Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Endothelin-3 stimulates the hypothalamic-pituitary-adrenal axis.

Endothelin-3 (ET-3) is a member of the novel vasoconstrictive peptide family, identified in porcine central nervous system. Intravenous bolus injection of 1000 pmol/kg of ET-3 in freely moving rats caused significant increases in plasma ACTH and corticosterone levels, almost equivalent to those of 100 pmol/kg of rat corticotropin-releasing hormone (rCRH). The action of ET-3 was virtually abolished by pretreatment of CRH-antagonist, alpha-helical CRH. When ET-3 was added to cultured anterior pituitary cells, neither direct stimulation of ACTH release nor potentiation of rCRH action was noted. The results indicate that ET-3 may function as a neuropeptide and stimulation of the CRH-neurons, direct or inderect, is mainly responsible for activation of ACTH and corticosterone release.     

Distribution and characterization of diglycosyldiacylglycerol isomers with different anomeric configuration in higher plants.

The novel diglycosyldiacylglycerol (DGDG), galactosyl(beta 1----6)-galactosyl(beta 1----3')-diacylglycerol (B isomer), present in Adzuki beans was found to be distributed together with the well-known galactosyl(alpha 1----6)-galactosyl(beta 1----3')-diacylglycerol (A isomer), in all (10) of the higher plants examined. The highest levels were found in leguminous seeds were the amounts were always less than 33% of the total DGDG of mature seeds. The highest proportion of the B isomer was found in Adzuki bean seed DGDG (26-33%), with the lowest in pea seed DGDG (2%). The amounts of the B isomer in DGDG of Adzuki and kidney beans cotyledons were almost equal to those in mature seeds. Immature seeds and hypocotyls of three kinds of beans also contained the B isomer in small amounts compared with the mature beans, while only trace amounts of the isomer was found in other organs such as leaves, stems, pods, roots and generative organs of plants, except for root from kidney beans. The molecular species composition of the principal diacylglycerol moieties in the A and B isomers of DGDG were found to be significantly different among several plant seeds, although the component diacylglycerol species were qualitatively similar to each other.     

Manganese, copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle

To clarify trace element distribution in red and white muscle, and to verify two populations of muscle mitochondria, the iron, zinc, copper, and manganese concentrations of whole muscle and their subcellular fractions were determined. The iron, zinc, copper, and manganese concentrations of red muscle were 1.83, 4.31, 2.05, and 1.67 times higher than those of white muscle, respectively. In skeletal muscle subcellular distribution or iron, zinc, and copper were entirely different and that of manganese was relatively similar as compared with those in liver reported previously. The pattern of mineral distribution in all fractions of red muscle was similar to that of white muscle, but their concentrations in some fractions were different between red and white muscle, e.g., iron, zinc, and manganese in supernatant fraction and copper in nuclear and microsomal fractions. The difference between subsarcolemmal and interfibrillar mitochondria were ascertained by the distribution of trace elements.     

Preparation of peptide mixture with high Fischer ratio from protein hydrolysate by adsorption on activated carbon.

A peptide mixture with a high Fischer ratio (a molar ratio of Val + Leu + Ile to Phe + Tyr) was prepared by the adsorptive separation of a casein hydrolysate by activated carbon. The effects of the pH and ethanol content of the hydrolysate on the Fischer ratio and on the yield of the resulting peptide mixture were examined. A peptide mixture with the Fischer ratio of 31.6 was obtained at pH 2.5 without the addition of ethanol. The Fischer ratio was close to the ratio of the infusion solution of free amino acids that is now used for patients with liver diseases.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.