Characterization of galactosyltransferases in spinach chloroplast envelopes

Abstract

Two galactosyltransferases involved in the galactolipid metabolism of spinach chloroplast envelopes were studied by specific assays: UDPgalactose: 1,2-diacylglycerol galactosyltransferase, which synthetizes monogalactosyldiacylglycerol from UDPgalactose plus diacylglycerol; and galactolipid: galactolipid galactosyltransferase (GGGT), which forms di- , tri- and tetragalactosyldiacylglycerol by dismutation of galactosyl groups between two galactosyldiacylglycerols. In the assay developed for UDPgalactose: 1,2-diacylglycerol galactosyltransferase, chloroplast envelope membranes were mixed with liposomes of phosphatidylcholine, and the mixture was treated with phospholipase C (from Bacillus cereus). The diacylglycerol produced from phosphatidylcholine is used by UDPgalactose: 1,2-diacylglycerol galactosyltransferase for synthesis of monogalactosyldiacylglycerol in the presence of UDPgalactose. Several characteristics of this enzyme were studied, including the effect of substrate concentrations, pH and temperature. UDPgalactose: 1,2-di-acylglycerol galactosyltransferase did not require cations for activity, but was stimulated by Mg 2+ and Mn 2+. Of the mono- and divalent cations tested only Zn 2+, Cd 2+ and Fe 2+ were inhibitory. Specific inhibitors for UDPgalactose: 1,2-diacylglycerol galactosyltransferase were UDP and N-ethylmaleimide. In contrast to UDPgalactose: 1,2-diacylglycerol galactosyltransferase, galactolipid: galactolipid galactosyltransferase was strongly stimulated by a series of mono- and divalent cations, most stimulatory being Mn 2+ Ba 2+, Ca 2+ and Mg 2+. Galactolipid: galactolipid galactosyltransferase was specifically inhibited by low concentrations of Zn 2+ (1 mM) and by chelating anions. UDPgalactose: 1,2-diacylglycerol galactosyltransferase synthetized monogalactosyldiacylglycerol from various molecular species of diacylglycerol; the highest activity was measured with distearoylglycerol and dioleoylglycerol. On the other hand, digalactosyldiacylglycerol synthesis by galactolipid: galactolipid galactosyltransferase proceeded most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol.