Properties of Glucosyltransferase and Glucan Transferase from Spinach

Abstract

A glucosyl and a glucosyl-glucan transferase activity from spinach (Spinacia oleracea L. var. Matador) leaves have been partially purified and characterized. The latter activity (fraction 1 after diethylaminoethylcellulose chromatography) is responsible for the transfer of glucosyl as well as of maltosyl, maltotriosyl, and higher homologous residues to glucose giving rise to maltose and the correspondingly larger molecules. This fraction also shows p-amylase activity. The transfer takes place only to glucose; maltose, as well as other ac-1,4-glucans, serve as donors. The enzyme fraction 2 is amylase-free and catalyzes only the transfer of glucosyl moieties, again with high acceptor specificity to glucose. Maltose and larger o-1,4-glucans, with the exception of maltotriose and maltotetraose, act as donors. The physiological function of these enzymes mav be the formation of oligosaccharide primers for starch synthetase or phosphorylase.Maltose was first recognized to participate in 1,4-to 1,4-glycosyl transfer reactions with extracellular enzymes of Escherichzia coli (1, 7), which synthesized members of the maltodextrin series from maltose. Enzymes catalyzing analogous transfer reactions have been reported in extracts of Bacillius inacerans (25), blood plasma (22), germinated green gram (24), and minced algae (6). Similarily, a recently reported cabbage leaf enzyme (17) produced several oligosaccharides from concentrated maltose solutions.In 1961 Edelman and Keys (8), described an enzyme from wheat germ which catalyzed the transfer of the nonreducing end glycosyl moiety of maltose to "C-glucose with reformation of "C-maltose, as in reaction 1.O-0 + 0* = 0-0*+In reactions 1 through 5, 0 represents a glucosyl unit, 0 represents a reducing glucosyl unit, 0* represents a "C-labeled glucosyl unit,-represents an a-1, 4-linkage, and represents continuation of such bonding as in amylose.The enzymatic activities described in this paper catalyzed reaction 1 as well as some reactions similar to, but not identical with, those of D-enzyme (EC 2.4.1 f; 1,4-glucan:a-1,4-glucan 4-glucosyltransferase) from potato (15,26,27,30,31). Other properties of the enzymatic reactions upon preliminary purification are described.MATERIALS AND METHODS Sources of Chemicals. Litner soluble starch and shellfish glycogen were purchased from Schuhardt, Munich. Amylopectin was prepared from fresh potato juice from which the amylose was precipitated with 1-butanol. Pure maltose was a generous gift from the laboratory of J. F. Robyt. Other maltodextrins were prepared from a fungal amylase digest of amylose precipitated three times (Sigma, St. Louis), separated on a charcoal-celite column (10), and purified by paper chromatography on formic acid and water-washed Whatman No. 1 paper. Other oligosaccharides were from commercial sources and were purified in the same way. Other materials were reagent grade.Radioactive compounds were obtained from the Radiochemical Centre. Amersham, with the exception of the following: 2-deoxy-glycose and a-D-glucos...

Characterization of Pea Chloroplast D-Enzyme (4-α-d-Glucanotransferase)

“…with 4-a-glucanotransferases from bacteria (7,23) and plants (8,17,18,26). Glucose has been found to be the best acceptor for potato D-enzyme.…”

“…Dextrins that have been demonstrated to be substrates for 4-a-glucanotransferases include maltose (1,2,7,18,20), maltotriose (11,26), larger maltodextrins (1 1, 25), and Schardinger (cyclic) dextrins (23). Chloroplast D-enzyme cannot utilize maltose as substrate (Table II, Fig.…”

“…It has been shown that 4-a-glucanotransferases can convert maltotriose into maltodextrins that are suitable substrates for starch phosphorylase (7,13). Also, it has been suggested that products of starch hydrolysis may be used by D-enzymes to produce maltodextrins that would be suitable substrates for starch phosphorylase (3,18,29) or starch synthase ( 18).…”

“…4-a-Glucanotransferases have been reported in spinach leaves (3,18,24), Phaseolus radiatus (22), sweet corn kernels (15), wheat germ (8), tomato fruits (19), carrot roots (19), potato tubers (11,26,27,32) and Arabidopsis leaves (17), with the most extensive studies conducted with D-enzyme from potato. There have been no characterization studies with a 4-a-glucanotransferase that has been demonstrated to be of plastid origin.…”

De Novo Maltotriose Biosynthesis from the Reducing End by Spinacia oleracea L. Chloroplasts

“…Finally, neither maltose synthase (19) nor maltose glucosyl transferase activities (15) were specifically isolated from extracts of spinach chloroplasts-whole leaf extracts were used in each case. The implication of maltose synthase in in vivo maltotriose biosynthesis by isolated spinach chloroplasts by results presented in this paper localizes the activity to the chloroplast.…”