Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach

Abstract

Cysteine synthase (O-acetylserine (thiol) lyase, EC 4.2.99.8) and β-cyanoalanine synthase (EC 4.4.1.9) have been isolated from leaves of Spinacea oleracea L. and separated by anion-exchange chromatography. Further separation of one minor and two major isoforms of cysteine synthase was achieved by hydrophobic interaction chromatography and high resolution native electrophoresis (PAGE). Analysis of root material indicated that amongst the multiple isoforms present, a single isoform predominated. Subcellular fractionation studies indicated that one of the major leaf forms, cysteine synthase B, was located in the chloroplast and the other, cysteine synthase A, occurred in the cytoplasm. No specific isoform of cysteine synthase was resolved in the mitochondria, while cyanoalanine synthase was predominantly located in the mitochondrial fraction. Sulphur deprivation decreased cysteine synthase activity, but not cyanoalanine synthase activity in both young and mature leaves, although cysteine synthase activity in the roots increased slightly. A selective decrease in cysteine synthase B (chloroplastic) abundance was observed in mature leaves. Patterns of expression of cysteine synthase in response to S-availability are discussed in relation to possible roles for this enzyme in controlling S-flux through the S-assimilatory pathway.