Immunochemical and Catalytic Characteristics of Adenosine Kinase ...

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Apr 29, 1988 - precipitated enzyme activity from both homogeneous and partially purified adenosine kinase from the par- asite. However, the antiserum did ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry andMolecular Biology, Inc.

Vol. 264,No. 8,Issue of March 15, pp. 4356-4361,1989 Printed in U.S.A.

Immunochemical and Catalytic Characteristicsof Adenosine Kinase from Leishmania donovani" (Received for publication, April 29, 1988)

Dipa BhaumikSand Alok K. Dattae From the LeishmaniaGroup, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta 700032, India

Polyclonal antibodiesto homogeneous preparation of made this enzyme a prospective target for chemotherapeutic adenosinekinasefrom Leishmaniadonovani were manipulations in various systems (6, 7). raised in rabbit. The antiserum was inhibitory and While the mechanism of substrate recognition and phosprecipitated enzyme activity from both homogeneous phorylation of various nucleosides/analogs by adenosine kiand partially purified adenosine kinase from the par- nase is far from clear, the immunological relationship of the asite. However, the antiserum did not immunoprecipi- enzyme from different sources is totally unknown. Henderson tate adenosinekinase of otherhighereukaryotic et al. (8),using 6-methylmercaptopurine riboside as substrate, sources tested so far. Immunoblot analysis of extracts showed that the enzyme from Ehrlich's ascites cells carries from L. donovani and other sources revealed specific out a reaction in which ATP is the first substrate to bind and reaction of the antiserum with only the parasite enAMP the last product to be released. In contrast, the results zyme. Under similarconditions, the enzyme monophospho- of others (9-11) are consistent with a reaction sequence in rylated adenosine and 7-amino-3[&~-ribofuranosyl]- which adenosine is the first substrate to bind and AMP the last product to be released. Chang et al. (12), using enzyme lH-pyrazol0[4,3-d]pyrimidine(formycin A) with almost equal efficiency, exhibiting K,,, values of 16 and from murine leukemia L1210 cells,demonstrated phosphoryl24 PM, respectively. The turnover number(Kcat)of the ation of adenosine via a two-site ping-pong mechanism. Reenzyme with both adenosine and formycin A was 24 cently Bone et al. (13),using bi-substrate analogs of adenosine kinase, showed that theenzyme carries outphosphoryl transs-', whereas Kcat/K,,, yielded valuesof 1.5 and 1.O PM" s-', respectively. Substrate competition experiments fer directly from ATP to adenosine. Previous studies from this laboratory (14) demonstrated indicated strong inhibition of [3H]formycin Aphosphorylation by adenosine.In contrast,[3H]adenosinephos- that the homogeneous preparation of adenosine kinase from phorylation was insensitive to formycin A except at Leishmania donouani, a purine auxotroph, displays qualitavery high concentrations. The inhibitions of [3H]for- tively differential biochemical and physical parameters commycin A and [3H]adenosine phosphorylation by aden- pared with the corresponding enzyme isolated from its natural osine and formycin A were noncompetitive with re- host. Since the enzyme is considered as one of the key enzymes spect to each other. Of the two nucleosides, adenosine of the purine salvage pathway, a detailed study of this enzyme, was found to be effective in eluting the enzyme from especially in parasitic protozoa (including L. donouani) which the 5'-AMP Sepharose 4B column. Phosphorylation of lack the de novo purine-synthesizing pathway, assumes pri[3H]formycin Awas strongly inhibited by N-ethylmal- mary importance. Keeping this view in mind we have undereimide at concentrations which exerted minimal effecttaken a detailed investigation of the immunological characon [3H]adenosine phosphorylation. Adenosine excluteristics and kinetic behavior of the enzyme. The specific sively, but not formycinA, protected the enzyme from purpose of this investigation is to look for selective exploitable N-ethylmaleimide-mediatedinactivation. Taken together the results suggest that ( a )adenosine unique differences (if any) amenable to chemotherapeutic manipulations. This paper deals with studies related to (i) kinase from L. donovani is immunologically distinct and ( 6 ) the enzyme possibly has two discrete catalyti- immunological characterization of the enzyme with respect to corresponding enzyme isolated from other eukaryotic sources cally activenucleoside interacting sites. and (ii) kinetic behavior of the enzyme with different structurally related adenosine analogs having anti-leishmanial property i n vitro (15, 16).These studies provided us with specific information which in the long run might be useful Adenosine kinase (ATP:adenosine 5'-phosphotransferase, from the viewpoint of drug designing. EC 2.7.1.20), an enzyme of the purine salvage pathway catalyzing the transfer of terminal phosphate from ATP to adenEXPERIMENTALPROCEDURES osine, has been shown to possess broad substrate specificity (1-5). This wide spectrum of substrate-utilizing property has Materials * This research was sponsored jointly by the Council of Scientific and Industrial Research, India and by Grant IND/87/018 from the United Nations Development Programme Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Junior Research Fellow supported by the Indian Council of Medical Research, New Delhi, India. To whom requests for reprints should be addressed.

