Metabolism of cystine by Merino sheep genetically different in wool production IV. Rates of entry of cystine into plasma, measured with a single intravenous injection of L-[35S]cystine, and the subsequent incorporation of 35S into wool fibres.

Ten, 2-year-old Merino ewes from a flock selectively bred for high clean fleece weight (Fleece Plus) and ten from a flock bred for low clean fleece weight (Fleece Minus) were randomly divided between two dietary treatments: 600 or 1100 g/day of pelleted lucerne hay. After 14 weeks, each ewe received an intravenous injection of L-[35S]cystine (66-4 muCi). Venous blood samples were collected at 15 specified times until 8 h after the injections, and wool fibres were plucked until 65-75 days after the injections. Protein-free filtrates prepared from blood plasma were bulked within sample times for ewes from the same flock and dietary treatment. Equations relating the specific radioactivity of free cystine isolated from the bulked filtrates to time after injection contained three exponential terms. The entry rate and pool size of cystine estimated from these equations were greater in Fleece Minus than in Fleece Plus ewes (by 25 and 44% respectively for entry rate and pool size). Both traits were also higher in ewes offered 1100 g lucerne/day than in those offered 600 g/day (58-7 v. 33-9 mg/h for entry rate and 19-2 v 11-8 mg for pool size). The concentration of free cystine in plasma was greater in ewes offered 1100 g lucerne/day (3-0 v 2-1 mg/1; P less than 0-05), and greater in Fleece Minus ewes (3-0 v. 2-1 mg/l; P less than 0-05). The percentage of the injected radioactivity recovered in the wool clipped to day 70 post-injection differed between genotypes and between dietary treatments (P less than 0.05), being greater in Fleece Plus than in Fleece Minus ewes, and greater in those offered 1100 g lucerne/day than in those offered 600 g/day. The relationships between 35S incorporated per 1000 fibres (R) and time after injection (t) were best fitted by equations of the form (formula: see text). For all sheep, n = 3. The coefficient of the second term was significantly greater (P less than 0-05) in ewes offered 1100 g lucerne/day, whilst the constant of this term was significantly greater in Fleece Minus ewes. The specific radioactivities of cystine incorporated into wool fibres (SRf) during various intervals of time after injection were derived from these equations and from the measured rates of output of cystine in wool. The equations computed to relate SRf to time after injection (t) were of the form (formula: see text). Again there were three components. The coefficient of the third component was significantly greater (P less than 0-05) in ewes offered 1100 g lucerne/day, whilst the constant of the second term was significantly greater in Fleece Minus ewes.     

Lysosomal cystine counter-transport in heterozygotes for cystinosis.

Heterozygotes for cystinosis exhibited approximately half the normal rate of cystine counter-transport into isolated leukocyte lysosomes. This gene-dosage effect strongly supports previous findings demonstrating that the basic defect in cystinosis is impaired cystine transport across the lysosomal membrane. The method was used to determine the cystinosis carrier status for siblings of affected children in two families with cystinosis.     

Evidence for lysosomal reduction of cystine residues.

Previously reported evidence for the existence of a thiol: protein disulphide oxidoreductase in rat liver lysosomes has been re-examined and ambiguous results obtained. However, incubation of purified rat liver lysosomes with ¹²⁵I-labelled insulin at pH 5.5 shows that cathepsin D and a thiol-dependent enzyme other than cathepsin B or L are important in its digestion. The latter enzyme is most probably a thiol: protein disulphide oxidoreductase.     

Methionine metabolism in mammals. The methionine-sparing effect of cystine

Cystine can replace approximately 70% of the dietary requirement for methionine. We used standard enzyme assays, determinations of the hepatic concentrations of metabolites and an in vitro system which simulates the regulatory site formed by the enzymes which utilize homocysteine in this study of the mechanism for this adaptation. A significant alteration in the pattern of hepatic homocysteine metabolism occurs following the substitution of cystine for methionine. The major change is a marked reduction in the synthesis of cystathionine. Decreases in both the level of cystathionine synthase and in the concentration of adenosyl-methionine, a positive effector of the enzyme, explain this finding. Despite significant increases in the hepatic levels of betaine-homocysteine methyltransferase and methyltetrahydrofolate-homocysteine methyltransferase, flow through these reactions remains relatively constant. The betaine enzyme may be essential for efficient methionine conservation. In the absence of choline, cystine cannot replace methionine in an adequate diet limited in the latter amino acid.     

Cysteine thiosulfonate in cystine metabolism

A sulfur-containing amino acid was observed in mammalian cystine metabolism, in vitro and in vivo, which we have characterized as 2-amino, 3-(thio-thiosulfonate)propionic acid (cysteine thiosulfonate). Its biosynthetic pathway appears to initiate with the cleavage of cystine by cystathionine γ-lyase to form thiocystine, which undergoes sulfinolysis to form cysteine thiosulfonate.     

Effect of cystine loading and cystine dimethylester on renal brushborder membrane transport

The effect of loading renal tubule cells with cystine was studied by incubating them with cystine dimethylester. Proline uptake into brushborder membrane vesicles isolated from the cystine loaded cells was not different from that observed into brushborder vesicles isolated from tubules incubated in buffer alone. Incubating brushborder membranes with 2 mM cystine dimethylester for 10 minutes reduced the uptake of proline by 27% after 15 seconds of incubation and by 21% after 60 seconds of incubation. There was no effect after 20 minutes of incubation. Pre-incubating brushborder membrane vesicles with cystine dimethylester had no statistically significant effect on the affinity of priline for the carrier, but did reduce the maximal rate of proline uptake by 49%.     

pH effects on cystine transport in lysosome-rich leucocyte granular fractions.

Inhibitors of lysosomal acidification (4,4'-di-isothiocyanostilbene-2,2'-disulphonate, NN'-dicyclohexylcarbodi-imide, carbonyl cyanide m-chlorophenylhydrazone, NH4Cl and methylamine hydrochloride) did not alter cystine egress or countertransport in polymorphonuclear-leucocyte lysosome-rich granular fractions at pH 7.0. Together, 2 mM-MgCl2/MgATP and 90 mM-KCl stimulated cystine egress 2-fold, but this effect also was not influenced by inhibitors of ATP-dependent lysosomal acidification. MgCl2/MgATP stimulated cystine transport at pH 5.5, but the effect also occurred with MgCl2, MgSO4 or MnCl2 alone, was prevented by chelation, and was not seen with NaATP; therefore, it was considered a bivalent-cation, not an ATP, effect. Proton-pump-mediated acidification of lysosomes does not appear to be required for cystine transport in normal polymorphonuclear-leucocyte granular fractions, as reported for lymphoblast lysosomes.     

Expression of the mouse macrophage cystine transporter in Xenopus laevis oocytes.

System xc- mediates transport of cystine and glutamate across the mammalian plasma membrane in a Na(+)-independent manner. This transport activity can be induced in mouse peritoneal macrophages during culture by diethylmaleate, a sulfhydryl-reactive agent. We injected mRNA from such macrophages into Xenopus oocytes and demonstrated the expression of System xc-, i.e., a Na(+)-independent, glutamate-inhibitable cysteine transport system. The expressed cystine transport activity depended on the assay temperature, in that cystine uptake measured at 37 degrees C was severalfold higher than that measured at 20 degrees C. Injection of size-fractionated mRNA indicated that the System xc- transporter of the mouse macrophage is encoded by mRNA of 1.5 to 2.9 kb.