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An adenosine kinase mutation in baby hamster kidney cells causing increased sensitivity to adenosine
Mutation Research, 129 (1984) 397-402
397
Elsevier MTR 03958
An adenosine kinase mutation in baby hamster kidney cells causing increased sensitivity to adenosine P. Juranlca, F. Meffe, S. Guttman, S.M. Archer and V.L. Chan Department of Microbiology, University of Toronto, Toronto, Ont. (Canada) (Received 9 November 1983) (Revision received 6 June 1984) (Accepted 27 July 1984)
Summary A class of arabinosyladenine (araA)-resistant mutants of baby hamster kidney ( B H K 21/C13) cells exhibits multiple phenotypes: resistance to araA and deoxyadenosine, extreme sensitivity to adenosine (Ado) and varying degrees of deficiency in adenosine kinase (AK) activity. One of these AdoS/araA r strains, ara-S10d, was isolated without mutagenesis and was shown to possess about 59% level of the wild-type A K activity. The A K from ara-Sl0d had an altered K m and p H optimum and was stimulated by K + cations. A number of Ado ~ to Ado ~ revertants were isolated from ara-S10d, and in all of the 7 examined, the A K activity was reduced to a nondetectable level. The altered kinetic parameters of the A K enzyme in ara-S10d cells suggest a mutation of the A K gene that leads to the synthesis of an altered enzyme. The loss of A K activity in the Ado ~ revertants suggests an association of the enhanced Ado sensitivity to the A K mutation.
Recently we (Chan and Juranka, 1981) reported the isolation of a large number of arabinosyladenine (araA)-resistant mutants of baby hamster kidney ( B H K 21/C13) cells. These araA-resistant ( a r a N ) mutants can be classified into 3 distinct phenotypic groups with apparently different mechanisms of resistance. All of the araA r mutants show cross resistant to deoxyadenosine (dAdo). The major group (class I) of araA r mutants are adenosine kinase (AK) deficient. The resistance to araA and to dAdo is probably due to lower intracellular levels of araATP and dATP as a consequence of the A K deficiency mutation. The resistance to araA and dAdo in the Class II araA ~
Correspondence to be addressed to: Dr. V.L. Chan, Department of Microbiology, FitzGerald Building, University of Toronto, Toronto, Ont MSS 1A8 (Canada). 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.
mutants is probably due to a mutation that altered the structure of the ribonucleoside diphosphate reductase (RDR) because R D R activity in resistant cells showed an increased resistance to inhibition by araATP and dATP (Chan et al., 1981). The Class III mutants, unlike those of Class I and II, exhibit extreme adenosine sensitivity (Chan and Juranka, 1981). All the AdoS/araA r mutants also exhibit an altered A K activity relative to the wild-type B H K cells. One of these AdoS/araA r mutants, ara-S10d, was derived spontaneously and cell-free extracts of this mutant possess 59% level of wild-type A K activity. This mutant was also shown to have an elevated rate of spontaneous mutation and the mutator property is associated with the Ado ~ mutation ( C h a n e t al., 1981). The apparent multiple alterations in the Ado s mutants raise an important question. Can the phenotype: Ado ~, araA r, dAdo r, A K deficiency
398 and it's demonstrated mutator activity, be attributed to a single mutation? In this communication we report results that implicate the involvement of a mutation in the A K gene, as being responsible for the increased adenosine sensitivity and the alterations of the kinetic behaviour of the AK enzyme in ara-S10d cells. Experimental
procedures
Materials and methods Chemicals and sera. AraA, Ado and dAdo were from Sigma (St. Louis, MI). erythro-9-(2-Hydroxy3-nonyl)adenine (EHNA) was from Burroughs Wellcome Co. Horse serum was from Animal Health Laboratory (Toronto, Canada) and fetal calf serum was from Flow Laboratory (Rockville, MD). Cell lines, media, and culture conditions. The origin of the wild-type B H K 21/C13 (BHK) and the isolation of a r a N mutant ara-S10d have been previously described (Chan and Juranka, 1981). B H K cells were cultivated in plastic tissue-culture flasks at 34 ° in alpha-MEM medium (Stanners et al., 1971) lacking ribonucleosides and deoxyribonucleosides, supplemented with 5% fetal calf serum. In all the plating experiments alpha-MEM medium supplemented with 10% horse serum was used. EHNA, an adenosine deaminase inhibitor, was included in all plating experiments which were performed to determine toxicity of adenosine and deoxyadenosine. The cell cultures were found to be free of mycoplasma using the uridine phosphorylase assay (Levine, 1974). Selection of Ado ~'to Ado r revertants. Single-step spontaneous revertants were isolated from newly derived subclones of ara-S10d. The selection procedure involved plating log-phase cells into 100m m dishes, each containing 15 ml medium with 50 or 100 t~M adenosine plus 5 t~M EHNA. Plates were replenished with fresh selective medium on the 5th or 6th day, and the surviving colonies were picked on the 14th or 15th day. The cells were recloned and maintained in non-selective medium. Enzyme extracts and protein determination. Extracts were prepared from log-phase cells as described previously (Chan and Juranka, 1981). Protein content of the cell-free extracts was de-