Formycin A' and formycin B were purchased from Sigma. [G-3H] Formycin A (6 Ci/mmol) was obtained from Moravek Bio-Chemicals, Inc, Brea, CA. Anti-rabbit goat y-globulin was the product of IndoMedix, Inc., TX. Protein Aand protein A-Sepharose CL-4B were the

' The abbreviations and trivial names used are: formycin A, 7amino-3-[~-~-ribofuranosyl]-l~-pyrazolo[4,3-d]pyrimidine; formycin B, 3-[~-~-ribofuranosyl]-pyrazolo[4,3-d]6H-7-pyrimidone; BSA, bovine serum albumin; NEM, N-ethylmaleimide; EGTA, ethylenebis(oxyethylenenitri1o)tetraacetic acid; TBS, Tris-buffered saline.

4356

Kinase Adenosine

from L. donovani

4357

tained was assayed for enzyme activity or for immunoblotting. Enzyme Neutralization Assay-The purified enzyme was diluted to 5-10 units/ml in enzyme dilution buffer. The diluted enzyme (30 pl) was incubated with varying amounts of antiserum for 30 min at 37 "C and further for 10 min in an ice bucket. After incubation, the Buffers total volume of the reaction mixture was appropriately diluted in The following buffers were used buffer A (250 mM potassium phosphate-buffered saline and assayed for residual enzyme activity. Zmmunoblotting-Sodium dodecyl sulfate-polyacrylamide gel phosphate, pH 7.5,1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 1 mM each of phenylmethylsulfonyl fluoride, benzamidine HCl, (10%) electrophoresis was carried out according to Laemmli (22). and iodoacetic acid); buffer B (same as buffer A but with 20 mM Proteins (approximately 200 pg) from the gels were transferred to potassium phosphate and 5% glycerol); buffer C (same as buffer B nitrocellulose paper as described by Burnette (23). Nitrocellulose but with 100 mM potassium phosphate, pH 7.5); buffer D (same as papers were soaked in 3% BSA in Tris-buffered saline (TBS). Antibuffer B with the difference that 100 mM Tris-HC1, pH 9.0, was used sera was added to BSA/TBS buffer at a dilution of 1500 and the instead of potassium phosphate and glycerol concentration was raised paper incubated for a further 2 hbefore washing in TBS for 2 h with to 20%). two or three changes. The paper was washed again six times with TBS buffer containing 0.2% Nonidet P-40, 0.1% sodium dodecyl Sources of Enzymes sulfate and 0.25% deoxycholate. Subsequently the paper was treated Adenosine kinase from L. donouani (MHOM/IN/1978/UR6) was with BSA/TBS buffer containing 2-5 X lo6 cpm/ml 12'I-protein A purified to homogeneity using the method described previously (14). (approximately 1 pg of protein A) for 2 h. The iodination of protein The enzyme preparation was routinely checked for purity. For all the A was carried out following published procedures (24). The filter was experiments, unless otherwise mentioned, enzyme preparation from then washed extensively with BSA/TBS containing 0.02% Nonidet a single batch was used. P-40 and autoradiographed. Adenosine kinase from human placenta (17)and livers of rabbit, rat, and hamster (18,19) were isolated as follows. The crude superRESULTS natant was passed through a DE52 column equilibrated with buffer Immunological Studies-Serum from rabbit immunized A. The flow-through fraction was made to 80% saturation with ammonium sulfate. The precipitate formed was collected, dissolved with purified enzymefrom L. donouani was tested for its in Buffer B, and dialyzed against the same buffer overnight. ability to immunoprecipitate adenosine kinase from various

