Adenylate cyclase from Coprinus macrorhizus was found in the particulate fractions containing plasma membrane, microsomes, and mitochondria. Glucose addition lead to inhibition of adenosine 3′:5′-cyclic monophosphate accumulation and fruiting body formation in vivo, but glucose did not inhibit the adenylate cyclase activity in vitro. The enzyme was solubilized by treatment with detergents or KCl. The localization of adenylate cyclase did not change in the course of fruiting body formation under various light conditions.
The tocopherol content of the yeast, Rhodotorula glutinis, was determined by spectrofluorometry and rotifer bioassay. Comparative tocopherol measurements were made on the mushroom, Agaricus campestris, and the alga, Euglena gracilis, strain Z. R. glutinis, previously reported to have a tocopherol content of <4.75μg/g dry weight, was shown to have <0.001μg/g dry weight tocopherol by the spectrofluorometric technique. The rotifer bioassay showed that there was <1.28pg tocopherol/g dry weight yeast. The tocopherol values for A. campestris and E. gracilis, strain Z, were 0.31-0.34 and 797μg/g dry weight, respectively, as determined by spectrofluorometry. These values are somewhat lower than previously reported determinations, possibly because of the greater specificity of the spectrofluorometric method. A slightly modified spectrofluorometric procedure for the analysis of tocopherol is presented; homogenization of tissue samples, lyophilization, lipid extraction with acetone, and one purification step precede spectrofluorometric determination. Use of a cold acetone fractional crystallization procedure for lipid purification removed 99.27% of the originally extracted lipid material. The average recovery of introduced tocopherol was 97.6%, indicating only minor loss of d-α-tocopherol during the extraction process. Along with rigorous solvent and glassware preparation, this technique has increased the sensitivity of the spectrofluorometric assay from the previously reported 0.01 to 0.005μg/ml tocopherol. Specificity of tocopherol fluorescence was determined by introducing known amounts of tocopherol into samples, examination of excitation and fluorescent spectra, and adding FeCl3.
Efflux transport reaction for 3-ketoglucose 1-phosphate (3KG-1-P) was investigated with a strain of Agrobacterium tumefaciens M-24 which is a mutant unable to metabolize 3KG-1-P. The efflux transport might be concluded to consist of two systems; a shaking-activated system and an influx transport-dependent system. As one of efflux transport systems, a novel system, which is revealed by physical treatment of shaking, was proved and designated as "shaking-activated" efflux transport system. The transport system was temperature-dependent and required an activation energy of 4, 600 cal/mol. Potassium cyanide and 2, 4-dinitrophenol did not show any inhibitory effect on the efflux reaction. Therefore, this transport might be energy-independent. From the observations obtained here, mechanism of the shaking-activated efflux reaction was discussed. As another efflux transport system, a system requiring a transport reaction of 3KG-1-P infflux, which is energy-dependent, was suggested.
The bacterium strain No. 12 isolated from a phenol-adapted activated sludge as a floc-forming bacterium was found to produce an exoenzyme which could deflocculate its own floc. As the floc of this bacterium was not deflocculated with EDTA and various enzymes such as cellulase, protease, and lysozyme, this exoenzyme was isolated and purified. The optimal pH of the enzyme for deflocculation was 3.8 to 5.0, and the optimal temperature 30°. The enzyme was unstable at a high temperature, and heating at 45° for 5min resulted in complete loss of the activity. The activity of the enzyme was inhibited by a relatively low concentration of salts, acidic and basic amino acids, uronic acids, and amino sugars. Purified preparation of this enzyme released sugars from the No. 12 floc during deflocculation, though it could not hydrolyze cellulose, pectin, and ethylene glycol chitin. These results indicate that the No. 12 floc is composed of some exopolysaccharides other than those so far reported and deflocculated only by the enzyme produced by the bacterium itself.
Numerous microbodies developed at the logarithmic phase of growth in cells of Candida tropicalis, when cultures were grown in a medium containing normal alkanes. From the homogenate of protoplasts prepared from 16-hr cells of Candida, biologically active microbodies were isolated by discontinuous sucrose density gradient centrifugation. The fractions recovered in the middle and lower-density regions of the gradient were associated with substantially high activities of three peroxisomal marker enzymes, catalase, D-amino acid oxidase, and urate oxidase, while a mitochondrial marker enzyme, cytochrome oxidase, was only slightly detected. Electron microscopic studies revealed that these fractions were largely comprised of morphologically well-preserved microbodies with a smaller number of more or less degraded organelles, but were virtually devoid of mitochondria. The microbody fractions contained an appreciable amount of DNA.