Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aminoacyl-tRNA protein transferases catalyze (posttranslational) aminoacylation of specific protein N-termini, using aminoacyl-tRNA as substrate. This modification targets the protein for ATP-dependent degradation; in eukaryotes, degradation occurs in the ubiquitin-mediated pathway. The eukaryotic transferase, which catalyzes Arg transfer to N-terminal Glu or Asp residues, is potently inhibited by phenylarsenoxides. The gene encoding Arg-tRNA protein transferase from the yeast Saccharomyces cerevisiae was subcloned and overexpressed in Escherichia coli to provide large amounts of homogeneous protein for a molecular analysis of this inhibition. The bifunctional reagent para-[(bromoacetyl)amino]-phenylarsenoxide is a potent and irreversible inactivator of the yeast transferase; the arsenoxide moiety of the reagent directs binding to the enzyme, while the alkyl halide moiety alkylates a residue(s) proximal to the arsenoxide site. One mole of 14C-labeled reagent was covalently incorporated during inactivation, with the side chain of Cys-315 representing the major site of alkylation. Mutation of Cys-315 to Ala yielded a fully active enzyme which was still subject to stoichiometric, irreversible inactivation by the bifunctional arsenoxide. With the C315A-enzyme, the major fraction of the 14C-labeled bifunctional reagent was associated with the side chain(s) of one or more of a stretch of Glu residues (Glu 339-341). These results show that phenylarsenoxides inhibit Arg-tRNA protein transferase by binding to a site that is either itself essential, or regulates an essential site. Inhibition appears to occur through a steric blockade mechanism.
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PMID:Inactivation of arginyl-tRNA protein transferase by a bifunctional arsenoxide: identification of residues proximal to the arsenoxide site. 781 89

Analysis of two isomeric cyclic hexapeptides of composition (Asp, Arg, Gly2, Pro, D-Pro) by a nuclear Overhauser effect constrained distance geometry conformation search yielded a narrowly defined backbone conformation for one and considerable ambiguity about the conformation in part of the other. Preliminary 13C relaxation studies of these peptides suggest that it is possible that this difference may correspond to a physical difference in internal mobility. In connection with this observation, other experimental evidence bearing on the backbone conformational mobility of cyclic oligopeptides with 4-10 residues, frequently considered to have well-defined backbones, is reviewed. Conformational heterogeneity involving rotation of a peptide bond plane relative to the overall ring plane is identified as a common phenomenon. Nuclear magnetic resonance line-shape studies at temperatures down to 200 K can detect backbone motions with activation free energy barriers down to about 10 kcal/mole, but conformational exchange with lower barriers, though detectable in other ways, will not be obvious from nmr spectra alone.
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PMID:Conformational mobility in cyclic oligopeptides. 810 73

Ornithine transcarbamylase (OTCase) has been purified from porcine liver by a simple four-step procedure that included chromatography on an affinity column to which the transition-state analogue, delta-N-phosphonacetyl-L-ornithine (PALO), was covalently bound. The procedures employed yielded an enzyme which was purified some 260-fold and was judged to be homogeneous by nondenaturing- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent homogeneity of the enzyme was confirmed by N-terminal sequence analysis. The molecular weight of the porcine enzyme was determined by Sephadex gel exclusion chromatography and sedimentation equilibrium. An approximate molecular weight of 107,000 was calculated by both procedures. The single band obtained by SDS-PAGE indicated a subunit molecular weight of 36,800 +/- 700; hence, the enzyme is a trimer of identical subunits. The sedimentation coefficient of the native enzyme was determined to be 6.47. At pH 8.0, the Km values for the substrates are 0.41 and 1.3 mM for ornithine and carbamyl phosphate, respectively. PALO is a competitive inhibitor and has a Ki of 0.13 microM, which suggests that it binds with about 10,000 times greater affinity than carbamyl phosphate. Amino acid analysis performed on acid hydrolyzed enzyme yielded 323 amino acids per monomer. Performic acid oxidation of the enzyme, followed by acid hydrolysis and amino acid analysis, showed three cysteine residues per subunit. A partial specific volume of 0.725 cc/g was calculated from the amino acid composition. Reaction of purified porcine OTCase with phenylglyoxal, an arginine-specific reagent, results in complete loss of catalytic activity. The decrease in enzymatic activity correlates with the modification of 1 mol of arginine per mole of OTCase monomer. In the presence of 20 mM carbamyl phosphate, 93% of the activity is retained during a 1-h reaction time. Other substrates and substrate combinations offer less protection.
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PMID:Purification and properties of porcine liver ornithine transcarbamylase. 813 41

