Kenneth S. Rogers

Variable sulfhydryl activity toward silver nitrate by reduced glutathione and alcohol, glutamate and lactate dehydrogenases.

Abstract Variable sulfhydryl activity towards silverions was detected with amperometric techniques for reduced glutatione, yeast alcohol dehydrogenase, bovine liver glutamate dehydrogenase, bovine heart and rabbit muscle lactate dehydrogenases. The second order reaction rate between thiol residues of protein and silver ions seemed dependent upon the state of protein conformation. Denaturation of the four enzymes with 7 mM sodium dodecyl sulfate removed the difference in kinetic activity observed for the thiol residues of the native proteins. NADH increased the reactivity of bovine liver glutamate dehydrogenase toward silver ions and decreased the reactivity of the sulfhydryl residues of the lactate dehydrogenases. No change was observed in sulfhydryl activity of reduced glutathione or yeast alcohol dehydrogenase by the presence of NADH.

3-Hydroxykynurenine, 3-Hydroxyanthranilic Acid, and o-Aminophenol Inhibit Leucine-Stimulated Insulin Release from Rat Pancreatic Islets

Abstract Individual islets were isolated from rat pancreas to study the effects of tryptophan and its metabolites on leucine-stimulated release of insulin. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol were inhibitors at concentrations below 10 mM whereas tryptophan, kynurenine, kynurenic acid, xanthurenic acid, and anthranilic acid were ineffective inhibitors at concentrations up to 10 mM. A structure-activity analysis of these metabolites demonstrated that vicinal aromatic hydroxy and amino groups with their concomitant electron donating properties are required for inhibition of insulin release. Inhibition of islet insulin release by the three kynurenine metabolites may be involved in the depressed insulin levels found in vitamin B6-deficient rats by other workers.

Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: A simple assay for tryptophan

Abstract Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds. Tryptophanyl fluorescence of these proteins was directly proportional to the tryptophan content of the reduced, detergent-protein complex. Denaturation of proteins by detergent alone, although causing pronounced changes in fluorescence intensity, was unable to provide a common micro-environment for all tryptophan residues. A simple fluorescence assay of tryptophan content in proteins was developed.

Melting point depression of DNA by tetraalkylammonium bromides

Abstract Varied concentrations of tetramethyl- (TMAB), tetraethyl- (TEAB), tetrapropyl- (TRAB), tetrabutyl- (TBAB), tetrapentylammonium bromide (TPAB) and ammonium bromide are monitored for effects on DNA sedimentation, viscosity and melting temperature. No changes are observed at 25–30°C for the hydrodynamic properties of DNA when treated with the quaternary ammonium bromide. At higher temperatures, 30–100°C, the tetraalkyl-ammonium bromides reduce the temperature required for conversion of the DNA double-strand helix to the coil (Tm). A form of Debye-Huckel equation ( T m =a mu; 2 + b ) accurately describes the results. Ionic strength of the quaternary ammonium compounds is represented by μ/2. The potency of tetraalkylammonium bromide in reducing Tm (“a” in the Tm equation) is correlated with the hydrophobicity of the molecule (number of carbon atoms), r = 0.999 and F = 526 (r represents the correlation coefficient and F represents the test value for significance of the equation evaluated by least squares analysis). The Tm of rat liver chromatin is also reduced in a similar fashion by TPAB. The Tm of poly(dG-dC) is not altered by 0.1 M TBAB whereas the Tm of poly(dA-dT) is greatly reduced by the compound. It is thought that the tetraalkylammonium bromide preferentially binds to the coil of DNA during heating and that a preferentially hydrophobic interaction may occur with the adenine-thymine bases.

Molecular interactions of six aromatic competitive inhibitors with bovine liver glutamate dehydrogenase

Abstract Six aromatic dicarboxylic acids (isophthalic acid, pyridine-3,5-dicarboxylic acid, and pyridine-2,6-dicarboxylic acid) and bromo-substituted aryl monocarboxylic acids (5-bromofuroic acid, m -bromobenzoic acid, and 5-bromothiophene-2-carboxylic acid) were tested with bovine liver glutamate dehydrogenase [ l -glutamate: NAD(P) + oxidoreductase (deaminating) EC 1.4.1.3] for inhibition against l -glutamate as a function of pH (6.6 to 9.0). These compounds were competitive inhibitors at all pH values tested and the inhibition remained competitive when either NADP + or NAD + was coenzyme. Maximum inhibitor potency (p K 1 ) for the dicarboxylic acids occurred at pH 7.8 and for the monocarboxylic acids occurred at pH 8.7. The relative inhibitor potencies were correlated with the inhibitors‘ “central atom” absolute charge densities | Q T | calculated from molecular orbital theory. This indicated that desolvation of this atom may be important for combination of inhibitor with enzyme. A high degree of solvation as indicated by the magnitude of charge density could have decreased the interaction of inhibitor with enzyme. Similar results had been obtained previously for 4 aliphatic dicarboxylic acid competitive inhibitors

Rabbit Erythrocyte Hemolysis by Lipophilic, Aryl Molecules

Summary Indole, skatole, and 17 additional aryl molecules can interact with the cellular membrane to produce hemolysis of the rabbit erythrocyte. The minimum concentrations of aryl molecules required to produce total lysis of 1.4 × 108 red blood cells are correlated with the lipophilicity of the molecules as measured by their partitioning across the interface barrier of n-octanol: aqueous phosphate buffer, 50 mM, pH 7.4, phases. Hemolysis requires lesser amounts of a molecule that is lipophilic than one that is hydrophilic.

Effects of pH on benzimidazole fluorescence

Four benzimidazoles (unsubstituted, 5-methyl, 2-ethyl, and 2-ethyl-5-methyl) have been characterized by fluorescence spectroscopy. At low pH (<6), activation at 270 nm caused fluorescence at 305 nm; at high pH (<8), activation at 270 nm caused fluorescence at 365 nm. The relative proportion of peak fluorescence at either 305 or 365 nm was correlated with the pKa values of the four benzimidazoles. It was concluded that the protonated specie of benzimidazole was fluorescent at 365 nm and the unprotonated specie was also fluorescent at 305 nm.

Some biological applications of regular solution theory

ABSTRACT There is firm experimental and theoretical justification for expecting that solution theories, in this case regular solution theory, can be applied in a semi-empirical manner to questions of biological interest. This background is outlined and one of the possible models resulting from the use of regular solution theory is applied to the analysis of erythrocyte haemolysis. Suggestive evidence arising from correlations of n -octanol-water partition coefficients with parameters appropriate to this approach tends to indicate that regular solution theory may apply to a wider variety of biological systems than has previously been thought.

Hydrazine stress in the diabetic: Ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Krebs cycle activity: the latter being required for protein synthesis.

Synthesis of 5-Substituted Isophthalic Acids and Competitive Inhibition Studies with Bovine Liver Glutamate Dehydrogenase

Summary Isophthalic acid, 5-carboxy-, 5-hydroxy-, 5-methoxy-, 5-fluoro-, 5-bromo-, 5-cyano-, and 5-methylisophthalic acid were inhibitors competitive with l-glutamate for bovine liver glutamate dehydrogenase. The extent of inhibition by the derived compounds was not much greater than that obtained with the parent compound, isophthalic acid. A plot of pKi versus pH showed the presence of an ionizable group (pKa 7.4-7.8) at the enzyme active site which interacted with the substitutent at the 5 position of the substituted isophthalates.