Thymidine kinase activity and thymidine salvage in adult Brugia pahangi and Dirofilaria immitis

Molecular and Biochemical Parasitology, 5 (1982) 361 - 370 Elsevier Biomedical Press

361

THYMIDINE KINASE ACTIVITY AND THYMIDINE SALVAGE IN ADULT BR UGIA P A H A N G I A N D DIR O F I L A R I A IMMITIS

JULIAN J. JAFFE, JOHN C.W. COMLEY and LYNN R. CHRIN

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, U.S.A. (Received 15 September 1981, accepted 13 February 1982)

Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective K m values for thymidine and ATP were 60 #M and 1.6 mM, and 5-iodo-2'-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5'-triphosphate. Adult female B. pahangi took up and incorporated [methyl -3 H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctionalin vitro. Key words: Brugia pahangi, Dirofilaria immitis, Thymidine kinase, Thymidine salvage pathway.

INTRODUCTION Chen and HoweUs [ 1 - 3 ] reported that neither adult Brugia pahangi nor Dirofilaria

immitis took up and incorporated thymidine in vitro under conditions that allowed the efficient uptake and incorporation of exogenous uracil and purines. They suggested that these filariae may be unable to use preformed thymidine (or thymine) as precursors of the corresponding deoxynucleoside triphosphate form that is incorporated into DNA, relying instead upon de novo synthesis of TMP. The demonstration of TMP synthetase (EC 2.1.2.45) activity in homogenates of adult B. pahangi and D. immitis [4] indicated tile capacity of these fflariae to produce TMP from dUMP. If it could be confirmed that adult filariae cannot use preformed thymidine, this would have important implications in the formulation of a strategy for antifilarial .chemotherapy, for it would in-

Abbreviations: DEC, diethylcarbamazine; TCA, trichloroacetic acid. 0166-6851/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

362 dicate that any strong and/or selective inhibitor of •arial TMP synthetase eventually should be able at least to induce infertility in these parasites. The objectives of the present study were: (1) to determine whether homogenates of adult B. pahangi and 1). immitis possessed thymidine kinase (EC 2.7.1.75) activity, this being the initial enzyme in the thymidine salvage pathway; and (2) to compare the fate of exogenous [methyl -3 H] thymidine presented to adult female B. pahangi in vitro and in vivo. MATERIALS AND METHODS The source of adult B. pahangi and D. immitis, the procedure for preparing cytosolic fractions of worm homogenates and in the case of B. pahangi, that for their short-term maintenance in vitro were as previously described, except that the original folate content of the incubation medium was not altered [5]. Sephadex G-200 gel f'titration was performed on dialyzed crude extracts as previously described [6], providing partially purified cytosolic thymidine kinases for further characterization and permitting estimation of their molecular weights.

Enzyme assay. Thymidine kinase activity was measured in a 0.2 ml reaction system containing 60 mM Tris-HC1 buffer (pH 7.5), 3.5 mM ATP, 7.0 mM MgC12, 0.8 IU creatine phosphokinase (EC 2.7.3.2), 24 mM creatine phosphate, 0.2 mM [2-14C] thymidine (57 Ci/mol) and fflarial cytosolic extract (0.05 ml). The reaction was stopped after 30 min at 37°C by placing the reaction vessel in boiling water for 2 min, and the contents were then centrifuged at 17 500 × g for 15 min. It was previously determined that the reaction was linear with time for 60 min and that its rate was proportional to the amount of extract. Reaction products in 20 #1 aliquots of the supernatant fraction were separated by thin-layer chromatography using Eastman Chromogram sheets (cellulose adsorbent with fluorescent indicator). Before development of the chromatogram, aqueous solutions of nonradioactive thymidine and TMP (5 ol of 1 mM solutions) were applied as overlays and served as carriers; the same amounts applied separately served as markers. Following development to 15 cm with a solvent system composed of 1.0 M ammonium acetate (pH 7.5)/95% ethanol (7:3, v/v), the R E of thymidine was 0.91 and that of TMP 0.79. Visualized by ultraviolet light at 254 nm, the TMP-containing spot was cut out and its radioactive content was measured by liquid scintillation spectrometry using a toluene/ ethanol (14:5, v/v) mixture containing 0.23% 2,5-diphenyloxazole. The amount of quenching was determined by means of an internal standard. Thymidine kinase activity is expressed as nmol TMP formed/h/mg protein. The protein content of the •arial extracts was determined by the method of Lowry et al. [7], using crystalline bovine serum albumin as a standard. Exposure of B. pahangi to [methyl-3H]thymidine in vitro. Two batches of 20 adult female B. pahangi, freshly removed from the peritoneal cavity of an infected jird (Mer-