termined using the method of Lowry et al. (1951). Adenosine kinase (A TP:adenosine 5'-phosphotransferase, EC 2.7.120) enzyme assays. The standard A K assay contained 50 mM Tris-maleic acid buffers (at various pHs), 1 m M dithiothreitol, 10/zM EHNA, 1.6 m M ATP, 1 m M MgC! 2 and 20 /~M [8-14C]adenosine (25 /~Ci//~mole). In experiments to determine the effect of cations, 100 mM NaCl or 100 mM KC1 was also included in the assay mixture. The reaction was initiated by the addition of the enzyme to the reaction mixture which was pre-equilibrated at 37 °. After incubation at 37°C for 5 or 10 min, an aliquot was removed and spotted onto DE81 filter discs. The discs were air-dried, washed in 2 m M ammonium formate, and counted in 10 ml AQUASOL. Results
In a previous paper we showed that the spontaneously derived Ado s mutant, ara-S10d, has about 59% level of wild-type A K activity (Chan and Juranka, 1981). The reduction in activity could not be attributed to the presence of an inhibitor as such inhibitory activity was not demonstrated in mixed mutant and wild-type cell-free extracts (Chan and Juranka, 198]). We questioned if the reduction in A K activity was due to a lower yield of the wild-type enzyme or alternatively due to a structural mutation of the AK enzyme. Hence we determined the p H optimum of the AK enzyme in cell-free extracts of wild-type B H K and ara-S10d cells. As can be seen in Fig. 1, the pH optimum of
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Fig. 1. Effect of pH on wild-type BHK and ara-S10d adenosine kinase activity. BHK (e) and ara-Sl0d (©).
399
the A K derived from B H K cells is 5.5, this is in agreement with the observations of other workers (Yamada et al., 1980; Miller et al., 1979a; Lindberg et al., 1967). In contrast the A K of the Ado s mutant, ara-S10d, has a p H optimum of 6.5; this is consistent with a structural change of the A K in ara-S10d cells. The K m for Ado was 11/~M and 50 btM for the A K enzymes of wild-type and ara-S10d cells respectively. The altered K m also suggests a structural change in the A K of ara-S10d cells. The g m for Ado obtained with purified A K enzyme is qualitatively similar to that obtained with crude enzymes (data to be published in a subsequent paper). In preliminary p H optimum experiments, we noted that the A K activity of ara-S10d cells was greatly elevated when assayed in the presence of K 2 H P O 4 but not N a 2 H P O 4 buffers (data not shown). The results presented in Fig. 2, reveal that K ÷ cations appear to stimulate ara-S10d A K activity 4-5-fold, whereas K ÷ cations have a slight inhibitory effect on wild-type A K activity. Arch and Newsholme (1978) have shown that K ÷ inhibits mouse brain A K activity. The Na ÷ cation does not affect ara-S10d A K activity. The B H K A K activity is stimulated 59% by Na ÷ at p H 5.6 (Fig. 2, panel A). Although we do not understand the biochemical basis for this stimulation of araS10d A K activity by K ÷ we do know that it is a property of the ara-S10d A K enzyme since the purified A K enzyme was also stimulated by K ÷
(Juranka and Chan, unpublished data). We reasoned that if the apparent multiple alterations (araAr/dAdor/AdoS/AK-deficiency) in the ara-S10d cells were due to a single mutation it should be possible to isolate Ado r revertants with alterations in A K levels and perhaps araA and dAdo resistance. To ensure independently derived revertants, subclones of ara-S10d were isolated. Using these subclones as the parents, single step Ado r colonies were isolated without mutagenic treatment (see Materials and Methods). The frequencies of Ado r colonies of various cultures and subclones of ara-S10d were in the range of 10 -6 and 10 -5. These Ado r colonies were picked and cloned in the absence of Ado. Cultures of these cells were plated in various concentrations of Ado in the presence of 5 # M EHNA, to determine if these clones had acquired inheritable resistance to Ado. The relative plating efficiency for 4 of these Ado r revertants are shown in fig. 3a. All the revertants, showed a significant increase in resistance to Ado. The wild-type B H K cells show a biphasic response to Ado. Adenosine at low concentration (1-5/~M) was found to be cytotoxic to B H K cells, however at higher concentration (10-50 /tM) Ado was relatively nontoxic until about 100 /~M. Ado toxicity in the 1-5 ~tM range can be prevented by the addition of 0.5 # M uridine and it is abolished by an AK-deficient mutation (Archer,
TABLE 1 R E L A T I V E A D E N O S I N E K IN A S E ACTIVITY ~
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B
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8
2
ilin C Na" K"
C Ncl" K"
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IGIG C
Cell strain
% of wild-type b
BHK (wild-type) ara-S10d R202A c R203A C7b C8a C7a C7c C9flR
100 59 < 0.02 < 0.13 < 0.45 < 0.09 < 0.01 < 0.01 1.9
Na" K +
Fig. 2. Effect of Na ÷ and K ÷ cations on wild-type BHK and ara-S10d adenosine kinase activity. The adenosine kinase activity of BHK (closed bars) and ara-S10d (open bars) was determined at pH 5.6 (panel A) and pH 7.4 (panel B) in the absence (C) or the presence of 100 mM NaCI (Na ÷ ) or 100 mM KCI (K ÷ ).