products of PharmaciaLKB Biotechnology, Inc. [1251]Iodidewas obtained from Bhabha Atomic Research Centre, Bombay, India. Sources of other products were as described in an earlier publication (14).

Enzyme Assays and Initial Velocity Determinations Radiochemical Assays-Standard reaction mixture (50 pl) contained 50 mM Tris-HC1, pH 7.5,1.5 mM MgCl2, 1 mM ATP, 50 mM KCI, 0.5 mM dithiothreitol, 0.005 mM erythro-9-(2-hydroxy1-3nonyl)adenine, 0.5 mM NaF, 100 pg/ml BSA, andthe indicated amounts of [2,8-3H]adenosine(50 p M ) or [G-3H]formycinA (210 p M ) (25-100 cpm/pmol). Enzyme (1.5-2 ng) was used depending on the type of assay. The rest of the procedure was the same as described previously (14). Spectrophotometric Assays-The activity was determined by a modification of the method of Lindberg et al. (1)using the pyruvate kinase-lactate dehydrogenase-coupled assay as described elsewhere (14).Nucleoside analogs used had no detectable effect on the efficiency of the coupling enzymes (data notshown). For determinations of kinetic constants of individual nucleosides or analogs, a spectrophotometric assay was used, while for competition and inhibition assays the radiochemical procedure was followed. Both assays agreed within 10-15% variations. The time-dependent linearity of all plots was checked graphically for all substrate concentrations. For determination of initial velocities of the reactions, kinetic data were fitted into Michaelis-Menten equations, i.e. rectangular hyperbola and double-reciprocal plots were done according to Cleland (20). Immunization Protocol-A homogeneous preparation of adenosine kinase from L. donovani was extensively dialyzed against 10 mM potassium phosphate, pH 7.5,and lyophilized. The sample (80-100 pg) in 0.5 ml was emulsified with an equal volume of Freund's complete adjuvant. Half of the emulsified sample was injected intradermally a t multiple sites on the back of a white female Belgian rabbit. The rest of the sample was injected subcutaneously. Three booster injections of the same amount of antigen in Freund's incomplete adjuvant were given at a month's interval. After collection from the marginal ear vein, the blood was kept at 4 "C for 12-14 h. and then centrifuged at 5000 X g for 15 min. The antiserum was collected, pooled, decomplemented, dialyzed, and stored at -20 "C. Prior to first antigen injection, rabbits were bled for normal control serum. The y-globulins from both immunized and preimmunized sera were purified according to a published method (21). Immunoprecipitation Assay-Adenosine kinase immunoprecipitation was carried out by adding enzyme (approximately 0.054 units in 30 pl) to sodium phosphate buffer, control sera, or serial dilutions of antisera in a final volume of 55 pl. Dilutions of the antisera were always made with control sera to maintain constant protein concentration. All the mixtures were incubated at 37 "C for 30 min. To the mixture was added anti-rabbit goat y-globulin or protein A-Sepharose CL-4B (at equivalence for rabbit y-globulin in order to facilitate immunoprecipitation); it was kept for 30 min at 37"C,followed by 30 min in ice. The mixture was centrifuged, and the supernatant ob-

