Frank Eisenberg

Inositol formation by cyclization of glucose chain in rat testis

Abstract A homogenate of rat testis capable of incorporating carbon from glucose-U-C 14 into inositol has been described in a previous publication ( Eisenberg and Bolden, 1963 ). In an attempt to differentiate 1 between a hexose moiety ( Imai, 1963 ; Loewus and Kelly, 1962 ) and hexose fragments ( Charalampous, 1957 ; Hauser and Finelli, 1963 ) as precursors of inositol, the conversion of specifically labeled glucose to inositol has now been studied in rat testis homogenate with the aid of a combined plant and chemical degradative system described previously ( Loewus et al , 1962 ). Experimental evidence to be presented in this paper points unequivocally to a pathway of biosynthesis of inositol in the mammal involving cyclization of a six-carbon unit.

Biosynthesis of inositol in rat testis homogenate

Abstract The observation of inositol formation in germ-free rats and mice ( Freinkel and Dawson, 1961 ) has established umequivocally the existence of a mammalian pathway for the biosynthesis of this cyclitol. Until now studies designed to elucidate this pathway have utilized the intact cell either in the whole animal ( Daughaday et al , 1955 ; Halliday and Anderson, 1955 ; Imai, 1963 ; Hauser and Finelli, 1963 ), tissue culture ( Eagle et al , 1960 ), or tissue slices ( Hauser and Finelli, 1963 ). Synthesis of inositol in intact non-mammalian cells has been observed in yeast ( Charalampous, 1957 ), chick embryo ( Daughaday et al , 1955 ), and plants ( Loewus and Kelly, 1962 ). The present communication describes experiments in which for the first time a homogenate, prepared from rat testis, has been found active in the synthesis of myo-inositol from glucose in yields about 10 times greater than in mammalian cellular systems. Under the same experimental conditions homogenates of rat liver, kidney, brain, spleen, lung, and lactating mammary gland failed to incorporate glucose carbon into inositol.

D-myo-inositol-l-phosphate, an intermediate in the biosynthesis of inositol in the mammal

Previous communications have reported an enzyme system in homogenate of rat testis ( Eisenberg and Bolden, 1963 ) which catalyzes the cyclization of the glucose chain to myo-inositol ( Eisenberg, Bolden and Loewus, 1964 ). A study of this reaction in yeast ( Chen and Charalampous, 1965 ) has shown that glucose-6-P and myo-inositol-1-P are intermediates in the cyclization. Since myo-inositol-1-P can exist in enantiomeric forms it was of interest in proposing a mechanism of cyclization to determine which optical isomer is involved. Although the L configuration has been observed repeatedly among the inositol phosphates derived from phosphoinositides from many natural sources ( Rapport and Norton, 1962 ), the D isomer has been known only synthetically ( Ballou and Pizer, 1959 ). Evidence that the intermediate in the formation of inositol in the rat is D-myo-inositol-1-P is presented in this paper.

Gas chromatographic assay of iduronic and glucuronic acids as aldonic acid butaneboronates

Abstract A gas chromatographic procedure has been developed for the analysis of uronic acids as aldonic acid butaneboronates. With these derivatives, aldoses frequently accompanying uronic acids in polysaccharide hydrolyzates are readily separated and measured. The method has been applied to the assay of iduronic and glucuronic acid released by enzymes associated with various mucopolysaccharidoses.

Gas chromatography of sugar phosphates and sugar nucleotides

Abstract Glucose 6-phosphate, various aldose 1-phosphates, and their corresponding sugar nucleotides have been converted into trimethylsilyl ethers and gas chromatographed. Glucose 6-phosphate separates into α- and β-isomers and can be recovered intact on a preparative scale from the column effluent after spontaneous hydrolysis of the ether linkages. The aldose 1-phosphates and sugar nucleotides are not recoverable intact from the column but lose phosphoric acid and nucleoside diphosphate, respectively, to yield the TMS ether of the sugar anhydride. Since the anhydride patterns are reproducibly characteristic, mixtures of nucleotides can be easily separated and their sugar components identified.

The formation of cyclic inositol 1,2-monophosphate, inositol 1-phosphate, and glucose 6-phosphate by brain preparations stimulated with deoxycholate and calcium: a gas chromatographic study.

Abstract A gas chromatographic method has been developed for the separation and isolation of water-soluble phosphates as trimethylsilyl ethers. With this method cyclic inositol 1,2-monophosphate and inositol 1-phosphate, derived from endogenous phosphatidylinositol, have been shown to increase when a particulate portion of brain homogenate is stimulated with deoxycholate and Ca ++ , confirming earlier observations of Lapetina and Michell (1). Concomitant with the appearance of inositol phosphates is the stimulated formation of glucose 6-phosphate in the whole homogenate. Although ATP can replace deoxycholate and Ca ++ in a dialyzed homogenate, glucose 6-phosphate apparently does not arise by any known metabolic pathway but from another unidentified source.

An improved method for the determination of deuterium in organic compounds.

Abstract A method for the determination of deuterium in organic compounds has been developed which is based on the oxidation of organic deuterium to deuterium chloride by HgCl2; subsequent reduction of DCl by zinc released deuterium for mass spectrometric analysis. The method is applicable to various types of organic compounds, including aliphatic, aromatic, and amino acids and gives quantitative results in the range 0.02 to 5 atom per cent excess deuterium.

Relative stability of the interglycoside bonds of sucrose to acid hydrolysis in H218O

Abstract Sucrose, a disaccharide with two interglycoside bonds, was hydrolyzed with acid in H 2 18 O. Through a kinetic study of the labeling of d -glucose with 18 O, an estimate was made of the relative stabilities of the two bonds. It was found in agreement with the known greater lability of fructofuranosides compared to glucopyranosides that for every glucopyranosyl-oxygen bond split three fructofuranosyl-oxygen bonds were hydrolyzed.