Masamichi Kusunose

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.

Structural analysis of molecular species of nocardomycolic acids fromNocardia erythropolis by the combined system of gas chromatography and mass spectrometry

Mycolic acids are high molecular fl-hydroxy fatty acids with long-chain branches in the o-position, which occur abundantly in certain groups of bacteria such as Mycobacteria [ 1, 2,3], Corynebacteria [4,5] and Nocardia [6,7,8] . The common structures of these acids have been well investigated. However, owing to the instability of mycolic acids at high temperature and the complexity of their homologues, gas chromatographic analysis of the individual acids has not yet been established. Recently, we have succeeded in separating the individual molecular species of the mycolic acids from Nocardia by the combination of gas chromatography and mass spectrometry as their trimethylsilyl derivatives. The present paper describes that the homologues of nocardomycolic acids from Nocardia erythropolis grown on a high glucose medium [9, lo] can be separated into 15 major acids according to their carbon numbers, which comprise saturated acids ran.l:;Ig from C,, to Ca6 with a smaller amounts of mon c 2s. In al! cases, the branches at the o-position are C, to C,,.

Purification and NH2-terminal amino acid sequences of human and rat kidney fatty acid ω-hydroxylases

A cytochrome P-450 (P-450), designated P-450HK omega, has been isolated and purified from human kidney microsomes to a specific content of 13 nmoles of P-450/mg of protein. P-450HK omega showed an apparent molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Absolute spectra of the oxidized form indicated that this P-450 was largely in the low-spin state and partly in the high-spin state. It catalyzed the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate, with no activity toward prostaglandin A1, benzphetamine, 7-ethoxycoumarin, or 7-ethoxyresorufin. The first 35 NH2-terminal amino acid sequence of P-450HK omega had about 70% homology with those of rabbit kidney fatty acid omega-hydroxylases of the P-450 IVA gene subfamily, P-450ka-1, P-450ka-2, and P-450kd, except for four undetermined residues. Moreover, Western blot and immuno-inhibition studies showed that P-450HK omega reacted with an antibody against the rabbit kidney fatty acid omega-hydroxylase. The results suggest that P-450HK omega is a member of the same P-450 gene family (IVA subfamily) as the rabbit enzymes. In addition, the terminal sequence of P-450HK omega also showed 54% homology with that of P-450k-2, a fatty acid omega-hydroxylase from rat kidney microsomes. To our knowledge, this is the first time that a P-450 specific for fatty acid omega-hydroxylase activity has been isolated to homogeneity from human tissues.

Superoxide dismutase from Mycobacterium species, strain Takeo

Superoxide dismutase from Mycobacterium species, strain Takeo, has been purified to homogeneity as judged by disc gel electrophoresis and ultracentrifugation. The enzyme was found to have a molecular weight of approximately 61 500 by sedimentation equilibrium and to contain manganese by atomic absorption and electron spin resonance spectra. The amino acid composition was also determined. The enzyme was considerably stable to the treatment with sodium dodecyl sulfate; unless incubating at 80°C for 2 min, it was not completely dissociated into the subunits. The molecular weight of the subunit was found to be approximately 21 000. Antibodies against the superoxide dismutase were produced by immunization of rabbits with the enzyme, and the γ-globulin fraction was purified. Superoxide dismutase preparations obtained from various species of mycobacteria and nocardia cross-reacted to different degrees with these antibodies on the Ouchterlony double diffusion plates. Comparative immunological studies indicated that strain Takeo might be most closely related to Myobacterium smegmatis among species of mycobacteria and nocardia tested. The antibodies against superoxide dismutase may be used as a valuable tool for the classification of mycobacteria.

2-hydroxy fatty acid-containing phospholipid of Arthrobacter simplex.

Abstract The major phospholipids of Arthrobacter simplex were separated into two types of phosphatidylglycerols and cardiolipin using thin-layer chromatography. The fast-ascending phosphatidylglycerol (phosphatidylglycerol-1) contained only nonhydroxylated fatty acids, whereas the slow-ascending phosphatidylglycerol (phosphatidylglycerol-2) contained 2-hydroxy fatty acids together with nonhydroxylated fatty acids. It was found that the principal fatty acid at the 1-position of both the phosphatidylglycerols was 10-methylstearic acid, while the fatty acids at the 2-position in phosphatidylglycerol-1 consisted largely of iso-C16, palmitic, iso-C17 and anteiso-C17 acids, and those in phosphatidylglycerol-2 were the corresponding 2-hydroxy fatty acids. As the growth proceeded from the early stage to the stationary stage, remarkable increases in 2-hydroxy fatty acids and phosphatidylglycerol-2 were observed to be concomitant with the decreases of the corresponding nonhydroxylated fatty acids and phosphatidylglycerol-1, respectively.

