72

Biochimica et Biophysica Acta, 1077(1991)72-78 © 1991 ElsevierSciencePublishersB.V.0167-4838/91/$03.50 ADONIS 016748389100129G

BBAPRO33862

Heterogeneity of chicken liver cytosolic aspartate aminotransferase Santiago Imperial, Carmen Quiroga, Montserrat Busquets and Antonio Cortes Departaraent de Bioq,"~..ica i Fisiologia, Facultat de Qubm'ca, Universitat de Barcelona. Barcelona (Spain)

(Received15 May 1990) (Revisedmanuscriptreceived30 October 1990) Key words: Aspartatcaminotransferase;Heterogeneity;Molecularform; Oxidation; Storage;(Chickenfiver) The isolated native molecular forms of chicken fiver cytosolie aspartate aminotransferase give rise to two kinds of generation processes: (a) on storage, molecnl~ f - q ~ s are trandonned into a series of variants with increasing anodic mobilities; and Ca) addition of thiol reagents not only avoids the process, but causes the partial trans[onnation of minor subforms into vm~ants with higher isodectric point values, in both cases the mohiiities oi each generated variant coincide with that of the corresponding native molecular form. The variants generated either by storage or in the presence of thiol reagents were separated by chromatofocusing. Several compm~live studies have demonstrated the strucPdral and fnnctional identity between native molecular forms and "in vitro' active generated variants of the enzyme. The results obtained suggest that native minor subforms arise from the major a form due to oxidation process and might represent intermediate species in the intracellular cytosollc aspartate amlnolransferase tlnnover. Introduction In the cytosolic aspartate aminotransferase (L-asparrate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) from several sources, mul~ple molecular forms (a, t , y . . . ) of increasing anodic mobility have been detected; a form predominates and i,.s specific activity is the greatest [l-V]. The behaviour of the minor forms (fl, y . . . ) has not been extensively studied, since their isolation is difficult and their activities are low as compared to that of the a form [2,3]. A comparative study carried out with preparations of the a form, and two fractions enriched in the fl and 7 form, respectively, from pig heart cytosolic aspartate aminotransferase [3,8] demonstrated that the difference among the specific activities of the forms is associated with the presence of coenzyme (pyridoxal 5:.phosphate) bound in an inactive mode. These results were confn'med by identification using X-ray difraction studies [9,10] of a modified coenzyme in one of the subanits of the fl form, the other subunit being unaltered. In the same study both subunits of a form appeared to be analogous to the unaltered subtmit in the fl form. Although these results

Correspondence: A. Con~s, Departamentde Bioquimicai Fisiologia, Facultat de Qulrnjca, Universitat de Barcelona, 08028 Barcelona, Spain.

explain the differences in specific activity detected for the molecular forms of cytosolic aspartate aminotransferase, other aspects of the structure of the molecular forms from pig heart and other sources remain to be elucidated, as well as the biological significance of the heterogeneity and the mechanisms for the generation of the subforms. The transformation of the molecular forms of cytosolic aspartate aminotransferase into variants with more anodic p l on storage, has already been observed for the enzyme from mammalian tissues [11,12]. However. the molecular basis of this transformation is currently under discussion. The most widely accepted hypothesis indicates that the variants generated on storage, are formed by hydrolysis of amido groups of glatamine and asparagine residues of the enzyme molecule [13,14]. We have observed that preparations of the isolated molecular forms of chicken liver cytosolic aspartate aminotransferase stored at 4 o C for long periods of time (up to 30 days) gave rise to additional active subforms with higher electrophoretic mobilities [15]. This process was avoided in the presence of 2-mercaptoethanol and other thiol-contalning compounds. In this study we have isolated the forms generated "in vitro' and we have determined the kinetic behaviour and some of the molecular properties of each individual generated subform. We compared these results with those obtained with the 'native' molecular forms of the