Each of the above enzymatic levels is a mean of duplicate assays from two or more experiments. b The specific A K activity of the wild-type BHK and ara-S10d cell-free extract was 4.34+0.63 and 2.56_+0.13 nmole A MP f o r m e d / m g prot e i n/ ra i n, respectively. c R202A and R203A were from ara-S10d; C7, C8, C9 were from subclones 7, 8 and 9 of ara-sl0d.
400
c
1.0
-
1.0
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v
0.1
t~J
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(o fie
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I 20
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I 60
40
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80
100
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Adenosine
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40
60
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Deoxyadenosine
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I 30
AraA
Ho, Juranka and Chan, unpublished data). None of the Ado r revertants show a biphasic response to Ado; a result similar to that of the Class I AK-deficient mutants (Archer, 1983). We measured the A K activity in cell-free extracts of each of the 7 revertants to determine if an Ado s to Ado r reversion would alter the A K level. As shown in Table 1, all 7 revertants contained no detectable A K activity. A deficiency in A K could not be attributed to the presence of an inhibitor as neither inhibition nor enhancement of A K activity was observed in all the 7 revertants and wild-type mixed extracts (data not included). Thus the Ado s to A d d reversion of ara-S10d is clearly associated with a complete loss of A K activity.
Fig. 3. Survival curve of wild-type BHK cells, the adenosinesensitive mutant ara-S10d and 4 adenosine-resistant revertants of ara-S10d in the presence of increasing concentrations of (a) adenosine plus 5 #M EHNA, (b) araA, and (c) deoxyadenosine plus 5 /~M EHNA. BHK (O), ara-S10d (i), C7a (zx), C7b (v), C8a (D) and C9flR ((3).
In order to determine if any of the A d d revertants showed a change in resistance to araA and dAdo, their relative plating efficiencies in increasing concentrations of araA (Fig. 3b) and dAdo (Fig. 3c) were determined. All the revertants tested displayed resistance to araA and dAdo as compared to the wild-type BHK cells and the level of resistance was not significantly different from that of the parental ara-S10d cells.
Discussion The observation that the A K enzyme of araS10d cells shows alteration in pH optimum, Km, and that it is stimulated by K ÷ cations, unlike the
401 wild-type enzyme, strongly suggests a structural change of the enzyme. The complete loss of A K activity in all 7 Ado r revertants provides further evidence that the Ado ~ phenotype is linked to the AK mutation in the ara-S10d cells. Since the 7 revertants were derived from 4 different subclones of ara-S10d, at least 4 of the 7 revertants are therefore from independent mutational events. The mechanism of extreme Ado sensitivity exhibited by ara-S10d and other Class III a r a N / A d o ~ mutants is still unresolved. A number of biochemical mechanisms have been suggested to explain adenosine toxicity in various mammalian cells (Fox and Kelley, 1978; Henderson and Scott, 1980). These include pyrimidine starvation (Ishii and Green, 1973; Green and Chan, 1973; Gudas et al., 1978), stimulation of adenylate cyclase by adenosine (Wolberg et al., 1975) and inhibition of S-adenosylmethionine-mediated methylation by the intracellular accumulation of S-adenosylhomocysteine (Kredich and Martin, 1977; Kredich and Hershfield, 1979). Studies in which high-performance liquid chromatography was used to analyze the level of Ado metabolites in Ado-treated Class III cells suggest that Ado toxicity in these mutants is associated with inhibition of pyrimidine nucleotide synthesis and with a marked elevation of adenine and guanine nucleotides, inosine monophosphate, S-adenosylmethionine and methylthioadenosine (manuscript in preparation). The mechanisms of resistance to araA and dAdo by the ara-S10d is not known. Both araA and dAdo are very poor substrates for AK (Miller et al., 1979b) presumably because of the absence of a 2'-hydroxyl group in trans to the fl-glycoside linkage, a conformation postulated to be required for substrates of adenosine kinase (Bennett and Hill, 1975). Thus, it is possible ara-S10d cells encode an altered AK enzyme which has a more stringent requirement for the trans-2'-hydroxyl group. We are currently purifying the A K enzyme from ara-S10d cells to determine its phosphorylating activity for araA and dAdo. The mechanism of resistance to araA and dAdo for the Ado ~ revertants is probably similar to that of the Class I araA r cells (Chan and Juranka, 1981) and is presumably due to the concurrent loss of phosphorylation activity for Ado, dAdo and araA (Ullman et al., 1978; Hershfield and Kredich, 1980; Hershfield et al., 1982).
