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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)

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.
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PMID:Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD. 0 Mar 95

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.
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PMID:Poly(adenosine dephosphate ribose) metabolism and regulation of myocardial cell growth by oxygen. 2 65

Glutamate decarboxylase, gamma-aminobutyrate-alpha-ketoglutarate aminotransferase and NAD-linked and NADP-linked succinic semialdehyde dehydrogenase, all constituting the GABA (gamma-aminobutyrate)-shunt pathway of glutamate metabolism are localized in the mitochondrial matrix in a streptomycin-bleached mutant of Euglena gracilis strain Z. Glutamate dehydrogenase, requiring NADP as the cofactor, was distributed in the cytoplasm. An improved version of the controlled digestion method for preparing Euglena mitochondria, which involves use of trypsin and a trypsin inhibitor and removal of broken cells before mechanical disruption of cells, is also described.
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PMID:Subcellular localization of the GABA-shunt enzymes in Euglena gracilis strain Z. 11 50

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.
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PMID:Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase. 17 55

The NAD-specific glutamate dehydrogenase of Neurospora crassa was S-carboxymethylated with [14C]iodoacetate, maleylated, and hydrolyzed with trypsin. The isolation and sequences of the resulting peptides are described. These peptides gave information on the structure of the protein that was previously unknown and gave many overlaps of previously isolated segments of the protein.
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PMID:Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VIII. Isolation and sequences of peptides from a tryptic hydrolysate of the maleylated protein. 19 3

Various serine proteases (e.g., trypsin, alpha-chymotrypsin, Pronase, and subtilisin) stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a membrane-enriched fraction of the rat ovary. Maximum stimulation is observed at protease concentrations ranging from 3 to 10 mug/ml. Higher protease concentrations inhibit ovarian adenylate cyclase in a dose-dependent manner. Protease stimulation causes a 6- to 8-fold increase in adenylate cyclase activity, which is comparable to the stimulation observed with human chorionic gonadotropin. Combinations of trypsin plus hormone or trypsin plus NaF stimulate ovarian adenylate cyclase activity to a greater extent than does any one of these alone. The mechanism of protease stimulation of adenylate cyclase involves limited proteolysis because zymogen precursors fail to activate the cyclase as does trypsin pretreated with trypsin inhibitors. Unlike cholera toxin, the serine protease stimulation is immediate (within the first 5 min) and requires no additional factors (e.g., NAD(+)). It is unlikely that protease stimulation of adenylate cyclase results from a proteolytic modification of the hormone receptor on the cell surface, because of the additive effects noted above and because protease stimulation is also observed in ovaries desensitized to hormone that lack this hormone receptor. Results with Lubrol-treated membranes also suggest that proteolytic enzymes do not directly activate the catalytic subunit of the cyclase or unmask new catalytic sites because the protease effect (like hormonal stimulation) is abolished by the detergent, whereas fluoride stimulation is enhanced. Other data suggest that serine protease and chorionic gonadotropin stimulation of adenylate cyclase result from activation of a membrane protease that then regulates adenylate cyclase in the ovary.
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PMID:Proteolytic enzyme activation of rat ovarian adenylate cyclase. 27 Jul 17

The purified preparations of glyceraldehyde-3-phosphate dehydrogenase isolated from frog and pike skeletal muscles were found homogenous under polyacrylamide gel electrophoresis. Their amino acid composition is similar to that of glyceraldehyde-3-phosphate dehydrogenase from other animal species. The interaction kinetics for frog and pike glyceraldehyde-3-phosphate dehydrogenase SH-groups with 5,5'-dithio-bis-(2-nitrobenzoate) (DTNB) were studied. A negative correlation between the thermal stability of the enzyme preparations from pig, pike, lamprey and frog muscles and the reactivity of their SH-groups with respect to DTNB was observed. NAD at saturating concentrations was found to protect the enzyme from lower vertebrates muscles against thermal inactivation in a lesser degree than does the pig muscle enzyme. The weaker protective effect of NAD was observed for lamprey and frog enzyme preparations, which are characterized by a low SH-group reaction ability. Frog and pike apoenzymes are considerably more resistant to trypsin proteolysis than the pig apoenzyme.
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PMID:[Comparative study of D-glyceraldehyde-3-phosphate dehydrogenase from frog and pike skeletal muscles]. 31 19

Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2M, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50 degrees or to 20 mM ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.
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PMID:Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase. 56 63

The role of cytosol components in the loss of rat liver adenylate cyclase activity which occurs during the preparation of particulate fractions from crude homogenates was studied. Epinephrine (5 micron)-, glucagon (10 micron)-, and fluoride (5 mM)- stimulated activities of twice-washed particulates were 31%, 58% and 67% of the homogenate activities, respectively. Addition of cytosol (100,000 X g supernatant devoid of adenylate cyclase activity) restored these activities to 82%, 88% and 80%. Cytosol also increased particulate basal activity from 60% of homogenate activity to 98%. The cytosol components capable of increasing adenylate cyclase activity were heat labile, nondialyzable, stable to freezing at -20 degrees, resistant to change of pH between 2 and 12, and unaffected by EGTA and NAD. Pretreatment with pepsin destroyed the effects of cytosol on both epinephrine- and glucagon-sensitive activities, whereas trypsin destroyed the effect of cytosol only on epinephrine-sensitive activity. The cytosol effect on adenylate cyclase was specific, since several purified proteins and ubiquitin, did not stimulate enzyme activity. Only part of the cytosol effect could be attributed to its GTP content. GTP at the concentration present in cytosol stimulated epinephrine-sensitive activity but significantly less than did cytosol, while GTP had no effect on glucagon-sensitive activity. Dialyzed cytosol retained its effectiveness even after removal of most (97%) of its GTP to a concentration where GTP had only a minimal effect on epinephrine-sensitive activity. Cytosol, unlike GTP, stimulated rather than inhibited activation by fluoride. Cytosol thus appears to contain at least two different protein components, which increase the activity of the two hormone-sensitive adenylate cyclases and presumably account in part for losses of adenylate cyclase activities seen during the preparation of particulates from homogenates.
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PMID:Activation of epinephrine and glucagon-sensitive adenylate cyclases of rat liver by cytosol protein factors. Role in loss of enzyme activities during preparation of particulate fractions, quantitation and partial characterization. 72 79

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.
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PMID:A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein. 132 15


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