The pH titration of nine amino acids (glycine, proline, valine, serine, glutamine, tryptophan, arginine, histidine and aspartic acid) in presence of urea in the concentration range 1-8 mole dm-3 has been performed. The results support suppression of the first dissociation constant (K1) of the amino acids and acceptance of H+ ions by the amide forming uronium ion (UH+). The second dissociation constant (K2) of the amino acids is affected relatively weakly by urea. Quantitative evaluation of different species existing in solution and the degree of dissociation of the acids as well as the degree of binding of H+ ion to the amide have been made. It has been found that the polarity of the aqueous-urea medium does not straight forwardly correlate with the altered pK1 of the amino acids. Urea can also affect the pH-titration behaviour of gelatin with an increase of the intrinsic pK of the acidic groups of the protein by 0.45 unit.
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PMID:Interaction of amino acids and gelatin with urea. 814 76

Chemical modification of cytochrome P450 was used to study the involvement of lysine and arginine residues in the interaction between cytochrome P450 and NADPH-cytochrome P450 reductase. Acetylation of 2.2 and 8.5 mol of lysine/mole of P450 by acetic anhydride led to 38.7 and 95% reductions, respectively, in benzphetamine demethylation activity by NADPH-dependent reconstituted P450/reductase complex, while modification of up to 8.5 mol of lysine/mol of P450 did not inhibit cumene hydroperoxide-supported P450-dependent benzphetamine demethylation. Acetylation of lysine residues by acetic anhydride does not grossly disturb the P450 protein conformation as revealed by absolute, CO-difference and fluorescence spectral studies. Modification of P4502B1 by acetic anhydride did not affect its substrate binding ability either. Lysine residues of P4502B1 putatively involved in the interaction with reductase have been identified by radiolabeling of lysine residues with [14C]acetic anhydride followed by trypsin digestion, HPLC separation, and amino acid microsequencing. Radiolabeled lysines occur at positions 251, 384, 422, 433, and 473. Modification of arginine residues in P4502B1 with phenylglyoxal and 2,3-butanedione seemed to have no significant effect on the benzphetamine demethylation activity of P4502B1 either reconstituted with reductase and NADPH or supported by cumene hydroperoxide. Studies of incorporation of [14C]phenylglyoxal showed no concentration- or time-dependent incorporation of phenylglyoxal into the P4502B1. These results support the hypothesis of a predominant role of lysine residues of P450 in the electrostatic interaction with NADPH-cytochrome P450 reductase.
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PMID:Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase. 832 89

We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pI of 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro-Phe-Arg, and Val-Leu-Arg-7-amino-4-methylcoumarin, gave K(m) values for mole kallikrein of 3.3, 46.1, and 2.8 microM, respectively, and specificity constants, kcat/K(m), of 3818, 165, and 8714 s-1 pM-1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family. S. aquaticus is recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research.
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PMID:Purification and characterization of salivary kallikrein from an insectivore (Scalopus aquaticus): substrate specificities, immunoreactivity, and kinetic analyses. 861 26

