Elaine Greenberg

Interaction of spinach leaf adenosine diphosphate glucose α-1,4-glucan α-4-glucosyl transferase and α-1,4-glucan, α-1,4-glucan-6-glycosyl transferase in synthesis of branched α-glucan

Abstract Chromatography of spinach leaf extracts on DEAE-cellulose resolves α-1,4-glucan, α-1,4-glucan 6-glycosyl transferase (branching enzyme) into two fractions. Branching enzyme fraction I which contains 10–20% of the total activity recovered from the DEAE-cellulose column is associated with the ADP-glucose α-1,4-glucan α-4-glucosyl transferase (α-glucan synthetase) fraction III. Separation of the branching enzyme I activity from the α-glucan synthetase III activity was achieved only by chromatography on an ADP-hexanolamine-Sepharose 4B column. The properties of the two branching enzyme fractions were very similar with respect to their activities toward potato amylose, amylopectin, and amyloses prepared with either rabbit muscle phosphorylase or spinach leaf α-glucan synthetase. Incubation of potato amylose with branching enzyme or incubation of phosphorylase or α-glucan synthetase with branching enzyme resulted in formation of glucan products resistant to complete hydrolysis by β-amylase (37–50%) and α-amylase (20–31%) but still quantitatively hydrolyzed by the combined action of pullulanase plus β-amylase. The only significant difference noted between the two branching enzyme activities is that fraction II has higher activity in citrate buffer than in other buffers in the pH range of 5.5–6.5 while fraction I has higher activity in bicine and phosphate buffers than with citrate buffer. Both branching enzyme fractions stimulate the previously described “unprimed activity” catalyzed by α-glucan synthetase fraction III about 11- to 14-fold. However, branching enzyme did not stimulate the primed activity. It was previously shown that the “unprimed reaction” was highly dependent on the presence of citrate and other anions. However, the K m of amylopectin and glycogen for α-glucan synthetase III which was found to be 0.53 mg/ml and 2.94 mg/ml, respectively, are decreased to 1.9 μ/ml and 0.86 μ/ml, respectively, in the presence of 0.5 m citrate. The levels at which primer are effective in the presence of 0.5 m citrate suggest that the “unprimed” synthetic activity previously reported may be due to the ability of the endogenous glucan associated with the α-glucan synthetase fraction to act as a primer when citrate or other anions are present. The stimulation of the “unprimed” activity by branching enzyme may then be explained by its catalysis of formation of an increased number of nonreducing chain ends in the growing glucan that are able to accept glucosyl residues from ADPglucose.

The effect of photosynthesis inhibitors on oxygen evolution and fluorescence of illuminated chlorella

Abstract Photosynthesis inhibitors, like symmetrical triazines, substituted ureas, and anilides, are able to stop oxygen evolution from illuminated Chlorella and cause a stimulation in fluorescence. These two phenomena seem to be inter-related as shown by partial inhibition studies. The exception to the rule was found to be cyanide which inhibited oxygen evolution but had no effect on fluorescence intensity.

Allosteric regulation of uridine diphosphoglucose: D-fructose-6-phosphate-2-glucosyl transferase (E.C.2.4.1.14)☆

Abstract Kinetic studies of wheat germ sucrose-P synthetase indicate that both substrates, fructose-6-P and UDP-glucose, exhibit sigmoidal saturation curves in the presence or absence of 22 mM MgCl2. MgCl2 stimulates the maximal velocity about 2-fold and decreases the apparent affinity of UDP-glucose, 3-fold. In view of these data, sucrose-P synthetase may be considered an allosteric enzyme involved in regulating the biosynthesis of sucrose.

Biosynthesis of starch in Chlorella pyrenoidosa: I. Purification and properties of the adenosine diphosphoglucose: α-1, 4-glucan, α-4-glucosyl transferase from Chlorella

Abstract The adenosine diphosphoglucose: α1 → 4 glucan transferase of Chlorella pyrenoidosa was purified 25-fold. Of a number of sugar nucleotides tested, only ADP-glucose and deoxy-ADP-glucose could serve as glucosyl donors. A number of α1 → 4 glucans can serve as primers for the reaction. Amylose or amylopectin were more effective than glycogen as acceptors of glucose from the sugar nucleotides. The enzyme had a pH optimum of 9.0 and was inhibited by p-chloromercuribenzoate. Glycolytic intermediates had no stimulatory effect on the transferase. The properties of this transferase were compared with the properties of other plant and bacterial AD Pglucose:α-1,4-glucan transferases.

