cleavage at or near the N-terminus of the helper component protein in the tobacco vein mottling virus polyprotein. 196 46

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z-Phe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possessed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase; it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.
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PMID:Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis. 199 77

H2ts125 is a fibre-defective, temperature-sensitive mutant of adenovirus serotype 2. H2ts125 fibre is unstable at the non-permissive temperature (ts phenotype), and does not migrate in the same way as the wild-type fibre in an SDS/polyacrylamide gel (elm phenotype). Sequence analysis has shown that H2ts125 carries two mutations on the fibre gene: Leu105 to Phe, and Ala434 to Val. Analysis of the structural modifications occurring in H2ts125 fibre was performed using peptide finger-printing and antipeptide sera as immunological probes. We found that all the detectable structural alterations in the mutant fibre were due to the substitution on codon 434. In addition, the ts phenotype was rescued by a wild-type DNA fragment containing the 3' moiety of the fibre gene and overlapping the 434th codon. Morphological analysis of fibre molecules observed under the electron microscope showed minor but statistically significant differences in the fibre length between mutant and wild-type. The mutant fibre was found to be slightly longer (308.8 +/- 1.9 A) than the wild-type fibre (300.1 +/- 2.1 A). Thus both ts and elm phenotypes were carried by the same Ala434 to Val mutation which probably resulted from a change in the three-dimensional structure of the fibre protein, and not from some proteolytic cleavage.
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PMID:Functional and structural effects of an Ala to Val mutation in the adenovirus serotype 2 fibre. 199 35

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.
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PMID:The presence of a covalently cross-linked matrix in human platelets. 200 80


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