Margaret L. Fonda

Reaction of phenylglyoxal with arginine. The effect of buffers and pH

Abstract Phenylglyoxal reacts much more rapidly with N2-acetylarginine than with either N2-acetyllysine or N-acetylcysteine. The rate of the reaction of phenylglyoxal with either N-acetylarginine or arginine increases with increasing pH from 7.5 to 11.5. The model reaction with arginine is much faster in bicarbonate, diethylamine, or triethylamine buffer than in N-ethylmorpholine, borate, phosphate, or Tris buffer. This activation by various buffers should be taken into consideration when glyoxal derivatives are used to modify arginyl residues.

The relationship of γ-aminobutyrate levels and its metabolism to age in brains of mice☆

Abstract The activities of glutamate decarboxylase, aspartate aminotransferase, and γ-aminobutyrate aminotransferase were determined in the brains of C57B1/6J mice of selected biological ages (10, 24, 33, and 37 months old). The glutamate decarboxylase activity was the same in the 10 and 24 month mice, but was decreased in the 30 and 37 month mice. The aspartate aminotransferase activity was constant in the 10, 24, and 33 month old mice and was slightly decreased in the 37 month mice. The γ-aminobutyrate aminotransferase activity was the same in the brains of the 10 and 24 month old mice and was increased in the 33 month mice and even more in the 37 month mice. The steady state levels of aspartate, glutamate, and γ-aminobutyrate were determined in the brains of mice 10, 18, 29, and 36 months old. The concentration of aspartate was the same in the 10, 18, and 29 month mice and was increased in the 36 month mice. The mice in all the age groups had the same brain concentration of glutamate. γ-Aminobutyrate concentration decreased somewhat with age. The weights of the brains did not vary with age from 10 to 33 months. The wet weights of the brains of the 36 and 37 month mice were lower than those of all the other age groups. The amount of protein per gram of wet weight of brain did not vary with age.

Identification of Lys190 as the primary binding site for pyridoxal 5′-phosphate in human serum albumin

The covalent binding of pyridoxal 5′‐phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high‐affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H]PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu‐Asp‐Glu‐Leu‐Arg‐Asp‐Glu‐Gly‐Xaa‐Ala‐Ser‐Ser‐Ala‐Lys which corresponds to residues 182–195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys325) and acetylation by aspirin (Lys199).

Glutamine and ammonia metabolism in the brains of senescent mice

Abstract The steady state levels of glutamine, glutamate, other amino acids, and ammonia were determined in the brains of mature and senescent C57B1/6J mice. Glutamine was the only amino acid which showed an age-related change. The glutamine concentration in the senescent mouse brain was approximately 50% greater than that in the mature mouse brain. The brain ammonia concentration was increased 60% in the old mice. Alos the activities of glutaminase and γ-glutamyltransferase were determined. These activities were changed very slightly as a function of age, and thus their levels probably do not account for the observed differences in glutamine and ammonia levels. These results indicate that in the brains of senescent mice, more glutamine is synthesized in an attempt to detoxify the higher levels of ammonia.

Vitamin B-6 metabolism in the livers of young adult and senescent mice

Abstract The steady state levels of pyridoxal-P and pyridoxamine-P, the activities of pyridoxal (pyridoxine) kinase and pyridoxamine-(pyridoxine)-P oxidase, and the metabolism of [3H]pyridoxine were determined in the brains of C57B1/6J mice of selected ages. The steady state concentratioons of the coenzymes and the activities of the enzymes required for pyridoxal-P synthesis did not change significantly as a function of age. The uptake and metabolism of vitamin B-6 by the brain was studied by injecting [3H]pyridoxine in the tail vein of young adult and senescent mice, killing the mice after 15 or 30 min, and separating the B-6 metabolites by ion exchange chromatography. More total radioactivity was accumulated in 15 min in the brains of the senescent mice than the brains of the young mice. The brains from both age groups rapidly synthesized pyridoxal-P from pyridoxine. However, less radioactive pyridoxamine-P and more radioactive pyridoxal were formed in the brains of the senescent mice than in the young mice killed 15 min after injection. These results are similar to those obtained for the metabolim of [3H]-pyridoxine in the liver of these senescent mice. The senescent mice appear to be vitamin B-6 deficient, have decreased brain amino acid transaminase activity, and either increased pyridoxal-P phosphatase activity or decreased protection of brain pyridoxal-P.

A correlated morphometric and biochemical study of estrogen-induced vitellogenesis in male Rana pipiens.