73 cytosolie aspartate aminotransferase i.e., those present in freshly prepared homogenates and proposed a possible physiological significance for the heterogeneity of this enzyme in the cell cytosoL Materials and Methods Substrates and inhibitors NAD +, NADH, ADP, INT (2( p-iodophenyl)-3-( pnitrophenol)-5-phenyltetrazolium chloride) and fumaric, glyoxylic, hydroxymalonic, oxalic, 2-oxoadipic, 2oxobutyric, oxomalonic and 2-oxovaleric acids were from Sigma. Adipie, L-malic, maleic, malonic, oxaloacefic, 2oxoglutaric, pyruvic and succinic acids, pyridoxal 5'phosphate and all amino acids used in this investigation were purchased from Merck. Enzyme preparations Glutamate dehydrogenase (EC 1.4.1.3) from bovine liver and malate dehydrogenase (EC 1.1.1.37) from porcine heart were from Boehringer-Mannheim. Diaphorase (lipoamide dehydrogenase, EC 1.6.4.3) from Clostridium kluyveri was from Sigma. The five molecular forms (a, ~8, 7, 0 and e) of chicken liver cytosolie aspartate aminotransferase were prepared as previously described [15]. The specific activities of the holoenzyme forms were 205 (a), 41 (/3), 35 (7), 23 (6) and 15 (c) l.U./mg. Enzyme assay Aspartate amlnotransferase activity was measured in 50 mM Tris-HCl buffer (pH 7.4) at 30_+0.1°C, with 0.8 mM 2-oxoglutarate and 10 mM L-aspartate. The oxaloactate produced was detected by the method of Karmen [16]. The initial reaction velocities were determined by measurement of the absorbance changes at 340 mo in a Hewlett-Packard 8450 A spectrophotometer, in 1-cm fight-path cells. Activities are expressed as international units (I.U.) defmed as the amount of enzyme producing 1/tmol of oxaloacetate per min under the conditions of the assay. The ketoacid substrate specificity of the enzyme was also studied by the method of Katmen [16] with 3 mM 2-oxoglutarate analogue and 10 mM L-aspartate. The amino acid substrate specificity was determined by the method of Rej [I,"] with 0.8 mM 2-oxoglutarate and 20 mM of the corresponding amino acid. The initial reaction velocities were determined by measurement of the absorbance changes at 500 nm due to the reduction of INT. Protein determination Protein content determinations were carried out by the method of Read and Northcote [18] using bovine serum albumin as standard.

Electrophoresis Polyaerylamide gel electrophoresis was performed at pH 8.8 using 8~ polyacrylamide gels according to the method of Maes [19]. The electrophoresis was carried out for 90 rain at 4 ° C in 50 mM (pH 8.8) Tris-giycine buffer. Aspartate aminotransferase activity was detected by specific staining according to the method of Sakakibara et al. [20]. Chromatofocusing lsoelectric points determinations and separation of the native and the 'in vitro' generated enzyme variants were carried out by chromatofocusing in the pH range 9-6 on a Polybuffer Exchanger 94 (Pharmacia) column (1 x 45 cm) equilibrated in 25 mM ethanolamine-acetic buffer (pH 9.4). Samples were dialysed overnight in the cold room against 100-fold equilibration buffer. Elution was carried out at a flow rate of 15 m l / h with 10 bed vols. of Polybuffer 96 (Pharmacia) (1:10), adjusted to pH 6.0 with acetic acid. 3 ml fractions were collected. Molecular weight determinations Apparent molecular weights were determined by the method of gel filtration described by Andrews [21] in a Sephacryl S-300 column. The elution buffer was, 0.1 M NaCI, 50 mM Tris-HCI buffer (pH 8.2). (1) Myoglobin ( M r 17800; Stokes Radius (RS)o20.8 A), (2) chymotripsinogen ( M r 2500(); Rs 22.4 A), (3) ovoalbumin ( M r 43000; Rs 30.5 A), (4) bovine serum albumin (Mr 67000; Rs 35.5 /~), (5) aldolase ( M r 158000; Rs 48.1 .A), (6) catalase (Mo, 232000; Rs 52.2 P,), (7) ferritin (Mr 440000; Rs 61.0 A) and (8) tyroglobulin (Mr 669000: Rs 85.0 A) were used as standard proteins (Sigma). The elution volumes of these markers were determined by recording the absorbance of the fractions at 280 nm. Aspartate aminotransferase molecular forms were detected by enzymatic assay. Kinetic studies Kinetic parameters of the native forms and generated variants of the enzyme were determined in 50 mM Tris-HCl buffer (pH 7.4). All initial rate data were plotted in the double reciprocal plot f l y vs. I / S and fitted to the appropriate rate equation [22] by non-linear regression using a BASIC program on a Olivetti M24 computer [23]. Parameter values are expressed as parameter :t: S.D. (due to regression). Results