A single mutation model for the multiple phenotypes: araAr/dAdor/Ado s and alteration in AK is consistent with the high frequency of isolation of araAr/Ado ~ Class III mutants (6 mutants out of 168 araN colonies tested; Chan and Juranka, 1981) and supported by the fact that ara-S10d was isolated without mutagenic treatment. The overall a r a N mutation frequency for nonmutagenized and mutagenized BHK cells were 6 X 10-s and 3.5 x 10 -4 respectively. Although we have shown that an AK mutation is linked to the Ado sensitivity of ara-S10d, there appears to be no correlation between AK levels and Ado sensitivity in the Class III cells. All of the Ado ~ mutants show similar sensitivity to Ado (Chan and Juranka, 1981), yet the level of AK determined at pH 5.5 (in the absence of KC1) in the four Ado ~ cells, ara-4c, ara-19a, ara-16c, and ara-S10d, were 0.6, 2, 7 and 59% of wild type activity, respectively (Juranka, 1981) while at pH 7.4 these activities were 4, 6, 20 and 170% respectively (Archer, 1983). A possible model for the Ado ~ mutation is that the AK enzyme has another activity or function in addition to the phosphorylation of Ado, araA and dAdo and the Ado s mutation alters this other function with a resulting increase in Ado sensitivity. Alternatively, the Ado ~ mutation, located in the AK gene, has a polar or enhancing effect on the adjacent gene(s) that control Ado sensitivity. Complete understanding of the pleiotropic AdoS/araA r mutations must await molecular cloning of the A K gene and DNA-sequence analyses of the mutations.
Acknowledgements This work was supported by the National Cancer Institute (NCI) and the Medical Research Council (MRC) of Canada. P. Juranka and S.M. Archer have a studentship from NCI and MRC respectively. We wish to thank Ms. Trudy Carroll for excellent typing.
References Arch, J.R.S., and E.A. Newsholme(1978) Activities and some properties of 5'-nucleotidase, adenosine kinase and adenosine deaminasein tissues from vertebratesand invertebrates in relation to the control of the concentration and the physiological role of adenosine, Biochem.J., 174, 965-977.
402 Archer, S.M. (1983) Mechanisms of adenosine toxicity in baby hamster kidney cells, M.Sc. thesis, Department of Microbiology, University of Toronto (Canda). Bennett, Jr., L.L., and D.L. Hill (1975) Structural requirements for activity of nucleosides as substrates for adenosine kinase: Orientation of substituents on the pentofuranosyl ring, Mol. Pharmacol., 11, 803-808. Chart, V.L., and P. Juranka (1981) Isolation and preliminary characterization of 9-fl-D-arabinofuranosyladenine-resistant mutants of baby hamster cells, Somat. Cell Genet., 7, 147-160. Chan, V.L., S. Guttman and P. Juranka (1981) Mutator genes of baby hamster kidney cells, Mol. Cell. Biol., 1, 568-571. Fox, I.H,, and W.N. Kelley (1978) The role of adenosine and 2'-deoxyadenosine in mammalian cells, Annu. Rev. Biochem., 47, 655-686. Green, H., and T.S. Chan (1973) Pyrimidine starvation induced by adenosine in fibroblast and lymphoid cells: Role of adenosine deaminase, Science, 182, 836-837. Gudas, L.J., A. Cohen, B. Ullman and D.W. Martin Jr. (1978) Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells, Somat. Cell Genet., 4, 201-219. Henderson, J.F., and F.W. Scott (1980) Inhibition of animal and invertebrate cell growth by naturally occurring purine bases and ribonucleotides, Pharmac. Ther., 8, 539-571. Hershfield, M.S., and N.M. Kredich (1980) Resistance of an adenosine kinase-deficient human lymphoblastoid cell line to effects of deoxyadenosine on growth, S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation, Proc. Natl. Acad. Sci. (U.S.A.), 77, 4292-4296. Hershfield, M.S., J.E. Fetter, W.C. Small, A.S. Bagnara, S.R. Williams, B. Ullman, D.W. Martin Jr., D.B. Wasson and D.A. Carson (1982) Effects of mutational loss of adenosine kinase and deoxycytidine kinase on deoxy ATP accumulation and deoxyadenosine toxicity in cultural CEM human T-lymphoblastoid cells, J. Biol. Chem., 257, 6380-6386. Ishii, K., and H. Green (1973) Lethality of adenosine for cultured mammalian cells by intereference with pyrimidine biosynthesis, J. Cell. Sci., 13, 429-439. Juranka, P. (1981) Isolation and preliminary characterization of
