Yu Hosokawa

Isolation and characterization of a cDNA for rat liver cysteine dioxygenase

Cysteine dioxygenase is a key enzyme of cysteine metabolism in mammals. The cDNA clones for rat liver cysteine dioxygenase were isolated by immunological screening and plaque hybridization from a rat liver cDNA library. The longest clone contained an insert of 1458 bp and encoded a polypeptide of 200 amino acids. The clone included the corresponding nucleotide sequence to amino acid sequences obtained from four lysyl endopeptidase-digested fragments of purified rat liver cysteine dioxygenase. The calculated molecular weight of rat liver cysteine dioxygenase was 23,025. Northern blot analysis revealed a single cysteine dioxygenase mRNA species of about 1.7 kb. A computer homology search indicated that this protein showed no homology with any known protein.

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase)

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.

Cysteine metabolism in vivo of vitamin B6-deficient rats.

The expirations of 14CO2 from DL-[1-14C]-, DL-[3-14C]- and L-[U-14C] cysteine used as isotopic tracers were estimated in order to determine the in vivo metabolic distribution of L-cysteine in pyridoxine deficient rats. The expired 14CO2 from L-[U-14C] cysteine was increased by pyridoxine deficiency. The loading of non-physiological dose of L-cysteine resulted in remarkable increase in the expiration of 14CO2 from each tracer in deficient rats as well as in controls. The in vivo metabolic distributions of L-cysteine were calculated from the expired 14CO2 from these isotopic tracers. The in vivo metabolic distribution of L-cysteine calculated showed that the remarkable lesion in taurine pathway occurred in pyridoxine deficient rats, and when non-physiological dose of L-cysteine was loaded the catabolism of L-cysteine of controls was markedly increased in either pyruvate or taurine pathway, whereas the L-cysteine catabolism in deficient rats was increased only in pyruvate but not in taurine pathway. The urinary excretions of 35S-labeled metabolites such as sulfate or taurine were also examined in deficient and control rats.

Dietary protein influences the responsiveness of splenocytes to Con A in growing mice

Abstract The effects of dietary proteins containing different levels of sulfur amino acid on the responsiveness of splenocytes to concanavalin A(Con A) were examined. The Con A-induced DNA synthesis of splenocytes in mice fed a purified egg protein(PEP) diet was significantly higher than those in mice fed a soy protein isolate(SPI) or casein diet. Reduced glutathione(GSH) and 2-mercaptoethanol(2-ME) markedly stimulated Con A-induced DNA synthesis in vitro, and the stimulatory effects of 2-ME were more marked in SPI and casein diet groups compared to the PEP diet group. In contrast, L-buthionine-( S,R )-sulfoximine(BSO) markedly inhibited Con A-induced DNA synthesis in splenocytes. The degree of inhibition was greater in the order SPI, casein and PEP diet groups. The Con A-induced IL-2 secretion from splenocytes in the SPI diet group was significantly lower than those in casein and PEP diet groups when BSO was added to the medium. IL-2 secretion from splenocytes in the SPI diet group was significantly increased by L-methionine supplementation in the diet. These results suggest that the sulfur amino acids content in the dietary protein may play a key role in the regulation of lymphocyte responsiveness to Con A in growing mice.