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)

Plasmin is a serine protease with trypsin-like specificity and is activated from plasminogen by several plasminogen activators. Since plasmin has lysine binding site in its heavy chain, lysine derivatives react with plasmin and then modify its activity. The effects of lysine derivatives such as epsilon-aminocaproic acid (EACA) and tranexamic acid on bovine plasmin activity were investigated. In the absence of lysine derivatives, the bovine plasmin activity which was evaluated as the amidolytic activity was reduced in a time- or temperature-dependent manner. However, the bovine plasmin activity became stable upon adding EACA or tranexamic acid. When plasmin was incubated at 4 degrees C for 1, 3 or 5 days without lysine derivatives, the plasmin activity decreased to 43.9%, 19.9% and 11.9% of the initial activity, respectively. On the other hand, when plasmin was incubated at 37 degrees C for 1, 3 or 5 days with tranexamic acid, its activity remained at 110%, 95.6% and 85.9%, respectively. After bovine plasmin had been incubated for 5 days at 4 degrees C in the absence of tranexamic acid, the plasmin activity declined to less than 20%. However, when bovine plasmin had been incubated for 5 days at 37 degrees C in the presence of tranexamic acid, the residual plasmin activity was more than 80%. A similar effect of EACA on bovine plasmin was observed, but it was weaker than that of tranexamic acid. Reversed-phase HPLC followed by SDS-PAGE demonstrated that bovine plasmin was degraded into several fragments. Amino acid sequencing of these fragments revealed that the Lys77-Arg78 or Arg78-Ile79, Arg342-Met343 and Arg557-Ile558 peptide bonds in the bovine plasmin molecule were cleft, respectively. Only the fragment consisting of the amino acid region from Met343 to the C-terminal amino acid, Asn786, exhibited amidolytic activity. In proportion to inactivation of the bovine plasmin, this fragment disappeared. The above findings suggest that lysine derivatives react with bovine plasmin and then stabilize the activity of plasmin by preventing the degradation of active fragment (Met343-Asn786).
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PMID:Stabilization of plasmin by lysine derivatives. 864 16

There is now extensive evidence that amyloid-beta peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-beta 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of alpha1-antitrypsin (alpha1-AT) and other serine protease inhibitors, also recognizes the amyloid-beta 25-35 domain. In fact, by recognizing the amyloid-beta 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-beta peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-beta peptide. A series of peptides based on the sequences of amyloid-beta peptide and alpha1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-beta peptide 25-35 and amyloid-beta peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-beta 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric ambeta/alpha1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-beta 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-beta peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-beta peptide, if soluble amyloid-beta peptide proves to be an in vivo precursor of the insoluble, toxic peptide.
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PMID:The serpin-enzyme complex receptor recognizes soluble, nontoxic amyloid-beta peptide but not aggregated, cytotoxic amyloid-beta peptide. 866 72

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.
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PMID:Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. 870 14

The ability of typical and atypical strains of Aeromonas salmonicida to utilize non-haem sources of protein-bound iron was evaluated. (i) In a plate bioassay, the suppression of growth imposed on typical and atypical A. salmonicida by addition of the high-affinity iron chelator ethylenediamine-di(o- hydroxyphenylacetic acid) (EDDA) to the growth medium was reversed by the addition of 30% or 90% iron-saturated bovine or human transferrin (Tf) or lactoferrin (Lf) to the growth medium. (ii) The mechanism of obtaining iron from Tf was investigated by the addition of bovine Tf contained within a dialysis bag. The reversal of iron-restricted growth suppression differed between the strains in that the atypical strains were unable to utilize Tf contained within a dialysis bag while the typical strains were able to do so. This suggested a siderophore-mediated uptake of iron from Tf by the typical strains, which are known to produce siderophores while atypical strains do not. (iii) A solid-phase binding assay using horseradish-peroxidase-conjugated or biotinylated Tf or Lf failed to detect Tf/Lf-binding activity using whole typical or atypical cells. (iv) When atypical extracellular products (ECP) plus bovine Tf or salmon serum were enclosed in a dialysis bag, diffusible products were released which could reverse the EDDA-imposed growth suppression of an atypical strain. This reversal was negated by inhibition of the ECP metalloprotease with EDTA. (v) Purified 70 kDa serine protease of a typical strain was able to digest bovine Tf to low molecular mass fragments as observed in SDS-PAGE. These results indicate that typical and atypical strains of A. salmonicida differ in their mechanism of utilization of non-haem protein- bound sources of iron. Typical strains utilize Tf via a siderophore-mediated mechanism and are also able to digest Tf with the extracellular serine protease. Atypical strains utilize Tf by a siderophore-independent mechanism probably involving the proteolytic degradation of Tf by the extracellular metalloprotease.
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PMID:Utilization of transferrin and salmon serum as sources of iron by typical and atypical strains of Aeromonas salmonicida. 870 95

