Partial Purification and Properties of a beta-d-Glucosyltransferase Occurring in Germinating Phaseolus aureus Seeds.

A soluble enzyme that transfers d-glucose from uridine diphosphate d-glucose to low molecular weight hydroxyl compounds has been discovered in germinating mung bean (Phaseolus aureus) seeds and purified 220-fold. Phenol and n-butyl alcohol are the most reactive aceptors examined. The reactivities of various acceptors are largely independant of hydroxyl group acidities or of the electronic properties of the acceptors. The space-filling properties or length of the acceptors, on the other hand, appear to be the predominant controlling factor which determines their relative abilities to accept d-glucose, the ideal size being that of phenol and n-butyl alcohol. The enzyme is deactivated by p-chloromercuribenzene sulfonate; dithiothreitol imparts considerable stabilization to the enzyme. It does not require exogenously added metal ions but is slightly stimulated by magnesium ion. On the other hand, the enzyme is partially inhibited by 10 mm manganous ion, cobalt ion, ferrous ion, and ethylenediaminetetraacetate. Zinc ion at 10 mm concentrations strongly inhibits the enzyme. The molecular weight of the enzyme, determined by gel filtration, is approximately 62,000.

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus.

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 x 10(-2)m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg(2+) was required for maximum activity of the enzyme. Mn(2+) and Zn(2+) were inhibitory, while Co(2+) had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg(2+) on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 x 10(-4)m and 7.1 x 10(-4)m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 x 10(-4)mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.

Substrate Activation of beta-(1 --> 3) Glucan Synthetase and Its Effect on the Structure of beta-Glucan Obtained from UDP-d-glucose and Particulate Enzyme of Oat Coleoptiles.

UDP-d-glucose, at a micromolar level in the presence of MgCl(2) and oat (Avena sativa) coleoptile particulate enzyme which contains both beta-(1 --> 3) and beta-(1 --> 4) glucan synthetases, produces glucan with mainly beta-(1 --> 4) glucosyl linkages. An activation of beta-(1 --> 3) glucan synthetase by UDP-d-glucose and a decrease in the formation of beta-(1 --> 3) glucan in the presence of MgCl(2) have been observed. However, at high substrate concentration (>/= 10(-4)m), the activation of beta-(1 --> 3) glucan synthetase is so pronounced that the formation of beta-(1 --> 3) glucosyl linkage predominates in synthesized glucan regardless of the presence of MgCl(2). These observations may explain the striking shift in the composition of glucan of particulate enzyme from a beta-(1 --> 4) to beta-(1 --> 3) glucosyl linkage when UDP-d-glucose concentration is raised from a low concentration (/= 10(-4)m).Besides UDP-d-glucose, CDP-d-glucose can also serve as substrate for the formation of beta-(1 --> 3) glucan in the presence of beta-(1 --> 3) synthetase.

The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings.

Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d-mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle-bound d-mannosyl-(14)C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d-mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose-(14)C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide.

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus.

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 mum. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.

Solubilization and Separation of Uridine Diphospho-d-glucose: beta-(1 --> 4) Glucan and Uridine Diphospho-d-glucose:beta-(1 --> 3) Glucan Glucosyltransferases from Coleoptiles of Avena sativa.

