Enzymic Reduction of Cystine by Subcellular Fractions of Cultured and Peripheral Leukoyctes from Normal and Cystinotic Individuals

Abstract

ExtractLysates of cultured and freshly drawn isolated peripheral leukocytes from normal and cystinotic individuals were examined for cystine-reducing activity, with particular emphasis on possible enzyme action in lysosome-containing fractions. Whole cell extracts of cultured leukocytes were found to possess strong glutathione-cystine transhydrogenase activity at both pH 7.5 and 5.5; no significant difference in this enzyme activity between extracts of normal and cystinotic cells was observable at either pH. Subcellular fractionation of these cultured leukocytes indicated that the major portion of transhydrogenase activity in the 5.5-7.5 pH range was confined to the highspeed supernatant (cytoplasmic) fraction, with little or no activity being detectable in the granular fractions. Comparative enzyme assays at both pH 4.5 and 7.5 with lysosomal and supernatant fractions of peripheral leukocytes freshly drawn from normal and cystinotic donors were consistent with the foregoing results in terms of the subcellular distribution of glutathione-cystine transhydrogenase activity; again there appeared to be no significant difference in the transhydrogenase activities of these fractions from normal and cystinotic leukocytes at either pH. The relation of these observations to the pathogenesis of cystinosis is discussed. Speculation

Uptake and Utilization of Exogenous Cystine by Cystinotic and Normal Fibroblasts

“…The data reported here are consistent with the previous publication of normal glutathione and cysteine levels in cystinotic fibroblasts (3). Indeed, we 2 along with Tietze, Bradley, and Schulman (28) have found cystinotic cells to have glutathione-cystine transhydrogenase and glutathione reductase activities.…”

Biochemical, Morphologic, and Gytogenetic Studies of Leukocytes Growing in Continuous Culture from Normal Individuals and from Patients with Cystinosis

“…The cultured white cells have been used for enzymologic studies of cystinosis which will be reported elsewhere [48]. The present study highlights certain other properties of these cells including the facility with which they may be used to investigate transport phenomena.…”

Pantetheinase Activity and Cysteamine Content in Cystinotic and Normal Fibroblasts and Leukocytes

“…There is no evidence for decreased enzymatically catalyzed cystine reduction or metabolism in cystinotic cells (21,(27)(28)(29)41) and little support for the presence of significant amounts of cystine-reducing activity in either normal or cystinotic lysosomes (35,40,46,47,49). Cystinotic cells have normal reduced glutathione content (27,34).…”

“…Studies of this and other disulfide reducing to confluency at 37OC in a 5% C02/95% air atmosphere in roller systems have failed to define the abnormality causing cystinosis bottles or 75 cm2 plastic Falcon tissue culture flasks. They were (28,29,(44)(45)(46)(47). cultured in Eagle's minimal essential medium with added nonesCysteamine [mercaptoethylamine (MEA)] is a naturally occur-sential amino acids, 2 mM glutamine, 10% fetal calf serum, and ring, freely soluble sullydryl compound which readily penetrates 100 pg/ml neomycin, penicillin, and streptomycin.…”