STUDIES ON MULTIMOLECULAR FORMS OF ALPHA GALACTOSIDASE OF CICER ARIETINUM SEEDS

Abstract

Three molecular forms of a-galactosidase, one high molecular weight form (a-galactosidase I) and two low molecular weight forms (a-galactosidase Ila and a-galac tosi dase lib) have been separated from the 6 days germinated cotyledons of Cicer-arietinum seeds using acetone treatment, pH precipitation, ammonium sulphate fractionation, gel filtration and ion exchange chromatography. The low molecular weight forms (a-galactosidase Ila and cr-galactosidase lib) showed a single protein band on polyacrylamide gel electro phoresis with electrophoretic mobilities, relative to bromophenol blue, of 0.24 and 0.32, respectively. The apparent molecular weights of a-galactosidase I, a-galactosidase Ila and a-galactosidase lib, as determined by Sephadex gel filtra tion method, were found to be 14 9,600, 56,000 and 50,000 dal tons, respectively. On SDS polyacrylamide gel electrophoresis, under completely reduced conditions, a-galactosidase Ila yielded three distinct protein bands corresponding to mole cular weight 36,000, 22,000 and 16,000 daltons and a-galactosidase lib yielded two bands of molecular weight 40,000 and 18,000 daltons, respectively, indicating that both enzymes have subunit structure.

The high and low molecular forms of a-galactosidase showed identical pH profiles. The substrate specificities in principle, were same with difference in quantitative values. The pattern of enzyme inhibition by metal ions and sugar analogus was similar, however, the degree of inhibition was significantly different for each form of the enzyme. The K values of a-galactosidase 1, a-galactosidase Ila m and a-galactosidase lib were found to be 0.56, 0.14 and 0.28 mM and V „ values were 5.3, 2.0 and 1.0 umol/min/mg, max respectively. The various a-galactosidase forms showed markedly different thermodynamic parameters. The purified cr-galactosidase Ila, a-galactosidase lib and the reconstituted high molecular weight a-galactosidase are glycoproteins contain ing 9.4/., 29.5/. and 25/. carbohydrate* respectively, composed of N-acetylglucosamine and mannose. The immobilized isoenzymes in calcium alginate beads exhibited higher pH tolerance and greater thermal stability as compared to the free enzyme. The immobilized enzyme did not follow the michaelis-Menten kinetics. Developmental studies of a-galactosidase isoenzymes during germination and maturation of seeds showed that the number of molecular forms of a-galactosidase and their relative concentra tions varied with physiological state of the seed. In green immature seeds (10 days after flowering), the total activity was very low, with only low molecular weight form (a-galacto sidase II). After 17 days of flowering high molecular weight (iii) form (a-galactosidase 1) made its appearance and a marked increase in total activity of a-galactosidase occurred. From 25th to 40 days after flowering, the level of high molecular weight form increased with a simultaneous decline in the level of a-galactosidase II. During germination relative level of a-galactosidase I activity which was pre dominant in dormant seeds continuously declined with a concomitant increase of a-galactosidase II level and finally on seventhday of germination when the metabolic activities were at peak the lower molecular weight a-galactosidase was highly predominant with only very small proportion of agalactosidase I. Thus, activity ratio of a-galactosidase I to a-galactosidase II was completely reversed during the process of germination. The conversion of low molecular weight forms into high molecular weight form a-galactosidase in vjtro on storage shows that these forms are derived from each other depending on the physiological state of the seed. Treatment with cycloheximide during germination inhibited a-galactosidase synthesis by 41/. and has reversed the pattern of evolution of multimolecular forms of a-galactosidase,which appeared to assume the isoenzyme pattern of the maturing seeds. The different varieties of Cicer-arietinum seeds with clearly distinguishable superfa.cial features have also been found to contain different isoenzymic patterns of a-galactosidase (iv) isoenzymes. For instance, in all white varieties of chickpea seeds the high molecular weight form of a-galactosidase was lacking. Instead, a very low molecular weight form (Mr=25,000 daltons) was found in these varieties which was not present in any of the black variety seeds. On the other hand all the black varieties of chickpea were characterized by the presence of high molecular weight form a-galactosidase (Mr=l49,600 daltons). The number of multimolecular forms and their relative intensity vary from variety to variety and is a function of the physiological state of the seed.