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)

The retinal capillary bed from 67 obese-hyperglycaemic mice and 64 lean litter mates was isolated by trypsin digestion and investigated with respect to structure and enzyme activities. There was no significant difference in the ratio between numbers of endothelial and mural cells. The capillary walls did not show any obvious structural differences and microaneurysms were not observed. The retinal vessels from the obese-hyperglycaemic mice, however, displayed significantly higher activities of the enzymes hydroxyacyl-CoA-dehydrogenase, asparate aminotransferase (ASAT) and adenylate kinase than their lean litter mates. The activities of glutathione reductase, glucose-6-phosphate dehydrogenase (G-6-PDH) and phosphofructokinase were similar in the two experimental groups. It is suggested that the present data reflect early metabolic disturbances related to diabetic retinopathy.
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PMID:Morphology and enzyme activities of the retinal capillaries in mice with the obese-hyperglycaemic syndrome (gene symbol ob). 15 2

HMG CoA reductase, which catalyzes the reaction, HMG CoA + 2 NADAPH2 leads to mevalonate + CoA-SH + 2 NADP, is considered to be the rate-limiting enzyme on cholesterol biosynthetic pathway. Since a degree in activity of this enzyme is almost proportional to the rate of cholesterol synthesis from acetate, elucidation of factors that regulate reductase activity would provide insight into the control mechanisms on the cholesterol biosynthesis. In the present study, attempts were made to establish standard assay conditions of HMG CoA reductase activiy, and to qualify the factors affecting the activity of the enzyme. The results obtained were as follows: (1) As standard assay conditions of HMG CoA reductase activity, 85, muM were chosen for substrate concentration, 25-80 mug for microsomal enzyme protein, and 20 min for incubation time in a final volume of 0.1 ml. (2) HMG CoA reductase activity of rat liver microsomes was exhibited diurnal variation. The level of reductase activity at night was 4 fold higher than that of at daytime. (3) Either ATP or insulin administration stimulated hepatic HMG CoA reductase activity. But, cyclic AMP had no effect on reductase activity. The stimulatory effect of ATP or insulin on reductase activity was inhibited by a preadministration of glucagon. These results suggested that an interplay of hormone might regulate reductase activity and consequently cholesterol biosynthesis. (4) HMG CoA reductase activity was increased by preincubation of microsomes with cytosol. Presence of ATP or Mg++ intensified this effect. When digested by trypsin or degenerated by heat treatment, cytosol lost the stimulating activity. These results suggested as existence of protein factors in cytosol, which might modulate the enzyme interconversion from inactive to active forms.
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PMID:[Studies on the regulatory factors of 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase) activity]. 18 33

The crystal structure of pig heart citrate synthase was analyzed at 0.35-nm resolution. Chain tracing was possible and an initial molecular model constructed. The dimensions of the dimer molecule (located on a crystallographic diad) are 7.5 x 6.0 x 9.0 nm. The chain folding is characterized by the predominance of helices and the absence of sheet structure. The electron density accounts for 355 residues per monomer, so that about 80 residues must be disordered in the crystal. The disordered segment in probably N-terminal. The ordered part consists of two closely associated domains, a large domain with 300 residues and a C-terminal domain of 55 residues consisting of 3(anti)parallel helices. The large domain is built from 12 helical segments, some of which are buried in the interior of the molecule. Inhibitor binding studies with citrate and CoA revealed citrate binding sites but showed no electron density for CoA. It is suggested that CoA binds to the disordered, flexible N-terminal domain. Experiments of limited proteolysis with trypsin showed that under conditions a segment of Mr 9000 is cleaved off selectively. The remaining 35 000-Mr part is dimeric.
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PMID:Crystal structure analysis of the tetragonal crystal form are preliminary molecular model of pig-heart citrate synthase. 43 30

3-Hydroxyacyl-CoA dehydrogenase was assayed for acetoacetyl pantetheine-reducing and acetoacetyl-CoA reducing activities in rat liver homegenates. Two isoenzymes of the enzyme, types I and II, were distinguished by the following procedures: trypsin treatment, heat treatment, CM-cellulose chromatography, antibody titration, and sucrose density gradient centrifugation of the light mitochondrial fraction. Type I enzyme was localized in mitochondria, and catalyzed the reduction of both acetoacetyl pantetheine and acetoacetyl-CoA. Type II enzyme was found mainly in peroxisomes, accompanied by a low activity in mitochondria or some other organelles, and was active with acetoacetyl-CoA but not with aceto acetylpantetheine. Both isozymes were induced by the administration to the rats of di-(2-ethylhexyl)phthalate, which enhances the peroxisomal beta-oxidation activity, but the extent of the induction of type II enzyme was much higher than that of type I enzyme. The activity of the former was found only in diethylhexylphthalate-treated rats.
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PMID:Occurrence of two 3-hydroxyacyl-CoA dehydrogenases in rat liver. 48 11

