Ornithine oxoacid aminotransferase found in AH 130 ascites hepatoma cells

356

Biochimica et Biophysica Acta, 714 (1982) 356-360

Elsevier BiomedicalPress BBA 21041 ORNITHINE OXOACID AMINOTRANSFERASE FOUND IN AH 130 ASCITES HEPATOMA CELLS TAKEO MATSUZAWAa, NAOFUMI SUGIMOTOa, MITSUKOSOBUEb and ISAO ISH1GURO a a Departments o f Biochemistry and b Pathology, Fu/ita-Gakuen University School of Medicine, Toyoake, Aichi 470-11 (Japan)

(Received July 30th, 1981)

Key words: Ornithine oxoacid aminotransferase; 2-Oxoglutarate; (Ascites hepatoma cell)

We found heterogenous ornithine oxoacid aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) in rat ascites hepatoma AH 130 cells. Compared with enzymes from normal rat tissues, this heterogenous enzyme showed larger Km values for 2-oxoglutarate, a different elution-profile upon affinity chromatography with 2-oxoglutarate, more anionic mobility upon polyacrylamide gel electrophoresis, and a clearly different salting-out property upon ammonium sulfate fractionation. Similar heterogeneity of this aminotransferase was found in human cancer cells.

rence of heterogenous ornithine oxoacid aminotransferase in AH 130 ascites hepatoma and human cancer cells, but it is entirely different from the ones suggested by Volpe et al. [8]. We employed affinity chromatography using 2-oxoglutarate Sepharose, polyacrylamide gel electrophoresis, and describe the kinetic properties determined by the rate-assay method.

Introduction Growing cells, e.g. cancer cells [1-3], mutageninduced transforming lymphocytes [4] and photoreceptor cells [5], provide an ornithine-proline pathway which presumably participates in the biosynthesis of proline-rich membranous proteins such as collagen and glycoproteins. The first-step of the ornithine-proline pathway is catalyzed by ornithine oxoacid aminotransferase, the next step by A 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2). Various cancer cell lines contain ornithine oxoacid aminotransferase in amounts comparable to normal tissues [1,6]. Tomino et al. [6] reported that the enzymes from hepatomas with different growth rates were hardly distinguishable in their kinetic, catalytic and electrophoretic properties from liver enzymes of control normal rats. We recently developed a precise spectrophotometric rate-assay for determining ornithine oxoacid aminotransferase activity, using Al-pyrroline-5-carboxylate reductase as the coupling enzyme [5]. With this assay, much smaller Km values for the substrates than those reported earlier [7] were obtained. Volpe et al. [8] suggested the occurrence of heterogenous ornithine oxoacid aminotransferase in cancer cells. Our present study confirms the occur-

Materials and Methods Preparation o f ornithine oxoaeid aminotransferase from various tissues. The enzyme was purified from

the liver, kidney and gut of Wistar strain rats as previously described [9]. Rat ascites hepatoma AH 130 cells were harvested from the ascites of SpragueDawley rats 1 week after intraperitoneal inoculation of cancer cells. Ornithine oxoacid aminotransferase from these AH 130 cells was purified according to a modification of our previous method [9] : the ammonium sulfate fractionation was different (see Results) and 2.oxoglutarate Sepharose was used as in affinity chromatography. KB (human rhinopharyngeal tumor), MKN-45 (poorly-differentiated human meduUary gastroadenocarcinoma) and MKN-29 (welldifferentiated human gastroadenocarcinoma) cell

0304-4165/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress

357

lines used in this study were maintained in culture medium, containing 20% calf serum in Eagle's minimal essential medium prepared in Hanks' balanced salt solution (GIBCO, Grand Island, N.Y.), to which had been added kanamycin (10 rag/100 ml) and penicillin (1000 U/100 ml). The glass culture bottles were maintained at 30°C; the medium was replaced twice a week. 1 week after initiation of the culture, proliferated cells were scraped off the glass surface and collected by centrifugation. They were then suspended in a small amount of saline solution containing 0.01 M potassium phosphate buffer (pH 7.5), sonicated for 2 min at 9 kHz, and then centrifuged at 1 2 0 0 0 × g for 10 min. The resulting supernatant was used as the enzyme solution. Ornithine oxoacid aminotransferase of human kidney was purified as previously reported [9], Enzyme assay. Ornithine oxoacid aminotransferase activity was routinely assayed using o-aminobenzaldehyde [7] ; for determining the Km values, a spectrophotometric method was used: In a total volume of 3.0 ml, the reaction mixture contained 100 /.tmol potassium phosphate buffer (pH 7.5), 0.33 /~mol NADH, 0.5 unit L-Al-pyrroline-5-carboxylate reductase (spec. act., 250 U/mg protein) [10], 30 ~mol 2-oxoglutarate, 20 pmol pyridoxal phosphate, 90 /,tmol ornithine, and an appropriate amount of enzyme solution. The protein concentration was determined by the dye method [ 11 ].

