Adenosine aminohydrolase activity in the regenerating rat liver

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 238, No. 1, April, pp. 259-262, 1985

Adenosine

Aminohydrolase

Activity in the Regenerating

BERTRUM Department

Rat Liver

SHEID

of Pharmacology, State University of New York, Downstate 450 Clarkson Avenue, Brooklyn, New York 11203

Medical

Center,

Received August 24, 1984, and in revised form December 14, 1984

The specific activity of adenosine aminohydrolase in the regenerating rat liver is significantly increased 12 h after partial hepatectomy. There is a twofold increase in enzyme activity at 48 h, after which the activity begins to decline. However, increased values still persist 7 days postsurgery. The enzyme is located mainly in the soluble supernatant (90-95%) of the cell. The purified enzyme from 48-h regenerating liver and control liver has similar kinetic properties (K, 54-58 PM for adenosine), similar molecular weights (30,000-35,000), and are equally inhibited by an irreversible transition-state analog and a reversible competitive inhibitor. It is concluded that adenosine aminohydrolase in regenerating liver is an integral component of a salvage pathway designed for the reutilization of nucleotides, and thus helps maintain a “growth state” for the regenerating liver. 0 1985 Academic Press, Inc.

Adenosine aminohydrolase (AAH; EC 3.5.4.4)’ is a ubiquitous enzyme that catalyzes the deamination of adenosine to inosine (1). Physiologically, the enzyme can either function in a salvage pathway (2, 3), or be involved in the metabolic degradation of adenosine (4,5). Clinically, congenital deficiency of the enzyme has been associated with a severe form of combined immunodeficiency disease (6), while inhibitors of the enzyme have been used in cancer chemotherapy to potentiate the cytotoxic actions of anticancer agents that are adenosine analogs (7). A number of investigators have demonstrated an increase in the specific activity of AAH in very rapidly growing malignancies as compared to their tissue of origin (g-11), while slow-growing, welldifferentiated tumors and fetal tissue have been shown to have normal to low levels of enzyme activity (8, 12, 13). These data suggest that AAH is not involved in carcinogenesis, but rather plays a metabolic role in supporting a rapid-growth state for appropriate tissues. In order to support 1 Abbreviation lase.

used: AAH, adenosine aminohydro-

this hypothesis, we have utilized the regenerating rat liver. It has been well established that partial hepatectomy (70% surgical removal of rodent liver) results in the intense proliferation of the remaining hepatocytes, resulting in DNA synthesis and cell division (14,15). Therefore, this tissue provides a unique model to study a rapidly growing differentiated tissue rather than a tissue undergoing dedifferentiation (a property of many rapidly growing malignancies). However, results from different investigators reporting AAH activity in regenerating liver have been grossly contradictory (8, 16). One group of investigators reported that AAH activity of regenerating rat liver was not significantly different from AAH activity values of normal adult liver (8), while another group of investigators reported a nearly twofold increase in AAH activity in regenerating mouse and rat liver which persisted for at least 2 weeks postsurgery (16). It is the purpose of the present study to reinvestigate AAH activity in regenerating rat liver, to determine some of the kinetic properties of the enzyme in normal liver and regenerating liver, and 259

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0 1985 by Academic Press, Inc. of reproduction in any form reserved

260

BERTRUM

to establish a physiological role for AAH in rapidly proliferating tissue. MATERIALS