sources. Results shown in Fig. lA indicate that theantiserum immunoprecipitated greater than 90% of total L. donouani adenosine kinase activity with 1%dilution of antiserum in the immunocomplex. In contrast, partiallypurified adenosine kinase from livers of rat, hamster, rabbit, and human placenta appeared to be essentially nonimmunoprecipitable. More or less identical patterns were obtained when purified IgG preparation was used as the source of antibody, confirming that the observed inhibition by the antisera was not due to other inhibitory material (Fig. 1B).Five micrograms of IgG inhibited 140 pmol of AMP-forming adenosine kinase activity from the parasite bymore than 80%. However, the equivalent amount of enzyme activity from rat, rabbit, hamster, and human sources remained more or less unaffected by the same amount ofIgG. The immunoprecipitated activity from L. donouani could not be recovered, suggesting that antiserum was inhibitory (Fig. 2). Furthermore, direct incubation of the antiserum with adenosine kinase from parasite resulted in

Antiserum dilution

1

AIgG

(~g/OSSOy)

FIG. 1. Immunoprecipitation of adenosine kinase from different sources by L. donovani adenosine kinase antibody. Equal amounts of adenosine kinase activity from various sources were incubated with varying amounts of antiserum (A) andanti-IgG (B). After immunoprecipitation with anti-rabbit goat y-globulin, as described under "Experimental Procedures," the activity remaining in each supernatant was determined. CS, control serum; C,&, control IgG; AI&, anti-IgG. Enzyme from L. donouani (O), rat (A), hamster (O),and rabbit (A) livers and human placenta (V)were used. Purified L. donouani enzyme and a DE52-purified preparation from other sources were taken for immunoprecipitation.

Kinase Adenosine

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from L. donovani TABLE I Kinetic parameters of L. donovani adenosine kinase with various nucleosides and analogs Inosine, formycin B, deoxyadenosine, araA, and 2-chloro-adenosine were found not to be substrates. .,V and K,,, determinations were done by spectrophotometric assay as described under “Experimental Procedures.” Kinetic constants Nucleosides

2

4

6

8

V,. V-IK,K, rmov min/mg

pM

Adenosine

38.5

2416

Formycin A

24 37 1.54

24

L t

liters/ min/mg

s-~

L t I K

p M ~

1.5

2.4

1

Antiserum (PI) FIG. 2. Neutralization of L. donouani adenosine kinase by the antiserum. Enzyme neutralization assays were carried out by adding indicated amounts of antiserum to a constant amount of purified adenosine kinase (0).A indicates enzyme activity recovered in the suspended immunoprecipitated pellet, while 0 represents enzyme activity remaining in the supernatant after immunoprecipitation with anti-rabbit goat y-globulin. Inset shows immunoblot analysis of enzyme samples remaining in the supernatant after treatment with varying concentrations of antiserum (0, 2, and 8 pl). Samples before ( I ) and after (II) protein A-Sepharose CL-4B precipitation were used. Details are described under “Experimental Procedures.”

b I

I

l“,

I

0.4 0.8 I 3 5 CATPI mM

-66K -45K

-36K -29K -24K

1 1 . 1

4

I

8ld 40 70 CKCII mM

FIG. 4. Effects of ATP ( A ) and KC1 ( B ) on adenosine (0) and formycin A (0) phosphorylating ability of L. donovani adenosine kinase. [MgZ‘] and pH were kept fixed at 1.5 mM and 7.5, respectively. One hundred percent represents 19 and 23 pmol/ min AMP and FMPformation, respectively.

