Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Synthetic gibberellin synergists in elongation of shoot growth ofOryza sativa L.

Previous studies showed that 2-ethyl-3-methoxycarbonyl-1-(p-tolylcarbamoyl) isourea acts as a potent GA₃-synergist in stimulating shoot growth of rice seedlings. Studies with several structurally related compounds show that the alkoxycarbonylcarbamoyl-isourea or -isothiourea skeleton is required for biological activity. Any chemical deletion from this skeleton causes complete loss of activity. From present and previous data it seems that alkoxycarbonylcarbamoyl-isourea or -isothiourea is converted by intramolecular cyclization in the rice seedlings into the corresponding triazinone that serves as the active form.Part 9 in the series Plant Growth-regulating Activities of Isourea Derivatives and Related Compounds; for Part 8 seeReferences, Ogawa et al. (1980b)     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

Development of the Formula-Detoxification System during Adaptation to Air of Submerged Rice Seedlings

Changes in activities of enzymes and levels of antioxidant substratesinvolved in the -detoxification system in seedlings of rice (Oryza sativa L. cv. Yamabiko) inresponse to variations in the oxygen environment were studied.Activities of superoxide dismutase, ascorbate peroxidase, monodehydroascorbatereductase, dehydroascorbate reductase, glutathione reductaseand catalase, expressed either on the basis of fresh weightof shoots or relative to levels of soluble protein were muchlower in seedlings germinated under water for 6 days than inthose germinated in air for the same period of time. When submergedseedlings were exposed to air, the activities of these six enzymesincreased to or exceeded the levels in aerobically grown controlsduring 24 h of adaptation to air. Ascorbate and glutathione,which act as antioxidant substrates in the -detoxification system, were present in submergedseedlings at nearly the same levels as those found in aerobicallygrown controls. On exposure of submerged seedlings to air, thelevel of ascorbate in creased slightly, but the level of glutathioneshowed a rapid increase, reaching 7 times that in aerobicallygrown controls within 12 h of adaptation to air. Levels of allsix antioxidative enzymes and of two substrates involved inthe detoxification of the superoxide radical increased withincreases in oxygen tension in the environment. Moreover, thedevelopment of this system consisted of two steps, namely, arapid increase in the level of glutathione and a subsequentslow increase in the activities of antioxidative enzymes. ¹ Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

Distribution of rhodanese in plants

To study the distribution of rhodanese (E.C. 2.8.1.1) in plants, rhodanese activity was assayed on 13 cyanogenic and 12 non-cyanogenic species. All the species tested had the enzyme activity. This phenomenon leads to a hypothesis that the enzyme is generally distributed in plants.     

Concentration-dependent inducing activity of activin A

Summary Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3–50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development. Offprint requests to: M. Asashima