Adult male Rana pipiens were administered estradiol-17 beta to induce vitellogenesis. Liver and blood were taken from control animals and experimental animals on Days 1, 2, 4, 8, 12, 16, and 120 following hormone treatment. Stereological analysis of livers showed that mitochondrial structural parameters remained constant while rough endoplasmic reticulum (RER) parameters increased significantly by 4 days and to more than four times control, or 120-day levels, by 8-16 days. Liver RNA concentration increased 2.5-fold and in parallel with RER, while liver protein and DNA concentrations did not change. Increases in total plasma protein and plasma vitellogenin (Vg) lagged behind increases in liver RER and RNA. Of the total plasma protein, Vg constituted 6% by 4 days, 40% by 12-16 days and less than 2% by 120 days. The half-life of plasma Vg was estimated to be no greater than 22 days. These studies provide the first quantitative correlations between ultrastructural and biochemical changes occurring in frog tissues following estrogen administration.

The effect of anions on the interaction of pyridoxal phosphate with glutamate apodecarboxylase

Abstract The rate of binding of pyridoxal phosphate to Escherichia coli glutamate apodecarboxylase was measured by absorption spectroscopy and enzyme activity in pyridine buffers containing a variety of anions. The change in absorbance at 428 nm using 20 μ m , apoenzyme and 50 μ m pyridoxal-P follows second-order kinetics. The apparent second-order rate constants obtained vary according to the anion present. Halides enhance the rate of binding in the order fluoride > chloride > bromide. Sulfate is a very potent inhibitor of binding. Phosphate, its methyl, ethyl, and phenyl esters, and pyrophosphate also inhibit pyridoxal-P binding to the apoenzyme. Monocarboxylic acids inhibit the rate of binding of coenzyme to apoenzyme in 50 m m pyridine-chloride, pH 4.6. Acetate is the best inhibitor, and there is a progressive decrease in inhibition with increasing chain length up to heptanoate. Multiple inhibition studies show that phosphate, sulfate, and acetate as inhibitors of pyridoxal-P binding are all competing for the same site. This anion-binding site appears to bind divalent anions tighter than monovalent anions and is sterically hindered for binding long chain carboxylic acids and phenylphosphate. Chloride, an activator of coenzyme binding, and sulfate, an inhibitor of binding, have no effect on the size of glutamate apodecarboxylase as determined by gel filtration. The holoenzyme and the apoenzyme both have a Stokes radius of 57 A and a molecular weight of approximately 310,000 in 50 m m pyridine-chloride or 50 m m pyridine-ulfate, pH 4.6. However, chloride, nitrate, and sulfate do have different effects on the heat denaturation of the apoenzyme.

Beef brain cytoplasmic aspartate aminotransferase. Purification, kinetics, and physical properties

Beef brain cytoplasmic aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was purified 380-fold. The existence of multiple forms (α, β, γ) of the enzyme was shown by chromatography on Sephadex CM-50 at pH 5.4 and by electrophoresis at pH 9.0 on plyacrylamide gel. Analytical ultracentrifugation yielded a sedimentation coefficient of 5.62 S and a mol. wt of 90 000 ± 6000 for the brain aspartate aminotransferase. A Stokes radius of 39.5 A and a mol. wt of 103 000 were determined for the enzyme by gel filtration on Sephadex G-200. Apparent Km values of 2 mM for l-aspartate and 0.17 mM for α-ketoglutarate were obtained with the brain aspartate aminotransferase. The enzyme was inhibited significantly by 1–3 mM α-ketoglutarate at pH 6.0. No inhibition of brain aspartate aminotransferase activity was noted with γ-aminobutyric acid. Most of these properties of the beef brain enzyme are the same as those obtained with beef heart cytoplasmic aspartate aminotransferase.

Glutamate decarboxylase. Inhibition by monocarboxylic acids

Abstract Aliphatic monocarboxylic acids are demonstrated to be substrate competitive inhibitors of bacterial glutamate decarboxylase. A chain-length effect is observed in the inhibition by the monocarboxylic acids with n -valeric acid functioning as the most effective inhibitor. The inhibitor dissociation constant for the enzyme complex with n -valeric acid is dependent on pH. It would appear that the protonated form of valeric acid is the ionic form which interacts most effectively with the enzyme. n -Valeric acid and glutaric acid, another substrate-competitive inhibitor of glutamate decarboxylase, compete for interaction with the enzyme. Chloride, an allosteric activator of the enzyme, inhibits the binding of glutaric acid, but not the binding of valeric acid to the enzyme.

The interaction of pyridoxal phosphate with aspartate apoaminotransferase

The rate of biniding of pyridoxal phosphate to the apoenzyme of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was measured by adsorption spectroscopy and by formation of active enzyme. At pH 5.1 and 8.3 the binding of coenzyme follows saturation kinetics. The binding process thus involves at least two steps. The rate of pyridoxal phosphate binding to the apoenzyme is dependent on the anion present in the pH 8.3 triethanolamine buffer. Chloride activates somewhat at very low concentrations. Phosphate and its methyl, ethyl, and phenyl esters are very effective inhibitors of the recombination in that 0.2--0.4 mM inhibit the rate of coenzyme binding by 50%. This is below the physiological concentration of phosphate. Sulfate also inhibits the rate of binding, but nitrate and acetate have little effect.