GLUCOSE DISSIMILATION IN USTILAGO MAYDIS

McKINSEY, RICHARD D. (U. Virginia, Charlottesville.) Glucose dissimilation in Ustilago maydis. Amer. Jour. Bot. 46(8): 566-571. Illus. 1959.-Studies were made of the manner in which glucose is dissimilated in Ustilago maydis. Radioactive isotope experiments utilizing glucoseI-C14 and glucose-6-C14 gave a C6 ratio of 0.23 indicating that the major portion of the glucose was being metabolized by a direct shunt mechanism. Spectrophotometric and manometric experiments indicated the probable presence of the following enzyme systems: aldolase, 3-phosphoglyceraldehyde dehydrogenase, phosphoglyceryl kinase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic acid dehydrogenase. Manometric and growth experiments indicated that Ustilago maydis is an obligate aerobe. STUDIES OF carbohydrate metabolism have demonstrated that glucose may be dissimilated by alternate pathways in a variety of organisms. Although the number of representatives studied has been small, alternate pathways have been shown to occur in such phylogentically diverse groups as the bacteria, fungi, higher plants and invertebrate animals (Horecker and Mehler, 1955; Axlerod and Beevers, 1956). In the fungi, the Basidiomycetes seems to have been somewhat neglected by students of carbohydrate breakdown. The neglect of the Basidiomycetes is strange in view of their abundance, phylogenetic significance and economic importance. The objective of the present investigation was to discover the general nature and relative importance of the pathways by which glucose is dissimilated by Ustilago maydis. The approach of the experiments was designed: (1) to obtain evidence of the probable existence or nonexistence of a sufficient number of intermediate steps in the Embden-Meyerhof-Parnas and pentose-phosphate pathways to give an indication of the presence or absence of each of the pathways and (2) to provide evidence that would allow some conclusions to be drawn as to the quantitative significance of each of the pathways, if present, in the general sugar metabolism of Ustilago maydis. MATERIALS AND METHODS. Fructose-1,6-diphosphate, glucose-6-phosphate, phosphoglyceric acid, pyruvic acid, adenosine triphosphate (ATP), adenosine diphosphate (ADP), triphosphopyridine nucleotide (TPN), and diphosphopyridine nucleotide (DPN), were purchased from Nutritional Biochemicals Corporation. Glucose-6-C14 was obtained from Nuclear Chicago Corporation and glucose-i-C14 was obtained from the National 'Received for publication March 9, 1959. This paper is a portion of a dissertation submitted to the Department of Biological Sciences and the Committee on Graduate Study of Stanford University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The writer is especially indebted to Professor R. M. Page of Stanford University for his encouragement and suggestions. Bureau of Standards. Wild type cultures (UZ5) of Ustilago maydis (DC.) Cda., obtained from Dr. D. D. Perkins (Department of Biological Sciences, Stanford University), were used in the biochemical studies. Cells were grown in liquid culture in a medium consisting of 1.5 g. of AC Broth (Difco) and 0.5 g. Tween #80 (Atlas Powder Co.) per 100 ml. of water. In each experiment, 50 ml. of this medium was placed in each of four 250-ml. Erlenmeyer flasks to which were added cells from 2-wk.-old cultures growing on complete medium (Perkins, 1949). The flasks were placed on a low-speed reciprocal shaker and incubated at 30?C. After 48 hr. of growth, the cells were harvested by centrifugation at 2000 X g and washed twice in distilled water. Cell-free extracts were prepared by suspending the damp cells in a minimum of solution containing 60 mg. cysteine hydrochloride dissolved in 10 ml. of 0.05 molar potassium phosphate buffer, pH 7.0. Cysteine has been reported (Cochrane, 1956) to protect enzymes from inactivation by heavy metals. The suspended cells were ground in a chilled mortar with 3 weights of powdered glass (40 mesh) in a 5?C. cold room. After 5 min. of grinding, additional suspending solution was added (10 ml.), and cell debris was removed from the extract by centrifugation at 3000 X g. RESULTS.-Oxygen requirement.-Ustilago maydis has been found to require aeration for rapid growth in liquid culture (Perkins, 1949). Attention was directed in the present investigation regarding whether Ustilago maydis is an obligate aerobe. Production of C02, which was used as an index of metabolic activity, was measured by means of a standard Warburg respirometer with oxygen-free nitrogen as the gas phase. Each Warburg flask contained approximately 400 mg. of wet whole cells, potassium phosphate buffer (0.05 molar, pH 7.0), 1.5 ml.; MgCl,, 10,u moles; glucose, 60,u moles; and water to 2.6 ml. No detectable CO2 was produced in 1 hr. under anaerobic conditions. Cells in the presence of air