Metabolic basis for disaccharide preference in a Cellvibrio.

Reagent grade chemicals were used where available. Adenosine triphosphate and coenzyme A were obtained from the Pabst Laboratories; riboflavin-5'-phosphate was obtained from the California Foundation for Biochemical Research; sugar phosphates were obtained from Nutritional Biochemicals Corporation. "Levigated Alumina," A-542, from Fisher Scientific Company, was used in preparing ground-cell suspensions and cell-free extracts. All chromatographic solvents were freshly distilled. The analytical procedures used were those described by Somogyi (1945) and Nelson (1944) for reducing sugars, Pan et al. (1953) modified for differential analysis of mixtures of cellobiose and glucose, Johnson (1941) for total nitrogen, Fiske and SubbaRow (1925) for total and inorganic phosphorus, and Lowry et al. (1951) for protein. Separation of sugar phosphates by paper chromatography was conducted using a modification of the procedures described by Mortimer (1952). A solvent consisting of methyl ethyl ketone, methyl cellosolve, ammonia, and water in the volume ratio of 7:2:3:1 was found to yield higher Rf values and greater resolution than Mortimer's solvent. The Rp values in the modified solvent were as follows: inorganic phosphate, 100; glucose-i-phosphate, 173; glucose-6-phosphate, 154; fructose-1 ,6-diphosphate, 4.6; and fructose-6-phosphate, 242. The Rp value is 1 Supported in part by a grant-in-aid from the National Science Foundation. 2Present address: Department of Microbiology, Yale University, New Haven, Connecticut. 100 times the ratio of the distance which a compound moves to the distance moved by inorganic phosphate, the measurements being made to the leading edge of all spots. Sugar phosphates were detected on paper chromatograms by using the procedure of Hanes and Isherwood (1949). Glucose-6-phosphate was distinguished from glucose-i-phosphate by spraying the papers first with aniline hydrogen phthalate (Partridge, 1949) and then with the sugar phosphate spray. Sugar phosphates were recovered from experimental systems as their barium salts (Umbreit et al., 1951) and then converted to their ammonium salts according to the method of Wade and Morgan (1955) prior to separation by paper chromatography. In isolating enzymaticallv synthesized glucose-i-phosphate from cell-free enzyme systems, the ion-exchange procedure of McCready and Hassid (1944) was used. Active cell suspensions were obtained from 3or 4-L broth cultures grown for 18 hr under forced aeration at room temperature. The medium was that used previously (Hulcher and King, 1958) supplemented with 0.025 g of enzymatic casein hydrolyzate per L and with 1 ppm Antifoam-A (Dow Chemical Company). Cell yields were 2 to 4 g of packed, wet cells per L. All cultures were tested for contamination by examining Gram stains and nigrosine stains before harvesting the cells by high speed centrifugation in the cold. Cell-free extracts were obtained by washing the packed cells twice in tris(hydroxymethyl)aminomethane (Tris) buffer (0.01 M or 0.03 M, pH 7.0) in the cold. The washed cells were then ground with twice their weight of alumina in a chilled mortar for 10 to 20 min. "Soluble enzyme" preparations consisted of the clear supernatants obtained by centrifugation at 20,000 X G for 30 min at 0 to 4 C. Such extracts contained several of the enzymes participating in the initial reactions of carbohydrate metabolism, but con-