363

iones unguiculatus) after washing were placed in 20 ml of medium containing 20/aCi [methyl.all] thymidine (specific activity = 64 Ci/mmol). After incubation at 37°C for 24 h under aseptic conditions, both batches of worms were transferred into the same volume of fresh medium with radiolabeled thymidine omitted, and they were incubated for an additional 24 h. The worms were removed, thoroughly washed and blotted dry, and each batch was placed in 0.5 ml of ice-cold 5% trichloroacetic acid (TCA) to prepare them for biochemical fractionation.

Exposure of B. pahangi to [methyl-3H] thymidine in vivo. Six jirds weighing around 80 g and harboring 4 0 - 2 0 0 adult male and female B. pahangi in their peritoneal cavities each received 5 intraperitoneal injections of a sterile saline solution of [methyl-3H]thymidine (specific activity = 64 Ci/mmol) at 2 h intervals, each at a dose of 0.33 pCi/g body weight in a volume of 0.2 ml. The jirds were killed 18 h after the last injection to provide the opportunity for tritiated water derived from [methyl-3H]thymidine [8] to leach from the parasites in situ and be excreted by the hosts. The worms were removed and thoroughly washed before batches of 10 females were selected at random for biochemical fractionation and further analysis.

Analysis of filariae exposed to [methyl-all] thymidine in vitro and in vivo. Worms were homogenized in ice-cold 5% TCA and fractionated by a slightly modified Schmidt-Thannhauser procedure [9] to separate acid-soluble substances, lipids, RNA, DNA and proteins. The radioactivity in each of these fractions was measured by liquid scintillation spectrometry. To determine whether the radioactivity in the acid-insoluble macromolecular fraction of worms exposed to [methyl -3HI thymidine in vivo was specifically localized in DNA, several such fractions were pooled and suspended in 2 ml of 0.1 potassium phosphate buffer (pH 7.0) containing 5 mM MgCI:. The suspension was divided into two equal parts, DNAase I (100/.tg) was added to one and RNAase A (200 pg) to the other. Each was incubated for 2 h at 37°C; the digests were rapidly cooled to 0°C and 1 ml of 10% ice-cold TCA was added to each. The distribution of radioactivity in acid-soluble and -insoluble fractions of both digests was compared. To confirm that the radioactivity in the DNA of worms exposed to [methyl -3 H]thymidine in vivo was specifically associated with thymidine nucleotides, several batches of DNA (with protein) remaining after removal of RNA from acid-insoluble fractions by alkaline digestion were pooled and freed of TCA by 5 successive extractions with ether. This pooled DNA, the radioactive content of which was predetermined, was subjected to sequential enzymic digestion by DNAase I and phosphodiesterase 1 (Crotalus adamanteus venom, Type II) according to the procedure of Buteau and Simmons [10], to obtain deoxynucleoside 5'-monophosphate forms of the constituent nucleotides. These were then extracted with chloroform/isoamyl alcohol (24:1, v/v) [10], freezedried, taken up in 200/gl of water, and 50 pl aliquots were spotted on thin-layer plates coated with MN 300F cellulose (Uniplate, Analtech). Before development of the chro-

364 matogram, TMP and dCMP (5 /A of 1 mM aqueous solutions) were applied as overlays and served as carriers; the same amounts applied separately served as markers. Following development to 15 cm with a solvent system composed of isobutyrate/NH4OH/water (66:1:33, v/v/v), the R v of TMP was 0.61 and that of dCMP was 0.68. The spots were visualized under ultraviolet light at 254 nm, scraped off the plate, and the amount of radioactivity in each was measured by liquid scintillation spectrometry.

