Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, a-ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mM extracellular a-ketoglutarate, but not 10 mM glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 lmolAE(L RBC) , while the initial rate for 0.8 mM a-ketoglutarate was 0.97 lmolAE(L RBC) )1 AEmin )1. However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with a-ketoglutarate (0.31 and 0.25 lmolAE(L RBC) )1 AEmin)1 , respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of a-ketoglutarate and ⁄ or glutamine. DatabaseThe mathematical models described here have been submitted to the Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/Whillier/ index.html free of charge.

The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)

The ventral, lateral, and dorsal lobes of the rat prostate produce secretions rich in protein. We examined these secretions for proteinase activities, using gelatin- and casein-containing SDS-polyacrylamide gel zymographies. The ventral lobe demonstrated both higher activities and more molecular forms of proteinase activities that cleaved these two protein substrates than did the other lobes. Ca(2+)-stimulated gelatinolytic activities of approximately 48, 51, 58, 64, 74, and 80 kDa were found in ventral prostate secretions in addition to activities detected in the absence of added Ca2+: a very strong 27-kDa activity; prominent 22-, 86-, 90-, and 94-kDa activities; and less active 36-, 41-, 100-, 130-, and 140-kDa activities. Ca(2+)-stimulated gelatinolytic activities of 51, 58, 74, 80, 86, 90, and 94 kDa were present in lateral prostate secretions (none were detected in secretions of the dorsal lobe), and Ca(2+)-independent activities of 25, 27, and 100 kDa were found in secretions of both the lateral and dorsal lobes. The Ca(2+)-stimulated activities were inhibited by EGTA and EDTA. Benzamidine inhibited all gelatinolytic activities except for the 22-, 25-, and 27-kDa Ca(2+)-independent forms when Ca2+ was not added to the reaction buffer. However, in the presence of 5 mM CaCl2, the Ca(2+)-stimulated forms of proteinase were unaffected by benzamidine, whereas the other activities sensitive to benzamidine were inhibited. Prominent Ca(2+)-independent caseinolytic activities of 20, 23, 31, 37, 83, 89, and 94 kDa were detected in ventral lobe secretions along with less active forms of about 39, 48, 53, 57, 60, 63, 80, 103, 110, 125, and 160 kDa. Caseinolytic activities of approximately 23, 31, 53, 89, 94, 103, 120, and 125 kDa were found in lateral prostate secretions, and 89, 94, and 103 kDa activities were found in secretions of the dorsal lobe. Quantitatively, most gelatinolytic and caseinolytic activities were present in the soluble portion of the secretion of each prostatic lobe. The ventral, lateral, and dorsal prostate lobes all secrete gelatinolytic and caseinolytic proteinase activities; however, quantitatively the ventral lobe is the most notable in this function since its secretions contain more molecular forms and greater activities of these proteinases.