Satish K. Srivastava

Accurate measurement of oxidized glutathione content of human, rabbit, and rat red blood cells and tissues☆

Abstract 1. 1. A method for the accurate measurement of oxidized glutathione (GSSG) in the erythrocytes and body tissues has been described. The oxidation of reduced glutathione to GSSG during protein precipitation by trichloroacetic acid is prevented by prior alkylation of the sulfhydryl groups with N-ethylmaleimide (NEM). Trichloroacetic acid and excess NEM are extracted with ether. The GSSG in the extract is measured enzymically using glutathione oxidoreductase. 2. 2. Oxidized glutathione content (average ± standard deviation) of human, rabbit, and rat erythrocytes has been shown to be 3.6 ± 1.4, 5.6 ± 0.7 and 6.1 ± 2.6 mμmoles/ml RBC, respectively. 3. 3. The GSSG content of rat liver, kidney, heart, lens, testes, and skeletal muscles is below the level of detection, i.e., 0.03 μmole/gm tissue.

A new fluorometric method for the determination of pyridoxal 5′-phosphate

Abstract A new fluorometric method using semicarbazide for the determination of pyridoxal and pyridoxal 5′-phosphate (PLP) in whole blood, red cells and plasma has been developed. Semicarbazide breaks the Schiff base of PLP and proteins by “trans-Schiffization” reaction and forms semicarbazone of PLP. The semicarbazone of PLP emits strongly at 460 nm when excited at 380 nm. Several metabolic intermediates were tested for the possible interference. Only pyridoxal was found to interfere. The interference can be corrected since pyridoxal emits at 380 nm when excited at 320 nm. Using this method we found that rabbit red cells in vivo are freely permeable to PLP.

Cleavage of lens protein-GSH mixed disulfide by glutathione reductase

Abstract Mixed disulfide complexing the bovine lens erystallins and GSH was prepared by incubating the soluble proteins with GSSG. GSH could be released from the mixed disulfide in the presence of NADPH or NADH. Mixed disulfide was purified using DEAE-Sephadex A-50 column chromatography and was found to be free of glutathione reductase. In such preparation of mixed disulfide NADPH failed to release GSH. However, addition of glutathione reductase along with NADPH released substantial amounts of GSH from the mixed disulfide. Bovine serum albumins also form mixed disulfide with GSH and the mixed disulfide could be cleaved by glutathione reductase. Alkylation of protein sulfhydryl groups by N -ethylmaleimide prior to the addition of GSSG for the preparation of mixed disulfide, almost completely prevents the formation of mixed disulfide indicating that the formation of mixed disulfide involves protein sulfhydryl groups. Evidence is presented that the cleavage of mixed disulfide is not mediated by GSH with cyclic reduction of GSSG by glutathione reductase, but rather proceeds enzymically through glutathione reductase. Using the borohydride method, small amounts of GSH were found to be bound to normal human lens proteins and a substantial amount of GSH was found to be bound to lens proteins from senile cataracts. Only a very small amount of GSH was bound to bovine lens proteins. Glutathione reductase failed to release a significant amount of GSH bound to lens proteins in human cataractous and normal lens. The significantly greater increase in the protein thiols after the borohydride treatment of lens proteins from normal and cataractous human lens and from bovine lens cannot be accounted for by release of GSH bound to proteins. This indicates significant intramolecular and/or intermolecular disulfide bridges in lens proteins.

Glutathione metabolism of the red cells effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress

Riboflavin deficiency results in diminished glutathione reductase activity of the red cells of human subjects and of rats. The effect of such glutathione reductase deficiency on the rate of hexose monophosphate shunt pathway metabolism has been studied by measuring the rate of 14CO2 production of red cells subjected to oxidative stress, measuring the rate of regeneration of GSH from GSSG, and by estimating the steady-state levels of GSSG and the rate of transport of GSSG from glutathione reductase-deficient red cells. No difference was observed between the red cells of normal subjects, of riboflavin-deficient (glutathione reductase-deficient) subjects, and the red cells of glutathione reductase-deficient subjects treated with riboflavin in vivo or in vitro to correct the glutathione reductase deficiency. n nThese studies indicate that, in the red cell, glutathione reductase does not play limiting role in the rate of metabolism in the hexose monophosphate pathway.

Hexose-6-phosphate dehydrogenase: Distribution in rat tissues and effect of diet, age and steroids☆

Abstract Hexose-6-P dehydrogenase is a microsomal enzyme which oxidizes glucose-6-P, galactose-6-P, 2-deoxy-glucose-6-P, and glucose using either NAD or NADP as hydrogen acceptor. Its distribution was studied in rat tissues; the enzyme was found in liver, adrenals, spleen, kidney, heart, lungs, gluteal muscle, testes, ovaries, seminal vesicles, prostate, uterus, and intestine. No enzyme was detected in the brain and mammary gland. Hexose-6-P dehydrogenase is not inhibited by various steroids which are known to inhibit glucose-6-P dehydrogenase. Also, feeding of a diet containing 68% galactose, glucose, or fructose does not alter the activity of hexose-6-P dehydrogenase in rat liver. Liver hexose-6-P dehydrogenase activity increased rapidly before birth, and then declined gradually to adult levels. It appears possible the hexose-6-P dehydrogenase serves as a source of reduced pyridine nucleotides for mixed-function oxidase systems, but definitive evidence for its role in metabolism is lacking.

