Carlos E. Cardini

The biosynthesis of glucosamine.

Abstract A partially purified enzyme has been prepared from Neurospora crassa which catalyzes the formation of glucosamine phosphate from hexose-6-phosphate and glutamine. The glucosamine phosphate was identified by colour reactions, by dephosphorylation and paper chromatography and by its behaviour towards an acetylating system. Quantitative analysis of amide nitrogen, glutamate, and hexosamine agreed with the following equation: Hexose-6-phosphate + glutamine → glucosamine-6-phosphate + glutamate Crude Neurospora extracts were found to phosphorylate glucosamine in the presence of ATP and, when suitably supplemented, to acetylate glucosamine or glucosamine phosphate.

[115] Characterization of phosphorus compounds by acid lability

Publisher Summary Measurements of the rate of hydrolysis of phosphoric esters have been carried out for analyzing mixtures, as a test of homogeneity, and as a criterion of identity. This chapter presents the procedure for the estimation of phosphate. Of the many methods available for the estimation of phosphate, that of Fiske and SubbaRow is one of the simplest and most widely used. In the Fiske and SubbaRow procedure, the extra-labile compounds are estimated as inorganic phosphate because of the relatively high acid concentration (pH 0.65) and because molybdate accelerates the hydrolysis of some organic phosphates. By measuring the color immediately after adding the reagents, however, Fiske and SubbaRow were able to estimate phosphocreatine. Better results are obtained by estimating the true inorganic phosphate by precipitating it with magnesium mixture or with calcium salts and ethanol. Other method is the Lowry and Lopez method in which the acid and molybdate concentrations are lower than in Fiske and Subba-Rows, so that some extra-labile compounds are hydrolyzed more slowly.

Mechanism of glucose transfer from sucrose into the starch granule of sweet corn

The mechanism of sucrose-starch transformation was studied in corn endosperm. Preparations of endosperm which contain the total protein and starch granules catalyze an incorporation of radioactivity from sucrose-C 14 to the granule in presence of ADP or UDP, ADP being more effective. No transfer of glucose-C 14 from glucose-1-P to the granule was observed unless Mg ++ and ATP were present. The K m values of the UDP-glucose-fructose glucosyl transferase for ADP, UDP, ADP-glucose, and UDP-glucose and their reciprocal inhibitions were measured From all these results the following two sequences for the glucose incorporation into the granule were proposed: (a) SucroseαADP-glucose (or UDP-glucose)→starch (b) SucroseαUDP-glucoseαglucose-1-PαADP-glucose→starch The enzymes which catalyze all these steps could be demonstrated in corn endosperm.

Enzymes acting on glucosamine phosphates

Abstract The chemical synthesis of α-acetylglucosamine- i -phosphate and some of its properties are described. From kidney or liver, enzymes have been obtained which catalyse the following reactions: 1. α- acetylglucosamine i - phosphate ⥮ acetylglucosamine -6- phosphate (1) 2. N - acetylglucosamine -6- phosphate ⥮ fructose -6- phosphate + NH 3 + acetate (2) 3. glucosamine -6- phosphate ⥮ fructose -6- phosphate + NH 3 (3) Reaction (1) was found to be activated by magnesium ions. The enzyme(s) responsible for reactions (2) and (3) were purified and it was observed that (3) requires catalytic amounts of N-acetylglucosamine-6-phosphate (or of the N-propionyl derivative) and that it is reversible. The possible mechanism of the reactions is discussed.

Enzymic phosphorylation of galactosamine and galactose.

Abstract The transfer of phosphate from adenosine triphosphate to galactosamine was found to be catalyzed by a liver enzyme. On the basis of the parallel distribution and from crossed inhibition experiments, it is suggested that the enzyme may be galactokinase. The optimum conditions for activity and a method for partial purification are described. Phosphorylation of galactosamine and of galactose was also found to be catalyzed by extracts from brain tissue and from a lactose yeast ( Saccharomyces fragilis ). Extracts from cells of the latter grown in lactose, which cells contain more galactokinase, were found to have a higher activity on galactosamine. Evidence is presented indicating that the reaction product is galactosamine 1-phosphate. This product was found to be more resistant to acid hydrolysis than aldose 1-phosphates.

The biosynthesis of plant glycosides. I. Monoglucosides

Abstract An enzyme from wheat germ which catalyzes the formation of glucosides from phenols and uridinediphosphate glucose has been partially purified. With hydroquinone as substrate, arbutin was found to be formed as follows: UDPG + hydroquinone → UDP + arbutin Arbutin was identified by paper chromatography in several solvents either directly or after hydrolysis with acid or β-glucosidase. The rate of reaction with different phenols decreased in the order: hydroquinone > hydroxyhydroquinone > methoxyhydroquinone > resorcinol > pyrogallol > pyrocatechol.

Studies on the biosynthesis of starch: I. Isolation and properties of the soluble adenosine diphosphate glucose: Starch glucosyltransferase of Solanum tuberosum☆

Abstract A soluble glucan synthetase (starch synthetase) has been isolated from potato tubers. This enzyme catalyzes the transfer of glucose from adenosine diphosphate glucose, deoxyadenosine diphosphate glucose, and to a lesser extent, from β-adenosine diphosphate glucose to phytoglycogen, amylopectin, and malto-oligosaccharides. β-Limit dextrins, dextran, and isomalto-oligosaccharides were inactive as primers. The freshly prepared enzyme also catalyzes the transfer of glucose from the sugar nucleotide to the intact starch granules, but this activity disappears with storage. The properties of the enzyme which acts on the soluble primers were studied.

The biosynthesis of plant glycosides. II. Gentiobiosides

Abstract An enzyme from wheat germ which catalyzes the reaction: UDPG + phenol β-glucoside → UDP + phenol β-gentiobioside has been purified and separated from the enzyme which leads to the formation of glucosides. The enzyme was found to be specific for phenol β-glucosides and substituted phenol β-glucosides (such as arbutin, salicin, p - and m -methoxyphenol glucoside, resorcinol glucoside, and mandelonitrile glucoside). Free sugars, disaccharides, or polysaccharides were not used as substrates.

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.