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W S Allison

Publications and source records attributed to W S Allison.

At least 73 records · Page 4Linked to original sources
PubMed1983

Modulation by ADP and Mg2+ of the inactivation of the F1-ATPase from the thermophilic bacterium, PS3, with dicyclohexylcarbodiimide.

The soluble F1-ATPase from the thermophilic bacterium PS3 (TF1) contains no endogenous adenine nucleotides and contains about 0.2 g ions of Mg2+/mol which resists removal by repeated centrifugation-elution on columns of Sephadex G-50. The isolated enzyme will not bind additional Mg2+ added in the absence of adenine nucleotides nor is the rate of inactivation of the isolated enzyme by dicyclohexylcarbodiimide (DCCD) affected by the addition of Mg2+. When ADP is added to isolated TF1, a 1:1 TF1 X ADP complex is formed which is stable to repeated gel permeation on columns of Sephadex G-50 subjected to centrifugation-elution. On formation of the 1:1 TF1 X ADP complex, the rate of inactivation of the enzyme by DCCD is accelerated 6-fold. The rate of inactivation of the 1:1 TF1 X ADP complex by DCCD is not further stimulated in the presence of 2 mM ADP which indicates that the binding of ADP to a single site in the enzyme is sufficient to promote maximal stimulation of the inactivation. Addition of Mg2+ to the 1:1 TF1 X ADP complex results in the binding of about 1 g ion of Mg2+/mol of enzyme. The 1:1:1 TF1 X ADP X Mg2+ complex thus formed is sluggishly inactivated by DCCD. When the Mg2+ is removed from the TF1 X ADP X Mg2+ complex by treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, the rate of inactivation of the enzyme by DCCD is accelerated 4-fold. Other divalent metal ions protect the 1:1 TF1 X ADP complex against inactivation by DCCD. Of these, Mn2+, Zn2+, Co2+, and Cd2+, which are about as equally effective as Mg2+ as cofactors for the hydrolytic reaction when present at 0.2 mM, offer about equal protection of the complex against inactivation by DCCD also when present at 0.2 mM. These results indicate that the binding site for ADP in the 1:1 TF1 X ADP complex is a catalytic site. TF1, inactivated by 92% with DCCD, has the same capacity to bind ADP as the active enzyme, forming a tight 1:1 TF1 X ADP complex which is stable to repeated centrifugation-elution on columns of Sephadex G-50. The 1:1 TF1 X ADP complex retains its capacity to bind Mg2+ to form the 1:1:1 TF1 X ADP X Mg2+ complex after it is inactivated by 88% with DCCD.

Adenosine Diphosphate↗
PubMed1983

Quinacrine mustard inactivates the bovine heart mitochondrial F1-ATPase with the modification of the beta subunit.

The bovine mitochondrial F1-ATPase is reversibly inhibited by quinacrine. The concentration of quinacrine which causes 50% inhibition at pH 7.0 is estimated to be 580 microM. Lineweaver-Burk plots constructed from kinetic data collected at pH 7.0 with equimolar concentrations of Mg2+ and ATP in the reaction mixtures indicate competitive inhibition by quinacrine. Uncompetitive inhibition by quinacrine is indicated by Lineweaver-Burk plots constructed from kinetic data obtained at pH 7.0 with variable ATP concentrations and a constant Mg2+ concentration of 3.0 mM. A KI of 440 microM was calculated from a replot of 1/Vmaxi versus quinacrine concentration using the intercepts of the Lineweaver-Burk plots constructed from the data obtained at a fixed concentration of 3.0 mM Mg2+. Quinacrine mustard is a potent inactivator of the F1-ATPase. The pseudo-first order rate constant, k1, for the inactivation of the enzyme by 500 microM quinacrine mustard is 0.161 min-1 at pH 7.0 and 23 degrees C. Under the same conditions in the presence of 5.0 mM ATP, 5.0 mM ADP, or 5.0 mM Mg2+ plus 5.0 mM ADP, k1 is, respectively: 0.082 min-1, 0.136 min-1, or 0.075 min-1. In the presence of 1.0 mM chlorpromazine or 5.0 mM quinacrine under the above conditions, k1, is 0.089 min-1 and 0.037 min-1, respectively. Free Mg2+ has no effect on the rate of inactivation of the enzyme by quinacrine mustard. The rate of inactivation of the F1-ATPase by quinacrine mustard as a function of pH revealed an apparent pK alpha of 8.0. Examination of the resolved subunits for fluorescence after inactivating the enzyme with quinacrine mustard demonstrated that only the beta subunit was labeled.

Adenosine Triphosphate↗
PubMed1983

Proton-translocating ATPase and lysosomal cystine transport.

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.

Animals↗
PubMed1983

Inhibition of an oligomycin-sensitive ATPase by cationic dyes, some of which are atypical uncouplers of intact mitochondria.

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.

Adenosine Triphosphatases↗
PubMed1981

Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit.