AND

METHODS

Materials. Sprague-Dawley male rats (loo-125 g) were purchased from the Camm Research Institute (Wayne, N. J.). [8-i4C]adenosine, 60 mCi/mmol, was purchased from Amersham (Arlington Heights, Ill.). Adenosine sulfate and enzyme-grade sucrose were products of the Sigma Chemical Company (St. Louis, MO.). Sephadex molecular sieve materials were purchased from Pharmacia (Uppsala, Sweden). Silica1 gel GF thin-layer plates were purchased from Analteeh (Newark, N. J.). Pentostatin was a gift from Warner Lamhert (Ann Arbor, Mich.), and 2(S)-O[l(R)-(9-adenyl)-2-(hydroxy)ethyllpropanediol was generously provided by Dr. L. Lerner, Department of Biochemistry, Downstate Medical Center, Brooklyn, New York. Tissue extro&. Partial hepateetomies (70% removal of the liver) were performed under ether by removal of the median and left lobes, using the standard technique of Higgins and Anderson (17). Sham operations (all procedures except removal of the liver) were also performed. Livers were removed at the designated time periods, trimmed free of extraneous material, washed in 0.20 M sucrose, 0.05 M Tris, pH 7.5, finely minced, and homogenized in 9 vol (w/v) of the sucrose-Tris buffer, using a Sorvall OmniMixer at 600 rpm for 90 s at 4°C. A soluble supernatant fraction was obtained by centrifugation at 105,0009 for 1 h in a Spinco ultracentrifuge. Enzyme assays were usually performed the same day. If this was not feasible, aliquots of the cytosol were stored at -70°C until use. The frozen and thawed samples had no significant loss of enzyme activity if used within 1 week. Protein was determined by the method of Lowry et al. (18). Enzyme purificatian In order to purify the enzyme, 4 g of liver was homogenized in 10 ml of sucroseTris buffer and the cytosol was obtained by centrifugation. The enzyme was then purified by essentially the method of Streeter et al. (19). Briefly, the protein precipitate from 60-90% ammonium sulfate saturation was dissolved in a minimal amount of 0.01 M Tris, pH 7.5, 0.01 M MgClz, 0.001 M dithiothreitol, 0.075 M KCl, and applied to a Sephadex G-100 column (2 X 100 cm). The column was eluted with the buffer and the eluant was collected in 2.5-ml fractions. The active fractions were pooled, frozen, lyophilized, dissolved in a minimal amount of buffer, and applied to a DEAE-cellulose column (7 X 20 cm). A linear gradient containing the buffer without KC1 and the buffer with 0.3 M KC1 was used to elute the enzyme. The active fractions were pooled, frozen, lyophilized, and stored at 4°C until use. An approximate 150fold purification of AAH from regenerating liver

SHEID (48 h postsurgery) and sham-operated liver was obtained by these procedures, The partially purified enzymes were used for kinetic studies and molecular weight determinations (20). Adenosine aminohydrolase activity. The enzyme activity was measured by two independent methods. A radiolabeled assay adopted from Pull and McIlwain (21) consisted of lo-100 ~1 (35-350 pg protein) of liver cytosol and 24-408 PM adenosine containing 40 PM [S-“C]adenosine/ml in 1.5-ml Eppendorf microfuge tubes. After a lo-min incubation at 37”C, the reaction was terminated with 50 ~1 of 4 M HCIOI. After centrifugation, 100 ~1 of the clear supernatant was added to 20 ~1 of inosine (3 mg/ml), adenosine (2 mg/ml), and hypoxanthine (2 mg/ml). A lo-p1 aliquot of 5 M K&O3 was then added to neutralize the perchlorate. A 20-~1 sample was then applied to a silica gel GF thin-layer plate which was developed in butanol:methanol:ethyl acetate:NH,OH (7:3:4:4). The resultant spots were scraped off the plates and placed in counting cocktail for 16 h prior to counting in a Beckman LSz liquid scintillation counter. Quenching and efficiency corrections were applied for all samples. Blanks, using 0.20 M sucrose, 0.05 M Tris, pH 7.5, in lieu of cytosol, were assayed concurrently. For inhibitor studies, the AAH inhibitors were preincubated with the cytosol for 10 min at 37°C before the adenosine was added (22). Enzyme assays were also performed by a spectrophotometric method which measures the deamination of adenosine to inosine at 265 nm (23). The reaction mixture (1 ml) contained 0.05 M Tris, pH 7.5, 0.031-0.126 pmol adenosine, and lo-100 ~1 of cytosol. Measurements were performed with a Beckman recording spectrophotometer. Initial rates of deamination were used, and no more than 25% of the substrate adenosine was allowed to be deaminated in either of the assay procedures.

RESULTS

Adenosine aminohydrolase activity in regenerating rat liver. Figure 1 is an illustration of the specific activity of AAH in regenerating rat liver for a period of 7 days. There was a significant increase in enzyme activity at 12 h. Peak levels of AAH activity were reached at 48 h, at which time they were approximately twofold greater than the AAH activity of sham-operated liver. The increase in AAH activity persisted for at least 1 week. There were no significant differences in enzyme activity between sham-operated liver and the liver from animals that did not undergo any surgery. Enzyme assays performed on mixtures of equal amounts

ADENOSINE

AMINOHYDROLASE

ACTIVITY

IN

REGENERATING

LIVER

261

procedures), while the radiolabeled assay was used to acquire all numerical data.