Transformation of the vative molecular forms of the enzyme on storage Isolated native a and p forms of chicken liver cytosolic aspartate aminotransferase were dialysed against a lO0-fold volume of 0.1 M sodium phosphate buffer (pH 7.4), 1 mM EDTA and allowed to stand at 4 ° C for 30

74 days. Under these conditions, a and fl forms were partially transformed inot more acidic variants ( a ~ t " and 7";/] -" T' and ~') as shown by polyacrylamide gel electrophoresis and specific stabfing (Fig. 1). The electrophoretic mobillties of the additional variants were analogous to those of the corr~;ponding native minor forms of the enzyme. The tran,.formation process described for the above a and fl forms of the enzyme (i.e., freshly isolated and dialysed against 0.1 M sodium phosphate buffer, pH 7.4, 1 mM EDTA) was not observed on storage of the purified forms solutions in the presence of 5 mM 2-mercaptoethanol (2-ME) under the same conditions (4°C, 30 days). Although 2-ME is more stable in solution than other thiol-containing reagents (e.g., dithiothreitol or glutathion) [24], to maintain a reducing environment and preserve its action during storage (30 days), the addition of this thiol reagent to the form solutions was repeated after 10 and 20 days of storage. In the presence of 2-ME, a form remained as a (Fig. 2B), fl form was partially transformed into one variant (a'.ME) with electrophoretic mobility similar to that of native a form (Fig. 2D) and the solutions of "r form had two additional variants (fl2-~E and a~.ME) with lower anodic mobilities (Fig. 2F), analogous to those of the native/] and a forms of the enzyme, respectively.

Separation of the variants generated on storage The different subforms present in the preparations of a and fl forms stored at 4 ° C for 30 days (see Fig. 1), were fractionated by chromatofocusing as described in the Materials and Methods section. Elution profiles are shown in Fig. 3. The active variants with lower anodic mobilities generated when fl and ~ forms of the enzyme were stored at 4 ° C for 30 days in the presence of 5 mM

NN ~,

d

N m c

o

s

F

?"i~,2. Effectof 2-mercaptoethanol(2-ME)on the generationof active variants of aspartate aminotransferaseon storage(30 days, 4°C). (a) Purified a-form,(b) purified a-form in the presenceof 5 mM 2-ME, (c) purified fl-fo..-m,(d) purified/]-formin pre.wnceof 5 mM 2-ME. (e) purified y-fort3 and (f) purified "g-formin presence of 5 mM 2-ME. For the conditionsof elcctrophorcsis,see Fig. L

2-ME (see Fig. 2), were also separated by chromatofocusing as shown in Figs. 4 and 5. Moreover, these figures reveal that the amount of these generated variants depends on the incubation time with the thiofic reagent at 4 ° C . Table I summarizes the p I values obtained for the native molecular forms of the enzyme and those of the variants derived from them.

Characterization of the native molecular forms and of the variants of the enzyme The five native molecular forms (a, t , ~, 8 and ¢) of cytosofic aspartate aminotransferase ant, the generated active variants a', fl', 7', a~oMEand ~-ME were used as

i

Fig. 1. Transfortaafionof the nativemolecularformsof chicken liver aspartate aminotransferaseon storage.(a) Freshlyobtainedmolecularforms and (b) molecularformsstored for 30 daysat 4°C. ~A) a-form.(13).O-formand (C) ~-form.Electrophoresiswas carried out for 90 m~nat 4°C in 250 mMTfis-Glycinebuffer(pH 8.8).Specificstainingfor aspartate aminotransferasewas performedby the methodof Sakakibaraet al. [20].

