Luis F. Leloir

The enzymatic transformation of uridine diphosphate glucose into a galactose derivative

Abstract Treatment of uridine diphosphate glucose (UDPG) with an enzyme of S. fragilis was found to produce about 25% of a galactose-containing compound. This compound is precipitated with mercuric ions like UDPG, and its migration in chromatography in acid-ethanol is similar. By alkaline treatment it gives, like UDPG, a doubly esterified hexose monophosphate. It is concluded that the compound is uridine diphosphate galactose, and the bearing of this finding on the mechanism of action of UDPG is discussed.

Biosynthesis of glycogen from uridine diphosphate glucose

Abstract An enzyme which leads to the formation of glycogen according to the equation: UDPG + primer → UDP + glucosyl α (1 → 4) primer has been studied. The optimal conditions for activity were determined with a partially purified preparation from rat muscle. The reaction requires the presence of a polysaccharide as primer and is strongly activated by hexose 6-phosphates. Using UDPG labeled in the glucose moiety, it was found that the radioactivity was transferred to the glycogen from which it could be removed as maltose with β-amylase or as G-1-P with phosphorylase. Thus it seems that the glucose residue becomes linked α (1 → 4) to the polysaccharide. Several inhibitors were tested as well as the occurrence of the enzyme in different organs.

Adenosine diphosphate glucose and starch synthesis

Received Septenber 19, 1961 In previous papers (Fekete et al., 1960; Leloir et al., 1961) an enzyme was described which catalyzes glucose transfer from uridine diphoaphate glucose (UD?G) to starch or oligosaccharides. Several synthetic nucleoside diphosphate sugars have now been tested with the same enzyme preparation. They were prepared following the procedures developed by Khorana and others (Roseman, Distler, Moffatt and Khorana, 1961) with alight modifications. The most interesting result was that adenosine diphosphate glucose (ADPG) reacts about tenfold faster than UDPG. A typical experiment is shown in figure 1.

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.

The role of dolichol monophosphate in sugar transfer.

Abstract The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate- N -acetylgalactosamine gave negative results. The optimal conditions for transfer from dolichol monophosphate glucose to endogenous acceptor was determined. Studies were carried out on the glucosylation of ceramide by brain extracts and of collagen by skin enzymes in order to find out if dolichol monophosphate glucose is an intermediate in these reactions. The results, while not definite, were not in favor of this assumption.

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.

Mannose transfer to lipid linked di-N-acetylchitobiose

Abstract Mannose was found to be transferred from guanosine diphosphate mannose to dolichol-P-P-di-N-acetylchitobiose when these substrates were incubated with hen oviduct or rat liver microsomes. The oligosaccharide moiety of the product appears to be the trisaccharide β-mannosyl-β-N-acetylglucosaminyl-(1→4)-N-acetylglucosamine as judged by the action of glycosydases. Under certain conditions further transfer of mannose occurs and larger oligosaccharides bound to lipid are formed.

The Biosynthetic Pathway of the Asparagine-Linked Oligosaccharides of Glycoproteins

This review deals with the structure and addition of the different types of oligosaccharides to asparagine residues in proteins. This process occurs in several steps, first an oligosaccharide which contains N-acetylglucosamine mannose and glucose is built up joined to dolichyl diphosphate. The oligosaccharide is then transferred to a polypeptide chain, loses its glucose, and is modified by removal of some monosaccharides and addition of others giving rise to a variety of saccharides.