Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide

Comp. Biochem. PhysioL Vol. 86B, No. 1, pp. 95-98, 1987 Printed in Great Britain

0305-0491/87 $3.00+0.00 Pergamon Journals Ltd

DISTRIBUTION OF ADENOSINE DEAMINASE IN SOME RAT TISSUES. INHIBITION BY ETHANOL A N D DIMETHYL SULFOXIDE JOSEP J. CENTELLES, RAFAEL FRANCO* and JORGE BOZAL*t Departamento de Bioquimica, Facultad de Quimica, Universidad de Barcelona, Diagonal 647, Barcelona 08028, Spain (Received 24 March 1986)

Abstract--1. The level of adenosine deaminase in various rat tissues has been tested. 2. The enzyme activity of cytosolic fractions decreased in the following order: lung > spleen > small intestine > stomach > kidney > heart > liver > skeletal muscle > forebrain > cerebellum. 3. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and K~ values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.

INTRODUCTION

(Stefanovich, 1982). It is known that adenosine regulates the cerebral blood flow ( W i n n e t al,, 1981) and that it is a modulator of the synaptic transmission (Mcllwain, 1973). It has been suggested that adenosine is a neurotransmitter and that adenosine deaminase modulates the synaptic transmission by catabolizing adenosine (see Schabe, 1981, for a review). Furthermore, the enzyme shows a specific localization in the brain, near the ectoenzyme 5'nucleotidase (Trams and Lauter, 1975, Franco et al., 1986). The enzyme has also an important role in iymphocytes since a congenital lack of adenosine deaminase is associated with severe combined immunodeficiency disease (Kredich and Hershfield, 1983) and with AIDS (Murray et al., 1985). In diabetic rats the level of the enzyme from rat heart also varies with respect to non-diabetic rats (Dr Jill Lincoln, personal communication). In this paper, the levels and some properties of adenosine deaminase from various rat tissues have been tested. This has been performed for checking differences in the enzymatic molecule depending upon the tissue. The inhibition of the enzyme by ethanol has also been studied since it might be correlated with the depressant effects that follow alcohol ingestion.

Adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4.) is an enzyme of purine metabolism, and because of its interest it has been studied from a wide variety of organisms: microorganisms (Koch and Vallee, 1959), plants (Brawerman and Chargaff, 1954), invertebrates (Ma and Fisher, 1968a; Harbison and Fisher, 1973; Aikawa et aL, 1977), vertebrates (Ma and Fisher, 1968b, 1972) and mammals (Phelan et al., 1970; Tritsch and Rosenfeld, 1976; Piggott and Brady, 1976; Fonoll et al., 1982) including human cells (Daddona and Kelley, 1977; Daddona et aL, 1984). Ma and Fisher (1968a) have shown that there are different types of adenosine deaminase, which have molecular weights of the order of 200,000, 100,000 and 35,000. These forms are designated as A, B and C forms, respectively. Adenosine deaminase from human and mammalian tissues appears as A and C forms. It is well established that the A form contains the C form and a complexing protein. The presence in a given tissue of A form, C form or both is very straightforward. It has been shown that, whereas normal human lung extracts show essentially a single peak corresponding to the larger deaminase molecule, extracts from lung cancer tissues exhibit an additional peak which corresponds to the smaller enzyme. Thus, the relative proportion of one form with respect to another form could reflect physiological or pathological changes of the ceils in which they function (Akedo et al., 1972). Adenosine deaminase and its substrate, adenosine, play a key role in other pathological states such as asthma (Ronchetti et al., 1984) and anoxia

Chemicals Chemicals and commercial proteins were: adenosine, Tris, imidazole, ethylenediaminetetraacetate and phenazine metasulphate (Merck); xanthine oxidase (20U/ml) and purine nucleoside phosphorylase (20 U/ml) (BoehringerMannheim); acrylamide and bis-acrylamide (EastmanKodak); agarose and Sephadex G-25 (Pharmacia Fine Chemicals) and MTT (Serva). All other chemicals were reagent grade.

*Membership of the Spanish Society of Biochemistry. tMembership of the European Society for Comparative Physiology and Biochemistry. Author to whom all correspondence should be addressed.

Enzyme assay Adenosine deaminase activity of the enzyme solutions was measured by following the decrease of absorbance at 265 nm (Kalckar, 1947) in a Unicam SP 1700 spectrophotometer. A unit of adenosine deaminase is defined as the

MATERIALS AND METHODS

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JOSEP J. CENTELLESet al.