Occurrence of α-hydroxy fatty acids in Actinomycetales

Long-chain cr-hydroxy fatty acids are well known to predominate in cerebroside and its related lipids in animals and yeasts. In contrast, little attention had been paid to the occurrence and metabolism of o-hydroxy fatty acids in bacteria. Recently, Asselineau and co-workers [ 1,2] found that a-hydroxy anteisopentadecanoic acid is present in the state of free fatty acid in StreptomJces. Subsequently, Kawanami and co-workers [3,4] reported that phosphatidylethanolamine from Streptomyces sioyaensis contained (Yhydroxy iso-pentadecanoic acid at the /I-position of the glycerol moiety. During the course of our study on the fatty acids of Nocardia, we have also found (Yhydroxy anteiso-pentadecanoic acid to be present as the component of the phosphatidylethanolamine from Nocardia Zeishmanii [5]. This finding led us to survey the distribution of cz-hydroxy fatty acids in bacteria. On the basis of thin-layer and gas-liquid chromatographic, and mass spectrometric analysis, the present paper describes that various o-hydroxy fatty acids occur abundantly in four different species belonging or closely related to Actinomycetales.

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.

Effect of triton X‐100 and trypsin on NADPH‐cytochromeC reductase reconstitutively active in fatty acid ω‐hydroxylation

In earlier papers [1 ,2] , we reported that a fatty acid ~-hydroxylation system in porcine kidney cortex microsomes was resolved into 2 protein fractions called Fraction I and II, which contained a CO-binding hemoprotein, and NADPH-cytochrome c reductase, respectively. Fraction II was replaced by the corresponding fraction from porcine liver microsomes (liver Fraction II) or spinach ferredoxin-NADP reductase with ferredoxin. Recently, liver Fraction II has been extensively purified. The present paper describes that the purified preparation of liver Fraction II exists in the monodisperse form in the presence of an appropriate concentration of Triton X-100, and that it can be easily transformed into a reconstitutively inactive form, the molecular weight of which is similar to that of trypsinextracted NADPH-cytochrome c reductase.

Some properties of a fatty acid ω-hydroxylation system solubilized from liver microsomes

Ichikawa and Yamano [ 1,2] reported that the purified cytochrome P-450 from rabbit liver microsomes was readily reduced by NADPH, ferredoxin-NADP reductase and ferredoxin (spinach electron transport system). However, the activities of aniline hydroxylation and aminopyrine demethylation could not be restored by the reduction of the cytochrome P-450 with such spinach electron transport system. We have recently found that the w-hydroxylation system of medium-chain fatty acids in pig kidney microsomes was resolved into two protein fractions, one of which was replaced by the spinach electron transport system [3] . In the present work, this finding was extended to the fatty acid w-hydroxylation system in rat liver microsomes. In addition, it was found that the treatment of the enzyme preparations with ether resulted in a great decrease of the activity, which could be markedly restored by Triton X-100.

Metabolism of Hydrocarbons in Microorganisms:Part I. Oxidation of p -Xylene and Toluene by Cell-Free Enzyme Preparations of Pseudomonas aeruginosa

The crude extract obtained from Pseudomonas aeruginosa grown on xylene as sole carbon source converted p-xylene-methyl-14C or toluene-methyl-14C to p-toluic or benzoic acid, respectively. However, addition of p-methylbenzyl or benzyl alcohol to the reaction mixture resulted in accumulation of p-methylbenzyl or benzyl alcohol. This indicates that in the crude extract, p-xylene or toluene is metabolized via its corresponding alcohol to p-toluic or benzoic acid, respectively. The enzyme system responsible for these reactions required NAD or NADH and FAD, and could be stabilized by the presence of glutathione. When the crude extract was fractionated by the use of DEAE-cellulose chromatography, it was demonstrated that two distinct protein fractions and two cofactors (NADH and FAD) were required for the step of the hydroxylation of p-xylene or toluene to its corresponding alcohol. NAD, NADP or NADPH had very few or little activity. FMN partially replaced FAD.