Naruto University of Education, Naruto, Tokushima 772, Japan. Received October 19, 1988. Summary: Highly purified lysosomes from the normal and leupeptin- ...
Vo1.157, No, 2,1988 December 15, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 440-442
DEGRADATION OF ASPARTATE AMINOTRANSFERASE IN RAT LIVER LYSOSOMES Atsushi Sato*, K.M. Faisal Khan*, Yasuo Natori*, and Mitsuko Okada** *Department of Nutritional Chemistry, School of Medicine, The U n i v e r s i t y of Tokushima, Tokushima 770, and **Department of Home Economics, Faculty of Health and Living Sciences, Naruto U n i v e r s i t y of Education, Naruto, Tokushima 772, Japan Received October 19, 1988
Summary: Highly p u r i f i e d lysosomes from the normal and leupeptin-treated rat livers were subjected to immunoblot analysis using antibodies against cytosolic and mitochondrial isozymes of aspartate aminotransferase (cAspAT and mAspAT). In the case of cAspAT (subunit M.W.= 46K), the leupeptintreated lysosomes showed a major band of 46K and a minor band of 36K while normal lysosomes showed a major band of 36K and a minor band of 41K. In the case of mAspAT (subunit M.W.= q4K), the leupeptin-treated lysosomes showed a 44K band and the normal lysosomes showed a 40K band. These observations suggest that both cAspAT and mAspAT are sequestered into lysosomes with the original subunit molecular weights and are degraded in the lysosomes by way of sequential formation of r e l a t i v e l y stable intermediates with distinct molecular weights. © z98~Acaa~micp..... ~nc.
Aspartate aminotransferase [EC 2.6.1.1] exists as two d i f f e r e n t isozymes in higher organisms; one located in the cytosol (cAspAT) and the other in the mitochondria (mAspAT).
A recent report from our laboratory (1) showed
that, in rat l i v e r , cAspAT and mAspAT are degraded with h a l f - l i v e s of 4.78 and 5.02 days, respectively.
However, the intracellular site of degradation
of these isozymes has remained unknown.
The present report provides
evidence that both isozymes are degraded in the lysosomal system of rat l i v e r . MATERIALS AND METHODS Preparation of Lysosomes from Rat L i v e r . Highly purified lysosomes were isolated from normal and leupeptin-treated rat l i v e r s by the method developed in our laboratory (2) with some modifications. Male Wistar rats, weighing about 200 g, were starved o v e r n i g h t before sacrifice. For leupeptin treatment, 1 mg of leupeptin per 100 g body weight was injected i n t r a peritoneally into rats and the animals were sacrificed 2 h later. The l i v e r s were homogenized i n 4 volumes of cold 0.25 M sucrose and the postnuclear supernatant fraction was prepared as o r i g i n a l l y described (2). The sucrose solution and all the subsequent media contained 50 pg leupeptin/ml to i n h i b i t protease action. The postnuclear supernatant fraction was made up to 1.8 mM Ca ++ by the addition of 0.01 volume of 180 mM CaCI 2 and incubated at 37°C for 10 rain. The incubated mixture was Centrifuged for 5 min at 340 x g to remove aggregated materials. The supernatant (3 ml each) was layered on 24 ml of iso-osmotic ( 0 . 2 5 M sucrose) Percoll (adjusted to pH 7.4) at a density o f ! . 0 8 g/ml in a Hitachi RP30 rotor t u b e . Centrifugation was performed for 15 min at 60,000 x g in a Hitachi RP30 rotor at 2°C. A f t e r 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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the centifugation, 3.5 ml of the gradient was collected from the bottom of the tube by an inserted tubing. This fraction contained practically all the lysosomes and no mitochondria. The pooled fractions were diluted with 10 volumes of cold 0.25 M sucrose and centrifuged at 10,000 x g for 10 rain. The supernatant was carefully removed with suction and the sedimented lysosomes were washed once more with cold 0.25 M sucrose by centrifugation under the same conditions. The washed pellet was finally suspended• in a small volume of 0.25 M sucrose • and used as the purified lysosomal fraction. This modified procedure gave lysosomes with similar yield and p u r i t y to the original procedure (2) in considerably shorter time. The lysosomal fraction was not contaminated with mitochondria as evidenced by the absence of the a c t i v i t y of a mitocbondrial marker enzyme (succinic-INT reductase). Immunoblot Analysis. Electrophoresis i n s o d i u m dodecyl sulfate (SDS)q to 20-~ linear gradient polyacrylamide slab gels was performed by the method of Laemmli (3). Immunoblot assay of AspAT was Carried out by staining the nitrocellulose paper after electrophoretic t r a n s f e r from the slab gel (4) with antibodies against rat liver cAspAT and mAspAT (1) and with a n t i - r a b b i t IgG-peroxidase conjugate {Cappel Laboratories I n c . ) . RESULTS AND DISCUSSION The lysosomal proteins from normal and leupeptin-treated rat livers were subjected to immunoblot analysis using antibodies against cAspAT or mAspAT. In the case of cAspAT (Fig. I ) , the immunoblot analysis of the lysosomes from leupeptin-treated rat liver showed a major 46K band and a minor 36K band.
The major 46K band had the same electrophoretic mobility as the marker
cAspAT whose subunit molecular weight is 46,295 (5).
The immunoblotting
of normal lysosomal proteins, however, showed a major 36K band and a minor 41K band.
The 46K band was not visible.
This observation indicates that
cAspAT, present in the cytosol, is sequestered into lysosomes and degraded in the lysosomes.
The degradation probably proceeds by way of relatively
stable intermediates of 41K and 36K. lysosomal thiol proteases (6).
Leupeptin is a potent inhibitor of
The intralysosomal degradation of cAspAT
appears to be catalyzed by leupeptin-sensitive proteases since the injection of the inhibitor causes an accumulation of apparently undegraded cAspAT in the lysosomes. As for mAspAT, the immunoblot analysis •revealed a 44K band in the leupeptin-treated lysosomes and a 40K band i n t h e
normal lysosomes ( F i g . 2 ) .
This result suggests that mAspAT, with the subunit molecular weight of 44,358 (7), is degraded in the lysosomes by way of a 40K intermediate. An accumulation of CAspAT in the leupeptin-treated lysosomes has been suggested by an earlier work of Kominami et al. (8).
The present report
is the f i r s t demonstration, to our knowledge, of the sequestration and degradation of well-characterized cytoplasmic enzymes i n t h e
normal lysosomes.
The present finding suggests that the subunit molecular weights of both cAspAT and mAspAT are essentially the same as the original isozymes when they are sequestered into lysosomes.
This is to say that p r i o r limited
h y d r o l y s i s b y an extralysosomal protease(s), at l e a s t t o the extent influencing 441
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