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A novel sulfatase hydrolyzing the sulfate ester bond in 3β-hydroxy-5-cholenoic acid 3-sulfate (Δ5-3β-sulfate) was purified from Pseudomonas testosteroniATCC 11996 as the second bile acid sulfatase. The molecular weight was 95,000 and the molecule was composed of a homodimer of a subunit of which the molecular weight was 46,000. This sulfatase hydrolyzed Δ5-3β-sulfate to 3α-hydroxy-5-cholenoic acid and sulfuric acid with inversion of β- to α-configuration of the hydroxyl group at the C-3 position of the substrate. The optimum pH and the stable pH of the enzyme were 8.5 and 6.5-9.7, respectively. 3β-Sulfate ester bonds of steroids such as isolithocholic acid, pregnenolone, and epiandrosterone, in which the side chain of the steroid ring was shorter than cholesterol, were also hydrolyzed to 3α-hydroxyl compounds corresponding to each steroid compound and sulfuric acid. We tentatively named this novel enzyme bile acid 3β-sulfate sulfohydrolyase (β-BSS).

N-Acetylneuraminate (NeuAc) synthase, which catalyzes NeuAc synthesis by condensation of N-acetyl-d-mannosamine (ManNAc) and phosphoenolpyruvate (PEP), was purified from a cell extract of Escherichia coliKI-M12 to electrophoretically homogeneity by serial column chromatographies. The molecular weight of native enzyme was estimated to be 106,000 by gel filtration. After denaturation in sodium dodecyl sulfate, the molecular weight was reduced to 52,000, indicating the existence of 2 identical subunits. The optimum pH was 7.5 and the stable pH range was 7.0 to 10.0. The enzyme was thermostable up to 30°C. No metal ion was required for the enzyme activity. SH-inhibitors such as p-chloromercuribenzoic acid and mercury chloride were potent inhibitors. The Kmfor ManNAc and PEP were 5.6 mmand 0.04 mm, respectively.

N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems. NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex. Escherichia colicells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem.265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence). That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity. These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP.

Tsukada, Yoji(Kyoto University, Kyoto, Japan), Tsunetake Sugimori, Kazutami Imai, and Hideo Katagiri. Action of cycloheximide on Zygosaccharomyces soja. J. Bacteriol. 83:70–75. 1962.—Cycloheximide is known to inhibit the growth of some species of Saccharomycesand other fungi. A new type of action of this antibiotic, with Zygosaccharomyces sojaas the test organism, is described in this paper. Growth of Z. sojawas completely inhibited under aerobic conditions by cycloheximide in concentrations of more than 5 μg per ml of culture solution. Continued shaking of the culture for more than 96 hr in the presence of an inhibitory amount of cycloheximide restored growth, accompanied by the excretion of riboflavin to the extent of 30 μg per ml of culture solution. In the cells recovering from cycloheximide inhibition, the type of glucose metabolism differed from that in the mother strain; a respiratory quotient of 4 with the mother strain fell to 1 in the progeny. Glucose metabolism (O2uptake and CO2evolution) by resting cells was investigated, and it was concluded that inhibition of glycolysis was not significant. This was verified by comparing various dehydrogenase activities in the cell-free extract. There were essentially no differences between riboflavin-forming and nonriboflavin-forming cells. The presence of the hexose monophosphate shunt was deduced from the fact that glucose-6-phosphate dehydrogenase activity was the strongest of the various dehydrogenase activities.