Enrico Cabib

The enzymic synthesis of yeast mannan

Abstract When guanosine diphosphate mannose (GDP-mannose) was first isolated from yeast, it was proposed that this compound might be the precursor of mannan ( Cabib and Leloir, 1954 ). Evidence supporting this hypothesis will be outlined in the present communication.

Guanosine diphosphate d-glucose glucohydrolase☆

Abstract A new enzyme has been found in yeast, which catalyzes the hydrolysis of guanosine diphosphate glucose to guanosine diphosphate and glucose. The enzyme was purified about 130-fold and its kinetic properties were studied. The enzymic activity seems to be strictly specific for guanosine diphosphate glucose, with a Km of 0.23 m m . The enzyme is active over a wide range of pH values, from 5 to 9. Guanosine diphosphate inhibits competitively and Mg++ partially relieves the inhibition. The enzyme appears to exist in soluble form in the cytoplasm. The possible significance of guanosine diphosphate glucose glucohydrolase and of sugar nucleotide phosphorylase in the control of the intracellular concentration of guanosine diphosphate glucose and guanosine diphosphate mannose is discussed.

Paper electrophoresis of sugars with cetyltrimethylammonium borate

Abstract Paper electrophoresis of sugars and other substances with cetyltrimethylammonium borate at pH 9.6 gives rise to a pattern of mobilities different from that found with potassium borate, thus making certain separations possible that are not obtained with the potassium salt. There seems to be a general trend towards a reversal in the order of mobilities with the cetyltrimethylammonium buffer. The observed mobilities have been interpreted as the net results of two contrary effects: a strong and uniform endosmotic flow towards the anode and an opposite and variable movement towards the cathode. The latter displacement would take place by association of the solutes with the positively charged ionic micelles that are present in the solutions of cetyltrimethylammonium salts. Applications of this behavior might lead to the separation of other substances.

[107] Isolation of uridine diphosphate glucose, uridine diphosphate acetylglucosamine, and guanosine diphosphate mannose

Publisher Summary This chapter describes the isolation of uridine diphosphate glucose, uridine diphosphate acetylglucosamine, and guanosine diphosphate mannose. The basic principle is that alcoholic extract from toluene-autolyzed yeast is fractionated on anion-exchange columns. The nucleotides are recovered by adsorption on charcoal and subsequent elution with aqueous ethanol. Ten kilograms of bakers yeast are brought to 36° and intimately mixed with 1000 ml of warm toluene. After incubation at 35° to 37° for 40 minutes, 10 liters of 95% ethanol are added, and the mixture is heated with stirring until it boils. After standing overnight in the refrigerator, it is filtered through a 32 cm Buchner funnel with a filter aid. The extract is brought to pH 7 with bromothymol blue as indicator just before chromatography. Column chromatography is then performed, followed by concentration of the product. The liberation of acetylglucosamine from UDP-acetylglueosamine in 0.01 N acid at 100° is practically complete in 15 minutes. The lability in acid of the phosphate linked to the sugar is the same as for UDPG. Mannose is very easily hydrolyzed from GDPM 4 by 0.01 N acid at 100°. Ten minutes are sufficient for quantitative splitting. About 50% of the total phosphate of GDPM is liberated by 1 N acid in 20 min or by 0.1 N acid in 120 minutes.

[34] Sugar nucleotide phosphorylases (“nucleoside diphosphate sugar: Orthophosphate nucleotidyl transferases”)

Publisher Summary This chapter discusses the synthesis of sugar nucleotide phosphorylases from yeast and from wheat germ. In the assay method for yeast, the reaction is carried out using guanosine diphosphate (GDP) mannose and 32 P. The radioactive GDP produced during incubation is separated from 32 P i by adsorption on charcoal. The charcoal is washed and directly counted. The sensitivity of the assay can be increased by using 32 P i of higher specific activity. At the concentration of GDP-mannose usually employed the enzyme is far from saturated. However, the relatively low concentration is used to conserve substrate. In the assay method for wheat germ, the enzyme is assayed by determination of the adenosine diphosphate (ADP) formed with phosphoenol pyruvate and pyruvate kinase. The assay method used for the yeast enzyme, should be also applicable to wheat germ phosphorylase. The reagents used, procedure, and the steps involved in the purification are also described in the chapter.