The effect of normal red cell constituents on the activities of red cell enzymes.

Abstract The effect of 2,3-DPG, pyridoxal-5′- P , pyridoxal, pyridoxine creatine and bicarbonate was studied on the activity of red cell glycolytic and pentose phosphate shunt enzymes at saturating and limiting substrate concentrations. 2,3-DPG strongly inhibits the activity of hexokinase, phosphoglucomutase, phosphofructokinase, aldolase and glyceraldehyde phosphate dehydrogenase. In the case of the first four enzymes, 2,3-DPG competes with the substrate and glyceraldehyde phosphate dehydrogenase is inhibited in a noncompetitive fashion. Pyridoxal-5′- P inhibits some of the enzymes inhibited by 2,3-DPG, including hexokinase, phosphoglucomutase, phosphofructokinase and aldolase; in addition, it also inhibits the activity of phosphoglucose isomerase, pyruvate kinase, glucose-6- P dehydrogenase and 6-phosphogluconate dehydrogenase. In some cases, the inhibition was competitive; in others, it was noncompetitive. Creatine and bicarbonate do not significantly inhibit any of the enzymes studied. The studies reported in this report indicate possible roles of 2,3-DPG and pyridoxal-5′- P in the regulation of glucose metabolism in red cells.

Purification and kinetic studies of adenine phosphoribosyltransferase from human erythrocytes

Abstract Adenine phosphoribosyltransferase was purified 2200-fold from human erythrocytes. The purification steps involved (NH 4 ) 2 SO 4 fractionation followed by Sephadex-G75 column chromatography, DEAE-Sephadex chromatography, and (NH 4 ) 2 SO 4 precipitation. Partially purified enzyme was stabilized by 0.3 m (NH 4 ) 2 SO 4 , which also protected the enzyme from heat denaturation. The enzyme was partially inhibited by sodium ions which were partially antagonized by magnesium. Divalent cation is an absolute requirement for high enzyme activity. Ca 2+ , Mn 2+ , and Mg 2+ supported high initial rates and Hg 2+ completely inhibited the enzyme. The kinetic data with initial velocity and the product inhibition studies are consistent with a mechanism in which the synthesis of nucleotide proceeds as an ordered sequential reaction.

Galactose cataract in riboflavin deficient rats

Abstract Galactose cataract was produced in riboflavin deficient and riboflavin supplemented rats by feeding a 68% galactose diet deficient or supplemented with riboflavin. Twenty-three or twenty-five-day-old rats were fed riboflavin deficient and riboflavin supplemented diet for 21 days before feeding high galactose diet. About 80% of riboflavin deficient rats develop mature cataracts in 16–18 days after feeding riboflavin deficient high (68%) galactose diet whereas about 10% of rats fed a riboflavin supplemented high galactose diet for the same period develop mature cataracts. Progress in the formation of cataracts was followed by measuring biochemical parameters such as GSH, total thiol, protein thiol, and the levels of glutathione reductase, glucose-6-P dehydrogenase, and 6-phosphogluconate dehydrogenase in the lens. The levels of glutathione reductase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were lower in the lenses of riboflavin deficient than in normal rats fed galactose but GSH, total thiol, and protein thiol were not appreciably different in riboflavin deficient and control animals. Riboflavin deficiency alone does not lead to cataract formation in rat lenses when the rats were fed a riboflavin deficient diet for 16 weeks. Attempts to correct the increased susceptibility of riboflavin deficient rats to galactose cataract by either topical application of riboflavin or by injecting riboflavin into the anterior chamber of the eye were not successful.

Antibody against purified human hexosaminidase B cross-reacting with human hexosaminidase A

Abstract Human hexosaminidase B has been purified to virtually homogeneous state from placenta. An anti-serum has been prepared in rabbits against the purified preparation. The serum reacted equally with human hexosaminidase B (free of hexosaminidase A) and with human hexosaminidase A (free of hexosaminidase B) as shown by immunodiffusion and by precipitation of enzyme activity from solution.

Permeability of Normal and Cataractous Rabbit Lenses to Glutathione

Summary Rabbit lenses were made cataractous by incubating in culture medium with added tyrosine and tyrosinase. The content of GSH was drastically lowered in the cataractous lens. The GSH which was lost from the lens could be partially recovered as GSSG in the medium. This finding may explain the fate of lens GSH, both normally and in cataract formation.