De Novo SYNTHESIS OF BACTERIAL GLYCOGEN AND PLANT STARCH BY ADPG:α‐GLUCAN 4‐GLUCOSYL TRANSFERASE*

One of the unsolved problems in glycogen and starch synthesis is the mechanism of initiation of the biosynthesis of these polyglucosides. It has been suggested that either phosphorylase or the sugar nucleotide a-glucan 4-glucosyl transferase is the enzyme involved in initiating starch or glycogen biosynthesis.I-’ Tsai and Nelson have found multiple forms of phosphorylase in maize endosperm; two of these can synthesize polysaccharide in the absence of added primer.2 Slabnik and Frydman have also found a phosphorylase in potato tubers that will catalyze polysaccharide synthesis without added primer. This has led to the suggestion that phosphorylase may be involved in the synthesis of starch de novo. The synthesis of glucans by phosphorylases from other tissues in the absence of added primer, however, has been shown to be due to small amounts of oligosaccharides present either in the substrate, G-1-P, or in the enzyme Gahan and Conrad lo have described an ADPG: a-1,4-glucan a-4-glucosyl transferase in extracts of Aerobacter aerogenes that synthesizes glycogen without added primer. They suggest that de novo synthesis differs from the elongation mechanism normally involved in glycogen biosynthesis. Recently our laboratory has separated multiple forms of ADPG : a-l,4-glucan a-4-glucosyl transferase from spinach maize endosperm,4 potato tubewe and Chlamydomonas rheinhardii.lR One of the transferases from each source was capable of catalyzing the synthesis of a glucan in the absence of added primer. The products contained principally a-1,4 linkages and some a-1,6 linkages. The unprimed activity was stimulated over 1,000-fold by bovine serum albumin (BSA) and high concentrations of some salts. The purpose of this paper is to describe the conditions required for the catalysis of the unprimed reaction by an ADPG : a-4-glucosyl transferase system isolated from a bacterial source (Escherichia coli B.), to show the effect of

Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-P adenylyltransferase (EC 2.7.7.27) from a glycogen-excess mutant of Escherichia coli B.

Abstract An Escherichia coli B mutant, CL1136 accumulates glycogen at 3.4 to 4 times the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to α- and β-amylolysis, chain length determination and I2-complex absorption spectra. The CL1136 mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and in contrast to the present strain enzyme studied previously, is highly active in the absence of the allosteric activator. The response of the CL1136 enzyme to energy charge has been determined and this enzyme shows appreciable activity at low energy charge values where the E. coli B enzyme is inactive. The response to energy charge for the CL1136 and E. coli B enzymes are correlated with the rates of glycogen accumulation observed in the microorganisms. The regulation of glycogen synthesis in E. coli is to a great extent at the level of ADPglucose pyrophosphorylase; varying concentrations of fructose-P2 and energy charge determine the rate of ADPglucose and glycogen synthesis. Both the allosteric regulation of ADPglucose pyrophosphorylase as well as the genetic regulations of the synthesis of glycogen biosynthetic enzymes (glycogen synthase and ADPglucose pyrophosphorylase) are involved in the regulation of glycogen accumulation in E. coli B.

TPNH and pyridoxal-5′-phosphate: Activators of ADP-glucose pyrophosphorylase of Escherichia coli B☆

Abstract TPNH and pyridoxal-5-P have been found to be potent activators of E. coli B ADP-glucose pyrophosphorylase. The concentrations required for 50% maximal activation was 0.15 mM TPNH and 8 μM pyridoxal-5-P. These values compare favorably to the value of 0.13 mM found previously for the activator, fructose diP. Kinetic evidence is presented to suggest all three activators bind to the same site(s) on the enzyme. PLP-activated ADP-glucose synthesis is less sensitive to 5′-adenylate inhibition than is TPNH-activated synthesis. The physiological functions of these activations with respect to glycogen storage is discussed.

Enzymic synthesis of GDP-mannose-14C from mannose-14C

A procedure synthesizing GDP-mannose-14C from mannose using Arthrobacter viscosus crude extracts has been described.

An ADP-glucose pyrophosphorylase with lower apparent affinities for substrate and effector molecules in an Escherichia coli B mutant deficient in glycogen synthesis☆

Abstract SG-14, a mutant strain of E. coli B that accumulates glycogen at one-half the rate observed for the parent strain, contains an altered ADP-glucose pyrophosphorylase with lower apparent affinities than that observed for the parent enzyme for its substrates, ATP and Mg2+ and for its effectors, fructose diphosphate, pyridoxal-P (PLP) and 5′adenylate. TPNH activates the native enzyme but not the mutant enzyme. In view of the reported physiological concentrations of ATP and Mg2+ and of the kinetic effects observed for FDP, PLP and AMP for the mutant enzyme, it is suggested that FDP is the important physiological activator for ADP-glucose pyrophosphorylase.

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

The ADPglucose pyrophosphorylases from Rhodopseudomonas sphaeroides and Rhodopseudomonas gelatinosa are activated by fructose-6-phosphate, pyruvate and fructose-1,6 biophosphate-P2. The effects of the activators are to increase significantly the Vmax of ADPglucose synthesis and to lower the S0.5 values (concentration of substrates giving 50% maximal velocity) for ATP and MgCl2. The R. sphaeroides enzyme is inhibited by Pi while the R. gelatinosa enzyme is inhibited by AMP as well as by Pi. The interaction between inhibitor and activator is complex. At very low concentrations of activator the enzyme is more sensitized to inhibition. However, at higher concentrations of activator there is a decrease in the sensitivity of the enzyme towards inhibition. The findings are discussed with respect to glycogen synthesis in these microorganisms and may be related to findings that indicate that Rhodopseudomonads have the ability to degrade sugars via the Entner-Duodoroff or Embden-Meyerhoff pathways.