Materials. [methyl. 3H] Thymidine was purchased from the New England Nuclear Corp., Boston, MA and [2 -14C] thymidine from Amersham Corp., Arlington Heights, IL. Creatine phosphokinase and creatine phosphate were from Calbiochem-Behring Corp., La JoUa, CA. All nonradioactive nucleosides and nucleotides were from P-L Biochemical Corp., Milwaukee, WI. DNAase I was from Worthington Biochemical Corp., Freehold, NJ; RNAase A, Type-I-A, and Crotalus admanteus venom, Type II, were from Sigma Chemical Co., St. Louis, MO. RESULTS

Activity and properties of filarial thymidine kinases. Thymidine kinase activity in crude cytosolic fractions of adult female D. immitis and B. pahangi homogenates was, respectively, 460 and 100 nmol/h/mg protein. Despite this four-fold difference, the activity of this thymidine salvage enzyme in both these filariae was markedly higher than that of TMP synthetase (0.3 nmol/h/mg protein) in identically prepared homogenates of both [4]. Various properties of the partially purified t'darial thymidine kinases were investigated, and the Findings are summarized in Table I. It is noteworthy that the specific activities of these partially purified enzymes from D. immitis and B. pahangi respectively were 6400 and 560 nmol/h/mg protein. Like other thymidine kinases, those from D. immitis and B. pahangi were most active when Mg2÷ was present. The activity of both enzymes was reduced by 15% when an equimolar amount of Mn2+ was substituted, and most of their activity was reduced when Mg2÷ was replaced by Fe 2+, Ca z+, Ba2+ or Zn 2+. However, the activity of the D. immitis enzyme was reduced by only 36% when Co 2+ was substituted for Mg2÷, whereas that of the B. pahangi enzyme was reduced almost completely in the presence of Co 2÷ (Table I). Both filarial thymidine kinases were dependent on the presence of both ATP and thymidine. Among six nucleoside 5'-triphosphates tested as phosphate donors in the reaction, ATP was the most efficient. Equimolar amounts of GTP, UTP and CTP were around 60% as efficient as ATP, dCTP was only 20% as efficient, and both enzymes were unable to use ITP. The ability of equimolar amounts of IdUrd, F3 dThd, dUrd, dCyd or Urd to act as inhibitors of the thymidine kinases in the presence of thymidine (0.2 mM) was investigated. IdUrd caused a 50% decrease in the rate of formation of TMP by both enzymes, F3dThd and dUrd a 10% decrease, whereas dCyd and Urd did not affect the reaction rate.

365

TABLE I Comparative properties of partially purified thymidine kinases from adult female Dirofilaria immitis and Brugia pahangi Property

D. immitis

Specific activity (nmol/h/mg protein) Molecular weight

B. pahangi

6400

560

180 000

185 000

pH optimum

7.0

7.0

Reaction promotion by 2* ions (% of optimal activity)

Mg (100); Mn (85); Co (64)

Mg (100); Mn (88); Co (6)

Efficacy of phosphate donors (% of optimal activity)

ATP (100); GTP (55); UTP (65); CTP (60); dCTP (20); ITP (0)

ATP (100); GTP (60); UTP (65); CTP (60); dCTP (20); ITP (0)

% Inhibition by deoxynucleosides (equimolar with thymidine)

IdUrd (50); F3 dThd (10); dUrd (10); dCyd (0)

IdUrd (50); F3 dThd (10); dUrd (10); dCyd (0)

Heat stability

-

84% at 42° C/15 min

-85% at 42° C/15 min

K m thymidine (mM)

0.06

0.06

K m ATP (mM)

1.6

1.6

% inhibition by TTP (equimolar with thymidine)

20

60

Suramin

50

50

Diethylcarbamazine

30

34

Sensitivity to drugs (% inhibition at 1 mM)

Preincubation of both fflarial thymidine kinases for 15 rain at 42°C in the absence of added thymidine caused a subsequent loss of over 80% of original activity. Both of these enzymes had the same sharp pH optL,n u m (pH 7.0), and both were eluted from a calibrated Sephadex G-200 colunm [6] in a single peak corresponding to a molecular weight of approximately 180 000. Apparent Michaelis constants (Kin) for thymidine and ATP were determined by double reciprocal plots [11], shown in Figs. 1 and 2. Both filarial thymidine kinases

366

n,

'z

0-2-

u.i

~E

_,<

0-1"

!

20

-20

,,~/'TH YM ID INE (mM)

!

40

i -0-5

!

!

0-5

1-0

YATP (raM)

Fig. 1. Double-reciprocal plot of the rate of formation of thymidine monophosphate as a function of thymidine concentration. Thymidine kinases from Brugia pahangi, (3--------o;and Dirofilaria immitis, A w

v .

