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)

A neutral protease with kininogenase activity was isolated from human polymorphonuclear (PMN) leukocytes by cation exchange chromatography and gel filtration. The protease appears heterogeneous by cation exchange chromatography, isoelectric focusing and cationic disc gel electrophoresis, but homogeneous by gel filtration, sucrose density gradient ultracentrifugation, SDS-disc gel electrophoresis and immunoelectrophoresis. By carrying out the electrophoresis of the protease in acrylamide gels of varying concentrations, it was shown that they represent charge isomers. The protease was stable at pH 4-10, but labile to heat, being almost completely inactivated when incubated for 30 min at 70 degrees C. It exhibited proteolytic activity between pH 5 and 9, being maximal at 7.5-8.5. The molecular weight of the PMN protease was estimated to be about 20,000 daltons by gel filtration in aqueous buffer and about 26,000-28,000 daltons by SDS-disc gel electrophoresis and gel filtration in Sepharose 6B in the presence of the dissociating agent guanidine HCl. Its sedimentation coefficient was about 2.7S. Corresponding to the charge heterogeneity, by isoelectric focusing, the kinin-generating and esterolytic activities of the PMN granule lysate focused between pH 6.0 and 11.5, whereas the isolated PMN protease focused between 10.0 and 11.8. With respect to kinin generation, caseinolysis, and alanine esterase activity, the protease was inhibited by DFP and certain chloromethyl ketone inhibitors, as well as the plasma protease inhibitory a1-antitrypsin, a2-macroglobulin and antithrombin III. Both bradykinin and a methionyl-lysyl-bradykinin-like peptide were generated from highly purified kininogens by a lysosomal lysate containing the PMN protease. However, this assay was done with a crude enzyme preparation which contains an aminopeptidase capable of converting lysyl-bradykinin or methionyl-lysyl-bradykinin to bradykinin. When injected intradermally, the protease induced hyperemia, hemorrhage, and moderate enhancement of vascular permeability, but the mixture of the protease and kininogen induced a marked enhancement of vascular permeability.
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PMID:Neutral proteases of human PMN leukocytes with kininogenase activity. 5 89

Serine proteases or esterases released from cell cultures into the growth medium were converted to radioactive derivatives by active site labeling with tritiated DFP, both in the presence and absence of other competing active site reagents. The individual labeled enzymes were then identified by SDS-polyacrylamide gel electrophoresis and scintillation autoradiography. Conditioned medium from embryonal mouse fibroblasts transformed by mouse sarcoma virus contained five serine enzymes that were not present in medium from normal cells; two serine enzymes were released by both cell types, and one serine enzyme was found only in medium from normal cells. Two of the enzymes released by transformed cells were identified as plasminogen activators; these accounted for most of the serine enzyme labeling in transformed culture media and for most of the serine enzyme difference between normal and transformed cultures. The culture fluids from two cell strains of human neoplastic origin were examined by the same method. A rhabdomyosarcoma strain released eight serine enzymes (mol wt ranging from 22,500 to 102,000), four of which were plasminogen activators; seven serine enzymes (mol wt 26,000-102,000), including two plasminogen activators, were detected in medium from human melanoma cultures. In terms of electrophoretic mobility two of the plasminogen activators from rhabdomyosarcoma were identical with those from melanoma cultures, while the remaining two rhabdomyosarcoma activators coincided with activators found in commerical urokinase.
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PMID:Serine enzymes released by cultured neoplastic cells. 63 49

A tri-, di-, and monoacylglycerol-hydrolyzing enzyme from rat adipose tissue has been detergent-solubilized and separated from monoacylglycerol lipase (H. Tornqvist and P. Belfrage, 1976, J. Biol. Chem. 251, 813-819) and lipoprotein lipase by use of ion-exchange chromatography, broad and narrow pH range electrofocusing and gel chromatography. The final preparation contained several different proteins. One of these, with an apparent minimum molecular weight of 86,000 by SDS-gel electrophoresis, was identified as the enzyme protein of hormone-sensitive lipase: a) the enzyme activity was reproducibly stimulated 50-100% by incubation with cyclic AMP-dependent protein kinase, cyclic AMP and ATP-Mg2+; b) the relative intensity of the Mw 86,000 protein band, and only this, closely paralleled the enzyme activity during narrow pH range electrofocusing and during subsequent gel chromatography of the electrofocusing enzyme peak fraction; c) only the Mw 86,000 protein extensively incorporated 32p from [gamma-32P]ATP after incubation with protein kinase and cyclic AMP. The pI of the enzyme was 6.7, it had the same Stokes radius on Sephadex G 200 as IgG and was 50% inactivated by 10 micron HgCl2, 20 micron PCMB, 50 micron DFP, 10 mM NaF and non-ionic detergents above their critical micellar concentration.
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PMID:Identification and some characteristics of the enzyme protein of the hormone-sensitive lipase from rat adipose tissue. 66 58

D was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl esters (BAME) was the most sensitive substrate for D among those tested. The substrate profile of D was dinstinct when compared to that of CIs, CIr, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.
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PMID:Human factor D of the alternative complement pathway: purification and characterization. 87 24

Neutrophil leukocyte lysosomes contain a neutral protease capable of cleaving a peptide from kininogen. The protease has a molecular weight of about 27,000-28,000, as determined by SDS-disc gel electrophoresis and gel filtration in the presence of guanidine-HCl. Its S-value is 2.7 and the pI 10.0-11.8. Although homogeneous by some criteria (sucrose density gradient ultracentrifugation, gel filtration in Sephadex G-75, SDS-disc gel electrophoresis, immunoelectrophoresis), the protease is heterogeneous by other criteria (cation exchange chromatography, cationic disc gel electrophoresis and isoelectric focusing). The heterogeneity is attributable to charge isomerism. The enzyme is inhibited by DFP and a number of chloromethyl ketone inhibitors. It is also inhibited by the plasma protease inhibitors alpha1-antitrypsin, alpha2-macroglobulin and antithrombin III. The generated peptide has chemical and pharmacological properties similar to the undecapeptide methionyl-lysyl-bradykinin, but probably contains one or two additional amino acids.
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PMID:Generation of a vasoactive peptide by a neutral protease of human neutrophil leukocytes. 94 93

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.
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PMID:Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682. 136 1

A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.
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PMID:Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease. 136 89

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.
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PMID:A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination. 148 36

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.
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PMID:Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase. 151 18

A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.
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PMID:Purification and characterization of a sea squirt beta-galactosidase. 193 20


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