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amount of enzyme which transforms 1 #mol of adenosine in 1 sec, under the assay conditions (30+ 0.1°C, 50#M adenosine in a 0.28 mM sucrose, 30 mM Tris-HCl buffer pH 7.4). Protein concentrations were determined by the method of Lowry et al. (1951). Bovine serum albumin (Sigma) was used as standard protein.

Analysis of kinetic data Initial velocity measurements were fitted to the appropriate equations by a non-linear regression program derived from Marquardt's method (Reich et aL, 1972) implemented in a Tektronix 4051 computer working in BASIC (Canela, 1984). Tissue samples and preparation of cytosolic fractions Tissues were removed from male Sprague-Dawley rats (250-300 g) which had been beheaded and exsanguinated; they were immediately washed in ice-cold solution A (0.28 M sucrose, 30 mM Tris, 1 mM EDTA-HCI, pH 7.4). Tissues were then homogenised in solution A [4°C, 6.5% (w/v)] as follows: cerebellum, forebrain, liver, kidney, spleen, heart and small intestine in a Potter-Elvehjem homogeniser (20 strokes, 500rev/min, 95-115#m clearance), and lung, stomach and skeletal muscle in a Waringblendor homogenizer. The homogenates were clarified by ultracentrifugation at 105,000 g for 90 min (Ultracentrifuge Beckman L5-75H, 35FA rotor). Supernatants (cytosoluble fraction) were collected and filtered through Sephadex G-25 (Pharmacia Fine Chemicals) equilibrated with solution A, before determination of adenosine deaminase activity. Inhibition by antibodies Antibodies against purified adenosine deaminase from rat brain (Centelles et al., 1986) were obtained from New Zealand White female rabbits using the technique described by Vaitukaitis (1981). IgG were further purified using the method described by Hurn and Chantler (1980). Aliquots of cytosoluble fractions were diluted with solution A until reaching an adenosine deaminase activity of 200#U/ml. 0.1 ml of these solutions corresponding to 20 #U of adenosine deaminase were placed in Eppendorf test tubes and incubated (30 +0.1°C, 30 min) with 0.1 ml of the purified IgG (3.3mg protein/ml) 0.1 ml of the mixture was then assayed for adenosine deaminase activity. Controls made with solution A (instead of cytosolic fractions) and purified IgG were devoid of adenosine deaminase activity. Electrophoresis Analytical electrophoresis was performed on 9% (w/v) polyacrylamide-disc-gels in Tris-HCl buffer, pH 8.9, by the method of Andrews (1981). A stacking gel prepared with Tris-H3PO4 buffer, pH 6.4, was used as concentrating gel. The upper buffer chamber contained 40 mM Tris-glycine buffer, pH 8.3, and the lower, 25mM Tris-HC1 buffer, pH 8.9. Electrophoresis was carried out at 75 V during 150 min.

RESULTS

Kinetic properties o f adenosine deaminase K m and V values are shown in Table 1. K~ values ranged from 30 to 50/~M and tissue content (in U / g tissue) decreased in the following order: lung > spleen > small intestine > stomach > kidney > heart > liver > skeletic muscle > forebrain > cerebellum. Dimethyl sulfoxide and ethanol were competitive inhibitors of the enzyme with Ki values similar in all tissues (Table 2).

Table 1. Maximum activities and Km values of adenosine deaminase in rat tissues Vmax Tissue

Km (p/4)

(mnol/s g wet weight)

Lung

49 +/- 8

Spleen

47 +/- 3

125+/- 13 123 +/- 42

Small intestine

41 +/- 2

115 +/- 38

Stomach

40 +/- 2

9 5 + / - 13

Kidney

41+/- 2

6 1 + / - 1o

Heart

43+/- 4

19 +/- 2

Liver

45+/- 1

17 +/- 2

~eletRl muscle

43 +/- 5

7 +/- 1

Forebrain

40 +/- 6

3.2 +/- 0.7

Cerebellum

38 +/- 2

3.1 +/- 0.9

The activitieswere measured as described in Materials and Methods and the results are expressed as means + S.D. of 6 animals. Range of adenosine concentration was 10-90 #M.

Electrophoretic behaviour Electrophoresis patterns of adenosine deaminase from forebrain, cerebellum, liver, kidney, spleen, heart, small intestine, lung, stomach and skeletal muscle were similar. In all cases, only one band of activity after specific staining was shown. Single and crossed applications of cytosolic fractions were performed.

