Predominant localization of dihydroxyacetone-phosphate acyltransferase activity in renal peroxisomes of male and female mice

Comp. Biochem. Physiol. Vol. 80B, No. 1, pp. 161-164, 1985

0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd

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P R E D O M I N A N T LOCALIZATION OF DIHYDROXYACETONEPHOSPHATE ACYLTRANSFERASE ACTIVITY IN RENAL PEROXISOMES OF MALE A N D FEMALE MICE MICHAEL I. MACKNESS* and MARTIN J. CONNOCK Department of Biological Sciences, The Polytechnic, Wolverhampton WVl 1LY, UK (Received 12 March 1984)

Aktraet--1. The subcellular localization of DHAPAT activity in male and female albino mouse kidneys was investigated by density gradient centrifugation. 2. DHAPAT has a predominantly peroxisomal distribution in both male and female kidneys; however some activity is also distributed in a less dense region of the gradient, predominantly containing microsomes. 3. Peroxisomal fractions also contain some lactate dehydrogenase activity and approximately 9% of the cellular NAD-dependent ~-glycerophosphate dehydrogenase activity.

INTRODUCTION

fatty acid free bovine serum albumin from Sigma Chemical Co. Ltd.; nycodenz from Nyegaard and Co., Oslo and fluorochemical FC-43 from 3M Chemical Co., Bracknell, England. All other reagents were of analytical grade.

Acyl-dihydroxyacetone phosphate (Acyl-DHAP) has been shown to be an important intermediate in the biosynthesis of glycerolipids containing ester and ether bonds (Hajra and Bishop, 1982; Hajra and Burke, 1978; Mannering and Brindley, 1972). AcylD H A P is biosynthesized by the enzymic acylation of D H A P with acyl-CoA (Hajra and Bishop, 1982; Hajra et al., 1979). The enzyme responsible, acyl coenzyme A: dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42), appears to be localized in the mitochondrial, peroxisomal and microsomal fractions of rat liver homogenatcs (Bates and Saggerson, 1979). However, at least some of the microsomal activity may be due to glycerol-3-phosphate acyitransferase as appears to bc the case in fetal and postnatal rat liver and prcadipocytes (Coleman and Haynes, 1983; Coleman and Bell, 1980). More detailed investigations into the subcellular localization of DHAP-acyltransferase (DHAPAT) suggest that the enzyme is primarily peroxisomal in rat (Hajra et al., 1979) and guinea-pig liver (Jones and Hajra, 1977, 1980). Activity of this enzyme is widely distributed in mammalian tissues but the subcellular localization has only been studied in liver and more recently brain (Hajra and Bishop, 1982). The present study was undertaken to investigate the subcellular distribution of D H A P A T activity in mouse kidney using the convenient assay developed by Bates and Saggerson (1979) which previously has only been applied to samples with complex mixtures of organelles. MATERIALS AND METHODS

Tissue enzyme activities

Mouse liver and kidney homogenates were prepared as previously described (Silcox et al., 1983) and used to assay for DHAPAT activity and protein. Subcellular fractionations (a) Dual centrifugation procedure. Preparation of kidney

post-nuclear supcrnatant (PNS) from kidneys of eight female albino mice, treatment of PNS with 100mM pyrophosphate (pH 8.2 in 10% sucrose) and rate and density dependent banding in gradients in a BXIV zonal rotor were performed as previously described (Silcox et al., 1982), except that the gradient for density-dependent separation comprised 5ml each of 19, 21, 25, 29, 33, 37, 41 and 45% (w/w) and 10ml each of 49 and 53% (w/w) nycodenz (pH 7.4), resting on a cushion of fluorochemical FC-43 (density = !.9 g/ml), which was also used to unload the rotor. (b) Single centrifugation procedure. Subeellular fractionation of PNS was also carried out in a single step procedure. Twenty-eight millilitres of pyrophosphate treated PNS (from the kidneys of ten male albino mice) was loaded, followed by an overlay of 7% sucrose into a BXIV zonal rotor, onto a 555 ml sucrose gradient comprising 30 ml of 15% (w/w) sucrose, 50ml each of 17, 19, 21, 23, 25, 27, 29, 31 and 33% (w/w) sucrose, 5ml each of 34, 36.5, 39, 41.5, 44, 46.5, 49, 51.5, 54, 56.5 and 60% (w/w) sucrose and 20 ml of 63% lying on a cushion of fluorochemical FC-43. Centrifugation was at 25,000 rpm for 2.5 hr, FC-43 was used to unload the gradients. The 34-60% portion of the gradient was fractionated into 5 ml fractions, the lower density portion of the gradient into larger fractions.