75 pa

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(pH 7.4). Satura0ag concentrations of 2-oxoglutarate and L-aspartate were 0.8 and 10 mM, respectively. Michaelis constants obtained for the variants were coincident within experimental error and very similar to those of the five native molecular forms of the enzyme. Minor forms (fl, y, 8 and c) of cytosolie aspartate aminotransferase and their corresponding variants (fl', y ' , 8' and fl~-ME) are far less active than the native a form or the generated "in vitro" subforms a" or a2.ME (Table I). (c) Subslrate specificity. The different molecular forms of chicken liver eytosolic aspartate aminotransferase showed a high substrate specificity. Of the 16 amino acids examined (Giy, L-AIa, L-Set, L-Thr, L-Cys, L-Met,

AAT (I.U/mL)

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Fractions Fig. 3. Chromatofocusingof a and fl forms of chicken liver aspartate aminotransferasestored 30 days at 4°C. (A) a-form and (B) fi-form. Chromatofocusingwas performed on PBE 94 (l x 4 0 crn) equilib,-ated with 25 mM (pH 9.4) ethanolamin¢-aoetic buffer. Fluent: 22.5 mM polybuffer 96 (pH 6.0); flow rate: 25 ml/h; 3 m! fractions ~,,.~e collected. (---) pH gradienL (I) Aspartate ammotransferase activity. The inserts show the pherogrmnsfor the different peaks. (A) a-form: (R) enzymebefore chromatofocusing:(!) a-form; (It) fl'; (lit) ¥'. (B) p-form: (R) enzyme before chromatofocusing; (1) ~-form; (H) y': (111) 8'.

enzymatic material for the different comparative studies performed. (a) Molecular weight. The enzymatic solutions were appfied to a Sephacryl S-300 colunm. The ten species assayed appeared as single peaks with identic, al elufion volumes and no significant differences in molecular weight were detected. By the representation given in Fig. 6A cytosolie asp =at:rate aminotransferase subforms had an Rs value of 38 A. This value, accordin~y to the plot of MW 1~ vs. erf - t (1-Kd) (Fig. 6B) [25] corresponded to an apparent molecular weight of 90000.

(b) Michaelis constants and maximum velocity values. Kinetic parameters of the different subforms of eytosolic aspartate aminotransferase were determined with 2-oxoglutarate (0.02-1.5 raM) and L-aspartate (0.3-20 raM) as variable substrates in 50 m M Tris-HCl buffer

pl,l -I '0.4

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Fig. 4. Time-dependent transformation of fi-form into a less acidic variant by storage h; the presence of 2-ME, Chromatofocusingand electrophoretic patterns of: (A) freshly prepared B-form; (B) ~-form stored for 15 days at 4°C in the presence of 5 mM of 2-ME and; (C) ~8.formstored for 30 days at 4°C in the prc~cnccof 5 mM of 2-ME. Chromatofocusingwas performed a,s in Fig. 3. (---) pH gradient. (e) Aspartate aminotransferaseactivity. For conditions of electrophoresis see Fig. 1. Specificstaining[20].

76 .02

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Fig. 5. Toue-dependent transformation of T-form into less acidic variants by storage in die presence of 2-ME. Chromatofocusing and eiectrophoretic patterns of: (A) freshly prepared v-form; (B) "),-form stored for 15 days at 4 ° C in the p ~ c e of 5 ram of 2-ME; and (C) "t-form stored for 30 days at 4 ° C in the presence of 5 mM of ?.-ME. Chromatofocusing was performed as in Fig. 3. (---) pH gradient. (0) Aspartate aminotransferase activity. For conditions of dectrophoresis see Fig. l. Specific staining [20].

20'

0:1

/ OS

B

erf'~(1-Kd) 10

Fig. 6. Apparent molecular weight detenni,lations of cytosolic aspartare aminotransferase molecular fetms ~nd "in vitro" generated varian~ Sephacryl S-300 (1 × 100 cm) was used as gel filtra~-rl support. Elution was carried out with 0.l M NaCI, 50 mm Tris-HCI buffer (pH 8.2). Numbers. indicate the proteins listed under "Materials and Methods'. (A) Plot of R s vs. erf-1 t l - Kd); (B) plot of MW t/3 vs. err - t (1 - Kd).

