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)

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.
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PMID:Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2. 398 90

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.
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PMID:Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties. 435 4

After the rat preputial gland was treated with collagenase and trypsin, five bands of cells were isolated by centrifugation in Ficoll gradients. Homogenates of the heavier cells (Bands IV and V) which contained less lipids, were more active than the homogenates of the lighter cells (Bands I, II and III) in transforming [1,2-3H]-dehydroepiandrosterone ([1,2-3H]-DHA) into [3H]-androstenedione and [3H]-testosterone and the latter into [3H]-dihydrotestosterone (DHT). In the presence of NAD, NADH and NADPH-generating system, [1,2-3H]-DHA was transformed into [3H]-DHT in 50-60% yield by homogenates of cells in Bands IV and V. DHT levels in the preputial gland were measured by radioimmunoassay. The levels in female rats reduced by 77% from 3.14 +/- 0.27 to 0.72 +/- 0.10 pg/mg tissue after adrenalectomy, and by 45% to 1.71 +/- 0.10 pg/mg tissue after ovariectomy. In male rats, the level reduced by 15% from 4.58 +/- 0.55 to 3.88 +/- 0.62 pg/mg tissue after adrenalectomy and by 40% to 2.74 +/- 0.21 pg/mg tissue after orchidectomy. These results demonstrated the transformation of DHA into DHT in the preputial gland of the rat, and that the adrenal is an important source of precursor steroid (DHA) for DHT formation in the preputial gland.
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PMID:Transformation of dehydroepiandrosterone into dihydrotestosterone by isolated cells from rat preputial gland. 622 75

After intracardial injection of [1,2-3H]dehydroepiandrosterone ([3H]DHA) into female rats, [3H]DHA was found to accumulate and was metabolized in the preputial gland, but not in the diaphragm. The identified metabolites of [3H]DHA in the preputial gland were delta 4-androstenedione-3 alpha, 17 beta-diol. Cells were isolated from the preputial gland after treatment with trypsin and collagenase III, and centrifugation in Ficoll gradients. Activity of the enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (3 beta-HSD) responsible for transforming DHA into delta 4-androstenedione was found mainly in the 105,000 g pellet (microsomal fraction) of homogenates of the isolated cells. It used preferentially NAD over NADP as a coenzyme, with a pH optimum at 8.5. The apparent Km for DHA was 5.5 X 10(-5) M, and the Vmax was 1.72 nmol/min/mg microsomal protein. These findings indicate that DHA is preferentially taken up by the preputial gland where it undergoes metabolism to form more potent androgens, and suggest that DHA may have important androgenic influence on the preputial gland.
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PMID:delta 5-3 beta-Hydroxysteroid dehydrogenase delta 4-5-isomerase activity and metabolism of dehydroepiandrosterone in rat preputial gland. 623 67

The alpha-oxidation of [1-14C]phytanic acid of high specific activity was studied in postnuclear and various subcellular fractions from rat liver. alpha-Oxidation in the postnuclear fraction required ATP, Mg2+, nicotinamide, and molecular oxygen for activity. alpha-Oxidation was inhibited by iron-specific chelating agents and respiratory chain inhibitors. Partial inhibition by carbon monoxide indicated a possible involvement of cytochrome P-450. However, phenobarbital-treated rat liver postnuclear fraction did not stimulate phytanic acid alpha-oxidation above that of control. Subcellular fractionation indicated that in addition to the mitochondrial fraction, cytosol was required for activity. The cytosolic factor appeared to be dialyzable; it was inactivated by heat treatment, but not affected by trypsin digestion. NAD, CoA, ascorbic acid, and catalase did not replace cytosolic activity nor did the recently characterized heat-stable factors in brain hydroxylation, namely, adenosine nucleotides and glutamate.
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PMID:Phytanic acid alpha-oxidation in rat liver. Requirement of cytosolic factor. 623 33

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.
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PMID:Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase. 624 65

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.
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PMID:Formation of an iso-1-cytochrome c-like species containing a covalently bonded heme group from the apoprotein by a yeast cell-free system in the presence of hemin. 624 50

The chromatin-associated enzyme poly(ADP-Rib) polymerase causes an NAD-dependent crosslinking of modified oligonucleosomes, as demonstrated by electrophoretic and sedimentation analysis [Butt, T. R. and Smulson, M. (1980) Biochemistry, 19, 5235-5242]. It was speculated that poly(ADP-ribosyl)ation of histone H1 and subsequent formation through crosslinking to an H1 dimer may be an important component of this phenomenon. To study this process, a method of complexing histone H1 to chromatin was required that promoted the restoration of accurate poly(ADP-ribosyl)ation of this histone. Previously we have established that two histone H1 molecules are crosslinked by a chain of poly(ADP-Rib) 15 or 16 units in length. In the current study, we made use of the ability of oligonucleosomes, reconstituted with H1, to carry out the synthesis of the poly(ADP-Rib)-H1 complex in order to monitor the accuracy of reconstitution. It appears that a specific distance and juxtaposition of adjacent H1 molecules along the polynucleosome fiber is required for the enzymatic synthesis of this modified histone complex. We established that a controlled trypsin digestion of oligonucleosomes removed H1 histone with minimal perturbation of other nuclear proteins associated with chromatin. In addition, poly(ADP-Rib) polymerase was partially removed from chromatin by this procedure. Subsequently, methods utilizing gradient salt dialysis have been employed to reconstitute both the polymerase and histone H1 to the depleted oligonucleosomes. The reassociation of H1 (and polymerase) to specific binding sites within oligonucleosomes was accomplished by the above procedures. Poly(ADP-Rib)--H1-dimer synthesis was not observed in depleted oligonucleosomes, but this capacity was found to be partially restored in the reconstituted chromatin. Similarly, the ability of NAD to promote crosslinking of nucleosomes was restored in the reconstituted samples. These results provide a basis for further studies on how the poly(ADP-ribosyl)ation of histones alters the structure of chromatin.
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PMID:The participation of poly(ADP-ribosyl)ated histone H1 in oligonucleosomal condensation. 629 27

We have identified a proteolytic system that selectively degrades histone H1 in normal human lymphocytes. Treatment of permeabilized human lymphocytes with a series of nucleotides produced a marked decrease in their histone H1 content compared to untreated cells. The nucleotide-stimulated process was selective for histone H1 because gel electrophoresis showed that almost all other lymphocyte protein bands remained constant while histone H1 disappeared. The elimination of histone H1 appears to be the result of proteolysis by a trypsin-like enzyme because it was inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor, and diisopropyl fluorophosphate. Proteolysis was stimulated by P1,P4-di(adenosine-5') tetraphosphate, P1,P3-di(adenosine-5') triphosphate, P1,P5-di(adenosine-5') pentaphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[alpha, beta-methylene]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or sodium phosphate did not show this stimulation of proteolysis. ATP, [alpha, beta-methylene]ATP, [beta, gamma-methylene]ATP, and pyrophosphate all stimulated proteolysis, suggesting that a pyrophosphate linkage was necessary for this process. Thus, resting human lymphocytes contain a trypsin-like protease that is stimulated by nucleotides or pyrophosphate to selectively degrade histone H1.
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PMID:Nucleotide-stimulated proteolysis of histone H1. 631 May 79

Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.
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PMID:Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid. 636 61


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