Materials

Assays

[U ~4C]fructose 1,6, diphosphate was obtained from Amersham International pie; aldolase (Cat. No. A7145), triosephosphate isomerase (Cat. No. T6258); palmitoyl-CoA and

Assays were performed according to the methods described in the accompanying references: non-specific esterase, acid and alkaline phosphatase, catalase, o-amino acid oxidase and succinic dehydrogenase (Small et al., 1981), sulphite cytochrome-c-reductase 0Vattiaux-de Coninck and Wattiaux, 1971), lactate dehydrogenase (McGroarty et al., 1974), glutamate dehydrogenase (Leighton et al., 1968), NAD-dependent ~-glycerophosphate dehydrogenase using

*Present address: Department of Physiology & Biochemistry, University of Reading, Whiteknights, Reading RG6 2AJ, UK. (Tel: (0734) 875123)

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MICHAEL I. MACKNESSand MARTINJ. CONNOCK

DHAP as substrate (Gee et al., 1974) and protein (Bradford, 1976). Dihydroxyacetone phosphate acyltransferase (DHAPAT) activity was measured using the method of Bates and Saggerson (1979). Twenty-five microlitres of enzyme source was used in a total volume of 500/~1. Assays were conducted at pH 5.5 and pH 7.4. The former pH was chosen because it appears to be the optimal pH of guinea-pig liver peroxisomal DHAPAT (Jones and Hajra, 1980), while the second pH was chosen as this is the pH at which the enzyme has been assayed in most subcellular localization studies. Presentation of fractionation results

The distribution of enzymes between fractions is given in the form of frequency histograms according to de Duve (1967). RESULTS AND DISCUSSION Table 1 shows D H A P A T activity in male mouse liver and kidney homogenates. It can be seen that kidney activity is slightly higher than liver at both pH 5.5 and at pH 7.4, while the activity in both organs is 23~o higher at the lower pH. The D H A P A T activity in mouse kidney is sufficient to investigate the subcellular localization of this enzyme. The subcellular markers used in this study were: catalase and D-amino acid oxidase for peroxisomes, succinic dehydrogenase, glutamate dehydrogenase and sulphite cytochrome-c-reductase for mitochondria, acid phosphatase for lysosomes, nonspecific esterase for endoplasmic reticulum, alkaline phosphatase for brush borders, lactate dehydrogenase and NAD-dependent ~-glycerophosphate dehydrogenase for the cytosol. Figure 1 shows the distribution of enzymes after density-dependent banding on a nycodenz gradient of a peroxisome-enriched sample prepared by ratedependent banding of PNS from kidneys of female mice (enzyme distributions and recoveries as given by Silcox et aL, 1982). A good separation of peroxisomes (markers catalase and o-amino acid oxidase) from other organelles is achieved on this nycodenz gradient, however some lactate dehydrogenase (a cytosolic marker) appears to be present in the peroxisomes as has been previously reported for rat kidney (McGroarty et aL, 1974). Peroxisomes (catalase) are purified ~33 times relative to the PNS. Lysosomes are better separated from peroxisomes on this nycodenz gradient than they are on sucrose gradients (Silcox et al., 1982, 1983; Small et aL, 1980). D H A P A T shows a clearly predominantly peroxisomal distribution in this gradient. D H A P A T activity has been previously attributed to the endoplasmic reticulum, mitochondria and peroxisomes (Bates and Saggerson, 1979; Coleman and Haynes, 1983; Jones and Hajra, 1977, 1980), therefore in order to examine the distribution in a sample Table 1. DHAPAT activity in male mouse liver and kidney homogenates. Enzyme activities are given as the mean+standard deviation of 10 samples Tissue Kidney Liver

DHAPAT specificactivity nmoles acyI-DHAPformed/min/mgprotein pH 5.5 pH 7.4 1.003 + 0.158 0.714 + 0.068 0.711 +0.155 0.519 ± 0.096

of whole PNS, a single step fractionation procedure was adopted which gave a reasonable separation of the bulk of these three organelles from each other (Fig. 2). It is clear from this experiment that the peroxisomes have NAD-dependent ~-glycerophosphate dehydrogenase activity. This and other experiments indicate that ~9~/o of the NADdependent ~t-glycerophosphate dehydrogenase activity is peroxisomal in mouse kidney. This result is consistent with the report of Gee et al. (1974) and Gee and Tolbert (1982) for rat kidney. We presume that as in other mammalian peroxisomes the enzyme favours the conversion of D H A P to ~-glycerophosphate. This enzyme has been suggested to function as part of a redox shuttle for the reoxidation of N A D H generated by peroxisomal//-oxidation. Peroxisomal fl-oxidation is very active in mouse kidney (Silcox et al., 1982). Thus a competition between fatty acid oxidation and glycerolipid synthesis is likely both for acyl-CoA and DHAP. D H A P A T activity is again localised predominantly in the peroxisomes (Fig. 2). However, some activity is associated with low density slow moving particles. This proportion represented 34%o of the recovered activity at pH 7.4 and 24~ of that recovered when the

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Fig. I. Density-dependent banding on nycodenz o f peroxisome enriched fractions prepared by rate-dependent banding o f female kidney PNS, Experimental conditions are

described in Materials and Methods. Enzyme recoveries from this gradient are as follows: Catalase 123%, succinic dehydrogenase 60~, glutamate dehydrogenase 97%, D-amino acid oxidase 45~, lactate dehydrogenase 92o,0, acid phosphatase 107~o, alkaline phosphatase 90%, esterase 114~, protein 106~o, DHAPAT 61°~,.

