Monkey Liver Cytosol Research Articles

Properties of 130 kDa Subunit of Monkey Aldehyde Oxidase

We previously demonstrated the existence of a minor 130 kDa subunit in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis of monkey liver cytosol and expressed monkey aldehyde oxidase (AO) in Escherichia coli. In contrast, the 130 kDa subunit was not observed in rat AO. In the current study, the properties of the 130 kDa subunit were investigated from the viewpoint of species differences in the presence of the subunit and AO activity. Monkey AO with His-tag at the N- and C-terminus were expressed, and were immunoanalyzed with anti-AO and anti-His-tag antisera. The results revealed that the minor 130 kDa subunit was produced by cleavage at the N-terminal side of the 150 kDa subunit. The cleavage point was shown to be located between 188Leu and 189Pro of 150 kDa AO subunit by the Edman degradation method. The two amino acids related to the cleavage are contained in the linkage between the 2Fe-2S and FAD domains in AO of human and monkey, but not in AO of rat and mouse. As a fact, the 130 kDa subunit was observed in AO of human and monkey, but not in AO of rat and mouse, suggesting the two amino acids might be one reason of a species difference in the formation of the 130 kDa subunit. However, the existence of the 130 kDa subunit is not associated with the species differences in AO activity, because the cleavage results in the loss of 2Fe-2S cluster domain essential for exertion of AO activity.

Cloning, Expression, and Characterization of Male Cynomolgus Monkey Liver Aldehyde Oxidase

In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

Cynomolgus monkey liver aldehyde oxidase: extremely high oxidase activity and an attempt at purification

Aldehyde oxidase (EC 1.2.3.1) in monkey (Macaca fascicularis) liver was characterized. Liver cytosol exhibited extremely high benzaldehyde and phthalazine oxidase activities based on aldehyde oxidase, compared with those of rabbits, rats, mice and guinea pigs. Monkey liver aldehyde oxidase showed broad substrate specificity distinct from that of the enzyme from other mammals. Purified aldehyde oxidase from monkey liver cytosol showed two major bands and two minor bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These bands were also observed in Western blotting analysis using anti-rat aldehyde oxidase. The molecular mass of the enzyme was estimated to be 130-151 kDa by SDS-PAGE, and to be about 285 kDa by HPLC gel filtration. The results suggest that isoforms of aldehyde oxidase exist in monkey livers.

Monkey liver indanol dehydrogenase. Purification, properties, and kinetic mechanism.

Indanol dehydrogenase was purified to apparent homogeneity from monkey liver cytosol. The enzyme was a monomer with a molecular weight of 36,000 and pI of 8.7. The amino acid composition was determined. The enzyme oxidized alicyclic alcohols including transdihydrodiols of benzene and naphthalene in the presence of both NADP+ and NAD+, and reduced several xenobiotic carbonyl compounds in the presence of NADPH, the 4-pro-R hydrogen atom of which was transferred to the substrate. The results of fluorometric binding and kinetic studies are consistent with an ordered sequential mechanism with NADP+ binding first. The enzyme was inhibited competitively versus NADP+ and uncompetitively versus 1-indanol not only by chelating agents such as 1,10-phenanthroline and 2,2'-bipyridine but also by a nonchelating isomer, 4,4'-bipyridine, which suggests hydrophobic interaction of the aromatic compounds with the enzyme, which did not contain zinc. The enzyme was also inhibited by Cibacron blue dye, synthetic estrogens, and delta 4-3-ketosteroids. The inhibition by Cibacron blue was competitive versus NADP+ and noncompetitive versus 1-indanol, whereas those by hexestrol, medroxyprogesterone acetate, and progesterone were uncompetitive versus NADP+ and competitive versus 1-indanol, corraborating the ordered addition of the coenzyme prior to 1-indanol.

Purification and properties of multiple forms of dihydrodiol dehydrogenase from monkey liver.

Two major and two minor forms of dihydrodiol dehydrogenase with similar molecular weights of around 36000 were purified from monkey liver cytosol. All the forms oxidized trans-dihydrodiols of benzene and naphthalene and reduced aromatic aldehydes, but showed differences in charge, specificity for other substrates and inhibitor sensitivity. One major (pI 8.7) and one minor (pI 7.9) form of the enzyme exhibited high activity for alicyclic alcohols and sensitivity to o-phenanthroline. The other major form (pI 6.2) oxidized 3 alpha-hydroxysteroids and was inhibited by dexamethasone and indomethacin, whereas the other minor form (pI 5.8) showed high reductase activity for aldehydes including D-glucuronate and sensitivity to barbital and sorbinil, and cross-reacted with human aldehyde reductase. The results indicate that the multiple forms of monkey liver dihydrodiol dehydrogenase are indanol dehydrogenases, 3 alpha-hydroxysteroid dehydrogenase and aldehyde reductase.

Microsomal and cytosolic epoxide hydrolases in rhesus monkey liver, and in normal and neoplastic human liver

The cytosolic epoxide hydrolase (EH-LC) was observed in rhesus monkey liver cytosol, and in both normal and neoplastic human liver. Microsomal epoxide hydrolase (EH-LM) was detected not only in the microsomes of normal and neoplastic human liver and normal rhesus monkey liver, but also in the cytosol of these tissues. No apparent differences were observed between the EH-LM in liver cytosol and that in microsomes. No major differences were observed between the levels of EH-LM in the cytosol of normal and that in neoplastic human liver.

Female and male green monkey liver estrogen receptor

Adult female and male green monkey liver cytosol contains tritiated 17β-estradiol ([ 3H]E 2) binding sites. The binding of [ 3H]E 2 to these sites is reduced substantially by diethylstilbestrol. Unlike adult male rate liver cytosol, neither male nor female green monkey liver cytosol contains moderate affinity high capacity [ 3H]E 2 binding sites. The green monkey liver cytosol [ 3H]E 2 sites are precipitated by ammonium sulfate at 30% of saturation. The properties of the redissolved ammonium sulfate precipitated female and male [ 3H]E 2 binding sites are similar: they have a high [ 3H]E 2 affinity, a low [ 3H]E 2 capacity, are estrogen specific, and are sensitive to sulfhydryl inactivation and protease digestion. Although the female and male sites are similar, there appears to be a 2- to 3-fold sex difference in the affinity (female K d , 0.8 to 1.3 × 10 −10 M; male K d , 0.4 × 10 −10M) and capacity (female, 2.3 to 3.7 fmoles/mg of liver; male, 1.2 fmoles/mg of liver)of the [ 3H]E 2 binding sites. Accordingly, adult female and male green monkey liver cytosol contains putative estradiol receptors.