When the bovine mitochondrial F1-ATPase is inactivated with dicyclohexyl[14C]carbodiimide and then gel-filtered, from 2 to 3 g atoms of 14C are incorporated/mol of enzyme. Prior inactivation of the enzyme by the modification of an essential tyrosine residue with 4-chloro-7-nitrobenzofurazan, a reaction that can be reversed by thiols, does not affect the irreversible inactivation of the ATPase by dicyclohexyl[14C]carbodiimide. During the large scale modification of the F1-ATPase by dicyclohexyl[14C]carbodiimide which led to 70% inactivation, 1.9 g atoms of 14C were incorporated/mol of enzyme. Isolation of the alpha, beta, and gamma subunits from this large scale inactivation revealed that the gram atoms of 14C bound per mol of each of the subunits was: alpha, 0.04; beta, 0.56; and gamma, 0.04. The majority of the radioactivity in a cyanogen bromide digest of the 14C-labeled beta subunit was isolated in a fragment that has the following amino acid sequence: Glu-Leu-Ile-Asn-Asn-Val-Ala-Lys-Ala-His-Gly-Gly-Tyr-Ser-Val-Phe-Ala-Gly-Val-Gly -Glu-Arg-Thr-Arg-Glu-Gly-Asn-Asp-Leu-Tyr-Glu*-His-Met; where Glu* represents the N gamma-glutamyl derivative of dicyclohexyl[14C]urea.

Adenosine Triphosphatases↗
PubMed1981

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3.

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.

Adenosine Diphosphate↗
PubMed1979

On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine.

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.

Adenosine↗
PubMed1978

Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine.

The mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analogue, p-fluorosulfonylbenzoyl-5'-adenosine. This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer containing 20% glycerol. Under these conditions, 4.2 g atoms of 14C are incorporated per 350,000 g of enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM p-fluorosulfonyl[14C]benzoyl-5'-adenosine. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was associated with the two largest subunits. The isolated alpha subunit contained 0.45 g atom of 14C/mol and the isolated beta subunit contained 0.88 g atom of 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the beta subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled beta subunit is contained ina tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-Tyr-Asp-Val-Ala-Arg, where Tyr is the radioactive derivative of the tyrosine residue that was sulfonylated during the inactivation.

Adenosine↗
PubMed1977

Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2.

Two novel heterobifunctional cross-linking reagents, which can be used to attach photoactivatable nitroaryl azides to primary amino groups of proteins, have been synthesized. The two compounds, N-5-azido-2-nitrobenzoyloxy-succinimide and ethyl N-5-azido-2-nitrobenzoylaminoacet-imidate-HCl, as well as ethyl 4-azidobenzimidate-HCl have been attached to lysine residues of cobra venom phospholipase A2 without a loss in enzymatic activity. Subsequent illumination of the modified forms of the enzyme at appropriate wavelengths under conditions in which the native enzyme exists in an aggregated state led to the formation of covalently linked dimers and large aggregates which could be separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate.

Elapid Venoms↗
PubMed1976

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.

Alcohol Oxidoreductases↗
PubMed1975

The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.

Amino Acid Sequence↗
PubMed1975

The conversion of glyceraldehyde-3-phosphate dehydrogenase to an acylphosphatase by trinitroglycerin and inactivation of this activity by azide and ascorbate.

Trinitroglycerin oxidizes the essential sulfhydryl group, Cys-149, of pig muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(phosphorylating) EC 1.2.1.12) TO A SLUFENIC ACID, NOT TO A DISULFIDE. This conclusion is based on the observation that the inactivation of the dehydrogenase activity of the enzyme by the organic nitrate induces the acylphosphatase activity which is catalyzed by the sulfenic acid form of the enzyme. Inorganic nitrite is released during this process which is stoichiometric with the degree of inactivation of the dehydrogenase. The acylphosphatase activity induced by trinitroglycerin, unlike the dehydrogenase activity, is sensitive to CN-. Treatment of the enzyme oxidized with trinitroglycerin with 14-CN- leads to the incorporation of protein-bound 14-CN-, which is stoichiometric with the degree of inactivation of the dehydrogenase. Treatment of the sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase at pH 5.3 with a 10-fold molar excess of azide over the concentration of enzyme subunit completely inactivates the acylphosphatase reaction catalyzed by the oxidized enzyme. Concomitantly, the dehydrogenase activity catalyzed by the sulfhydryl form of the enzyme reappears which indicates that excess azide reduces the sulfenic acid which is required for the acylphosphatase. Treatment of the oxidized enzyme with a stoichiometric amount of azide at pH 5.3 stimulates the acylphosphatase activity and does not lead to the reappearance of dehydrogenase activity. When the sulfenic acid form of the enzyme is incubated with 20 mM L-ascorbate at pH 5.3, the acylphosphatase activity is completely inactivated and the dehydrogenase activity catalyzed by the reduced form of the enzyme is recovered. Thus, L-ascorbate also reduces the protein sulfenic acid which is required for the acylphosphatase activity.

Animals↗