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Fig. 2. Single step fractionation of male kidney PNS on a sucrose gradient. Experimental conditions are described in Materials and Methods. Enzyme recoveries from this gradient are as follows: Catalase 103%, p-amino acid oxidase 62~, succinic dehydrogenase 111~, sulphate cytochrome-c-reductase 90~, esterase 83~, e glycerophosphate dehydrogenase 90~, lactate dehydrogenase 60~, alkaline phosphatase 80~, acid phosphatase 63~, protein 82~, DHAPAT at pH 5.5, 37~, DHAPAT at pH 7.4, 45~.

enzyme was assayed at pH 5.5. This activity, however did not have a distribution corresponding to the endoplasmic reticulum marker (esterase). No DHAPAT activity in this gradient could be attributed to mitochondria. Mitochondria of greater purity, prepared by rate sedimentation (Fig. 1 of Silcox et al., 1982) lacked detectable D H A P A T activity. Because of the relatively low recoveries of D H A P A T activity from our gradients we cannot rule out the possibility of some endoplasmic reticulum activity. However, if there is such activity it seems to be undetectable with this assay in microsomal fractions that contain relatively small amounts of other organelles. The low recoveries we observed for D H A P A T appear to be due in part to the effect of pyrophosphate treatment. Pyrophosphate was found to activate the enzyme in PNS samples but is left behind in the sample layer on sedimentation of subcellular particles during centrifugation. From our results we can conclude that in kidneys of both male and female mice, D H A P A T is predominantly a peroxisomal enzyme, as it also appears to be in mammalian liver (Hajra et aL, 1979; Jones and Hajra, 1977, 1980). Acknowledgement--This work was supported by a National

Kidney Research Fund Postdoctoral Fellowship.

REFERENCES

Bates E. J. and Saggerson E. D. (1979) A study of the glycerol phosphate acyltransferase and dihydroxyaeetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Biocbem. J. 182, 751-762. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biocbem. 72, 248-254. Coleman R. A. and Bell R. M. 0980) Selective changes in enzymes of the sn-glycerol 3-phosphate and dihydroxyaeetone-phosphate pathways of triacylglycerol biosynthesis during differentiation of 3T3-L1 preadipocytes. J. biol. Chem. 255, 7681-7687. Coleman R. A. and Haynes E. B. (1983) Selective changes in microsomal enzymes of triacylglycerol and phosphatidylcholine synthesis in fetal and postnatal rat liver. J. biol. Chem. 258, 450--456. de Duve C. (1967) In Enzyme Cytology (edited by Roodyn D. B.), pp. 1-26. Academic Press, New York. Gee R., McGroarty E., Hsieh B., Wied D. M. and Tolbert N. E. (1974). Glycerol phosphate dehydrogenase in mammalian peroxisomes. Archs Biochem. Biophys. 161, 187-193. Gee R. and Tolbert N. E. (1982) Glycerol phosphate dehydrogenase in rat and mouse liver peroxisomes. Ann. N.Y. Acad. Sci. 386, 417-419.

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M1CHAEL I. MACKNESSand MARTIN J. CONNOCK

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Mannering R. and Brindley D. N. (1972) Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyaeetone phosphate pathways of glycerolipid biosynthesis in rat liver. Biochem. J. 139, 1003-1012. Silcox A., Burdett K, and Connock M. J. (1983) Reduced levels of peroxisomal enzymes in the kidney of the genetically obese (ob/ob) mouse. Contrast with liver. Biochem. Int. 7, 273 280. Silcox A., Small G. M., Burdett K. and Connock M. J. (1982) Detection of carnitine acyltransferases and acylCoA fl-oxidation enzymes in renal peroxisomes of normal and clofibrate treated mice. Biochem. Int. 5, 359-366. Small G. M., Brolly D. and Connock M. J. (1980) Palmitoyl-CoA oxidase: detection in several guinea pig tissues and peroxisomal localisation in mucosa of small intestine. Life Sci. 27, 1743-1751. Small G. M., Hocking T. J., Sturdee A. P., Burdett K. and Connock M. J. (1981) Enhancement by dietary clofibrate of peroxisomal palmitoyl-CoA oxidase in kidney and small intestine of albino mice and liver of genetically lean and obese mice. Life Sci. 28, 1875-1882. Wattiaux-De Coninck S. and Wattiaux R. (1971) Subcellular distribution of sulfite cytochrome c reductase in rat liver tissue. Eur. J. Biochem. 19, 552-556.