Trypsin treatment of intact cells or isolated plasmalemmae from embryonic chick neural retinae leads to an accumulation of lysophospholipids in the plasmalemmae. Trypsin was used at activities commonly used in cell disaggregation techniques. This accumulation appears to result from the decrease in acyltransferase activity in the plasmalemma produced by enzyme treatment. Plasmalemmal CoA ligase activity is not affected by trypsin treatment. Trypsinization has little effect on plasmalemmal phospholipase A2 activity. These results are discussed in relation to (a) the effects of trypsinization on cell adhesion, and (b) the theory that cells cannot adhere to lecithins because of their fluidity or surface-free-energy values. We propose that the effects of trypsinization on adhesion may in large part be due to the effects on other plasmalemmal proteins. Similarly the inability of cells to adhere to lecithin substrates is simply explained as being due to the lysolecithin that contacting cells release from these substrates.
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PMID:Cell surface lipids and adhesion. IV. The effects of trypsin on lipid turnover by the plasmalemma. 52 67

Male lean mice belonging to the obese-hyperglycemic strain were made diabetic by intravenous injection of streptozoticin. The retinal capillary bed freed by trypsin digestion was studied with regard to morphology and the activity of some enzymes. There was a significant increase in the ratio between the endothelial and mural cells which was interpreted as indicating mural pericyte disappearance. The activities of adenylate kinase, aspartate-aminotransferase and hydroxyacyl-CoA-dehydrogenase in the retinal vessels of the diabetic animal were significantly higher than in vessels from the control animals. No differences were found in the activities of glucose-6-phosphate dehydrogenase, glutathione reductase and phosphofructokinase between the two animal groups. It is suggested that these results reflect early morphological and metabolic changes of the retinal vessels, preceding the well known clinical picture of diabetic retinopathy.
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PMID:Morphology and enzyme activities of the retinal capillaries in streptozotocin-diabetic mice. 54 3

The results presented here show that isolated subunits of transcarboxylase specifically catalyze the two partial reactions of transcarboxylation as shown in eq 1-3. The 12S central subunit is active in the transcarboxylation with methylmalonyl-CoA but inactive with oxalacetate and the peripheral metallo 5S subunit is active in the transcarboxylation with oxalacetate but inactive with methylmalonyl-CoA. These subunits, likewise, are specific for the reverse partial reactions; the central subunit catalyzing transfer from the carboxylated biotinyl group to propionyl-CoA to yield methylmalonyl-CoA and the peripheral subunit to pyruvate to yield oxalacetate. Thus, the central subunit contains the sites for the CoA esters (methylmalonyl-CoA and propionyl-CoA) and the peripheral metallo subunits for the keto acids (oxalacetate and pyruvate). In the overall reaction the biotinyl carboxyl carrier protein acts as a shuttle to carry the carboxyl groups between the two subunits. Biotin and certain biotin analogs are inactive in these partial reactions but the similar to 40- or similar to 66-residue biotinyl peptides, which are derived from the carboxyl carrier protein, are active. Transcarboxylase can be reconstituted from its isolated subunits and a comparison was made of the rate of the overall reaction when the subunits were assembled, as in the intact enzyme, with that obtained when the reaction was catalyzed by the nonassembled subunits. In the latter case, since the biotinyl carboxyl carrier subunit must diffuse from one subunit to the other, the overall reaction is much slower than with the assembled subunits. The reaction with trypsinized transcarboxylase from which the similar to 66-residue and similar to 40-residue biotinyl peptides have been stripped, likewise, was slow even though the biotinyl peptides were added to the reconstitution mixture. The 12SH and 5SE subunits remain assembled after trypsin treatment but the biotinyl peptides apparently do not combine firmly or properly with the trypsinized enzyme and the biotinyl group apparently must oscillate as a carboxyl carrier between the two sites on the subunits by diffusion.
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PMID:Evidence that the two partial reactions of transcarboxylation are catalyzed by two dissimilar subunits of transcarboxylase. 112 91

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.
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PMID:Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors. 155 74

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
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PMID:3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus). 156 81


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