Characterization of a renal medium chain acyl–CoA synthetase responsible for glycine conjugation in mouse kidney mitochondria

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Abstract

Glycine conjugation of a series of benzoic acid derivatives was investigated in mouse kidney mitochondria. The chlorine and methyl substitutions in the para- and meta-positions of the benzene ring yielded an increase in glycine conjugation. The acids with a methoxy group showed a low degree of glycine conjugation. In addition, the acids with nitro or amino groups were conjugated to a slight extent with glycine. The in vitro conjugation of salicylic acid with glycine occurred not in liver but in kidney. The specificity of the renal medium chain acyl–CoA synthetase catalyzing the first reaction of glycine conjugation was also examined. The enzyme accepted not only medium chain fatty acids but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with alkyl and alkoxyl groups in the para- and meta-positions of the benzene ring. An ortho-substituted acid exhibited no activity. In addition, the enzyme was less active with valproic acid, tranexamic acid, indomethacin and ketoprofen. The enzyme was inhibited by diflunisal, 2-hydroxydodecanoic acid and salicylic acid, which did not act as substrates. There was a poor correlation between the activity of the medium chain acyl–CoA synthetase and glycine conjugation of eleven substituted benzoic acids. These findings suggest that the present medium chain acyl–CoA synthetase is involved in glycine conjugation of the substituted acids in mouse kidney mitochondria, but there may be a larger contribution of another isoenzyme.

Introduction

Amino acid conjugation is one of the most important routes for the detoxification, not only of many xenobiotic carboxylic acids but also of endogenous acids. It is restricted to small carboxylic acids such as aliphatic, aromatic, heteroaromatic, arylacetic and aryloxyacetic acids, and varies with the animal species and the structure of the carboxylic acids [1], [2], [3], [4]. Conjugation with glycine is the most common for xenobiotic aromatic acids in a wide range of species. Glutamine conjugation is restricted to arylacetic acids such as phenylacetic, naphthylacetic and indolylacetic acids in man and other primate species. Taurine conjugation occurs with arylacetic and aryloxyacetic acids at random but particularly evident in carnivores. As a first step, we have been investigating glycine conjugation which is observed frequently in the number of animal species in order to elucidate the factors that regulate amino acid conjugation.

Besides xenobiotic carboxylic acids, several endogenous branched medium chain fatty acids are specifically subjected to glycine conjugation in subjects with medium chain acyl–CoA dehydrogenase [5]. Glycine conjugation also provides a pathway for the disposal of ammonia which accumulates in patients with genetic defects in the urea cycle [6].

The liver is the main site of glycine conjugation in the body. However, the kidney is also an important site for glycine conjugation. The relative importance of the liver and kidney for glycine conjugation varies with the species and substrates. In man, 68% of the glycine conjugation of salicylic acid was renal and 32% hepatic [7]. In rhesus monkeys, the conversion of salicylic acid to salicyluric acid occurs exclusively in the kidney [8]. In rabbit, 54% of the glycine conjugation of benzoic acid is carried out by the liver, with 46% in the kidney [9], whereas for p-aminobenzoic acid the kidney accounts for 70% of total glycine conjugation [10]. Although the mass of kidney is considerably smaller as compared with that of liver, the kidney is quantitatively more important than the liver for salicylic acid and p-aminobenzoic acid.

The pathway of glycine conjugation consists of two sequential reactions that involve the activation of the acid with subsequent linkage to the amino acid at the amino functional group [11]. The latter is known to occur in two distinct forms that have been purified to high homogeneity and extensively characterized [12], [13], [14], [15], [16]. However, little information is available about the medium chain acyl–CoA synthetases catalyzing the first reaction of glycine conjugation. Although glycine conjugation has been known as a biotransformation reaction for a long time, at least three medium chain acyl–CoA synthetases have been partially purified from liver mitochondria [17], [18], [19], [20]. Recently, we have purified one medium chain acyl–CoA synthetase from bovine [21], [22] and mouse liver mitochondria (F. Kasuya, K. Igarashi, M. Fukui, manuscript submitted for publication) to high homogeneity and characterized. However, no attempt has been made to separate and characterize the medium chain acyl–CoA synthetase from the kidney.

We purified and characterized the medium chain acyl–CoA synthetase from mouse kidney mitochondria. In addition, we investigated the glycine conjugating activity for the substituted benzoic acids in mouse kidney mitochondria and the extent to which the medium chain acyl–CoA synthetase is involved in their glycine conjugation.

Section snippets

Materials

Sephadex G-25 and DEAE-Sepharose were purchased from Pharmacia (Piscataway, USA); hydroxylapatite was obtained from Nacalai Tesque (Kyoto, Japan), and Reactive-Green 19 was from the Sigma Chemical Co. CoA, NADH and ATP were obtained from Kohjin Co. Ltd. (Tokyo, Japan), pyruvate kinase, lactate dehydrogenase, myokinase and phosphoenolpyruvate were from Oriental Yeast Co. Ltd. (Tokyo, Japan). Other chemicals used were analytical grade and were used as received.

Purification of a medium chain acyl–CoA synthetase

The medium chain acyl–CoA synthetase

Results

The medium chain acyl–CoA synthetase from mouse kidney mitochondria was purified by using the same method as with the liver. The addition of 10–20% glycerol stabilized the enzyme. The active fractions from a Reactive-Green 19 column were analyzed by SDS-PAGE. The protein bands were visualized by silver staining, corresponding to one major band of 65 kDa and one very weak band of 39 kDa. Enzymatic activity was not observed in the band of 39kDa as described previously [21]. Therefore, active

Discussion

In the present study, a medium chain acyl–CoA synthetase has been purified 180-fold from mouse kidney mitochondria. The purification procedure was the same as we previously used for the purification of medium chain acyl–CoA synthetase from bovine liver [21]. The purified kidney enzyme was similar to a medium chain acyl–CoA synthetase purified 12-fold from bovine liver by Mahler et al. [17]. However, it did not utilize salicylic acid as a substrate. A separate acyl–CoA synthetase that uses

Acknowledgements

This work was supported by the Grant-in-Aid for Scientific research and by Grant for research from Kobegakuin University.

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