Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

Metalloproteinase of Legionella pneumophila is the major extracellular proteinase of this bacterial species which splits human immunoglobulin G in the hinge region to form the (Fab')2 fragment. This fragment is relatively stable and undergoes further proteolysis at a slow rate. The c' fragment is unstable and is apparently split down to fragments CH2 and CH3. The metalloproteinase splits human serum albumin down to products having lower molecular masses. Another bacterial metalloproteinase, thermolysin, produces a similar effect, although at a slower rate.
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PMID:[Limited proteolysis of human albumin and immunoglobulin G by Legionella pneumophila metalloproteinase]. 163 17

Conjugates have been prepared from glutaraldehyde-activated linear polyacrylamide and bovine serum albumin, casein, or gelatin. Incorporation of these conjugates into sodium dodecyl sulfate-polyacrylamide gels has provided a simple and general method for the analysis of proteases following electrophoresis. The conjugates did not migrate during electrophoresis or development, but remained susceptible to proteolytic action following regeneration of enzyme activity. The sensitivity of this procedure was such that 2 pg of trypsin or chymotrypsin, 39 ng of elastase, and 2 ng of thermolysin could be detected. Results obtained with trypsin and chymotrypsin are 5 to 10 times more sensitive than previously reported techniques for protease detection following electrophoresis.
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PMID:Detection of proteases in polyacrylamide gels containing covalently bound substrates. 637 42

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent ('inactive') state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A2 and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A2 resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A2 is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of the endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.
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PMID:Topology of glutathione-insulin transhydrogenase in rat liver microsomes. 687 Nov 88

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
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PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84

Whey protein was digested with one of seven kinds of proteases at 37 degrees C (trypsin, proteinase K, actinase E, thermolysin, or papain) or at 25 degrees C (pepsin or chymotrypsin) for 24 h. The digested samples were assayed for the inhibitory activity of angiotensin-converting enzyme and for changes in the systolic blood pressure caused in spontaneously hypertensive rats after gastric intubation. The strongest depressive effect on the systolic blood pressure (-55 mm Hg) was observed at 6 h after gastric intubation of the whey protein that was digested by proteinase K. Finally, six peptides were chromatographically isolated from the proteinase K digest by a combination of hydrophobic reversed-phase HPLC and gel filtration. The amino acid sequences and their origins were clarified as follows: Val-Tyr-Pro-Phe-Pro-Gly [beta-casein (CN); f 59-64], Gly-Lys-Pro (beta 2-microglobulin; f 18-20), Ile-Pro-Ala (beta-lactoglobulin; f 78-80), Phe-Pro (serum albumin; f 221-222; beta-CN, f 62-63, f 157-158, and f 205-206), Val-Tyr-Pro (beta-CN; f 59-61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro (beta-CN; f 80-90). Chemical synthesis of these six peptides confirmed that all peptides, except an undecapeptide, have antihypertensive activity in spontaneously hypertensive rats. The synthetic tripeptide Ile-Pro-Ala, originating from beta-lactoglobulin, showed the strongest antihypertensive activity (-31 mm Hg).
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PMID:Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. 989 Dec 60

We investigate the feasibility of using the luminescence response of polymerized liposomes incorporating ethylenediaminetetraacetate europium(III) (EDTA-Eu(3+)) for monitoring protein concentrations in aqueous media. Quantitative analysis is based on the linear relationship between the luminescence enhancement of the lanthanide ion and protein concentration. Analytical figures of merit are presented for carbonic anhydrase, human serum albumin, gamma-globulins, and thermolysin. Qualitative analysis is based on the luminescence lifetime of the liposome sensor. This parameter, which follows well-behaved single exponential decays and provides characteristic values for each of the four studied proteins, demonstrates the selective potential for protein identification. Then partial least squares-1 and artificial neural networks are compared toward the quantitative and qualitative analysis of human serum albumin and carbonic anhydrase in binary mixtures without previous separation at the concentration levels found in aqueous humor.
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PMID:Artificial neural networks for qualitative and quantitative analysis of target proteins with polymerized liposome vesicles. 1718 20

