Since the galactosylglycerides are confined (almost exclusively) to chloroplasts, it would seem natural to seek an active enzyme preparation from these organelles. First, it was shown that carefully prepared isolated chloroplasts could incorporate 14C-galactose into the lipids. The nature of the substrates involved was not clear until Ongun and Mudd in California showed that an acetone powder of spinach chloroplasts would catalyse the incorporation of galactose from UDP-galactose into monogalactosyldiacylglycerol if the acetone-extracted lipids were added back. The acceptor proved to be diacylglycerol or, for the synthesis of digalactosyldiacylglycerol, a monogalactosyl-diacylglycerol acceptor was needed. Further work on the enzymes involved was carried out by Joyard and Douce in Grenoble who showed that the reactions were confined to the chloroplasts envelope -hence the need for carefully prepared chloroplasts.
The reactions are, therefore:
1,2-diacylglycerol + UDP-galactose ^
monogalactosyldiacylglycerol + UDP
monogalactosylglycerol + UDP-galactose
^ digalactosyldiacylglycerol + UDP
The UDP-galactose is produced in the cytosol. In contrast, the diacylglycerol is synthesized by a phosphatidate phosphohydrolase localized in the envelope. The two galactosyltransferases have slight differences in their enzymic characteristics and, of course, result in the formation of P- and a-glycosidic bonds.
More recently, in Wintermanns' laboratory in The Netherlands, it was noticed that isolated chloroplast envelopes were capable of forming digalactosyldiacylglycerol from labelled mono-galactosyldiacylglycerol by a reaction that did not require UDP-galactose. Further examination revealed that inter-lipid galactosyltransfer was involved thus:
2 monogalactosyldiacylglycerol ^ digalactosyldiacylglycerol + diacylglycerol
This enzyme is also capable of generating higher homologues by further transfer of galactose from monogalactosyldiacylglycerol. The trigalactosyl-and tetragalactosyldiacylglycerols thus formed are detected in small quantities in many isolated chloroplasts. It is, however, unclear at present whether such higher homologues are actually present in vivo or are artefacts of the isolation and analytical processes. The relative rate of formation of digalactosyldiacylglycerol by the two pathways is also an area of controversy.
One difference in the fatty acyl compositions of galactosylglycerides, which has been noticed repeatedly, in the analysis of plants is the presence of hexadecatrienoate (16:3) in monogalactosyldiacylglycerol (but little in digalacto-syldiacylglycerol) from certain plants. The presence of 16:3 in some plants seems to be related to the provision of palmitate at the sn-2 position of monogalactosyldiacylglycerol where it acts as a substrate for fatty acid desaturases. In such plants the diacylglycerol for galactolipid biosynthesis is generated by phosphatidate phosphohydrolase within the chloroplast. In contrast, the source of diacylglycerol for galactolipid biosynthesis in other plants comes from outside the chloroplast and it does not contain 16-carbon acids at the sn-2 position. A glance at Table 7.4 will also reveal that even in plants, like spinach, which do contain hexa-decatrienoate in their monogalactosyl-diacylglycerol, little is present in the digalactosyl derivative. It is presumed that this is due to substrate specificity of the second galactosyltransfer-ase, as well as to the desaturation of palmitate to hexadecatrienoate on monogalactosyldiacylglycerol referred to above.
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