Fig. 2. Double reciprocal plot of the rate of formation of thymidine monophosphate as a function of ATP concentration. Thymidine kinases from Brugiapahangi, O-----o;and Dirofilaria immitis, = =.

had K m values of 60 ~M for thymidine and 1.6 mM for ATP, values that were unaffected by a wide range o f concentrations o f added dCDP or dCTP. Thymidine kinase from B. pahangi was significantly more sensitive to feedback inhibition by TTP than that from D. immitis. The activity of the former was inhibited 60% when equimolar amounts of thymidine and TTP, and excess Mg2÷, were present whereas that o f the latter was inhibited only 20%, even in the presence of 20 mM sodium fluoride. The ability o f the standard antifilarial drugs suramin and diethylcarbamazine (DEC) to inhibit the activity of both filarial thymidine kinases was tested by preincubating the enzymes with 1 mM concentrations of each drug for 5 rain at 37°C before the reaction was started by the addition of [2-14 C] thymidine. At this high concentration, both enzymes were inhibited 50% by suramin and 30% by DEC, indicating their relative insensitivity to these drugs.

Fate of [methyl-3 H] thymidine presented to B. pahangi females in vitro and in vivo. Uniformly low amounts o f radioactivity were found in the lipid, RNA, DNA and protein fractions of female worms following their incubation for 24 h at 37°C in a medium containing 1 /aCi/ml of [methyl -3 H]thymidine (specific activity = 64 Ci/mmol) and their subsequent 24 h incubation in thymidine-free medium (Table II). The amount of radioactivity in the acid-soluble fraction was appreciably higher than in the other subcellular fractions; there was no attempt to determine the extent to which the radioactivity in this fraction was associated with thymidine or thymidine nucleotides. Uniformly low amounts o f radioactivity were found in the lipid and protein fractions of female worms randomly selected from intraperitoneally located populations in jirds

367

TABLE II Radioactivity in different fractions of adult Brugia pahangi females exposed to [methyl-~ H] thymidine in vitro Group No. a

1 2 a

RadioactNity (dpm/~action/20 worms) Add-solub~

L~id

RNA

DNA

Protein

250 208

36 73

35 36

3 12

45 9

Two groups of 20 adult females, freshly removed from the peritoneal cavity of an infected jird, were exposed in vitro to [methyl-a H]thymidine (specific activity = 64 Ci/mmol) for 24 h under conditions described in Materials and Methods. They were fractionated after an additional 24 h incubation in a thymidine-free medium.

18 h after the hosts received 5 intraperitoneal injections o f [methyl-3H]thymidine (specific activity = 64 C i / m m o l ) , 0.33 /aCi/g b o d y weight, at 2 h intervals (Table III). The average a m o u n t o f radioactivity in the R N A fraction also was relatively low, although the a m o u n t in this fraction varied considerably in individual batches o f worms. The average a m o u n t s o f radioactivity in the acid-soluble and D N A fractions were conspicu o u s l y higher than in the o t h e r subcellular fractions, although these t o o were quite variable in individual batches o f worms. When acid-insoluble m a c r o m o l e c u l a r fractions o f these w o r m s were treated w i t h RNAase, a p p r o x i m a t e l y 4% o f the original radioactivity was r e m o v e d ; in contrast, t r e a t m e n t w i t h DNAase r e m o v e d 96%. Essentially all the radioactivity in the D N A fraction was associated w i t h t h y m i d i n e (Table IV). These TABLE II1 Radioactivity in different fractions of adult Brugia pahangi females exposed to [methyl-a H]thymidine in vivo. Group No. a

Radioactivity (dpm/fraction/10 worms) Acid-soluble

Lipid

RNA

DNA

Protein

1 2 3 4 5 6

1227 1165 215 230 1140 600

26 17 53 68 56 50

47 3 127 100 10 57

2164 108 227 280 300 365

39 35 29 35 42 47

Mean_+ S.E.

763 _+ 194

45 _+8

57 -+ 20

574 -+ 320

38 +- 3

a Populations of adult males and females in the peritoneal cavities of six jirds were exposed to intraperitoneally administered [methyl.a Hlthymidine (specific activity = 64 Ci/mmol) as described in Materials and Methods. Groups of 10 females from each treated jird were selected at random for fractionation 18 h after the last injection of radiolabeled thymidine.