Botulinum neurotoxins (BoNTs) and their fragments are targets of therapeutic developments and are increasingly used as therapeutic, prophylactic, and research reagents. However, published data on their properties vary widely. In order to gain a better understanding of these variations, we initiated a systematic investigation of the stability parameters of catalytic light chains (Lc) as well as of cell surface binding domains (Hc) of the neurotoxin. When followed by CD spectroscopy, we noticed that the recombinant light chains of serotypes A (LcA), B, D, E, and G rapidly lost their secondary structures by mild stirring. Denaturation of LcA increased with stirring speed and temperature resulting in a catalytically inactive precipitate. Reducing agents or an anaerobic environment were ineffective in the denaturation. Under identical conditions, bovine serum albumin, ovalbumin, carboxypeptidase B, and of thermolysin, a structural and functional analogue of LcA, remained unchanged. Hc domains of serotype A, B, C, E, and F were also denatured by mild stirring. Adding the nonionic detergent Tween-20 to LcA completely prevented the denaturation. We speculate that the BoNT domains undergo surface denaturation due to rapid exposure of hydrophobic residues by mechanical agitation. This study has important implications for handling BoNT proteins used in therapeutic development.
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PMID:Extreme sensitivity of botulinum neurotoxin domains towards mild agitation. 1922 30

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.
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PMID:Tissue dissociation enzyme neutral protease assessment. 2069 5

Free, non-protein bound asymmetrically guanidine-dimethylated arginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis. Human erythrocytic membrane comprises considerable amounts of large (>50 kDa) ADMA-containing proteins. Location in the erythrocyte membrane and identity and physiological functions of ADMA-containing proteins are unrevealed. In healthy subjects, the concentration of free ADMA in heparinised plasma is almost identical to that of serum. We hypothesised that the robustness of free ADMA concentration in human blood is due to a remarkable resistance of erythrocytic ADMA-containing proteins against proteases. In vivo, we investigated the course of the concentration of ADMA in serum and EDTA plasma of a critically ill patient with acute kidney injury during extended haemodialysis. In vitro, we studied the effects of thermolysin, a useful experimental proteolytic enzyme of erythrocyte membrane proteins, on erythrocytic ADMA. The protein binding (PB) of ADMA to human serum albumin (HSA) was also determined. In these studies, ADMA was measured by a previously reported, fully validated GC-MS/MS method. We measured almost identical ADMA concentrations in plasma and serum samples of the patient. During dialysis, the circulating ADMA concentration decreased slowly and moderately indicating removal of this substance, which was however much less than expected from its low molecular weight (202 Da) and high water solubility. After dialysis, circulating ADMA concentration increased again, a phenomenon called rebound, and ADMA reached higher levels compared to the baseline. The PB value of ADMA to HSA was about 30 %. This surprisingly high PB value of ADMA to HSA may be an explanation for the rather poor dialysance of ADMA. Washed human erythrocytes suspended in phosphate-buffered physiological saline were found not to release appreciable amounts of free and ADMA-containing proteins. The lack of effect of coagulation or anticoagulation on the concentration of circulating free ADMA in humans is likely to be due to a remarkable resistance of ADMA-containing proteins in the erythrocyte membrane against proteases in vivo in humans. Our study suggests that free ADMA is released in the circulating blood at relatively high rates. The considerable PB of ADMA to HSA is likely to add to the apparently poor dialysability of ADMA. Other contributing factors could be redistribution of free ADMA between plasma and erythrocytes in favour of plasma ADMA and parallel formation of free ADMA from erythrocytic ADMA-containing proteins during haemodialysis.
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PMID:Asymmetric dimethylarginine (ADMA) in human blood: effects of extended haemodialysis in the critically ill patient with acute kidney injury, protein binding to human serum albumin and proteolysis by thermolysin. 2592 52


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