Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thermal transitions were measured by differential scanning calorimetry for rabbit cardiac sarcolemma in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.5, in glycerol-buffer and dimethyl sulfoxide - buffer mixtures, after heat denaturation, and after enzymatic degradation of the proteins. Specific solvent effects on the protein transitions were observed. Glycerol stabilized some of the four protein transitions, while dimethyl sulfoxide destabilized all protein transitions. The thermal transitions in the lower temperature range were studied for both the membranes and the lipid extracted from the membranes. A very small endotherm was observed for both the lipid extracted from the sarcolemma and the intact membrane (0.1-0.2 cal/g; 1 cal = 4.1868 J). A larger endotherm was observed in both the glycerol-buffer and dimethyl sulfoxide - buffer mixtures. Major perturbation of the protein by enzymatic degradation (papain or trypsin digestion), by heat denaturation, or by reaction with excess N-ethylmaleimide all produced larger endotherms near 20 degrees C. The very small magnitude of the endotherm near 20 degrees C suggests that it is not a typical gel - liquid crystalline transition of the bilayer. However, the occurrence of an endotherm in the extracted lipid suggests that some reorientation of lipid is involved.
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PMID:Differential scanning calorimetry studies of rabbit cardiac sarcolemma. 295 74

Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (-)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane-bound high-affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10-min incubation at 50 degrees C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited (-)desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,w of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin. Solubilization reduced the affinity of nimodipine for its high-affinity site slightly from 0.35 nM to 1.2 nM and that for its low-affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low-affinity site was completely abolished by 0.1 microM nitrobenzylthioinosine. After solubilization only the high-affinity site for (-)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd = 15.3 nM) but unchanged specific density. Binding to the solubilized high-affinity site for nimodipine and (-)desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200-110 inhibited allosterically (-)desmethoxyverapamil binding. d-cis-Diltiazem stimulated nimodipine binding at 20 degrees C 1.2-fold, reduced the dissociation rate from 0.018 min-1 to 0.0083 min-1 and had no effect on the association rate (0.173 min-1. nM-1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min-1. The association and dissociation rate constants for (-)desmethoxyverapamil were 0.024 min-1. nM-1 and 0.025 min-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers. 300 37

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.
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PMID:NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin. 302 8

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. 309 25

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.
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PMID:Polypeptide elongation factor Tu from Halobacterium marismortui. 312 12

Rat hepatocytes in primary culture are stimulated to synthesize DNA by high-molecular-weight fractions from rat serum. This activity has been previously given the name hepatopoietin A (HPTA). HPTA, with an apparent molecular weight of 150,000 to 250,000, stimulated the incorporation of [3H]thymidine into DNA, quantitated by autoradiography as percentage of nuclear labeling. Properties of HPTA include: sensitivity to heat; stability in minimal essential media at 4 degrees C; in lyophilized form; or in 25% glycerol at -20 degrees C; and instability at 4 degrees C in isotonic buffer. Trypsin digestion of HPTA resulted in an increase in biological activity. Both the trypsinized and native forms of this activity were not inhibited by antiserum against mouse epidermal growth factor in this bioassay. Treatment of HPTA with trypsin resulted in a shift of its apparent molecular weight on a Sephadex G-50 column to less than 6000. This trypsinized HPTA activity did not comigrate with 125I-labeled epidermal growth factor on the same column. These results demonstrate that HPTA exists in normal serum as a large precursor to a more active moiety, generated by proteolytic cleavage, which is not identical to epidermal growth factor. Fractions from human serum and plasma of molecular weight similar to that of HPTA have also been shown to stimulate DNA synthesis in rat hepatocytes.
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PMID:Hepatopoietin A: partial characterization and trypsin activation of a hepatocyte growth factor. 315 46

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.
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PMID:Two phosphorylated forms of myosin in thrombin-stimulated platelets. 335 50

