Search PubMed⌕ Search

Biomedical subjects

C L Borders

Publications and source records attributed to C L Borders.

At least 37 records · Page 2Linked to original sources
PubMed1983

Affinity inactivation of bovine Cu,Zn superoxide dismutase by hydroperoxide anion, HO2-.

Bovine liver Cu,Zn superoxide dismutase (SOD) is inactivated by hydrogen peroxide at alkaline pH, and full inactivation correlates with the loss of 1.1 histidine/subunit. At each pH utilized, saturation of the rate of inactivation is observed. This process is characterized by a half-saturation constant for peroxide and a maximum pseudo-first-order rate constant for inactivation. At 25 degrees C, the former decreases from 15.7 to 3.2 mM as the pH is increased from 9.0 to 11.5, while the latter increases from 0.83 to 2.43 per min over the same pH range. We have previously (Arch. Biochem. Biophys. 224, 579 (1983] proposed that the true affinity reagent for the inactivation of yeast SOD is the hydroperoxide anion, and we now believe the same is true for bovine SOD. However, a subtle difference between the two enzymes exists, for while the maximum pseudo-first-order rate constant for inactivation of bovine SOD increases with increasing pH, the same parameter for the yeast enzyme is pH-independent.

Animals↗
PubMed1983

Hydroperoxide anion, HO-2, is an affinity reagent for the inactivation of yeast Cu,Zn superoxide dismutase: modification of one histidine per subunit.

Yeast Cu,Zn superoxide dismutase is inactivated by H2O2 at alkaline pH, and complete inactivation correlates with the modification of 1.0 histidine per subunit. At elevated concentrations of H2O2, a saturation process is evident and is characterized by kmax, the maximum pseudo-first-order rate constant for inactivation, and Kinact, the total hydrogen peroxide concentration at which the enzyme is half-saturated. In the pH range from 9.0 to 11.5 at 25 degrees C, kmax remains constant at 0.54 +/- 0.03 min-1, but Kinact decreases progressively with increasing pH, from 15.5 mM at pH 9.0 to 1.11 mM at pH 11.5. It is proposed that the reason for the observed increased affinity with increasing pH is that the reactive species is not H2O2 per se, but rather the HO-2 anion (the pKa for H2O2 is 11.6). An increase in pH would thus lead to an increased concentration of HO-2 at a fixed total peroxide concentration, and saturation would occur at a lower total peroxide concentration. By analogy with other anions, it is proposed that HO-2 coordinates directly to the Cu ion to form the reactive complex. Once the enzyme-peroxide complex is formed, however, the rate-determining step leading to modification of histidine and loss of activity is independent of pH between 9.0 and 11.5.

Affinity Labels↗
PubMed1983

Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Chloride--bicarbonate exchange in red blood cells: physiology of transport and chemical modification of binding sites.

About 80% of the CO2 formed by metabolism is transported from tissues to lungs as bicarbonate ions in the water phases of red cells and plasma. The catalysed hydration of CO2 to bicarbonate takes place in the erythrocytes but most of the bicarbonate thus formed must be exchanged with extracellular chloride to make full use of the carbon dioxide transporting capacity of the blood. The anion transport capacity of the red cell membrane is among the largest ionic transport capacities of any biological membrane. Exchange diffusion of chloride and bicarbonate is nevertheless a rate-limiting step for the transfer of CO2 from tissues to lungs. Measurements of chloride and bicarbonate self-exchange form the basis for calculations that demonstrate that the ionic exchange processes cannot run to complete equilibration at capillary transit times less than 0.5 s. The anion exchange diffusion is mediated by a large transmembrane protein constituting almost 30% of the total membrane protein. The kinetics of exchange diffusion must depend on conformational changes of the protein molecule, associated with the binding and subsequent translocation of the transported anion. We have characterized the nature of anion-binding sites facing the extracellular medium by acid-base titration of the transport function and modification of the transport protein in situ with group-specific amino acid reagents. Anion binding and translocation depend on the integrity and the degree of protonation of two sets of exofacial groups with apparent pK values of 12 and 5, respectively. From the chemical reactivities towards amino acid reagents it appears that the groups whose pK = 12 are guanidino groups of arginyl residues, while the groups whose pK = 5 are likely to be carboxylates of glutamic or aspartic acid. Our studies suggest that the characteristics of anion recognition sites in water-soluble proteins and in the integral transport proteins are closely related.

Amino Acids, Dicarboxylic↗
PubMed1982

Irreversible inactivation of red cell chloride exchange with phenylglyoxal, and arginine-specific reagent.

Chloride exchange in resealed human erythrocyte ghosts can be irreversibly inhibited with phenylglyoxal, a reagent specific for the modification of arginyl residues in proteins. Phenylglyoxal inhibits anion transport in two distinct ways. At 0 degrees C, inhibition is instantaneous and fully reversible, whereas at higher temperature in an alkaline extracellular medium, covalent binding of phenylglyoxal leads to an irreversible inhibition of the transport membranes system. Indiscriminate modification of membrane arginyl residues was prevented by reacting the with phenylglyoxal in an alkaline extracellular medium while maintaining intracellular pH near neutrality. The rate of modification of anion transport depends on phenylglyoxal concentration, pH, temperature, and the presence of anions and reversible inhibitors of the anion transport system in fashions that are fully compatible with the conclusion that phenylglyoxal modifies arginyl residues that are essential for anion binding and translocation. Phenylglyoxal reacts rapidly with the deprotonated form of the reactive groups. It is proposed that the effects of anions and of negatively charged transport inhibitors on the rate of irreversible binding of phenylglyoxal are related to the effects of the anions on a positive interfacial potential. This potential determines the local pH, and thereby the concentration of deprotonated groups, in an exofacial region of the anion transport protein.

Aldehydes↗
PubMed1979

Chemical modification of histidyl and lysyl residues in yeast enolase.

Modification of yeast enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) by diethyl pyrocarbonate at either pH 6.1 or 6.6 caused a biphasic inactivation of the enzyme. In the presence of excess Mg2+, either an equilibrium mixture of substrates or 3-phosphoglycerate, a competitive inhibitor, prevented the second slower phase of inactivation, but had no effect on the first rapid phase. Complete inactivation by diethyl pyrocarbonate correlates with the modification of six histidyl residues/subunit, while 3-phosphoglycerate protects two histidyl residues/subunit from modification. Modification of enolase by two lysine-specific reagents, 2,4,6-trinitrobenzenesulfonate and pyridoxal 5'-phosphate, at pH 8.3 caused a slow loss of enzyme activity. However, substrates did not significantly protect against inactivation by either reagent, and inactivation with 2,4,6-trinitrobenzenesulfonate correlates with the modification of 18 lysyl residues/enzyme subunit.

Binding Sites↗
PubMed1978

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.

Arginine↗
PubMed1977

Arginyl residues: anion recognition sites in enzymes.

Chemical modification with 2,3-butanedione in borate buffer indicates that nine of ten glycolytic enzymes studied contain arginyl residues at their active sites. Fructose-1,6-diphosphatase also has arginines at its binding site for the allosteric inhibitor, adenosine monophosphate. These and other data suggest that, as a general rule, enzymes acting on anionic substrates or cofactors will probably contain arginyl residues as components of their ligand binding sites. This could account in part for the relatively infrequent occurrence of arginine in proteins.

Adenosine Monophosphate↗
PubMed1976

Phosphoglycerate mutase has essential arginyl residues.

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.

2,3-Diphosphoglycerate↗
PubMed1975

An essential arginyl residue at the nucleotide binding site of creatine kinase.

Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.

Adenine Nucleotides↗