Erythroid 5-aminolevulinate synthetase from trout (Salmo gairdneri R.)

Comp. Biochem. Physiol. Vol. 85B, No. 3, pp. 675-678, 1986 Printed in Great Britain

0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd

ERYTHROID 5-AMINOLEVULINATE SYNTHETASE F R O M T R O U T (SALMO GAIRDNERI R.) J. FERN,~NDEZ,* O. GONZ,~LEZ,* M. MARTiN~" and M. R u I z AMIL:~ *Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain; and ~Departamento de Bioquimica Facultad de Farmacia Universidad Complutense, 28040 Madrid, Spain (Received 13 December 1985)

Abstract--1. A mitochondrial and cytosolic erythroid ALA-synthetase have been found in trout. 2. The polypeptide existant in the cytosol is probably a precursor of the 90,000 mol. wt mitochondrial ALA-synthetase. 3. The erythroid ALA-synthetase is about 20,000 mol. wt larger than the hepatic enzyme. 4, The differences in mol. wt and catalytic properties between erythroid and hepatic enzyme support the existence of two forms of the ALA-synthetase in teleostei.

INTRODUCTION

MATERIALS AND METHODS

ALA-synthetase, the enzyme catalyzing the condensation of glycine and succinyl C o A to form A L A , is the first and rate-limiting enzyme in the heme biosynthetic pathway of animals (Granick, 1966; Granick and Sassa, 1971); this mitochondrial enzyme contains pyridoxal-5'-phosphate as a coenzyme. Previous studies revealed that synthesis of A L A synthetase in the liver is subject to feedback regulation by heme at both transcriptional and translational steps (Whiting, 1976; Srivastava and Borthwick, 1982). This would represent a regulation mechanism of heme biosynthetic pathway acting on the translocation of the enzyme into mitochondria, the site of its physiological functioning (Hayashi et al., 1983). In the trout, the enzyme has been identified in liver (Fern~mdez et al., 1985), suggesting that ALA-synthetase is present in the cytosol as a precursor of the mitochondrial enzyme. On the other hand, erythroid ALA-synthetase from mammalians and birds show a different behaviour of hepatic enzyme. Bishop et al. (1981) found two forms: erythroid and non-erythroid A L A synthetase in guinea pigs, with different mol. wt, catalytic properties and regulation. Also A L A synthetase isozymes in the liver and erythroid cells of chicken were found by Watanabe et al. (1983). Immunochemical comparison of two isozymes imply that the erythroid ALA-synthetase differs from the hepatic enzyme. In the current study, the existence of A L A synthetase in haematopoietic tissue of trout ( S a l m o gairdneri R.) is reported and optimal conditions for its assay determined. The results compared with a previous report realized in trout liver suggest also the existence of different forms of ALA-synthetase in Teleostei.

Adult rainbow trout (Salmo gairdneri R.) obtained from several fish farms, weighing 150 g were used for the assays. All biochemicals were aquired from Sigma, Boehringer Mannhein or Merck. Preparation of cytosolic and mitochondrial fraction. Erythroid mitochondrial and cytosolic ALA-synthetase used in the characterization experiments were partially purified from adult trout. Kidney (hematopoietic tissue in Teleostei) was rapidly excised, washed and homogenized with 5 times their volume of ice-cold 50 mM Tri~HC1 buffer (pH 7.5) containing 0.25M sucrose, 0.1 mM EDTA and 0.1raM pyridoxal-5'-phosphate, using a Braun Potter-S homogenizer operating at 1000 rpm (5-10 min). The mitochondria were isolated by differential centrifugation at 6000g for 15 min and 9000 g for 15 rain. The mitochondrial fraction was prepared as described in Fern~indez et al., 1985). The resulting supernatant was used as the mitochondrial fraction. The postmitochondrial supernatant was centrifuged at 105,000g for 2 hr and the resulting supernatant was used as the cytosolic fraction.

tTo whom correspondence should be addressed.