TABLE 1

Comparison of several properties of native forms and active variants of aspartate aminotransferase Molecular

Parameter

form

pi

K m (L-Asp) (mM)

Km (2-oxo) (~M)

I'm (%)

MW

a ,8 !' 8 • a'

8.50 8.25 7.95 7.30 7.!0 8.45 8.25 7.95 8.50 8.20

130 ± 0.07 1.35+0.10 1.25±0.09 1.40+0.10 1.25+0.12 1.25+0.09 1.304-0.10 1.20+0.10 1.405:0.08 1.30+0.10

25 + 2.0 28+1.7 30+2.5 264-2.0 32±1.8 28+2.0 244-1.5 31+0.8 295:1.0 28+2.0

100.0 20.0 16.5 11.0 7.2 99.0 22.0 17.0 100.0 21.0

90 ~ 0 90000 90000 90000 90000 90000 90000 90000 90000 90000

fl' y' a[.ME /~-ME

L-Pro, L-Val, L-Leu, L-lie, L-Lys, L-Arg, L-His, L-Phe, L-Tyr and L-Trp) none of them produced reactions greater than 0.5~ of that obtained with a saturating concentration (10 raM) of L-aspartate. No enzyme transamination activity was observed using a series of different 2-oxodicarboxyfic acids (oxomalonic and oxoadipic) or 2-monocarboxylic acids (glyoxylic, pyruvic, oxobutyric and oxovaleri-'c) analogues of 2-oxoglutarate as substrates at a 3 mM concentration. These rest,its are in good agreement with those obtained using porcine and human enzymes [17,26] and show that the several subforms of chicken liver cytosolic aspartate amino transferase share this narrow range of substrate specificity.

77 TABLE !1 Inhibition of aspartate aminotransferase (a form) by dicarboxylicacids Enzymeactivity was detenained with 0.8 m.*.!2-oxoglutarateand 10 mM L-aspartate in 50 mM Tris-HCI buffer (pH 7.4). The values shownare the averageof the resultsof fiveexperiments.The variability observedin the inhibitionpercentageswhvnine other native forms and active variantsof the enzymewereu..cedis about 5~. Dicarboxylicacids Control Oxalate Malonate Hydroxymalonate Saccinate Malate Maleate Fumarase Adipate

%Inhibition 0 26 12 22 8 16

42 9 4

(d) Inhibition by 2-oxoglutarate analogues. Cytosolic aspartate aminotransferase molecular forms are inhibited to a similar extent by several 2-oxoglutarate analogues that are incapable of undergoing transamination. It is clear from the results (Table II) that the enzyme-inhlbitor interaction is influenced by the intercaboxyl group distance. In this sense, it has been indicated [27], that dicarboxylic acids have an allowable conformation one in which the carboxyl groups are close together. Maleic acid is a good model for this particular conformation. It justifies that this compound is the strongest inhibitor of the enzyme (see Table ll). Ano~er steric restriction is dependent upon the polarity of the substituent groups in the dicarboxylic acid molecule. It agrees on the fact that hydroxymalonate and r-malate bind to the enzyme more strongly than malonate and succinate, respectively. The kinetic stud-

TABLE 1II Inhibition by maleote and oxalate of aspartate aminotransferase (a form) Ki~, inhibition c6astant determined from the slopevs. inhibitor concentration linear replot: K~, inhibitionconstantdetermined fromthe intercept vs. inhibition concentration linear reploL C, competitive inhibition; M, mixedinhibitinn.The variabilityobservedin the inhibition constantvalueswith the other native formsand active variantsof the enzymeis a~ut 5~. Inhibitor

2-Oxoglutarate(0.02-0.8 mM) L-aspattate I mM L-aspartatel0mM (non-satusating) (saturating)

