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Biomedical subjects

R F Colman

Publications and source records attributed to R F Colman.

At least 145 records · Page 8Linked to original sources
PubMed1981

Modification of two essential cysteines in rabbit muscle pyruvate kinase by the guanine nucleotide analogue 5'[p-(fluorosulfonyl) benzoyl] guanosine.

Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.

Adenosine Diphosphate↗
PubMed1981

Re-evaluation of molecular weight of pig heart NAD-specific isocitrate dehydrogenase.

NAD-specific pig heart isocitrate dehydrogenase was earlier reported, on the basis of gel filtration experiments, to have a molecular weight of approximately 340,000. In the present study, the enzyme is shown by equilibrium ultracentrifugation to have a weight average molecular weight of approximately 224,000 which can be attributed to a rapidly associating-dissociating protein system. The results of light-scattering measurements are consistent with the lower value of molecular weight. The enzyme exhibits an average frictional ratio, f/f0, of 1.39 as determined from ultracentrifuge experiments, and this deviation from typical proteins may account for the previous high molecular weight estimates. An average Stokes radius of 6.0 nm was calculated from the present gel filtration experiments. By use of this value and a sedimentation coefficient of 9.1 S, an average molecular weight of 245,000 has been calculated. Previous studies (Ramachandran, N., and Colman, R. F. (1980) J. Biol. Chem. 255, 8859-8864) have indicated that the enzyme is composed of three different subunits, present in the ratio 2:1:1, each of which has a molecular weight of about 40,000. These results, together with the present observations, lead to the conclusion that, under stabilizing conditions in solution, the NAD-dependent isocitrate dehydrogenase predominantly exhibits a minimum, molecular weight of 160,000 but behaves as a mixture of oligomeric species with an average Mr of about 224,000.

Animals↗
PubMed1981

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.

Adenosine↗
PubMed1980

Affinity labeling of the active site of yeast pyruvate kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot of kinact versus the 5'-p-fluorosulfonylbenzoyl adenosine concentration yields a hyperbolic curve indicative of binding of the analog prior to reaction. Marked protection is afforded by phosphoenolpyruvate + fructose 1,6-diphosphate + Mg2+ or MgATP suggesting that reaction occurs within the active site. When assayed at less than saturating phosphoenolpyruvate concentrations, the inactivation caused by the reagent in the absence of added ligands appears slower, and reaction in the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+ produces an activation of the enzyme, the extent of which is dependent on the assay concentration of phosphoenolpyruvate. The rate constant for activation was observed to be 0.113 min-1. The activated enzyme exhibits both a lowered K0.5 and Hill coefficient compared to native pyruvate kinase. Subsequent addition of 5'-p-fluorosulfonylbenzoyl adenosine to activated pyruvate kinase in the absence of added ligands leads to inactivation with the rate constant independent of the assay concentration of phosphoenolpyruvate. Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites. In the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+, incorporation of tritiated 5'-p-fluorosulfonylbenzoyl adenosine is linearly proportional to the extent of activation of the enzyme, with 4 mol of reagent bound/mol of tetrameric pyruvate kinase for maximally activated enzyme. In the absence of added ligands, approximately 4.5 mol of reagent are incorporated/mol of enzyme at 15 min of reaction, while 80% of the original activity remains. Subsequent incorporation is proportional to the extent of inactivation with 8 mol bound at 100% in activaton. In the presence of phosphoenolpyruvate, fructose 1,6-diphospate, and Mg2+, 3 tyrosines and 1 lysine residue, and in the absence of ligands, 6 tyrosines and 2 lysine residues are modified, suggesting that both amino acids are within the two nucleotide sites.

Adenosine↗
PubMed1980

Chemical characterization of distinct subunits of pig heart DPN-specific isocitrate dehydrogenase.

Pig heart DPN-dependent isocitrate dehydrogenase is heterogeneous on isoelectric focusing in 6 M urea. Under these conditions, three types of subunits (termed alpha, beta, and gamma), which have isoelectric points of about 5.7, 6.6, and 7.2, respectively, can be separated. On the basis of densitometric scans of analytical isoelectric focused gels stained with Coomassie blue, it is estiated that the subunits are present in the whole enzyme in the approximate ratio of 2 alpha:1 beta: 1 gamma. The three isolated subunits have distinct amino acid compositions and the amino acid composition of the total enzyme, when expressed as residues per average polypeptide chain of 40,000 daltons, is consistent with contributions of alpha, beta, and gamma subunits in the ratio of 2:1:1. Each isolated subunit yields a readly distinguishable tryptic peptide map which is much simpler than that of the total enzyme, and is consistent with the number of peptides expected from the lysyl plus arginyl residues for the subunit. The alpha and beta chains both have alanine as the NH2-terminal amino acid, whereas phenylalanine is the NH2-terminal residue of the gamma subunit. Since the alpha subunit exhibits a molecular weight of 39,000 and the beta and gamma subunits have indistinguishable molecular weights of 41,000, the two-band pattern observed on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is understandable. These results suggest that a complete DPN-specific isocitrate dehydrogenase would have a minimum molecular weight of 160,000.

Amino Acids↗
PubMed1980

Adenosine 5'-(2-bromoethyl)-phosphate. A new affinity label for adenine nucleotide sites in proteins.

A new adenosine analogue adenosine 5'-(2-bromoethyl)-phosphate has been synthesized. The reactive moiety, a bromoalkyl group, has the ability to react with the nucleophilic side chains of several amino acids. This compound reacts with NAD-dependent isocitrate dehydrogenase from pig heart, causing inactivation. Addition of the allosteric regulator ADP to the reaction mixture protects the enzyme from loss of activity. A second adenosine analogue has been synthesized, adenosine 5'-(n-propyl)-phosphate, which is used to assess any effects that might arise from the noncovalent interaction of adenosine 5'-(2-bromoethyl)-phosphate with the enzyme. It is proposed that adenosine 5'-(2-bromoethyl)-phosphate reacts with an adenine nucleotide site on isocitrate dehydrogenase and that this compound may have general applicability as an affinity label of catalytic and regulatory adenine nucleotide sites in proteins.

Adenine Nucleotides↗
PubMed1980

Identification of two distinct adenosine diphosphate receptors in human platelets.

(1) FSBA, an inhibitor of platelet shape change and aggregation, inhibits and reverses the binding of fibrinogen to washed platelets induced by ADP in the presence of calcium ion; (2) FSBA does not inhibit either aggregation of or fibrinogen binding to platelets treated with chymotrypsin; (3) additional evidence is provided that FSBA does not inhibit aggregation by stimulating cyclic AMP accumulation; (4) FSBA does not inhibit PGE1-induced cyclic AMP accumulation and has little ability to inhibit the action of ADP on cyclic AMP or the binding of the ADP analog [beta 32P] 2-MeSADP; and (5) these results provide additional support for the view that the effects of ADP on aggregation and on cyclic AMP are mediated by distinct receptors, one of which, designated "A", is labeled by FSBA; the second receptor designated "C", has selective affinity for 2-MeSADP.

Adenosine↗