ALA-synthetase assay ALA-synthetase activity in subeellular fraction of adult kidney trout was assayed by the method of Mauzerall and Granick (1956). The reaction mixture contained the following reagents in a volume of 0.7 ml: 100 mM Tris-HC1 buffer (pH 7.5), 150 mM glycine, 0.07 mM succinyl CoA, 0.71 mM pyridoxal-5'-phosphate, 1.87 mM EDTA, 15 mM MgCI 2 and 0.5 ml enzyme extract. Mixtures were shaken in a incubator for 60 min at 15"C. Reactions were stopped by the addition of 0.2 ml of a 10% solution of trichloroacetic acid containing 0.1 M HgCI 2. In order to estimate the enzyme reaction product (ALA), this compound was converted to 2-methyl-3-acetyl-4-propionic acid pyrrole by reaction with modified Ehrlich's reagent. All assays were carried out in duplicate using zero time samples as reference. Proteins were determinated by the method of Lowry et al. (1951). Gel filtration chromatography An estimation of the mol. wt of determined using a Sephacryl S-200 (40 × 2.6 cm) equilibrated with 0.5 M 7.5). Elution was carried out with the 675

ALA-synthetase was gel filtration column Tris-HCl buffer (pH same buffer. Samples

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of 2.5 ml were collected and the protein concentration was determined by measuring the absorbance at 280 nm. Reference proteins and their tool. wts were: urease (473,000), lactate dehydrogenase (240,000), aldolase (158,000), amino glucosidase (97,000), enolase (82,000) and serum albumin (67,000). RESULTS AND DISCUSSION

Mitochondrial and cytosolic form activities o f erythroid ALA-synthetase The cytosolic fraction of ALA-synthetase of haematopoietic tissue showed a specific activity of 3.63 x 10 -4 mU/mg protein which represents 30% of the activity found for the mitochondrial fraction (1.38 × 10 -3 mU/mg protein). Previously we reported that the mitochondrial form of trout hepatic ALAsynthetase had a higher specific activity than the cytosolic form. Ohashi and Kikuchi demonstrated that the cytosolic form of ALA-synthetase in cock liver represents a 25% of the activity in the liver. The same phenomenon was observed during the induction of ALA-synthetase in mouse and rat liver (Ades and Harpe, 1981). Similar values have been reported by Aoki et al. (1971) for rabbit reticulocytes (1.2x 10-3mU/mg protein), Jordan and Shemin (1972) for chicken reticulocytes (35 x l0 -3 mU/mg protein) and for human erythrocytes (70 x 10 -3 mU/mg protein). No activity of mitochondrial ALA-synthetase was detected when Lubrol WX-4 was omitted. This fact suggests that the enzyme is loosely bound to the inner mitochondrial membrane. Molecular weights The mol. wts of the both forms of ALA-synthetase were estimated by gel filtration chromatography and the values of 90,000 and 120,000 were obtained for the mitochondrial and cytosolic form respectively. The lesser mol. wt of the mitochondrial enzyme front to the cytosolic form has been reported by Fern~mdez et al. (1985), in trout liver. In both fractions a proteic fraction of 46,000 with catalytic activity was always obtained. The existence of enzymatic forms of low mol. wt with activity has been found by Whiting (1976) who considered them as subunits of ALA-synthetase. Subsequently, Borthwick et al. (1983) suggested that the enzyme could be degraded proteolytically to a smaller form of mol. wt around 50,000 retaining full enzymatic activity; more recently we report a proteic fraction of ALAsynthetase, in mitochondria and cytosol from trout liver, which had enzymatic activity (Fernandez et al., 1985). In view of the findings in chick liver (Ades and Harpe, 1981) and rat liver (Raymond and Shore, 1979; Yamauchi et al., 1980) that several mitochondrial proteins are synthesized in the cytoplasm as precursors and that precursors become cleaved to their respective mature tool. wts at some point during their translocation into mitochondria, the 120,000 mol. wt polypeptide existent in the cytosol is probably a precursor to the 90,000 mol. wt mitochondrial ALA-synthetase. The erythroid ALA-synthetase was about 20,000 mol. wt larger than the hepatic enzyme from trout.

