Nelly Lavintman

The formation of branched glucans in sweet corn

Abstract The properties of a preparation of α-glucan branching glycosyl transferase (α-1,4-glucan:α-1, 4-glucan 6-glycosyltransferase E.C. 2.4.1.18) from sweet corn were studied by using amylose and amylopectin as substrates. The preparation behaved differently toward each substrate, with respect to pH optimum, activation by citrate, and the effect of Hg ++ and temperature. Similar extracts obtained from starchy maize, wheat, and barley were shown to have only Q-enzyme activity i.e., no action on amylopectin. Studies on substrate specificity showed that maltodextrins of average DP 2 above 22 served as substrates for sweet corn, starchy maize, and liver branching preparations. Electron micrographs of natural phytoglycogen are shown.

Particulate UDP-glucose: Protein transglucosylase from potato tuber

We have previously shown [ 1] that a particulate fraction constituted mainly of proplastids could be isolated from potato tuber juice by differential centrifugation. This fraction contained besides phosphorylase, a starch synthetase activity with similar specificity for the glucosyl donor to that demonstrated for the starch synthetase attached to the starch grain [2]. These transglucosylase activities could be demonstrated without primer addition, since primer was already present in the preparation [ 11. This paper reports the results of further studies on the proplastid particulate fraction and presents information about its capacity to transfer glucose, specifically from UDP-glucose, to a trichloroacetic acid (TCA)-insoluble product which shows the properties of a glucoprotein. This glucoprotein can act as acceptor for the synthesis of a 1,4-cu-glucan from ADP-glucose or glucose l-phosphate with the same enzymatic system.

Biosynthesis of starch. Formation of a glucoproteic acceptor by a potato non-sedimentable preparation.

1. A non-sedimentable fraction of potato tuber has been found to catalyze [14C]glucose transfer from [14C]glucose 1-phosphate to an endogenous proteic acceptor in the absence of added primer. This transfer is activated by Mn2+. 2. The labeled glucosylated product formed is trichloroacetic acid insoluble and sensitive to proteolytic and amylolytic digestions. It appears to be a glucoprotein with glucosyl chains bound to the peptide portion of the molecule through an unknown linkage. 3. The carbohydrate portion of the glucoprotein can be released by prolonged incubations with the enzymatic preparation, and becomes in turn, trichloroacetic acid soluble and alcohol precipitable. 4. Both products, the glucoprotein as well as the alpha-1,4-glucan that seems to arise from the enzymatic cleavage of the former, can be used as primers by the transglucosylating system with ADP[14C]glucose, UDP[14C]glucose or GDP[14C]glucose as glucosyl donors. The results presented in this paper are the first demonstration of soluble glucosyl transferases with the same glucose donor specificity to that of the particulate starch synthetase. 5. This report presents further evidence in favor of the assumption of a glucoproteic intermediate in alpha-a,4-glucan synthesis initiation.

Enzymatic glycosylation of steroid alkaloids in potato tuber

Abstract The glycosylating activities of two enzymatic preparations from potato tubers were compared, using β-sitosterol and β-solanidine as substrate. The 25 000 g fraction synthesized steryl glucoside (SG) and acylsteryl glucoside (ASG) when β-sitosterol as well as β-solanidine was included in the reaction mixture. The AS fraction formed SG but not ASG from β-sitosterol and glycosylated β-solanidine forming six different derivatives. We tentatively related these products to the six components of solanine: α-, β- and γ-solanine and α-, β- and γ-chaconine.