9-fl-D-arabinofuranosyladenine-resistant mutants of baby hamster kidney cells, M.Sc. thesis, Department of Microbiology, University of Toronto (Canada). Kredich, N.M., and M.S. Hershfield (1979) S-Adenosylhomocysteine toxicity in normal and adenosine kinase deficient lymphoblasts of human origin, Proc. Natl. Acad. Sci. (U.S.A.), 76, 2450-2454. Kredich, N.M., and D.W. Martin Jr. (1977) Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells, Cell, 12, 931-938. Levine, E.M. (1974) A simplified method for the detection of mycoplasma, Meth. Cell. Biol., 8, 229-248. Lindberg, B., H. Klenow and K. Hansen (1967) Some properties of partially purified mammalian adenosine kinase~ J. Biol. Chem., 242, 350-356. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. Miller, R.L., D.L. Adamczyk and W.H. Miller (1979a) Adenosine kinase from rabbit liver, I. Purification by affinity chromatography and properties, J, Biol. Chem., 254, 2339-2345. Miller, R.L., D.L. Adamczyk, W.H. Miller, G.W. Koszalka, J.L. Rideout, L.M. Beacham I11, E.Y. Chao, J.J. Haggerty, T.A., Krenitsky and G.B. Elion (1979b) Adenosine kinase from rabbit liver, II. Substrate and inhibitor specificity, J. Biol. Chem., 254, 2346-2352. Stanners, C.P., G.L. Eliceiri and H. Green (1971) Two types of ribosome in mouse-hamster hybrid cells, Nature (London) New Biol., 230, 52-54. Ullman, B., L.J. Gudas, A. Cohen and D.W. Martin Jr. (1978) Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: A model for immunodeficiency disease, Cell, 14, 365-375. Wolberg, G., T.P. Zimmerman, K. Hiemstra, M. Winston and L.C. Chu (1975) Adenosine inhibition of lymphocyte-mediated cytolysis: Possible role of cyclic AMP, Science, 187, 957-959. Yamada, Y., H. Goto and N. Ogasawara (1980) Purification and properties of adenosine kinase from rat brain, Biochim. Biophys. Acta, 616, 199-207.
397
Elsevier MTR 03958
An adenosine kinase mutation in baby hamster kidney cells causing increased sensitivity to adenosine P. Juranlca, F. Meffe, S. Guttman, S.M. Archer and V.L. Chan Department of Microbiology, University of Toronto, Toronto, Ont. (Canada) (Received 9 November 1983) (Revision received 6 June 1984) (Accepted 27 July 1984)
Summary A class of arabinosyladenine (araA)-resistant mutants of baby hamster kidney ( B H K 21/C13) cells exhibits multiple phenotypes: resistance to araA and deoxyadenosine, extreme sensitivity to adenosine (Ado) and varying degrees of deficiency in adenosine kinase (AK) activity. One of these AdoS/araA r strains, ara-S10d, was isolated without mutagenesis and was shown to possess about 59% level of the wild-type A K activity. The A K from ara-Sl0d had an altered K m and p H optimum and was stimulated by K + cations. A number of Ado ~ to Ado ~ revertants were isolated from ara-S10d, and in all of the 7 examined, the A K activity was reduced to a nondetectable level. The altered kinetic parameters of the A K enzyme in ara-S10d cells suggest a mutation of the A K gene that leads to the synthesis of an altered enzyme. The loss of A K activity in the Ado ~ revertants suggests an association of the enhanced Ado sensitivity to the A K mutation.
Recently we (Chan and Juranka, 1981) reported the isolation of a large number of arabinosyladenine (araA)-resistant mutants of baby hamster kidney ( B H K 21/C13) cells. These araA-resistant ( a r a N ) mutants can be classified into 3 distinct phenotypic groups with apparently different mechanisms of resistance. All of the araA r mutants show cross resistant to deoxyadenosine (dAdo). The major group (class I) of araA r mutants are adenosine kinase (AK) deficient. The resistance to araA and to dAdo is probably due to lower intracellular levels of araATP and dATP as a consequence of the A K deficiency mutation. The resistance to araA and dAdo in the Class II araA ~
Correspondence to be addressed to: Dr. V.L. Chan, Department of Microbiology, FitzGerald Building, University of Toronto, Toronto, Ont MSS 1A8 (Canada). 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.