Similar results have been reported by Watanabe et aL (1983) for the ALA-synthetase isozymes in the liver and erythroid cells of chicken. These results also demonstrated that both hepatic and erythroid enzymes from different sources are made as larger precursors which are transported into mitochondria in association with proteolytic processing. Effect o f p H on ALA-synthetase activity The effect o f p H was tested over the range pH 5-10 at 15°C, using acetate, Tris-maleate, Tris-HCl and diethanolamine buffers at the concentration of 0.66 M. Both fractions show a pH optimum of 7.5 when assayed in Tris-HCl buffer. No differences in the effect of pH on the ALA-synthetase activity were observed between these erythroid forms and the hepatic forms, pH optimum 7.5 (Fernandez et al., 1985). Similar values have been reported by Jordan and Shemin (1972) for R. spheroides, pH optimum 7.4, Aoki et al. (1971) for rabbit reticulocytes, pH optimum 7.6 and by Hayashi et al. (1983) for embryo chicken liver, pH optimum 7.6. Effect o f temperature The enzyme reaction showed a maximum activity at 40°C for both types of fractions. Over 40°C the activities decrease and no aciivity is detectable between 50 and 60°C (Fig. 1). Their activation energies are 16.Skcal/mol for the cytosolic form and 11.8 kcal/mol for the mitochondrial form. The values of QJ0 (middle in a temperature range from 10 to

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40°C) have proved to be 1.46 and 1.48 for the cytosolic and the mitochondrial fractions respectively. The effect of temperature over the erythroid ALAsynthetase forms is different from that found by us (Fernfindez et al., 1985) over the hepatic ALAsynthetase forms. The hepatic forms showed a maximum activity at 30°C and low activity is detectable at 50 and 60°C. This fact along with the differences in mol. wts of the hepatic and erythroid ALAsynthetase suggests that these proteins could be isozymes, as occurs in Mammalia (Bishop et al., 1981) and birds (Watanabe et al., 1983). Kinetics with respect to substrates and cofactor

ALA-synthetase in both subcellular forms exhibits an absolute requirement for the substrates, glycine and succinyl CoA and for the cofactor pyridoxal 5'-phosphate. When any of these substances is omitted from the assay media, no formation of ALA is detectable. All preparations were treated with antipain and Lubrol WX-4 (when required) as described in Materials and Methods. Figure 2 shows the effect

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Fig. 3. Activity and substrate succinyl CoA concentration: (a) cytosolic and (b) mitochondrial form. Results are expressed as mU/mg.

of substrate glycine on the two forms of ALAsynthetase. In both, the kinetic was sigmoid and S 0.5 values for the glycine (Table 1) were 14.8 mM and 20.4 mM for mitochondrial and cytosolic forms respectively. Both subcellular forms of erythroid ALAsynthetase showed positive coooperativity. Glycine demonstrated substrate inhibition at concentrations in excess of 150mM (Fig. 2). The S 0.5 values for succinyl CoA were estimated to be 0.01 mM for mitochondrial fraction and 0.005 mM for the cytosolic fraction (Table 1). As can be observed in Fig. 3 the kinetic was sigrnoid with a slight inhibition by excess of substrate at concentrations above 0.008 mM. The Km of the mitochondrial enzyme is two-fold higher than that of the cytosolic form which indicates a greater affinity of the mature ALA-synthetase for the succinyl CoA. This fact could be explicable because the succinyl CoA is not present in the cytosol and therefore the cytosolic precursor would not be functional. The kinetic behaviour of erythroid ALAsynthetase forms of trout (Fig. 3) showed a different kinetic for the substrate succinyl CoA than the hepatic forms (Fern~indez et al., 1985). The erythroid enzyme presents sigmoid curves of activity and the

Table 1. Kineticproperties of ALA-synthetaseforms Glycine Succinyl CoA Subcellularfraction S05(mM) nn Sos(mM) nn Mitochondrial 14.8 1.2 0.01 1.6 Cytosolic 20.4 1.6 0.005 1.4

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that the m R N A for erythroid 5-aminolevulinate synthetase differs from the m R N A for the hepatic 5-aminolevulinate synthetase. However, it is not clear at present whether the hepatic and erythroid A L A synthetases are specified by the two independent genomic sequences.