The particulate glucan synthetase preparation isolated from a homogenate of oat coleoptiles at 4 C lost 65% of its original activity after 1 day when the UDP-d-glucose substrate concentration was 5 x 10(-7)m to 1.0 x 10(-6)m. Storage of the particulate enzyme at -20 C or in liquid nitrogen did not prevent the enzyme from losing its activity. Incorporation of 0.5% hovine serum albumin into the medium stabilized the particulate enzyme at 0 C for 6 days and for at least 2 weeks in liquid nitrogen.When the particulate enzyme was treated with 8% digitonin, 40 to 50% of its activity appeared in the 100,000g supernatant fraction. The particulate and digitonin-solubilized enzyme preparations synthesized both beta-(1 --> 4) and beta-(1 --> 3) glucosyl linkages from UDP-d-glucose, but beta-(1 --> 3) glucan was the main product at 1 x 10(-3)m UDP-d-glucose substrate. The activity of beta-(1 --> 4) glucan synthetase was stimulated at least 10-fold in the presence of MgCl(2). A separation of beta-(1 --> 4) and beta-(1 --> 3) glucan synthetase activities could be achieved at 1 x 10(-3)m UDP-d-glucose when the digitonin-solubilized enzyme was adsorbed on a hydroxylapatite gel and then eluted with concentrated potassium phosphate buffer. The results indicate that the particulate enzyme contains two enzymes, one responsible for the synthesis of beta-(1 --> 4) and another beta-(1 --> 3) linkages in the glucan or glucans synthesized from UDP-d-glucose.

Determination of linkages in beta-D-Glucans from Phaseolus aureus by exo-beta-(1 to 3)-D-glucanase.

An alkali-insoluble glucan synthesized from UDP-d-glucose by the particulate enzyme system from Phaseolus aureus is hydrolyzed by a highly purified exo-beta-(1 --> 3)-d-glucanase to d-glucose, to the extent of 91% in 24 hr. The alkali-insoluble glucan from GDP-d-glucose formed by the particulate enzyme system from the same plant which is known to be (from chemical data) a beta-(1 --> 4)-d-glucan (cellulose) is not acted upon by this glucanase.

Biosynthesis of Insoluble Glucans From Uridine-Diphosphate-d-Glucose With Enzyme Preparations From Phaseolus aureus and Lupinus albus.

Particulate, and digitonin-solubilized, enzyme systems from Phaseolus aureus and Lupinus albus catalyze the biosynthesis of aqueous-insoluble glucans from UDP-d-glucose. The digitonin treatment greatly increases the enzymic activity of (per unit protein) both the 34,000g pellet and the supernatant liquid as compared with that of the original particles. Most of the polymer produced (90-95%) is soluble in hot, dilute alkali; the interglucosidic linkages of the alkali-soluble and alkali-insoluble polymers are identical. The optimum concentration for the incorporation of radioactivity from UDP-d-glucose-(14)C into soluble glucan is high; at 10(-3)m at least 50% of the added radioactive glucosyl donor is incorporated.Careful examination of the products of degradation of the polymers produced by various enzymic preparations showed that beta-(1-->3)-glucans are produced. No evidence was obtained for any measurable amount of beta-(1-->4)-d-glucose linkages.

Pathway of Uridine Diphosphate N-Acetyl-d-Glucosamine Biosynthesis in Phaseolus aureus.

Studies with extracts obtained from mung beans (Phaseolus aureus) showed that UDP-N-acetyl d-glucosamine is formed from d-fructose 6-phosphate by a series of the following enzymic reactions: [Formula: see text]UDP-N-acetyl-d-glucosamine inhibits the first reaction in the multistep pathway leading to its biosynthesis.

Biosynthesis of Alkali Insoluble Polysaccharide from UDP-d-Glucose with Particulate Enzyme Preparations from Phaseolus aureus.

A particulate enzyme system from Phaseolus aureus seedlings catalyzes the synthesis of alkali insoluble polysaccharide material from UDP-d-glucose. 80 to 90% of the d-glucose units are joined by beta-1,4 linkages, the remainder being combined by beta-1,3 linkages. It is not known whether the material is a single polysaccharide or a mixture.With a substrate concentration of 4 x 10(-3)m the enzyme system catalyzes the formation of alkali insoluble polysaccharide from UDP-d-glucose at a rate 23 times greater than that from GDP-d-glucose. The modifications introduced also increase the formation of cellulose from GDP-d-glucose to 3 times the rate previously reported.None of the glycosyl nucleotides containing bases other than guanine or uracil (adenine, cytosine, thymine) served as substrate for the production of alkali insoluble polysaccharide with this enzyme system. dUDP-d-glucose could also not serve as substrate.