Biosynthesis of a glycolipid in starch grains from sweet corn

Starch grains, isolated from immature endosperm of sweet corn 1, incubated with UDP-[14Clglucose brought about the formation of a butanol-extractable radioactive compound as well as the incorporation of [14C]glucose into the grain by starch synthetase = (UDP-glucose:starch glucosyltransferase). Unlike starch synthetase which can utilize several sugar nucleotidesL the enzyme responsible for the formation of the butanol-soluble product was highly specific for UDP-glucose. Other sugar nucleotides tested, such as ADP-glucose, GDP-glucose, GDP-mannose and UDPgalactose as well as glucose 1-phosphate together with ATP, glucose or sucrose, were not active as glycosyl donors. The Km for UDP-glucose was about o.I mM. The grinding of grains which increases the activity of starch synthetase with ADP-glucose and suppresses its activity with UDP-glucose a prevented the formation of the butanol-extractable product. Disintegration with 7 M urea had the same effect. The capacity to synthesize the butanol-soluble product was also destroyed by washing the grains with acetone or butanol. Table I shows the action of some cations and nucleotides upon both activities with UDP-glucose. Cations increased the butanol-extractable incorporation as well as the starch synthetase activity. UDP or UTP inhibited the former activity; ADP was without effect. The formation of the butanol-extractable compound was also found in the 25 ooo ×g and IOOOOO × g particulate fractions of endosperm. I t is worth pointing out that in this latter fraction phytoglycogen was obtained 4.

(1→4)-α-d-glucan phosphorylase [(1→4)-α-d-glucan:orthophosphate glucosyltransferase] isoenzymes from sweet corn seed embryo

Abstract The presence of multiple forms of phosphorylase [(1→4)-α- d -glucan:orthophosphate glucosyltransferase] in sugary maize seeds was demonstrated by polyacrylamide-gel-disc electrophoresis. The patterns of phosphorylase isoenzymes from immature and from germinating seeds were different. Most of the isoenzymes from embryo of germinated seeds precipitated at an ammonium sulfate concentration above 45% of saturation. The most cathodic band of the zymograms appeared on the third day of germination, then disappeared. This form of phosphorylase occurred only in the embryo of germinating seeds and it was absent both in the immature embryo and in the endosperm at any stage of development. The slow-moving embryo isoenzyme was purified through chromatography on DEAE-cellulose. Its kinetic properties and enlargement mechanism were studied.

Effect of isopropanol on the activity of particulate starch synthetase

Abstract The effect of several alcohols on particulate starch synthetases from potato tuber and sweet corn endosperm has been studied. High concentrations of isopropanol in the incubation mixture produced a great increase of the enzyme activity. The action of this alcohol on kinetic constants and on the distribution of incorporated glucose between amylose and amylopectin has been studied.

A primer independent activity of rabbit muscle phosphorylaseb

SummaryRabbit muscle phosphorylaseb was found to be capable of forming protein bound±-1,4 glucosyl chains upon incubation of the enzyme with appropriate concentrations of glucose-1-phosphate with no primer addition (unprimed synthesis). This activity would only be present in a small fraction of the total muscle phosphorylaseb activity, as judged from the high concentrations of enzyme which are required to demonstrate the occurrence of unprimed synthesis. Polyacrylamide gel electrophoresis shows the presence of a phosphorylase isoenzyme capable of accepting glucosyl moieties, giving rise to a glucosylated protein enzymatically active in the chain lengthening of its own glucan.

Activation of particulate starch synthetase from Zea mays embryo

Abstract The effect of spermine on particulate ADP-glucose: starch synthetase from the developing embryo of sweet corn has been studied. Spermine induces a considerable increase of glucose incorporation from ADP-glucose into the starch granules. The change in kinetic constants, the distribution of incorporated glucose between amylose and amylopectin and the pattern of incorporation into starch granules or malto-oligosaccharides has been studied. The data were compared with those obtained with citrate ions.

A Nitroxide Diuron Analog as a Probe for the Mode of Action of Herbicides

A new spin label nitroxide which is also a herbicide of the urea family has been synthesized. The nitroxide spin label derivative of diuron (ditempuron, DTPU) inhibits electron flow at the site of diuron inhibition of photosystem II (PSII) activity, and it is also reduced by electron flow from one of the first acceptors of PSII. The reduction of the nitroxide group of DTPU is inhibited by diuron or by trypsin treatment of the thylakoids but not by DNP-INT or DBMIB. It is suggested that DTPU binds specifically to and accepts electrons at the Qв site.