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 properties of the molybdenum iron-sulfur flavoprotein, aldehyde oxidase from rabbit livers, have been further investigated in comparison with bovine milk xanthine oxidase. In agreement with earlier work, the ultraviolet/visible spectra indicate that the flavin and iron-sulfur centres of the enzymes are quite similar to one another. The molybdenum centres have been compared by EPR spectroscopy of molybdenum(V) and regarding re-insertion of the sulfido ligand of molybdenum into the desulfo enzyme forms. The pH optimum for sulfide insertion is approximately 2 lower for aldehyde oxidase than for xanthine oxidase. A detailed comparison of molybdenum(V) EPR signals has been made for the signals known as Arsenite, Slow and Rapid. Computer simulation of spectra in 1H2O and 2H2O, at 9 and 35 GHz was used. Slow signals from the two enzymes are scarcely distinguishable from one another. Under the conditions used, aldehyde oxidase yielded only the Rapid type 2 signal, whereas xanthine oxidase gives both the Rapid type 1 and 2 signals. The nature of the structural difference between the Rapid type 1 and type 2 signal-giving species is discussed. It is concluded that the molybdenum centres of xanthine oxidase and aldehyde oxidase are indeed similar to one another and that such differences as exist between their molybdenum(V) EPR signals and re-sulfuration properties are related to differences only in the substrate-binding sites. N-terminal amino acid analyses have been performed on peptides obtained by trypsin cleavage of aldehyde oxidase. Comparison with a sequence previously deduced [Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S. & Repine, J. E. (1993) Proc. Natl Acad. Sci. USA 90, 10690-10694] makes it clear that the latter is not, as was assumed, that of a xanthine dehydrogenase but of an aldehyde oxidase. In contrast to the situation with xanthine oxidase, attempts to convert non-proteolysed aldehyde oxidase to a dehydrogenase form by treatment with dithiothreitol were unsuccessful. The reason for this is considered in the light of sequence data in the literature. The location of the NAD(+)-binding site is discussed, and the sequence data are also discussed in relation to the molybdenum, iron-sulfur and substrate-binding sites.
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PMID:Properties of rabbit liver aldehyde oxidase and the relationship of the enzyme to xanthine oxidase and dehydrogenase. 755 19

The paper presents results of scientific activity of the Department of Metabolism Regulation. The main sections are: carbamates formation and their role in metabolism regulation; metabolic system of acid-base homeostasis in animals; polyamines metabolism in the extremal states; mechanisms of metabolic adaptation in mammals. Experimental data are presented which evidence for the fact that tissue proteins in vivo are subjected to nonenzymic carboxylation with formation of carbominic groups. In this case a charge variation in definite sites of protein molecule is observed, which specifies variation of the protein conformation and biological properties. Basic regularities of protein carbamate formation reactions are revealed with factors affecting their intensity. It is shown that the presence of carbonic acid in the medium increases the rate of reactions catalyzed with lactate dehydrogenase from the rabbit liver, glucose-6-phosphate dehydrogenase from yeast and trypsin. Under the same conditions the reaction velocity rate catalyzed with glucose-6-phosphate dehydrogenase from the rabbit liver and with ATP-citrate (pro-35)-liase is considerably decreased. Changes in the concentration of carbonic acid within the physiological limits are found to have no effect on lactate dehydrogenase from the cattle heart and chymotrypsin. The rate of the reaction catalyzed by NAD-dependent malate denydrogenase was studied as affected by carbon dioxide. It is shown that acceleration of the catalysis in these systems depends on the presence of both a bicarbonate anion and soluble carbon dioxide. IR spectra of NAD-dependent malate dehydrogenase in the deuterium oxide solutions were studied in the CO2-free solutions and solutions saturated with it.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Role of low molecular weight metabolites as natural regulators of metabolism]. 757 Oct 78

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.
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PMID:Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway. 760 58

The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta. Trypsin digestion of the purified enzyme generates fragments of the alpha subunit. The beta subunit is uncleaved unless NADP(H) is present (Tong, R.C.W., Glavas, N.A. and Bragg. P.D. (1991) Biochim. Biophys. Acta 1080, 19-28). Purified transhydrogenase bound to either NAD- or NADP-agarose was treated with trypsin. The alpha subunit was cleaved to 16, 29 and 43 kDa fragments in both cases. The beta subunit remained bound to NAD-agarose but was released as two cleavage fragments (25 and 30 kDa) from NADP-agarose. The beta subunit of the transhydrogenase bound to NAD-agarose was cleaved by trypsin in the presence of NADP(H) to yield 25 and 30 kDa fragments of the beta subunit. These results suggest that the beta subunit contains two pyridine nucleotide-binding sites.
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PMID:Evidence for the presence of two pyridine nucleotide-binding sites on the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase. 766 84