mutants is probably due to a mutation that altered the structure of the ribonucleoside diphosphate reductase (RDR) because R D R activity in resistant cells showed an increased resistance to inhibition by araATP and dATP (Chan et al., 1981). The Class III mutants, unlike those of Class I and II, exhibit extreme adenosine sensitivity (Chan and Juranka, 1981). All the AdoS/araA r mutants also exhibit an altered A K activity relative to the wild-type B H K cells. One of these AdoS/araA r mutants, ara-S10d, was derived spontaneously and cell-free extracts of this mutant possess 59% level of wild-type A K activity. This mutant was also shown to have an elevated rate of spontaneous mutation and the mutator property is associated with the Ado ~ mutation ( C h a n e t al., 1981). The apparent multiple alterations in the Ado s mutants raise an important question. Can the phenotype: Ado ~, araA r, dAdo r, A K deficiency
398 and it's demonstrated mutator activity, be attributed to a single mutation? In this communication we report results that implicate the involvement of a mutation in the A K gene, as being responsible for the increased adenosine sensitivity and the alterations of the kinetic behaviour of the AK enzyme in ara-S10d cells. Experimental
procedures
Materials and methods Chemicals and sera. AraA, Ado and dAdo were from Sigma (St. Louis, MI). erythro-9-(2-Hydroxy3-nonyl)adenine (EHNA) was from Burroughs Wellcome Co. Horse serum was from Animal Health Laboratory (Toronto, Canada) and fetal calf serum was from Flow Laboratory (Rockville, MD). Cell lines, media, and culture conditions. The origin of the wild-type B H K 21/C13 (BHK) and the isolation of a r a N mutant ara-S10d have been previously described (Chan and Juranka, 1981). B H K cells were cultivated in plastic tissue-culture flasks at 34 ° in alpha-MEM medium (Stanners et al., 1971) lacking ribonucleosides and deoxyribonucleosides, supplemented with 5% fetal calf serum. In all the plating experiments alpha-MEM medium supplemented with 10% horse serum was used. EHNA, an adenosine deaminase inhibitor, was included in all plating experiments which were performed to determine toxicity of adenosine and deoxyadenosine. The cell cultures were found to be free of mycoplasma using the uridine phosphorylase assay (Levine, 1974). Selection of Ado ~'to Ado r revertants. Single-step spontaneous revertants were isolated from newly derived subclones of ara-S10d. The selection procedure involved plating log-phase cells into 100m m dishes, each containing 15 ml medium with 50 or 100 t~M adenosine plus 5 t~M EHNA. Plates were replenished with fresh selective medium on the 5th or 6th day, and the surviving colonies were picked on the 14th or 15th day. The cells were recloned and maintained in non-selective medium. Enzyme extracts and protein determination. Extracts were prepared from log-phase cells as described previously (Chan and Juranka, 1981). Protein content of the cell-free extracts was de-
termined using the method of Lowry et al. (1951). Adenosine kinase (A TP:adenosine 5'-phosphotransferase, EC 2.7.120) enzyme assays. The standard A K assay contained 50 mM Tris-maleic acid buffers (at various pHs), 1 m M dithiothreitol, 10/zM EHNA, 1.6 m M ATP, 1 m M MgC! 2 and 20 /~M [8-14C]adenosine (25 /~Ci//~mole). In experiments to determine the effect of cations, 100 mM NaCl or 100 mM KC1 was also included in the assay mixture. The reaction was initiated by the addition of the enzyme to the reaction mixture which was pre-equilibrated at 37 °. After incubation at 37°C for 5 or 10 min, an aliquot was removed and spotted onto DE81 filter discs. The discs were air-dried, washed in 2 m M ammonium formate, and counted in 10 ml AQUASOL. Results
In a previous paper we showed that the spontaneously derived Ado s mutant, ara-S10d, has about 59% level of wild-type A K activity (Chan and Juranka, 1981). The reduction in activity could not be attributed to the presence of an inhibitor as such inhibitory activity was not demonstrated in mixed mutant and wild-type cell-free extracts (Chan and Juranka, 198]). We questioned if the reduction in A K activity was due to a lower yield of the wild-type enzyme or alternatively due to a structural mutation of the AK enzyme. Hence we determined the p H optimum of the AK enzyme in cell-free extracts of wild-type B H K and ara-S10d cells. As can be seen in Fig. 1, the pH optimum of
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55
6
6.5
7
7.5
1 8
8.5
pH
Fig. 1. Effect of pH on wild-type BHK and ara-S10d adenosine kinase activity. BHK (e) and ara-Sl0d (©).
