Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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An acid protease from Monascus kaoliang was purified by consecutive applications of fractional acetone precipitation, batchwise CM-cellulose method and DEAE-cellulose column chromatography. The preparation was homogeneous on disc polyacrylamide gel electrophoresis at pH 4.5 and 7.5. The yield was about 30% with overall increase in specific activity of about 6-fold. The molecular weight as determined by SDS gel electrophoresis was about 34,000. The enzyme was a glycoprotease as indicated by specific carbohydrate staining on gels. It possessed the nature of an acid protease with a pH optimum at 3.0 toward heat-denatured casein and was stable over the range of pH 3.0 to 6.0. Reducing agents and thiol poisons had no effect on this enzyme, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inactivate this protease, indicating the probable absence of serine residue in the active site. The enzyme was inactivated by reaction with the carboxy-group specific reagent, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP). Pepstatin, a specific inhibitor for pepsin, was shown to inhibit this enzyme strongly. However, biacetyl (2,3-butadione) had little effect on this protease, although it inactivated pepsin to an 85% activity loss. Also, p-bromophenacyl bromide, another specific inhibitor of pepsin, failed to inactivate this acid protease.
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PMID:Purification and characterization of an acid protease from Monascus kaoliang. 74 88

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
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PMID:Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. 142 18

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

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.
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PMID:Diisopropyl fluorophosphate labeling of sperm-associated proteinases. 211 Aug 39

Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.
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PMID:Purification and characterization of bradykinin-hydrolyzing enzyme from 2-day-old rat epidermis. 336 52

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca. 811 Jul 87

A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.
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PMID:Characterization of a thrombin-like enzyme, grambin, from the venom of Trimeresurus gramineus and its in vivo antithrombotic effect. 853 42

Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells. However, in yeast little is known about presence and function of acyl-CoA thioesterase activity. Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity. Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE. The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate. YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail. Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested. The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM. The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type. Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with [3H]DFP. p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity. N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.
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PMID:Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa. 883 45

Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.
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PMID:Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family. 899 22

A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 degrees C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A2, lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a approximately 150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of approximately 38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1988) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase.
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PMID:Purification and characterization of a catalytic domain of rat intestinal phospholipase B/lipase associated with brush border membranes. 944 64


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