368

TABLE IV Analysis of radioactivity in nucleic acids of adult Brugia pahangi females exposed to [methyl-3 H]thymidine in vivo Fraction

Radioactivity (dpm) Original

After RNAase

After DNAase

After DNAase + phosphodiesterase TMP

dCMP

Acid-insolublea (RNA + DNA + protein)

4080

3930

150

DNAb

2040

-

-

1 9 9 0

50

DNAb

1610

-

-

1570

40

a Equal aliquots of a single batch of pooled acid-insoluble fractions were enzymically digested with either RNAase A or DNAase I as described in Materials and Methods, and the radioactivity remaining in the acid-insoluble fractions of both digests was compared. b Two separate batches of pooled DNA fractions were sequentially digested with DNAase 1 and phosphodiesterase I (Crotalus adamanteus venom, Type 11) as described in Materials and Methods. The pyrimidine deoxynucleoside 5'-monopho.sphates extracted from the digests were separated by MN 300 F cellulose thin-layer chromatography, and the radioactivity in TMP and dCMP was determined. findings indicate that at least B. pahangi females possess a thymidine salvage pathway that can operate efficiently when the worms are within their mammalian hosts but that functions marginally if at all as soon as they are placed under presently employed in vitro culture conditions. DISCUSSION These and previous findings demonstrate that adult f'flariae possess both de novo and salvage pathways leading to the synthesis of thymidine nucleotides. The reason why the thymidine salvage pathway in B. pahangi females apparently can operate efficiently only when the worms are within their mammalian hosts remains to be determined, but it is probably linked to the physiological state of these parasites. One likely variable is the comparative rate of DNA synthesis in populations of ceils within the reproductive system (gonadal, viteUarial, embryonic) of macrofflariae in vivo and in vitro. There is an established association between high thymidine kinase activity and DNA replication in animal ceUs [12]. The bladder fluke of frogs, Gorgoderina attenuata, which has a thymidine salvage pathway that can function when the worms are maintained in vitro for short periods, took up and incorporated radiolabeled thymidine preferentially into the nuclei of ovarian gonial cells, scattered cells in the vitellaria and cells of developing miracidia within eggs [13]. Essentially the same sites of thymidine localization were

369 found in another digenetic trematode, Philophthalmus megalurus [14]. The DNA in mitochondria as well as that in cell nuclei can replicate, and it was recently reported that in vitro yet another digenetic trematode, Schistosoma mansoni, took up and incorporated radiolabeled thymidine into the mitochondria of mature oocytes [15]. The few published studies on the reproductive capacity of adult fdariae in vitro indicate that oogenesis and development of embryos invariably are impaired, although the rapidity by which such impairment becomes prominent can vary among species [16, 17]. We found that intrinsic thymidine kinase activity in adult B. pahangi females in vitro declined progressively in time: 100 nmol/h/mg at the onset, 80 nmol/h/mg after 6 h and 40 nmol/h[mg after 24 h. Such a decline in the activity of this inducible enzyme [12] in f'flariae living under suboptimal conditions could reflect a similarly progressive decline in the number of cells wherein DNA synthesis is occurring. Moreover, the failure of Chen and Howells [1 ] and ourselves to detect significant uptake and incorporation of thymidine by adult B. pahangi females maintained in vitro for short periods ( 1 - 2 4 h) during which time intrinsic thymidine kinase activity is still appreciable might be attributed to the sensitivity of this enzyme to feedback inhibition by TTP. This possibility would become realistic if it could be shown that DNA synthesis in cycling B. pahangi ceils ceases abruptly when the worms are transferred to incubation media, leading to the rapid accumulation of TTP, the end product of thymidine salvage. The relative importance of de novo and salvage pathways for providing thymidine nucleotides to dividing fflarial ceils remains to be determined. Regarding the latter pathway, it seems hardly likely that adult f'tlariae depend to a significant degree upon hostderived thymidine, in view of the very low amounts (around 0.1/aM) of this pyrimidine that are present in the extracellular fluids of mammals with the exception of rodent species [18]. It remains to determine whether filariae can recycle endogenous stores of thymidine, perhaps released from the DNA of autolysing nuclei and mitochondria of gonadal nurse cells and those in the vitellaria, to provide thymidine nucleotides for gametogenesis and developing embryos. Finally, it now appears that it would be much more difficult to render f'flariae infertile by inducing a deficiency in their thymidine nucleotide stores than was previously believed. Ostensibly, both a strong and/or selective inhibitor of TMP synthetase and thymidine kinase would be required for the purpose. Nevertheless, further research on fllarial thymidine metabolism and its association with DNA synthesis may reveal ways to disrupt this aspect of filarial biochemistry. ACKNOWLEDGEMENT This investigation was supported in part b y funds from the Filariasis component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and in part by a bridging grant from the Burroughs Wellcome Fund.

370

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18

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