were 16 and 24 p~ for the phosphorylation of adenosine and formycin A, respectively. The enzyme had Kat of 24 s” with both of the substrates, whereas Kat/K,,, for adenosine and FIG.3. Autoradiogram of immunoblot analysis of extracts formycin A were 1.5 and 1.0 s” p ~ ” respectively. , In contrast, from various sources containing adenosine kinase activity. Samples of crude preparation of adenosine kinase (200 pg each) from formycin B, in which the 6-amino group of formycin A is the livers of rat ( I ) , hamster ( I I ) , rabbit (III),and human placenta changed to 6-oxo,could not beused as a substrate. The ( I V )were analyzed along with crude ( V ,76 pg)and purified adenosine enzyme also did not use inosine, araA, and deoxyadenosine kinase (VI, 60 ng) from L. donovani. The positions of molecular as substrates. 2-Chloroadenosine was found to be ineffective weight markers are shown on the extreme right. as asubstrate. It should be pointed out thatraising the pH of the incubation mixture to 9.0 also did not help these cominhibition of enzyme activity, confirmingthe inhibitory effect pounds to be substrates. Mf-ATP and KC1 Requirements-Earlier studies from of the antibody on the enzyme. ZmmunoblotAnalysis-The experiments described above this laboratory (14) demonstrated that atpH 7.5 the enzyme indicated that the antibody was effective in immunoprecipi- phosphorylated adenosine maximally when the ATP/Mg+ tating adenosine kinase activity from L. donouani and did not ratio was set at 0.66 with the concentration of ATP being 1 cross-react with adenosine kinase from other sources tested mM. Results of the present experiment (Fig. 4A)indicate that so far. However, it was not clear whether the antibody had formycin A phosphorylation was most efficient at an ATP/ cross-reactivitywith proteins other thanadenosine kinase. In Mg2+ ratio of0.13 at 0.2 mM ATP. An increase in ATP order to get this information we analyzed the crude extract of concentration above 0.2 mM resulted in decreasing activity the enzyme from various sources including L. donouani by with formycin A. Formycin A phosphorylation by adenosine immunoblot technique and compared them with the homo- kinase was also stimulated by the addition of KC1 (Fig. 4B). geneous preparation of adenosine kinase from L. donouani. In contrast, adenosine phosphorylation was neither inhibited Results (Fig. 3) indicated that the antibody is absolutely nor stimulated by KC1 even up to a concentration of 70 mM. specific foradenosine kinase (M, = 38,000) (14) of the parasite Differential requirements of adenosine kinase for optimum origin and did not cross-react with any of the proteins present phosphorylation of adenosine and formycin A indicated difin the extract,including adenosine kinase, regardless of their ferential modes of recognition of each substrate by the enorigin. zyme. In order to investigate this behavior further, a series of Substrate Status of Adenosine and Its Analogs-Table I experiments was carried out. Substrate Competition Experiments-Results presented in shows results obtained with regard to kinetic parameters of L. donouani enzyme with adenosine and its various analogs Fig. 5 indicate that [3H]formycin A phosphorylation was as substrates. Comparative analysis at pH 7.5 revealed that strongly inhibited by increasing concentrations of adenosine, the rates of phosphorylation ( VmaX) of adenosine and formycin and almost 100% inhibition was observed at an adenosine A were 38.5 and 37 pmol/min/mg protein, respectively. The concentration of 50 pM. In contrast, phosphorylation of [3H] K, values calculated from Lineweaver-Burk reciprocal plots adenosine was weakly inhibited even if the concentration of I I1 111 IY Y YI

from L. donovani

Kinase Adenosine

eluting the enzyme from the column (Fig. 7 B ) . With 1 mM formycin A only 5-10% of the totalactivity could be recovered. Increasing the concentration of formycin A to 5 mM also did not elute the enzyme. The residual activity was completely eluted only when 5 mM adenosine replaced formycin A in the elution buffer. Effect of NEM on Adenosine and Formycin A Phospho& ation-Results presented in Fig. 8 indicate differential effects of NEM on adenosine kinase-mediated phosphorylation of adenosine and formycin A. In the presence of an increasing concentration of NEM the phosphorylation of formycin A was inhibited to a much greater extent than the phosphorylation of adenosine. In one 30-min incubation assay, 0.5 mM NEM inhibited formycin A phosphorylation to the extentof 95%, whereas adenosine phosphorylation was reduced by 38% (Fig. 8A). Results presented in Fig. 8B indicate thatthe rate of inactivation of adenosine kinase by NEM was pseudo-first order. Adenosine protected the enzyme from NEM-mediated inactivation in a concentration-dependent manner, whereas formycin A had no protective effect. These results were interpreted to indicate that the"SH group responsible for overall activity of the enzyme resides at or is controlled by the adenosine binding site.

