Studies with plant α-galactosidases

• Main Author: Hustler, Margaret Joan
• Published: Royal Holloway, University of London 1976
• Subjects: 572; Plant Sciences
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.460173

The two molecular forms, I and II, of α-galactosidase from immature, mature (resting) and germinated vicia faba seeds have been studied. The enzymes have been purified by a multistage procedure and, in particular, the effect these stages have upon the relative isoenzyme levels has been investigated. Upon purification in Mcllvaine buffer the relative isoenzym pattern for mature seeds was seen to change from the low molecular weight enzyme being the major form in the crude extract, through the two molecular forms being almost equal after the citric acid precipitation and ammonium sulphate fractionation, to the high molecular weight enzyme being the major form after the final purification stage of dialysis. A parallel purification carried out in Acetate buffer introduced a time lag between the change in isoenzyme levels found upon acid precipitation in the McIlvaine system. Purification of extracts of whole (testas intact) immature seeds in Mcllvaine buffer showed enzyme to be the dominant form throughout all the purification stages, while beans devoid of testas produced extracts where enzyme j was the greater. Drying the green immature beans in the presence of testas gave the normal mature bean isoenzyme patterns, while drying in the absence of testas, or where the testa has been replaced by a non-physiological covering produced extracts where enzyme remained the major isoenzyme throughout all stages of purification. Enzyme was the more abundant isoenzyme in the crude McIlvaine buffer extracts from germinated seeds, with small amounts of enzyme appearing upon purification. A preparation of α-galactosidase when incubated with a 30 - 65% ammonium sulphate fraction from green bean testa was observed to convert in vitro to an enzyme with very similar physical properties to enzyme from mature seeds. Some factors affecting this conversion have been investigated and it appears that the active component in the green testa is proteinaceous. The physiological role of CX-galactosidases in seeds has been considered. Attempts were made to modify galactomannans by hydrolysis with CX-galactosidases, so as to produce galactose-depleted products which would form stronger gels with K-carrageenan than would the parent galactomannans. vicia faba enzymes failed to produce any significant hydrolysis whereas coffee and clover enzymes increased the mannose/galactose6ratio but also appeared to hydrolyse the mannan backbone so that gel formation was inhibited. This was believed to be due to the presence of contaminating fB-mannanase. Unsuccessful attempts were made to remove this activity from clover enzyme by several purification stages culminating in affinity chromatography.