A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.
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PMID:Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). 879 81

A novel hyaluronan-binding protein (PHBP) was purified from human plasma by affinity chromatography on hyaluronan-conjugated Sepharose. The contaminating IgM and albumin in the partially purified preparation were removed with anti-IgG antibody-conjugated Sepharose and anti-albumin antibody-conjugated Sepharose, respectively, and no other contaminant was observed. Finally, 800 micrograms of PHBP was isolated from 500 ml of human plasma. PHBP gave a single 70-kDa band on SDS-PAGE under non-reducing conditions, and 50-kDa and 17-kDa bands under reducing conditions. Thus, PHBP was a heterodimer composed of 50-kDa and 17-kDa subunits, bridged by a disulfide linkage. Both subunits had novel N-terminal amino acid sequences, indicating that PHBP was a novel hyaluronan-binding protein in human plasma. The amino acid sequence deduced from the nucleotide sequence of the cloned PHBP cDNA exhibited significant homology to that of hepatocyte growth factor activator (HGFA). The results of Northern blot analysis indicated that liver, kidney, and pancreas expressed PHBP mRNA. The predicted structure of PHBP showed three epidermal growth factor (EGF) domains, a kringle domain and a serine protease domain, from its N-terminus, although HGFA has a fibronectin type II domain, an EGF domain, a fibronectin type I domain, an EGF domain, a kringle domain, and a serine protease domain, from its N-terminus.
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PMID:Purification and characterization of a novel hyaluronan-binding protein (PHBP) from human plasma: it has three EGF, a kringle and a serine protease domain, similar to hepatocyte growth factor activator. 882 52

Activated astrocytes have been identified as the main source of the serine protease inhibitor alpha 1-antichymotrypsin (ACT), an acute phase protein that is tightly associated with amyloid plaques in Alzheimer's disease (AD) and in normal aged human and monkey brain. We analyzed the synthesis of ACT by cultured murine astrocytes in vitro. The murine astrocytes expressed an ACT-like antigen that crossreacted with antibodies to human ACT. The murine ACT-like protein is secreted by the astrocytes and is able to form an SDS-resistant complex with the serine protease cathepsin G, indicating that the secreted ACT is biologically active. We conclude that cultured primary astrocytes synthesize and secrete murine ACT in an active form. We, therefore, suggest that the ACT present within AD plaques is locally derived from plaque-associated activated astrocytes as a part of a glia-mediated local inflammatory response that is associated with the neurodegeneration seen in AD.
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PMID:Synthesis and secretion of active alpha 1-antichymotrypsin by murine primary astrocytes. 889 50

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth--a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkaline pH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.
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PMID:Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds. 892 6

A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.
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PMID:Geotrichum candidum P-5 produces an intracellular serine protease resembling chymotrypsin. 893 Jan 36

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.
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PMID:Factor VII central. A novel mutation in the catalytic domain that reduces tissue factor binding, impairs activation by factor Xa, and abolishes amidolytic and coagulant activity. 894 45


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