formycin A was raised to 1.7 mM. The results were identical regardless of M p - A T P concentration. In view of nearly similar K,, V,.,, and turnover number of the enzyme with adenosine and formycin A, the above observation could not be explained according to the general principle of inhibition by alternative substrates (25). Kinetic analysis of the inhibition indicated that adenosine inhibited phosphorylation of [3H]formycin A noncompetitively at a saturating concentrationof M$+-ATP with KCand K, of 28 and 27 p ~ respectively , (Fig. 6 A ) . Similarly, the inhibition of [3H]adenosine phosphorylation by a very high concentration of formycin A was noncompetitive as well (Fig. 6 B ) . Replots of the data yielded a linear intercept replot, while the slope replot is parabolic. KEwas determined as 2 mM. The noncompetitive inhibition of formycin A phosphorylation by adenosine and vice versa is suggestive of the fact that probably each of the nucleosides following binding alters the stereochemical conformation of the enzyme in such a way that thephosphorylation of the other nucleoside is somehow affected. Elution Behavior of the Enzyme from the 5"AMP-Sepharose 4B Column-The enzyme bound strongly to the5'-AMPSepharose 4B column and was eluted completely with 1 mM adenosine (Fig. 7 A ) . This result is expected in viewof the finding that AMP is a competitive inhibitor of adenosine phosphorylation (11).However, formycin A was ineffective in CFormycin AlmM(-o-) 0.5 I 1.5 2 1

._ 0

501';

,

I

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DISCUSSION

This work reports on (i) the successful preparation and characterization of polyclonal monospecific antibody against adenosine kinase from L. donouani and(ii) the catalytic characteristics of the enzyme. Specific antibodies against adenosine kinase from Chinese hamster ovary cells have been raised previously (26). However, cross-reactivity of the antibody with adenosine kinase from other sources has not been reported. The high degree of antigenic specificity displayed by the leishmanial adenosine kinase-specific antibody both in immunotitration and immunoblot analysis points to the fact that the antibody is directed toward an epitope(s) distinct from the conserved sequences (if any) present among adenosine kinase from various sources. Inhibition of enzyme activity by the antibody might either be due to binding of IgG at the active site of the enzyme or due to conformational change or masking of the active site as aresult of IgG binding. Thus the present findings on the immunospecificity of the enzyme further support our previous conclusion (14) that theparasite

7

u 0.1 0.2 0.3 0.4 CAdenosineI mM(+)

Re. 5. Effeets of adenosine (0)and formycinA (0),respectively (asinhibitors), on radiolabeled formycinA and adenosine phosphorylation by L. donovani enzyme at saturating ATP concentration.

FIG. 6. Kinetics of in~bitionof [SH]formycinA ( A ) and [$H]adenosine ( B ) phosphorylation by unlabeled adenosine and formycin A, respectively. ATP-Mg2' concentration

was kept at the saturating

level. The resultsare displayed in LineweaverBurk plots. Insets show slope (e)and intercept (0) replots versus concentration of alternative substrates as inhibitors.

L

I

I

0.025 0.05

i

I

0.075 0.1

l/CFormycin A J p &

I

I

0.1

I

I

I

0.2

0.3

0.4

IICAdenosineJ p$vi'

4360

Adenosine Kinasefrom L. donovani

5

IO 15 20 Fraction nu~rnber

25

FIG. 7. Elution characteristics of adenosine kinase from 5 ' AMP Sepharose 4B column by adenosine ( A )and formycin A ( B ) containing buffers as eluants. Equal amounts of enzyme activity from G-100 column fractions (14)were loaded on two identically sized 5'-AMP Sepharose 4B columns. After extensive washing with buffer C containing 1 M KCl, the columns were run identically with buffer D containing indicated nucleosides. Substrate concentrations were adjusted while assaying each fraction.