Inhibition by antibodies to adenosine deaminase Percentage of inhibition of the enzyme from various tissues by antibodies anti-adenosine deaminase is shown in Table 3. Inhibition was similar for all the cytosolic fractions tested. DISCUSSION

It has been shown that adenosine deaminase from rat brain appeared as a single molecular form whose apparent molecular weight (35,000) is similar to that of the C form of frog (Ma and Fisher, 1968a), chicken (Ma and Fisher, 1968b), mammals (Ma and Fisher, Table 2. Competitive inhibition of adenosine deaminase by ethanol and dimethyl sulfoxide Ki (M)

Tissue

Ethanol

Dimethyl sulfoxide

Lung

0..58 +/- 0.05

Spleen

0 . 6 0 + / - 0.06

1.36 +/- 0.07 1.7 +/- 0.1

Small intestine

0.69 +/- 0.06

1.40 +/- 0.07

Stomach

0.71 +/- 0.05

1.26 +/- 0.09

Kidney

0.78 +/- 0.09

1.5 +/- O.t

Heart

0.88 +/- 0.06

1.4 +/- 0.1

Liver

0.50 +/- 0.08

1.2 +/- 0.1 1.10 +/- 0.09

Skeletal rauscle

O, S4 +/- 0.04

Forebrain

0.66 +/- 0.07

1.1 +/- 0.1

Cerebellum

0.48 +/- 0.09

0.99 +/- 0.06

The activities were measured as described in Materials and Methods and the results are expressed as means + S.D. of 6 animals. Range concentrations used in the assays were: 0-1.1 M for ethanol and 0 -I.9M for dimethyl sulfoxide.

Adenine deaminase from rat tissue Table 3. Inhibitionof adenosinedeaminaseof rat tissuesby

anti-(rat brain adenosinedeaminase)immunogtobulins Anti-(adenosine d eamin as e )immunoglobulin s Tissue

Lung

(~ inhibition)

6~, +/- 2

Spleen

56 +/- 14

Small IntestLne

70 +/- 2

~toraaeh

72 +/- 7

Kidney

80 +/- 2

Heart:

74 +/- 6

Liver

69 +/- 4

Skeletal muscle

62 +/- 20

Forebrain

43 ~-/- 7

Cerebellum

48 +/- 8

The inhibitionwas measuredas describedin Materialsand Methods. Percentage of inhibitionof antibodieswith respect to eachcytosolicfractionis shown(mean+ S.D. of 5 animals).

1969; Maguire and Sim, 1971; Fonoll et al., 1982) and human tissues (Daddona and Kelley, 1977). The kinetic, immunochemical and electrophoretic results shown in the present paper indicate that adenosine deaminase is similar in all tissues and corresponds to the C form of the enzyme. Ma and Fisher (1969), when describing the different forms (A and C) of the enzyme from various sources, found that in rat liver it appears mainly as C form. Though they are not analogues of adenosine, ethanol and dimethyl sulfoxide, which are competitive inhibitors of the enzyme, might compete with water, which is the other substrate of the enzyme. Inhibition by ethanol can be correlated with the depressant effect that follows alcohol ingestion. Since the enzyme is presumably located at the external side of membranes in brain cells (Franco et al., 1986), its inhibition might increase the extracellular adenosine concentration; it is well established that adenosine has a depressant effect upon neurones and modulates the cerebral blood flow (Winn et al., 1981). The levels of the enzyme are very different from tissue to tissue being high in the lung, spleen, small intestine and stomach. Working with rabbit tissues Conway and Cooke (1939) found that the adenosine deaminase level is high in part of the intestinal tracts and in the spleen, whereas in the kidney, liver, skeletal muscle, heart, lungs and brain the values are lower. In human tissues the level of the enzyme is high (more than 1 U/g fresh tissue) in the digestive tract (except for large intestine) and in the spleen; whereas tissues such as skeletal muscle, bladder, lung, kidney, heart and brain contain a low activity (less than 0.5U/g fresh tissue). These similarities and differences between rat, rabbit and human tissues suggests that the enzyme has an important role in the function of some tissues. The high activity found in intestine comes, mainly, from the laminapropia (Chechik et al., 1984; Chechik et al., 1985) as it has been demonstrated by immunohistochemical studies. This specific localization can be correlated with the effect of inhibitors of adenosine deaminase on the relaxant action of adenosine in the C.B.P. 86/1B--G

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smooth muscle of intestine (data not shown). In brain subfractions, the enzyme is located with the 5' nucleotidase in the external face of the vesicles and synaptosomes (Franco et al., 1986). Thus, although the enzyme activity is low in the whole brain (forebrain or cerebellum) its specific localization can have an important effect degrading adenosine, a putative neurotransmitter or neurohormone. Thus, besides biochemical experiments, which indicate that there is one molecular form of the enzyme, immunocytochemical studies are required for localizing the enzymes within different cells and hence, improving our knowledge about its specific function in each tissue. REFERENCES

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