Catabolism of Sulpholipid by an Enzyme from the Leaves of Phaseolus multiflorus

Little is known about the catabolism of plant sulpholipid (sulphoquinovosyldiacylglycerol), a major component of green leaves. Several plant lipases have been examined, but none has absolute specificity towards a particular lipid class, unlike the phospholipid acyl hydrolases found in animals and micro-organisms (Dawson, 1973). For example, the so-called galactolipase (EC 3.1.1.26) fromPhaseolus leaves (Sastry & Kates, 1964; Helmsing, 1969) is actually more active with monoacylglycerols and lysophospholipids (Galliard & Dennis, 1974). A ‘sulpholipase’ from Scenedesrnus (Yagi & Benson, 1962) catalysed the breakdown of sulphoquinovosyldiacylglycerol via a monoacyl intermediate, but, in addition, hydrolysed phospholipids. Non-specific lipolytic acyl hydrolases have also been isolated from potato tubers (Galliard, 1971 ; Hirayama et al., 1975), and these are most active with oleoylglycerol (mono-olein). In this present study we attempted to test whether there was a sulphoquinovosyldiacylglycerol acyl hydrolase present in leaves of Phaseolus and to determine the properties of such an enzyme. A crude enzyme preparation was isolated from the 104000g supernatant of homogenized Phaseolus multzyorus (runner-bean) leaves by precipitation with (NH4)#04 to 75 % saturation. The preparation was fractionated on an anion-exchange column (DEAE-cellulose DE-32) at pH7 and the activity was eluted with a salt gradient in 0.01 Mphosphate buffer, pH7. The active fractions were combined and further fractionated by gel filtration on a Sephadex G-200 column and eluted with 0.01 M-phosphate buffer, pH7. The active fractions were combined and used in all the subsequent experiments as a partially purified enzyme preparation. Enzymic activity was measured by quantifying the fatty acid products by g.l.c., with an internal pentadecanoate standard. The enzyme preparation had acyl hydrolase activity towards sulphoquinovosyldiacylglycerol, galactosyldiacylglycerol (monogalactosyl diglyceride) and galabiosyldiacylglycerol (digalactosyl diglyceride), which agreed with the findings obtained by Galliard & Dennis (1974). The enzyme had a very broad pH curve with sulphoquinovosyldiacylglycerol. The optimum was at pH5, and 50% of the activity was retained at pH7.6. All subsequent incubations were carried out at pH5. The ‘apparent’ K , for sulphoquinovosyldiacylglycerol was determined and found to be 0.15 mM. A substrate competition experiment with sulphoquinovosyldiacylglycerol, galactosyldiacylglycerol and galabiosyldiacylglycerol suggested there was one enzyme present that would hydrolyse all three substrates. The hydrolysis of the three substrates was not inhibited by 2m~-cysteine, unlike the preparation investigated by Helmsing (1969). The fatty acid pattern obtained from the hydrolysis of both Swiss-chard and springcabbage sulphoquinovosyldiacylglycerol was different from the fatty acid composition in the original substrates. There was no agreement with the fatty acids located on either the 1or the 2-position, and the absence of a monoacyl intermediate confirmed that fatty acids esterified to both positions were hydrolysed. From the results it appeared that linolenic acid was released in preference to palmitic acid, and, in consequence, that the enzyme preparation would be more active towards molecular species of sulphoquinovosyldiacylglycerol containing a high proportion of linolenic acid. Sulphoquinovosylglycerol was separated on AgN0,-impregnated t.1.c. plates into three molecular species, which differed in their degree of saturation. The species were predominantly saturated, palmitoyl/linolenoyl and dilinolenoyl, representing 13, 71 and 16 % respectively of the orginal sulphoquinovosyldiacylglycerol. Incubation of equal amounts of these species with the enzyme showed clearly that the dilinolenoyl species was hydrolysed at the fastest rate (Burns et al., 1977). All the purification procedures and the competition experiment suggested there was one enzyme that would deacylate sulphoquinovosyldiacylglycerol, galactosyldiacyl-