mixture as well (28). The substrate activity of formycin B in mouse L-cells was thus explained as due to the presence of nitrogen at the 8-position (29). However, from the results of our in vitro experiments this conclusion cannot be extended in the case of the parasite enzyme. Likewise, araA, 2-chloroadenosine, and deoxyadenosine, which are weak substrates for eukaryotic adenosine kinase (7, 30), could not be used at all by the parasite enzyme. Therefore, comparison of our results with others indicate that the substratespecificities of this enzyme vary from source to source and depend upon additional factors other than N-1nitrogen protonation. In one of our previous papers (11)we reported studies on the substratedependence of labeled adenosine in the absence and presence of alternate substrates6-methylmercaptopurine riboside and 7-deazaadenosine (tubercidin). On both occasions the effect turned out to be competitive. However, the identical type of competition experiments with adenosine and formycin A yielded different results. The inefficient inhibition of [3H]adenosine phosphorylation by formycin A as opposed to strong inhibition of [3H]formycin A phosphorylation by adenosine could not be explained simply as competition between the substrates. Rather, it raised the possibility as to whether adenosine and formycin A were phosphorylated by (i) anenzyme with a single substrate interacting site, (ii) the same enzyme with different substrate interacting sites,or (iii) different enzymes. Since the enzyme preparation, according to most of the criteria of protein purity, was apparently pure, the thirdpossibility was ruled out. Thus, of the two remaining mechanisms, our results were consistent with the second possibility because of the following experimental facts: (i) requirements of adenosine and formycin A phosphorylations were different; (ii) kinetic constants of the enzyme with both substrates were nearly similar; (iii) NEM inhibited phosphorylation of formycin A much more strongly than thephosphorylation of adenosine; (iv) adenosine, exclusively, protected the enzyme from NEM-mediated inactivation; (v) theenzyme could be eluted from the 5'-AMP-Sepharose 4B column by only adenosine, whereas formycin A was not aseffective; and finally (vi) the inhibition of phosphorylation of formycin A by adenosine was noncompetitive and vice versa. Differential activity patterns of adenosine kinase with different nucleoside substrates were observed previously (3032). Mehta and Gupta (33), using formycin A-resistant mutants of Chinese hamster ovary cells, observed near normal uptake of radiolabeled adenosine but no incorporation of [3H] formycin A. In contrast, mutants selected in the presence of toyocamycin (N-nucleoside) showed a reduced level of [3H] adenosine uptake and no incorporation of [3H]formycin A. It was also noted that these cell extracts were completely devoid of adenosine kinase activity. Since most of the eukaryotic cells possess multiple alternative pathways for purine nucleotide biosynthesis, namely de novo as well as various salvage pathways including phosphorylation, deamination, phosphorolysis of nucleosides, and purine phosphoribosyltransferase systems, there can be multiple interpretations of the in vivo observations. In anotherstudy, adenosine kinase from human erythrocytes which unlike the parasite enzyme displays substrate inhibition and stimulation by a low concentration of AMP was shown to have an additional noncatalyticadenosine binding site. Binding of adenosine at thissite imparts protection to theenzyme (10). Distinct binding sites for deoxyguanosine and deoxyadenosine on monomeric deoxyguanosine/ deoxyadenosine kinase from Lactobacillusacidophilus have also been reported (34). Protection of leishmanial adenosinephosphorylating activity from NEM-mediatedinactivation exclusively by adenosine and notby formycin A suggests that

:YL!A2w 0

0.2

0.4 0.6 CNEMJmM

0.0

5

IO 15 Minutes

20

FIG. 8. Effect of NEM on the phosphorylation of adenosine (A) and formycin A (0) ( A ) and effects of adenosine and formycin A on the rateof inactivation of adenosine kinase by NEM ( B ) .A , the enzyme was preincubated with varying concentrations of NEM for 2 min at 30 "C prior to starting the reaction by addition of labeled substrates. B, the enzyme with or without adenosine or formycin A as indicated and NEM a t a final concentrationof 0.2 mM were preincubated at 30 "C. At various time intervals aliquots were removed, diluted, and assayed for adenosine phosphorylating activity. When various concentrations of adenosine were used as the protecting ligand, the assay mixture was modified so as to keep a fixed substrate concentration. Adenosine concentrations were 0 (O), 50 (A), and 100 SM (O), while formycin A concentration was 100 SM (0).Notation (A)indicates rate of inactivation without NEM.