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REFERENCES

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Fig. 4. Effect of cofactor PLP on ALA-synthetase activity: (a) cytosolic and (b) mitochondrial form. Results are expressed as mU/mg.

hepatic enzyme showed a Michaelian kinetic. Bishop al. (1981) suggested the existence of A L A synthetase isozymes on the basis of differences in kinetic and ligand-binding properties observed between the enzymes isolated from erythroid and nonerythroid tissues of guinea pig. The effect of cofactor P L P on the erythroid A L A synthetase (Fig. 4) showed a Michaelian kinetic and the K m value for the mitochondrial form (0.014 mM, Table l) was three- to four-fold higher than that of the hepatic enzyme (0.005 m M ) (Fern~indez et al., 1985). In both fractions, the cofaetor became inhibitory at concentrations greater than 0 . 0 1 0 m M . Several investigators suggested that the regulatory mechanism acting on erythroid ALA-synthetase may be different from that for the non-erythroid enzyme. Watanabe et al. (1983) provided convincing evidence with their inmunodiffusion and inmunotitration experiments that in chicken the erythroid and hepatic ALA-synthetase are different tissue-specific entities. The differences in mol. wt and kinetic properties observed in this work between erythroid and hepatic ALA-synthetase from adult trout support the existence of erythroid and non-erythroid forms of the enzyme in Teleostei tissues with their appropriate regulatory mechanism. These observations suggest et

Ades J. Z. and Harpe K. G. (1981) Biogenesis of mitochondrial proteins. J. biol. Chem. 256, 9329-9333. Aoki Y., Wada O., Urata G. and Takaku F. (1971) Purification and some properties of 5-ALA synthase in rabbit reticulocytes. Biochem. biophys. Res. Commun. 42, 568-575. Bishop D. F., Kitchem H. and Wood W. A. (1981) Evidence for erythroid and non-erythroid forms of ALA-synthase. Archs Biochem. Biophys. 206, 380-391. Borthwick I. A., Srivastava G. and Brooker J. D. (1983) Purification of 5-ALA synthase from liver mitochondria of chicken embryo. Eur. J. Biochem. 129, 615--620. Fernfindez J., Gon~lez O., Martin M. and Ruiz Amil M. (1985) Some properties of cytosolic and mitochondrial forms of 5-ALA synthetase from trout liver (Salmo gairdneri R.). Comp. Biochem. Biophys. (submitted). Granick S. (1966) The induction in vitro of the synthesis of the 5-ALA synthase in chemical porphyria: a response to certain drugs, sex hormones and foreign chemicals. J. biol. Chem. 241, 1359-1375. Granick S. and Sassa S. (1971) Metabolic Pathways, Vol. l, (Edited by Vogel, H, J.), pp. 77-141. Academic Press, New York. Hayashi N., Watanabe N. and Kikuchi G. (1983) Inhibition by hemin of in vitro translocation of chicken liver 5-ALA synthase into mitochondria. Biochem. biophys. Res. Cornmun. 115, 700-706. Jordan P. and Shemin D. (1972) 5-Aminolevulinic acid synthetase. In The Enzymes Vol. 7, 3rd edn, (Edited by Boyer, P. D.), pp. 339-356. Academic Press, New York. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 256-275. Mauzerall D. and Granick S. (1956) The occurrence and determination of 5-ALA acid and porphobilinogen in urine. J. biol. Chem. 219, 435-446. Ohashi A. and Kikuchi G. (1979) Purification and some properties of 5-ALA synthase from rat liver cytosol. J. Biochem. 85, 239-248. Raymond Y. and Shore G. (1979) The precursor for carbamyl phosphate synthetase is transported to mitochondria via a cytosolic route. J. biol. Chem. 254, 9335-9338. Srivastava G. and Borthwick I. A. (1982) Purification of rat liver mitochondrial 5-ALA synthase. Biochem. biophys. Res. Commun. 109, 305-312. Watanabe N., Kikuchi G. and Hayashi N. (1983) 6-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken. Biochem. biophys. Res. Cornmun. 113, 377-383. Whiting M. J. (1976) Synthesis of 6-ALA synthase by isolated liver polyribosomes. Biochem. J. 150, 478-486. Yamauchi K., Hayashi N. and Kikuchi G. (1980) Translocation of ~-ALA synthase from the cytosol to the mitochondria and its regulation by hemin in rat liver. J. biol. Chem. 255, 1746--1751.