Properties

OL

I I

2

3 4 DAYSAFTERSURGERY

5

6

7

FIG. 1. Adenosine aminohydrolase activity in regenerating rat liver and sham-operated rats. Each point on the graph represents two to four animals, and the vertical lines indicate the SE; (0) regenerating liver; (0) sham-operated rats. The velocity of the reaction was proportional for 35-105 pg of cytosol protein, and linear with time up until 4 min. No more than 25% of the substrate was allowed to be deaminated in all assays.

of cytosol from 48-h regenerating liver and sham-operated liver gave the expected additive results, thus eliminating the possibility of the existence of inhibitors or activators of the enzyme in either tissue. Other experiments, utilizing differential centrifugation to isolate the various cell organelles, demonstrated that 90-95% of the AAH activity resided in the soluble supernatant of liver. Most (>95%) of the remaining activity was found in a 10,OOOg precipitate. When the precipitate was washed with 0.05 M Tris, pH 2.5, buffer containing 1% Triton X-100, the AAH activity increased fivefold. In all of these experiments, two separate methods of assaying AAH activity were employed, as described under Materials and Methods. The spectrophotometric assay was found to be more economical and convenient; however, it lacked the reproducibility of the radiolabeled assay. This was probably due to the fact that the products of the deamination: inosine, the substrate, adenosine, and the tissue extract, all absorb at the 265nm wavelength. Therefore, the spectrophotometric assay was used only for screening purposes (such as assaying the individual fractions during purification

of adenosine aminohydrolase.

The partially purified enzymes from 48-h regenerating liver and sham-operated liver were used for all kinetic studies. Figure 2 is a double-reciprocal plot for AAH activity versus varying adenosine concentrations. The Km for adenosine was 55-58 PM for the enzyme from either source, while the maximum velocity was achieved in all assay mixtures. Molecular weight determinations showed that both enzymes, after separation from a specific binding protein, had a molecular weight of 30,000-35,000. The same IDS0 values (total inhibitor concentration at which the enzymatic reaction velocity is 50% of the uninhibited reaction under the given conditions of the assay) were obtained when the enzyme from either source was inhibited with the irreversible transitionstate analog, pentostatin (24), or the reversible competitive inhibitor, 2 (S)-O[l(R)-(9-adenyl)-2-(hydroxy)-ethyl]propanediol (25). The IDbO value for pentostatin was 1 X lo-l1 M, while the IDS0 value for the competitive inhibitor was 6 x lo-7

M.

-20I/S

IO’/ADENOSINE

(/A &I-‘)

FIG. 2. Double-reciprocal plot of 2-day-old regenerating liver AAH activity versus varying substrate concentrations. A X0-fold purified enzyme preparation was used, and assays were performed as described in the text. The reaction was linear for 4 min at all substrate concentrations. A double-reciprocal plot of sham-operated liver AAH activity versus varying substrate concentrations was approximately the same (within experimental error) as the plot for regenerating liver.

BERTRUM DISCUSSION

2. BALIS,

Evidence is presented which demonstrates that in the regenerating rat liver there is a significant increase in AAH activity 12 h postsurgery. The increase in activity peaks at 48 h, at which time it is approximately twofold higher than control levels. These data are consistent with the results of Rothman et al. (16), but are diametrically opposed to the data of Jackson et al. (8). At the present time we cannot offer any reasons for the differences in results with the latter investigators. The present data, demonstrating the timing of the increase in AAH activity in regenerating liver, is related to the timing of the increase in the biosynthesis of RNA and DNA precursors. The synthesis of the nucleotides is known to be initiated between 12 and 24 h after regeneration, and then to peak at 24-48 h (2628). The enzyme was found to be localized mainly in the soluble supernatant of the cell (90-95s). The rest of the activity was associated with a 10,OOOg fraction and increased fivefold after Triton X-100 treatment. This may indicate a separate plasma membrane-bound AAH isozyme. This possibility is now being investigated. Partially purified AAH from 48-h regenerating liver and sham-operated liver had similar Michealis-Menton constants and molecular weights. The enzyme from either source also behaved in a similar fashion when inhibited with an irreversible transition-state analog and a reversible competitive inhibitor. From these data it is concluded that adenosine aminohydrolase in regenerating liver plays a role in the reutilization of nucleotides via a metabolic salvage pathway. While fetal rat liver displays lower AAH activity, as compared to normal adult liver (8, 29), it may not require increased AAH activity, as does regenerating liver, since there is a very slow turnover of nucleic acids in the embryonic liver, therefore not necessitating a fully functional salvage pathway (29). REFERENCES 1. GYBRGY,

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