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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.
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PMID:Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox. 152 Mar 24

Two fibrinolytic enzymes, jararafibrase I and jararafibrase II, were purified from Bothrops jararaca venom. The purified jararafibrase I and jararafibrase II ran as single protein bands on analytical polyacrylamide gel electrophoresis and had mol. wts of 47,000 +/- 2000 and 21,400 +/- 500, respectively, by SDS-polyacrylamide gel electrophoresis. The isoelectric points of jararafibrase I and jararafibrase II were 4.6 and 6.5, respectively. The specific activities of jararafibrase I and jararafibrase II were 2.2 units/mg protein and 6.3 units/mg protein, respectively. Both enzymes exhibited no detectable plasminogen activating activity. The activity of the enzymes was completely inhibited by 1,10-phenanthroline and ethylenediaminetetraacetate, suggesting that both enzymes were metalloproteinases. Jararafibrase I and jararafibrase II had single-chain protein compositions, and the amino acid sequence up to the 49th amino acid from the NH2-terminal of jararafibrase II was: Leu-Pro-Glu-His-Gln-Arg-Tyr-Ile-Glu-Leu-Phe-Ile-Val-Val-Asp-His-Gly-Met- Phe-Met-Lys-Tyr-Asn-Gly-Asn-Ser-Asp-Lys-Ile-Arg-Arg-Arg-Ile-His-Gln- Met-Val-Asn-Ile-Met-Lys-X-Ala-Tyr-Arg-Tyr-Leu-Tyr-Ile-(X = not confirmed).
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PMID:Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom. 152 77

Wistar rats from the same litter were randomly divided into four groups and received subcutaneously from the 6th to 28th day post partum one of the following drugs: L-proline, methylmalonate, L-phenylalanine plus alpha-methylphenylalanine, or equivalent volumes of 0.9% (w/v) saline (controls). On day 30, the animals were killed, the brain was removed and the cerebral cortex and cerebellum was immediately dissected. Total intermediate filament fraction (IF) was obtained from cerebral cortex and cerebellum by using a high-salt phosphate-buffered solution supplemented by 1% Triton X-100. The pellet contained the bulk of the IF proteins. Following SDS-polyacrylamide gel electrophoresis, these proteins were identified as the 200, 150 and 68 kD subunits of neurofilaments (NF-H, NF-M and NF-L, respectively), the 66 kDa associated protein, the 57 kDa intermediate filament-like protein and the 52 kDa glial fibrillary acidic protein (GFAP). They were further scanned through densitometry from enriched fractions of controls and of animals treated with the various drugs in order to determine the effects of the treatments on their concentration. Our results showed that the concentration of IF protein in cerebellum was not affected by the treatments, whereas chronic administration of all drugs significantly decreased NF-H subunit concentration in rat cerebral cortex.
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PMID:Diminished concentration of the NF-H subunit of neurofilaments in cerebral cortex of rats chronically treated with proline, methylmalonate and phenylalanine plus alpha-methylphenylalanine. 152 92

The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.
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PMID:Characterization of a new kallikrein-like enzyme (KLP-S3) of the rat submandibular gland. 153 57

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
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PMID:The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. 157 91

In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.
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PMID:The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture. 157 92

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.
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PMID:Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600. 159 45

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.
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PMID:Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer. 160 52

1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.
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PMID:Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom. 161 86

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.
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PMID:Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox. 161 87


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