Maleate

M Kh ~ 0.29 + 0.0l m M Kti ~ 6 . 9 ± 0 . 3 m M

Ox~a~

M Kts ~ 3.8:t:0.7 m M Kti ~ 21.5 ~ 1.8 m M

C Kh = 0.35+0.02 mM

Kt~= 2.9+ 0.2 mM

ies of the inhibitions induced by oxalate and maleate were performed in a 50 mM Tris-HCi buffer (pH 7.4). According to the general rules for predicting the effect of dead-end inhibitors on the slope and intercept of double-reciprocal plots for a given varied substrate [28], the different inhibition patterns (Table III) show that the dicarboxylic acids used can bind both to the pyridoxal and pyridoxamine forms of the enzyme and that their binding to the pyridoxal form is hindered by saturating concentrations of L-aspartate in the reaction medium. From the inhibition constants it is clear that maleate and oxalate have a higher affinity for the pyridoxamine form of cytosolic aspartate aminotransferase. Moreover, these results indicate that the mechanisms of inhibition of each native subform and "in vitro" generated variants by maleate and oxalate are analogous. Discussion

Cytosolic aspartate aminotransferase from chicken liver shows molecular heterogeneity in polyacrylamide gel electrophoresis. The enzyme exists as various molecular forms of increasing anodic mobilities (a, fl, y . . . ). These multiple forms appear by post-transcriptional modifications as deduced from 'in vitro' experiments carried out with isolated molecular forms, specially a and fl forms. On storage at 4 ° C of a and fl forms, for long periods of time (30 days) each individual form is partially transformed into a set of variants, (fl', ¥', 8 ' ) with electrophoretic mobilities coincident with those of the other more acidic native molecular forms of the enzyme (fl, y,8...). The same results were obtained on storage in the simultaneous presence of various antiproteinase reagents (data not shown), thus, the possibility that the enzyme variants might arise by modifications in the polypeptide size of the enzyme by trace proteinases seems unlikely. This generation process does not proceed in the presence of thiol reagents (e.g., 2-ME or dithiothreitol) allowing the maintenance of preparations of a form for long periods of time but partially transforming/~ and "r forms into less acidic variants, a~.ME and a~.Me-/~.ME, respectively, which also show mobilities identical to the corresponding native molecular forms a and ft. These two opposite processes occur in non-reducing and reducing conditions, resFectively, suggesting that the transformations observed are not due to deamidation of asparagine or giutamine residues, but m oxidation and reduction of certain nuriber of amino acid residues of the enzyme. We show here that, for a give molecular form, the variants with the same electrophorefic mobility, generated by storage either in the presence or in the absence of 2-ME (e.g., for a --+a ' and a[.ME) show analogous isoelectric point, molecular weight, substrate specificity, mechanism of inhibition by substrate analogues,

