Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Fertilization Potential of the Medaka Egg

Fertilization of the medaka egg in 10% Ringer's solution generates a depolarization of 4 mV just before the appearance of a characteristically longer hyperpolarization (25). The depolarization appears to be the result of a nonspecific leak triggered by sperm stimulation and the amplitude of the depolarization is thought to be independent of [Ca²⁺]_o (25). We have investigated the ionic dependence of this depolarization. An initial small depolarization (3–4 mV; duration, 5–8 sec) is followed by a rising phase of a spike-like depolarization in the range of 10–60 mV. The amplitude of this spike-like depolarization is propodional to log [Ca²⁺], ranging from 0.33–18 mM. Calcium antagonists, e.g. 10 mM cobalt or 10 μg/ml verapamil in 10% Ringer do not block the depolarization in the presence of 1.1 mM CaCl₂.     

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.     

Purification to homogeneity of aromatase from human placenta

Aromatase cytochrome P-450 has been purified from human placenta to homogeneity, as demonstrated by electrophoresis on polyacrylamide gels with SDS, and by double diffusion against an antibody raised in rabbits. The enzyme converts androstenedione to estrone (Vmax 13.3 n moles/min/n mole P-450; Km 30 microM) and testosterone to estradiol. Aromatase activity requires P-450, P-450 reductase and NADPH. Enzyme activity is inhibited by anti-aromatase antibodies and by 4-hydroxyandrostenedione. The enzyme shows a molecular weight of 55,000, is extremely unstable and spontaneously forms P-420 with a half-life of 2.5 days.     

Reverse Changes in Plasma Membrane Properties upon Deacclimation of Mulberry Trees (Morus bombysis Koidz.)

To gain more insight into the relation between plasma membranechanges and cold hardiness in mulberry trees (Morus bombysisKoidz. cv Goroji), biochemical and biophysical changes in theplasma membrane were studied during cold deacclimation in spring.The majority of the changes in the plasma membranes that occurredduring the cold acclimation process in the fall/winter werereversed following deacclimation in the spring. Significantdecreases in phospholipid content, degree of unsaturation inphospholipid fatty acids, and membrane fluidity were observedin the plasma membranes during cold deacclimation. The sterolto phospholipid ratio increased with decreasing cold hardiness.Reverse changes were also detected in the majority of proteinand glycoprotein components. These reversible changes in theplasma membranes are considered to be involved in the mechanismof cold hardiness of plants. ¹Contribution No. 2766 from the Institute of Low TemperatureScience. (Received July 10, 1985; Accepted October 25, 1985)     

Cytochrome b5 promotes the synthesis of delta 16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis

Conversion of progesterone to 17 alpha-hydroxyprogesterone plus androstenedione (17 alpha-hydroxylation) and to androstadienone (delta 16 synthetase activity) by microsomes from neonatal pig testis, were both inhibited by antibodies raised against homogeneous cytochrome P-450 C21 side-chain cleavage. Inhibition of the two activities showed the same relationship to the concentration of antibody added. Analogous results were obtained with pregnenolone as substrate. In a reconstituted enzyme system consisting of the homogeneous cytochrome P-450 C21 side-chain cleavage enzyme, P-450 reductase and NADPH, addition of cytochrome b5 resulted in the synthesis of the corresponding delta 16-C19-steroid from progesterone (androstadienone) and pregnenolone (androstadienol). The effect of cytochrome b5 was concentration-dependent and prevented by anti-cytochrome b5. It is concluded that the cytochrome P-450 C21 side-chain cleavage enzyme from pig testicular microsomes is also capable of synthesizing delta 16-C19-steroids and is, therefore, likely to be responsible for the large amounts of the pherormone androstadienone produced by male pigs.     

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.