Cathepsin H (EC 3.4.22.16) from cow brain, purified to approximately 1800-fold with approximately 26% activity yield, hydrolysed BANA, Leu-2-NNap, Arg-2-NNap, and Met-2-NNap maximally at pH 6.5, 6.8, 7.0 and 7.2, respectively. It was activated by sulphydryl compounds and EDTA while sulphydryl alkylators and blockers were found to inhibit the enzyme activity. Met-2-NNap was found to be the best substrate followed by Thr-2-NNap, His-2-NNap, Leu-2-NNap, Arg-2-NNap and Ala-2-NNap, respectively. The Km values for hydrolysis of various substrates viz., Met-2-NNap, Leu-2-NNap, Arg-2-NNap, Arg-NNapOMe, Thr-2-NNap, His-2-NNap, BANA, Arg-pNA and Lys-pNA were 0.128, 0.167, 0.169, 0.288, 0.428, 0.500, 0.667, 0.195 and 0.476 mM, respectively. The temperature optima for hydrolysis of BANA and Leu-2-NNap were approximately 45 degrees C and approximately 50 degrees C with activation energies of approximately 13.7 and approximately 11.0 kcal mole-1, respectively. The enzyme was fairly stable upto 50 degrees C and between pH 4.0-7.5.
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PMID:Physico-chemical properties of brain cathepsin H. 871 50

Loss of heterozygosity of chromosome region 9p21 occurs commonly and early in sporadic melanoma, suggesting the involvement of a tumor suppressor gene at this locus in the pathogenesis of this neoplasm. Although germline mutations and deletions of the p16INK4 gene located at 9p21 have been reported in familial melanoma, the relative contributions of mutation and deletion in sporadic melanoma are at present unclear. In this study, we investigated 26 cases of sporadic cutaneous melanoma (14 of which demonstrated loss of heterozygosity at 9p21) for mutations of p16INK4. One tumor with allelic loss of 9p contained a CC-->TT mutation at codons 57/58, altering an arginine to a stop codon, consistent with bi-allelic inactivation of p16INK4 in this case. No mutations were identified in any of the other melanomas, or in one benign intradermal nevus with atypical features and two Spitz nevi that also showed loss of heterozygosity of 9p. The inactivation of both copies of p16INK4 in the one case of melanoma adds support to the theory that p16INK4 is important in the development of sporadic cutaneous melanoma, although allelic loss or other methods of inactivation of p16INK4 rather than point mutation appears to be numerically more important. The low frequency of mutation of p16INK4 in cases of sporadic melanoma with loss of heterozygosity of 9p is, however, also consistent with there being another tumor suppressor gene near this locus that is involved in some cases of sporadic melanoma.
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PMID:Infrequent mutation of p16INK4 in sporadic melanoma. 918 29

Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.
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PMID:Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli. 877 Dec 8

The interaction of the two N-terminally myristoylated isoforms of Dictyostelium hisactophilin with lipid model membranes was investigated by means of the monolayer expansion method and high-sensitivity titration calorimetry. The two isoforms, hisactophilin I and hisactophilin II, were found to insert with their N-terminal myristoyl residue into an electrically neutral POPC monolayer corresponding in its lateral packing density to that of a lipid bilayer. The partition coefficient for this insertion process was Kp = (1.1 +/- 0.2) x 10(4) M-1. The area requirement of the protein in the lipid membrane was estimated as 44 +/- 6 A2 which corresponds to the cross sectional area of the myristoyl moiety with an additional small contribution from amino acid side chains. The interaction of hisactophilin I (hisactophilin II) with negatively charged membrane surfaces is modulated in a pH-dependent manner by charged amino acid residues clustered around the myristoyl moiety. The electrostatic binding site consists of three lysine (one arginine and two lysine), seven (nine) histidine, and four (four) glutamic acid residues and has an isoelectric point of 6.9 (7.1). For small unilamellar POPC/POPG (75/25 mole/mole) vesicles, an apparent binding constant, K(app) = (8 +/- 1) x 10(5) M-1, was measured at pH 6.0 by means of high-sensitivity titration calorimetry. Electrostatic interactions hence increase the binding constant by about 2 orders of magnitude compared to hydrophobic binding alone. With increasing pH, the electrostatic attraction decreases and turns into an electrostatic repulsion at pH > 7.0 +/- 0.1. The area occupied by the cluster of charged residues constituting the membrane binding region was 280 +/- 20 A2 as derived from monolayer measurements in close agreement with molecular modeling data derived from the NMR structure of hisactophilin I [Habazettl et al. (1992) Nature 359, 855-858].
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PMID:Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch. 878 May 5


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