399
the A K derived from B H K cells is 5.5, this is in agreement with the observations of other workers (Yamada et al., 1980; Miller et al., 1979a; Lindberg et al., 1967). In contrast the A K of the Ado s mutant, ara-S10d, has a p H optimum of 6.5; this is consistent with a structural change of the A K in ara-S10d cells. The K m for Ado was 11/~M and 50 btM for the A K enzymes of wild-type and ara-S10d cells respectively. The altered K m also suggests a structural change in the A K of ara-S10d cells. The g m for Ado obtained with purified A K enzyme is qualitatively similar to that obtained with crude enzymes (data to be published in a subsequent paper). In preliminary p H optimum experiments, we noted that the A K activity of ara-S10d cells was greatly elevated when assayed in the presence of K 2 H P O 4 but not N a 2 H P O 4 buffers (data not shown). The results presented in Fig. 2, reveal that K ÷ cations appear to stimulate ara-S10d A K activity 4-5-fold, whereas K ÷ cations have a slight inhibitory effect on wild-type A K activity. Arch and Newsholme (1978) have shown that K ÷ inhibits mouse brain A K activity. The Na ÷ cation does not affect ara-S10d A K activity. The B H K A K activity is stimulated 59% by Na ÷ at p H 5.6 (Fig. 2, panel A). Although we do not understand the biochemical basis for this stimulation of araS10d A K activity by K ÷ we do know that it is a property of the ara-S10d A K enzyme since the purified A K enzyme was also stimulated by K ÷
(Juranka and Chan, unpublished data). We reasoned that if the apparent multiple alterations (araAr/dAdor/AdoS/AK-deficiency) in the ara-S10d cells were due to a single mutation it should be possible to isolate Ado r revertants with alterations in A K levels and perhaps araA and dAdo resistance. To ensure independently derived revertants, subclones of ara-S10d were isolated. Using these subclones as the parents, single step Ado r colonies were isolated without mutagenic treatment (see Materials and Methods). The frequencies of Ado r colonies of various cultures and subclones of ara-S10d were in the range of 10 -6 and 10 -5. These Ado r colonies were picked and cloned in the absence of Ado. Cultures of these cells were plated in various concentrations of Ado in the presence of 5 # M EHNA, to determine if these clones had acquired inheritable resistance to Ado. The relative plating efficiency for 4 of these Ado r revertants are shown in fig. 3a. All the revertants, showed a significant increase in resistance to Ado. The wild-type B H K cells show a biphasic response to Ado. Adenosine at low concentration (1-5/~M) was found to be cytotoxic to B H K cells, however at higher concentration (10-50 /tM) Ado was relatively nontoxic until about 100 /~M. Ado toxicity in the 1-5 ~tM range can be prevented by the addition of 0.5 # M uridine and it is abolished by an AK-deficient mutation (Archer,
TABLE 1 R E L A T I V E A D E N O S I N E K IN A S E ACTIVITY ~
E "x .c
B
16
I
T_
E ~
8
2
ilin C Na" K"
C Ncl" K"
C
No + K °
IGIG C
Cell strain
% of wild-type b
BHK (wild-type) ara-S10d R202A c R203A C7b C8a C7a C7c C9flR
100 59 < 0.02 < 0.13 < 0.45 < 0.09 < 0.01 < 0.01 1.9
Na" K +
Fig. 2. Effect of Na ÷ and K ÷ cations on wild-type BHK and ara-S10d adenosine kinase activity. The adenosine kinase activity of BHK (closed bars) and ara-S10d (open bars) was determined at pH 5.6 (panel A) and pH 7.4 (panel B) in the absence (C) or the presence of 100 mM NaCI (Na ÷ ) or 100 mM KCI (K ÷ ).
Each of the above enzymatic levels is a mean of duplicate assays from two or more experiments. b The specific A K activity of the wild-type BHK and ara-S10d cell-free extract was 4.34+0.63 and 2.56_+0.13 nmole A MP f o r m e d / m g prot e i n/ ra i n, respectively. c R202A and R203A were from ara-S10d; C7, C8, C9 were from subclones 7, 8 and 9 of ara-sl0d.
400
c
1.0
-
1.0
v
•
hl
:
c
ft.
v
0.1
t~J
\
(o fie
I
I 20
I
I 60
40
)uM
I
I
80
100
0.1 o
Adenosine
1.0
0.01 c o w
I o
g
0.!
2o
juM
o
i
I
l
I
40
60
8o
~oo
Deoxyadenosine
o
0.01 I
I
I
0
10
20
~M
I 30
AraA
Ho, Juranka and Chan, unpublished data). None of the Ado r revertants show a biphasic response to Ado; a result similar to that of the Class I AK-deficient mutants (Archer, 1983). We measured the A K activity in cell-free extracts of each of the 7 revertants to determine if an Ado s to Ado r reversion would alter the A K level. As shown in Table 1, all 7 revertants contained no detectable A K activity. A deficiency in A K could not be attributed to the presence of an inhibitor as neither inhibition nor enhancement of A K activity was observed in all the 7 revertants and wild-type mixed extracts (data not included). Thus the Ado s to A d d reversion of ara-S10d is clearly associated with a complete loss of A K activity.
Fig. 3. Survival curve of wild-type BHK cells, the adenosinesensitive mutant ara-S10d and 4 adenosine-resistant revertants of ara-S10d in the presence of increasing concentrations of (a) adenosine plus 5 #M EHNA, (b) araA, and (c) deoxyadenosine plus 5 /~M EHNA. BHK (O), ara-S10d (i), C7a (zx), C7b (v), C8a (D) and C9flR ((3).