Affinity chromatography with 2-oxoglutarate Sepharose. AH-Sepharose 4B which contains a spacer arm of 1,6-diaminohexane and a free amino terminal, was reacted with 2-oxoglutarate in the presence of 1-ethyl-3-(3-dime thylaminopropyl) carbodiimide-HC1 (EDC) at pH 4.7.7.5 g of AH-Sepharose 4B gel was washed and swollen with 1.51 of 0.5 M NaC1 on a glass filter, washed with reagent-grade water and adjusted to pH 4.7 by adding a small amount of 0.5 M HCI. To this we added 50 ml of 0.18 M 2-oxoglutarate solution (pH 4.7), the final volume was 90 ml. EDC (1.72 g) was added and the pH was maintained at 4.7 during the 24-h coupling reaction at room temperature. Thereafter, the Sepharose was washed with 0.1 M acetate buffer (pH 4.0) and 0.1 M NaHCO3 buffer (pH 8.3), each of which contained 0.5 M NaC1. The Sepharose was packed into a 1.3 × 20 cm column and equilibrated with 0.01 M potassium phosphate buffer (pH 7.5) (buffer 1). The en-

zyme solution, which was converted to the pyridoxamine form by an addition of ornithine and dialyzed, was applied to the column; elution was with 100 ml of buffer 1, then with a linear gradient of 0-0.3 M salt concentration in buffer 1 (200 ml of buffer 1 + 200 ml of 0.1 M potassium phosphate buffer containing 0.5 M KC1). Polyacrylamide gel electrophoresis was done using the method of Ornstein and Davis

[121. Results

K m values determined by the rate-assay method. Fig. 1. shows the K m values at various pH determined by Lineweaver-Burk plots of the values obtained with the rate-assay method. The K m values

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358 TABLE I K m VALUES FOR SUBSTRATES OF ORNITHINE OXOACID AMINOTRANSFERASE FROM CANCER CELLS AND NORMAL TISSUES All K m values were determined by the rate-assay method at pH 7.5, with 2xl-pyrroline-5-carboxylate reductase and NADH in 3 ml. Values in parenthesis indicate the number of experiments that were performed. - K m value (mM) 2-oxoglutarate

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Fig. 2. 2-Oxoglutarate Sepharose affinity chromatography of ornithine oxoacid aminotransferase prepared from AH 130 cells and normal rat tissues. The linear oblique lines indicate the linear density gradient of the salt concentration in elution. - - , enzyme activity; . . . . . . , absorbance at 280 nm. I; apoenzyme, II; holoenzyme. Each tube contained 3.5 ml eluate.

against o m i t h i n e decreased, t h o s e against 2-oxoglutarate increased, w i t h increasing pH. T h e K m values obt a i n e d in t h e p r e s e n t e x p e r i m e n t s were m u c h smaller t h a n t h o s e r e p o r t e d earlier [ 1 , 3 , 6 - 9 , 1 3 - 1 9 ] . T h e K m values o f o m i t h i n e o x o a c i d a m i n o t r a n s ferase f r o m various cells are listed in T a b l e I. T h e K m values for 2 - o x o g l u t a r a t e a n d o r n i t h i n e o f e n z y m e s f r o m n o r m a l rat liver, k i d n e y a n d gut were indistinguishable f r o m each o t h e r . T h e K m value for 2-oxog l u t a r a t e o f e n z y m e f r o m rat ascites h e p a t o m a A H 130 cells was significantly larger t h a n t h e values o f e n z y m e s f r o m n o r m a l rat tissues. I n MKN-45 cells, t h e Km value for 2 - o x o g l u t a r a t e was a b o u t 10 t i m e s larger t h a n t h a t o f n o r m a l h u m a n k i d n e y e n z y m e . T h e K m values for o r n i t h i n e were very similar in all e n z y m e s tested.