enzyme is different from eukaryotic adenosine kinase. It is interesting that an enzyme which is so ubiquitous and abundant in nature displays such distinct variable properties from source to source. Nonetheless, the development of specific antibody should facilitate studies on as yet unexplored questions on regulation of synthesis, degradation,and physiological functions of the enzyme, which is known to express in a stage-specific manner (14). Studies on the substratespecificity of L. donovani adenosine kinase revealed several unique features. Formycin A, an extremely poor substrate for rabbit liver adenosine kinase (7), was used efficiently by the parasite enzyme (TableI). In addition, formycin B, which is phosphorylated partly by mouse L-cell adenosine kinase (27), was not at all utilized. Bennett et al. (28), using adenosine kinase from H.Ep.2 cells, demonstrated that theionization of the N-1proton of purine nucleoside/analogs plays a major role in determining the substrate specificity. Furthermore, it was shown that the ionization of the N-1proton was dependent upon the presence of nitrogen at the 8-position and high pH of the incubation

Kinase Adenosine probably NEM-sensitive “SH group(s) are present notat the active sites per se but somewhere near the vicinity of the adenosine catalyzing site. It is our belief that NEM, by binding with --SH group(s), alters the stereochemical configuration of the two sites differentially. Additional proof should await chemical analysis and determination of the stereochemical relation between loss of activity and degree of modification; however, such studies require much larger amounts of pure enzyme than are presently available. Physiological implications of the occurrence of two substrate interacting sites are not clear at present. It is possible that “syn” conformation of formycin A, because of its C-C glycosidic linkage in contrast to “high anti” conformation of adenosine (C-N glycosidic linkage), might be instrumental for recognition of such a kind of unique binding site on the enzyme (35-37). While two differential mechanisms of substrate recognition, uiz. lock and key specificity of adenosine as opposed to induced fit recognition of formycin A or vice versa by the same interactingsite,arestill possible, the chances for these mechanisms to be operative seemed small in viewof the kinetic data obtained with both of the substrates. Thus in conclusion it may be said that the immunological ~i distinctjvity and catalytic characteristics of L. d o ~ v aadenosine kinase are probably related to some obvious uniqueness in the protein molecule. Moreover, the demonstration of discrete C- and N-nucleoside-specific catalytic sites on adenosine kinase, besides having implications in drug designing, opens the way for studying the role of different amino acid residues in nucleoside binding, catalysis, and other as yet unclarified questions in adenosine kinase enzymology in general. Work in theseareas is currently in progress in our laboratory. Acknowledgments-We are indebted to Dr. S. K. Niyogi of Oak Ridge National Laboratory, Oak Ridge,TN, for his generous material help. We wouldalso like to thankDr. D. Sarkar of the Indian Institute of Chemical Biology for his advice while we raised the antibody. Acknowledgment is also due to Ruma Chattejee for herinitial assistance. Finally we thank H. N. Datta and S. Sahu for artwork and S. K. Chaudhuri for typing. REFERENCES 1. Lindberg, B., Klenow, H., and Hansen, K. (1967) J. Biol. Chem. 242,350-356 2. Schnebli, H. P., Hill, D. L., and Bennett, L. L., Jr. (1967) J. Biol. Chem. 242,1997-2004 3. Divekar, A.Y., and Hakala, M. T. (1971) Mol.Pharmacol. 7, 663-673 4. Meyskens, F. L., and Williams, H. E. (1971) Biochim. Biophys. Acta 2 4 0 , 170-179 5. Krenitsky, T. A., Miller, R. L., and Fyfe, J. A. (1974) Biochem. Pharmacol. 23, 170-172 6. Murray, A. W., Elloitt, D.C., and Atkinson, M. R. (1970) in Progress in Nucleic Acid Research and MolecularBiology (Dav-

from L. donovani 7.

8.

9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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