In order to determine if any of the A d d revertants showed a change in resistance to araA and dAdo, their relative plating efficiencies in increasing concentrations of araA (Fig. 3b) and dAdo (Fig. 3c) were determined. All the revertants tested displayed resistance to araA and dAdo as compared to the wild-type BHK cells and the level of resistance was not significantly different from that of the parental ara-S10d cells.
Discussion The observation that the A K enzyme of araS10d cells shows alteration in pH optimum, Km, and that it is stimulated by K ÷ cations, unlike the
401 wild-type enzyme, strongly suggests a structural change of the enzyme. The complete loss of A K activity in all 7 Ado r revertants provides further evidence that the Ado ~ phenotype is linked to the AK mutation in the ara-S10d cells. Since the 7 revertants were derived from 4 different subclones of ara-S10d, at least 4 of the 7 revertants are therefore from independent mutational events. The mechanism of extreme Ado sensitivity exhibited by ara-S10d and other Class III a r a N / A d o ~ mutants is still unresolved. A number of biochemical mechanisms have been suggested to explain adenosine toxicity in various mammalian cells (Fox and Kelley, 1978; Henderson and Scott, 1980). These include pyrimidine starvation (Ishii and Green, 1973; Green and Chan, 1973; Gudas et al., 1978), stimulation of adenylate cyclase by adenosine (Wolberg et al., 1975) and inhibition of S-adenosylmethionine-mediated methylation by the intracellular accumulation of S-adenosylhomocysteine (Kredich and Martin, 1977; Kredich and Hershfield, 1979). Studies in which high-performance liquid chromatography was used to analyze the level of Ado metabolites in Ado-treated Class III cells suggest that Ado toxicity in these mutants is associated with inhibition of pyrimidine nucleotide synthesis and with a marked elevation of adenine and guanine nucleotides, inosine monophosphate, S-adenosylmethionine and methylthioadenosine (manuscript in preparation). The mechanisms of resistance to araA and dAdo by the ara-S10d is not known. Both araA and dAdo are very poor substrates for AK (Miller et al., 1979b) presumably because of the absence of a 2'-hydroxyl group in trans to the fl-glycoside linkage, a conformation postulated to be required for substrates of adenosine kinase (Bennett and Hill, 1975). Thus, it is possible ara-S10d cells encode an altered AK enzyme which has a more stringent requirement for the trans-2'-hydroxyl group. We are currently purifying the A K enzyme from ara-S10d cells to determine its phosphorylating activity for araA and dAdo. The mechanism of resistance to araA and dAdo for the Ado ~ revertants is probably similar to that of the Class I araA r cells (Chan and Juranka, 1981) and is presumably due to the concurrent loss of phosphorylation activity for Ado, dAdo and araA (Ullman et al., 1978; Hershfield and Kredich, 1980; Hershfield et al., 1982).
A single mutation model for the multiple phenotypes: araAr/dAdor/Ado s and alteration in AK is consistent with the high frequency of isolation of araAr/Ado ~ Class III mutants (6 mutants out of 168 araN colonies tested; Chan and Juranka, 1981) and supported by the fact that ara-S10d was isolated without mutagenic treatment. The overall a r a N mutation frequency for nonmutagenized and mutagenized BHK cells were 6 X 10-s and 3.5 x 10 -4 respectively. Although we have shown that an AK mutation is linked to the Ado sensitivity of ara-S10d, there appears to be no correlation between AK levels and Ado sensitivity in the Class III cells. All of the Ado ~ mutants show similar sensitivity to Ado (Chan and Juranka, 1981), yet the level of AK determined at pH 5.5 (in the absence of KC1) in the four Ado ~ cells, ara-4c, ara-19a, ara-16c, and ara-S10d, were 0.6, 2, 7 and 59% of wild type activity, respectively (Juranka, 1981) while at pH 7.4 these activities were 4, 6, 20 and 170% respectively (Archer, 1983). A possible model for the Ado ~ mutation is that the AK enzyme has another activity or function in addition to the phosphorylation of Ado, araA and dAdo and the Ado s mutation alters this other function with a resulting increase in Ado sensitivity. Alternatively, the Ado ~ mutation, located in the AK gene, has a polar or enhancing effect on the adjacent gene(s) that control Ado sensitivity. Complete understanding of the pleiotropic AdoS/araA r mutations must await molecular cloning of the A K gene and DNA-sequence analyses of the mutations.
Acknowledgements This work was supported by the National Cancer Institute (NCI) and the Medical Research Council (MRC) of Canada. P. Juranka and S.M. Archer have a studentship from NCI and MRC respectively. We wish to thank Ms. Trudy Carroll for excellent typing.
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