17 beta-Hydroxysteroid oxidoreductase, as well as estrone sulfate and dehydroepiandrosterone sulfate sulfatases, were found in the plasma membrane of microvilli of the fetal syncytiotrophoblast. Because of their location, these enzymes may influence feto-maternal transfer of steroids circulating as sulfates, the utilization of sulfated estrogen precursors and the proportion of estrone and estradiol delivered towards fetal and maternal circulations. Microvillar vesicles isolated from human term placentas were disrupted in hypotonic medium to obtain a membrane preparation. A fraction of the estradiol 17 beta-oxidoreductase (E2DH) activity in the vesicle remained associated to the membrane after disruption and treatment with 2 M NaCl. The membrane-associated activity was resistant to inhibition with trypsin and did not react with a polyclonal antibody which neutralized cytosolic E2DH activity. The membrane-associated enzyme was solubilized with a cholate-glycerol buffer solution and purified on Sephadex G-100. The estimated molecular weight of the solubilized enzyme (137 kDa) appears to correspond to a tetramer since it was found to be about twice the size of the cytosolic enzyme. Both enzymes focused in polyacrylamide gels at pH 5.2. The Km relative to E2 of the membrane-associated E2DH (1.3 microM) differs from those of mitochondrial (0.43 microM), microsomal (0.69 microM) and cytosolic (11 microM) fractions. The cytosolic and the microvillar membrane associated 17 beta-hydroxysteroid oxidoreductases also differ in their specificity for C18 and C19 steroid substrates and in their pH dependence patterns. Sulfatases acting on estrone sulfate and dehydroepiandrosterone sulfate in microvillar membranes were insensitive to trypsin and as resistant to washes with 2 M NaCl as alkaline phosphatase. This data indicated that steroid sulfatases are also microvillar membrane associated enzymes of potential physiologic importance in the hydrolysis of estrogen precursors.
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PMID:Steroid metabolizing enzymes associated with the microvillar membrane of human placenta. 345 41

Available androgen binding to soluble proteins from the cytosol of human endometrium was studied using the dextran coated charcoal adsorption method and sucrose density centrifugation analysis. Specific binding of [3H]-5 alpha-dihydrotestosterone ([3H]-DHT) was observed with both methods. The apparent dissociation constant (Kd), for DHT binding is 1.3 +/- 0.2 (SEM) nM and the binding capacity 177 +/- 42 (SEM) fmol/mg protein. Sucrose density ultracentrifugation identifies specific [3H]-DHT binding that sediments at 4S and 8S. The stability of the androgen receptor in human endometrium is increased by the addition of 10% glycerol to the homogenization buffer. The addition of trypsin or pronase and heating at 60 degrees C reduces specific binding which demonstrates that the specific [3H]-DHT binder is a protein. The uptake of [3H] DHT in endometrial tissue minces indicated that 20% of the bound radioactivity was nuclear. Steroid specificity suggests that the binding protein from the uterus is specific for androgens. These observations indicate that androgen binding protein in the human uterus has the characteristics of the androgen receptor.
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PMID:The androgen receptor of the human endometrium. 358 78

The activities, properties, and steady-state kinetics of the five enzymes catalyzing the synthesis of 1-acyl- and 1-alkyl-sn-glycerol 3-phosphate in the cultured skin fibroblasts from Zellweger syndrome patients and normal controls were studied in detail. Judging from their Km and Vmax values, glycerol phosphate acyltransferase (EC 2.3.1.15), acyl/alkyl dihydroxyacetone phosphate reductase (EC 1.1.1.101), and acyl coenzyme A reductase (long-chain alcohol forming), appear to be affected only slightly by the absence of peroxisomes characteristic of the Zellweger syndrome. Glycerophosphate acyltransferase also showed no differences in N-ethylmaleimide sensitivity nor in inhibition by dihydroxyacetone phosphate between these cell types. Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) and alkyl dihydroxyacetone phosphate synthase (EC 2.5.1.26) have altered activity and kinetic constants in homogenates from Zellweger syndrome fibroblasts. Dihydroxyacetone phosphate acyltransferase has similar Km (DHAP) values in both control and Zellweger syndrome cells; however, the value for the Vmax in Zellweger syndrome cells is only 6% of that found in the controls. This is interpreted as indicating that this enzyme is not defective in this disease but is simply present at a depressed level. Also, this enzyme activity has a maximum rate at pH 7.0-7.5 in the mutant cells as opposed to pH 5.4 in the controls. Acylation of dihydroxyacetone phosphate by control cell homogenate was stimulated by N-ethylmaleimide at both pH 5.7 and 7.5 whereas this activity from Zellweger syndrome cells was slightly inhibited at pH 5.7 and strongly inhibited at pH 7.5. In the absence of detergent, dihydroxyacetone phosphate acyltransferase in the Zellweger syndrome cells was much more labile to trypsin than in the control cells. Alkyl dihydroxyacetone phosphate synthase had a slightly higher Km (33 vs 17 microM) for palmitoyl dihydroxyacetone phosphate and a lower Vmax (0.07 vs 0.24 mU/mg protein) in the Zellweger syndrome cells as compared to controls. Although this is a substantial decrease in activity, it probably contributes little to the decreased rate of ether lipid synthesis in these cells. The major problem in this respect is apparently the loss of dihydroxyacetone phosphate acyltransferase activity. All of these enzymes, in both control and Zellweger syndrome cell homogenates, are sedimentable by centrifugation at 100,000g. Also, with the exception of dihydroxyacetone phosphate acyltransferase they had similar patterns of inactivation by heat in both cell types.
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PMID:Properties of the enzymes catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls. 364 70


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