359

Affinity chromatography with 2-oxoglutarate Sepharose. The rat liver and kidney enzymes gave the same elution profile; peaks 1 and 2 were identified as the apo- and holoenzyme, respectively (Fig. 2). The elution profile of AH 130 enzyme was somewhat different; the enzyme eluted in the front position indicating a lower affinity for 2-oxoglutarate. The quotient of amounts of the apoenzyme by the holoenzyme was about 2 in AH 130 enzyme; it was 0.5 in the enzymes from normal rat liver and kidney. Polyacrylamide gel electrophoresis. Fig. 3 shows the electrophoretic patterns of ornithine oxoacid aminotransferase from AH 130 and rat liver. The AH 130 enzyme was isolated from rat liver enzyme by polyacrylamide gel electrophoresis. The dye marker, Bromophenol Blue, migrated toward the anode with some activities of the AH 130-, but not the rat liver enzyme. Ammonium sulfate fractionation. Ammonium sulfate fractionation of the enzyme from rat liver, kidney and gut recovered all catalytic activity at 0 35% saturation; in AH 130 it yielded 94% of the enzyme activity at 35-50% saturation.

increasing growth rates, and in rat hepatomas, Katunuma et al. [21] described a phylogenically abnormal nitrogen metabolism (uricotelism) which is assumed to be repressed in normal mammalian cells. Thus, the ornitine metabolism of cancer cells may be directed at polyamine synthesis rather than urea formation. The kinetic or chromatographic natures of AH 130 enzyme, which show a larger K m value for 2-oxoglutarate or a lower affinity for 2-oxoglutarate Sepharose, may contribute to the suppression of ornithine degradation, since in vivo, ornithine oxoacid aminotransferase seems to act exclusively on ornithine degradation. The fact that genetic defect of this enzyme results in severe hyperornithinemia [22], supports this hypothesis of in vivo ornithine catabolism. The smaller Km value for 2-oxoglutarate suggests the effective inhibition of omithine formation from Al-pyrroline-5-carboxylate (unpublished data) and virtually no in vivo ornithine formation from proline in the postprandial rise of the hepatic ornithine level was observed [23]. As in cancer cells, ornithine oxoacid aminotransferase from proliferating microorganisms, yeast [24] and Bacillus sphaericus [25], showed much larger Km values for 2-oxoglutarate.

Discussion Acknowledgements The present experiments gave four indications that ornithine oxoacid aminotransferase from rat ascites hepatoma AH 130 cells is different in its protein nature from the same enzyme derived from normal rat tissues; i.e. the larger K m value for 2-oxoglutarate, the different elution profile in the 2-oxoglutarate Sepharose column, the more anionic behavior upon polyacrylamide gel electrophoresis, and the saltingout characteristics in much higher ammonium sulfate fractionation. Ornithine oxoacid aminotransferase prepared from rat liver, kidney and gut was indistinguishable with respect to the kinetic properties and the elution profiles obtained upon affinity chromatography with 2-oxoglutarate Sepharose. These findings are consistent with those reported by Sanada et al. [ 17]. We obtained no evidence suggesting that two types of ornithine oxoacid aminotransferase, the 'liver' and 'kidney' forms, appear in the cell cycle of cancer cells as suggested by Volpe et al. [8]. In a series of hepatomas, Weber et al. [20] however, have noted a dramatic increase in the ornithine decarboxylase/ornithine transcarbamoylase quotient with

We thank Professor Nobuhiko Katunuma (Tokushima University) for his valuable discussions during the course of this study. We also thank Dr. Katsuhiko Ono (Department of Viral Oncology, Aichi Cancer Center) for generously providing the AH 130 cells, Dr. Mikihiro Shamoto (Department of Pathology, Fujita-Gakuen University School of Medicine) for supplying human kidneys from autopsy materials, and Mrs. Ursula A. Petralia for reading this manuscript. This investigation was supported by the FujitaGakuen University Research Fund.

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