78 Michaelis c o n s t a n t s a n d m a x i m u m velocity values (see Tables I, II a n d III). A c c o r d i n g to the a b o v e m e n t i o n e d criteria o n e m a y speculate t h a t in the cytosol m o s t a s p a r t a t e a m i n o t r a n s ferase activity exists as a , the fully active a n d n o n - m o d ified f o r m o f the enzyme, whereas m i n o r s u b f o r m s (/~, y, 6 a n d c) represent species w h i c h w o u l d c o r r e s p o n d to a f o r m with increasing n u m b e r o f oxidized a m i n o a c i d residues. A l t h o u g h o n l y k n o w l e d g e o f the s u l p h y d r y l status o f e a c h v a r i ~ t f o r m m a y defini:ely p r o v e this hypothesis, the n u m b e r o f eight to ten free ¢ysteine residues p e r e n z y m e molecule o f the cytosolic a s p a r a t e a m i n o t r a n s f e r a s e f r o m different sources [2,3,7,29,30] is c o m p a t i b l e with this suggestion as well as the decrease in the n u m b e r o f the e n z y m e titrable cysteines r e p o r t e d for "aged' pig h e a r t e n z y m e p r e p a r a t i o n s [3]. T h e det e r m i n a t i o n o f the total a n d reactive thiol g r o u p s as well as the identification of oxidized f o r m s o f cystein side c h a i n s in the molecular f o r m s a n d g e n e r a t e d v a r i a n t s w h i c h are essential steps to u n d e r s t a n d the precise n a t u r e of this process are e x p e r i m e n t s c u r r e n t l y in p r o gress in o u r l a b o r a t o r y . T h e r e is increasing evidence t h a t several c o v a l e n t modifications are i m p o r t a n t in the initial stages o f the d e g r a d a t i o n o f intracellular p r o t e i n s in p r o k a r y o t i c a n d e u k a r y o t i c cells [31,32]. O n e o f the different types o f these modifications is the o x i d a t i o n o f certain a m i n o a c i d r e s i d u ~ b y m i x e d - f u n c t i o n systems [33,34] t h a t convert the e n ~ a n e s to less active o r catalytically i n a c tive f o r m s t h a t a r e m o r e accessible to p r o t e o l y t i c a t t a c k [35,36]. A s the m i n o r s u b f o r m s o f cytosoHc a s p a r t a t e a m i n o t r a n s f e r a s e are f a r less active t h a n the m a j o r a - f o r m of the enzyme, their presence ' i n vivo' w o u l d represent t r a n s i t i o n a l states o f the n o r m a l t u r n o v e r o f the protein. It has b e e n i n d i c a t e d t h a t the rate o f proteolysis in m a m m a l i a n cells increases with the degree o f o x i d a t i o n (redox state) o f cells a n d this is reflected in c h a n g e s in t h i o l / d i s u l f i d e ratios a n d possibly the state o f oxidation of p r o t e i n sulfhydryl g r o u p s [37]. A c c o r d i n g to this, future investigations should include studies with liver perfusions in the presence of disulfide c o m p o u n d s to e x a m i n e the effects o f these s u b s t a n c e s o n the t u m o e e r o f cytosolic aspa.~ate a m i n o t r a n s f e r a s e a n d o n cellular proteinases. References 1 Decker, L.E. and Ran, F-M. (1963) Proc. Soc. Exp. Biol. Med. 112, 144-149. 2 Bertland, L.H. and Kaplan, N.O. (1968) Biochemistry 7, 134-142.

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Kagamiyama, H. (1983) Clin. Chim. Acta 133, 119-123. Andrews, P. (1965) Biochem. J. 96, 595-606. Cleland, W.W. (1963) Binchim. Biophys. Acta 67, 104-137. Canela. E.i. (1984) lnL J. Bio-med. Comput. 15. 121-130. Stevens, IL, Stevens, L. and Price. N.C. (1983) Biochem. Educ. IL 70. 25 Hooriike., K., Tojo, H., Yamano, T. and Nozaki, M. (1983) J. Biochem. 93, 96-106. 26 Novogrodsky, A. and Meister, A. (1964) Biochim. Biophys. Acta 81, 605-608. 27 Bonsib. S.M~ Harru~f, ILC. and Jenkins, W.T. (1975) J. Biol.

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Chem. 250, 8635-8641. 28 SegeL I.H. (1975) Enzyme kine,~ics.Ch. 9, pp. 506-845. London Wiley Intersct~-'ncePublications,London. 29 PetrtllLP~ Pucci, P~ Gan.Jllo,A.M, Sannia, G. and Marino, G. O981) M o L Cell.Biochero. 35, 121-128. 30 Sldyapnikov, S.V, Myaslmikov, AN., Severin, E.S., Myagkova, M.A.. Tov.'hinsky.Yu, M. and Braunstein,A.E. (1979) FEBS LetL 106, 385- 388.

31 Bemlohr, D. and Switzer, ILL. (1981) Biochemistry 20, ~675-5681. 32 Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827. 33 Levine~ILL., Oliver. C.N~ Fulks. ILM. and Stadtman, E.R. (1981) Proc. Natl. Acad. Sci. USA 78, 2120-2124. 34 Fucci, L~ Oliver, C.N, Coon, M.J. and Stadtman, F-R. (1983) Proc. Natl. Acad. ScL lISA 80, 1521-1525. 35 Rivett, AJ. (1985) J. BinL Chem. 260, 300-305. 36 Roseman, J.E. and Levine, ILL. (1985) Fed, Proc. 44,1092 (Absl.). 37 Tischler, M.E. and Fagan, J. (1982) Arch. Biochem. Biophys. 217, 191-201.