Active-site amino acid residues of human type II inosine 5'-monophosphate dehydrogenase (IMPDH) were investigated using the covalent modification reagents 6-chloroinosine 5'-monophosphate (6-Cl-IMP) and iodoacetamide. IMPDH was incubated with these reagents in the presence and absence of IMP, NAD, and NADH, and the activity of the enzyme for IMP dehydrogenation or 2-Cl-IMP dehalogenation was followed. IMPDH activity was rapidly lost when the enzyme was incubated with the IMP analog, 6-Cl-IMP, or with iodoacetamide. The enzyme was protected against inactivation in the presence of the substrate IMP. It was not protected against inactivation by NAD alone. Saturating concentrations of IMP and NADH reduced the inactivation rate by about the same amount as with IMP alone. IMPDH samples labeled with 6-Cl-IMP and an unlabeled control were alkylated with iodoacetamide, digested with trypsin, and analyzed by HPLC-mass spectrometry (HPLC-MS). All eight cysteines of human type II IMPDH were found to exist as free sulfhydryls on the active, unlabeled form of the enzyme. At an enzyme/inactivator ratio of 1:4, only one cysteine residue, Cys-331, was found to be covalently modified by 6-Cl-IMP. From the results of the substrate protection experiments and HPLC-MS data, it is concluded that 6-Cl-IMP binds in the IMP binding site of IMPDH and reacts covalently with Cys-331 to form a purine riboside 5'-monophosphate-enzyme adduct.
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PMID:Probing the active site of human IMP dehydrogenase using halogenated purine riboside 5'-monophosphates and covalent modification reagents. 790 43

Chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent malate dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.
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PMID:Essential histidine at the active site of sorghum leaf NADP-dependent malate dehydrogenase. 796 39

The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the alpha polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the alpha polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli beta polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.
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PMID:Cloning and sequencing of the genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and the implications for the domain structure of the enzyme. 807 1

The two active-site tryptophans of diphtheria toxin, Trp-50 and Trp-153, were individually or jointly replaced with phenylalanine or alanine by directed mutagenesis of a synthetic gene for the toxin's catalytic A fragment. Substitution of Trp-50 with alanine (W50A) decreased the ADP-ribosyltransferase activity by nearly 10(5)-fold and reduced NAD-glycohydrolase activity beyond the limits of our detection. Effects of the W153A mutation on these activities were less dramatic, < 40-fold decrease in ADP-ribosylation and < 10-fold decrease in NAD glycohydrolysis. The W50F and W153F substitutions caused only minimal reductions (< 2-fold) in enzyme activities and NAD affinity. Decreases in affinity for NAD in the initial, ground state complex, as measured by intrinsic protein fluorescence, correlated well with the reductions in enzyme activity. None of the mutations caused greater than a 10-fold decrease in NAD affinity for the ternary Michaelis complex in the ADP-ribosylation reaction; and none caused significant increase in susceptibility to proteolytic digestion by trypsin. The results indicate that Trp-50 is a major determinant of NAD affinity. Also, they identify this residue as a candidate for modification in the development of inactive forms of the toxin for use in vaccine development.
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PMID:Active-site mutations of diphtheria toxin. Tryptophan 50 is a major determinant of NAD affinity. 808 36

Treatment of the Rho-ADP-ribosylating C3-like transferase from Clostridium limosum by ultraviolet irradiation in the presence of [carbonyl-14C]NAD incorporated 1 mol of label/mol of exoenzyme. Concomitantly, the transferase and NAD glycohydrolase activity was impaired. A peptide containing the radiolabel was obtained by proteolysis with either staphylococcal protease V8 or trypsin. Their amino acid sequences were Ala/Asp-Gly-Tyr-Ile-Glu-Pro-Ile-Ser-Thr-Phe-Lys-Gly-Gln-Leu-X-Val-Leu-Le u-Pro- Arg and Gly-Gln-Leu-X-Val-Leu-Leu-Pro-Arg, respectively. These sequences correspond with regions Ala-160 through Arg-179 and Gly-171 through Arg-179, respectively, of the very similar Clostridium botulinum C3 transferase, with X being Glu in the unlabeled enzyme. This identifies the glutamic acid residue that corresponds to Glu-174 of C. botulinum C3 transferase as part of the NAD-binding site of the catalytic center of the C. limosum exoenzyme.
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PMID:NAD-binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum. 822 42

An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.
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PMID:Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells. 824 57


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