Deoxycytidylic Acid Deaminase in Ehrlich Ascites Tumor Cells1'2
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Deoxycytidylic Acid Deaminase in Ehrlich Ascites Tumor Cells1'2 SILvI0 FIALA AND ANNA E. FIALA (Laboratory of Cell Physiology, Veterans Admini8tration Hospital, San Fernando, California and the Deparlment of Biochemistry, University of Southern California, Los Angeles, California) SUMMARY Ehrlich ascites tumor cells contain a highly active dCMP3-deaminase, but no de tectable dCMP-phosphatase. A stable dCMP-deaminase could be obtained in ex tracts, provided the ascitic fluid was washed from the cells. Inactivation occurred when the extracts were diluted. The experiments indicated that this was due to the dissociation of the enzyme followed, eventually, by an irreversible rearrangement of the protein moiety of the enzyme molecule. This dissociation seems to be preventedby the substrate, by dCTP or by heated extract. The linearity of dCMP-deaminase macti vation, with respect to time, indicated an interaction between the enzyme and inacti vating factors. This led to the discovery of 2 protein factors capable of inactivating dCMP-deaminase. One of the factors was found in the microsomes of various tissues, the other was a pseudoglobulin fraction from the ascitic fluid. In addition, orthophos phate and pyrophosphate were found to inactivate dCMP-deaminase. The inacti vating effect of all these compounds could be antagonized, but not reversed, by dCTP. dTTP not only inhibited dCMP-deaminase, in the presence of substrate, but also pro gressively inactivated dCMP-deaminase in the absence of substrate. In contrast to the above irreversible inactivators, however, the action of dTTP could be reversed by dCTP. The factors, encountered in extracts, may provide a rapid regulatory control of the activity of dCMP-deaminase. In addition, the reciprocal relationship between dCMP-deaminase and dCMP-phosphatase activities found in other tissues, indicates a slower regulation of these enzymatic activities by the genetic apparatus of the cell. Deoxycytidylic acid deaminase, the enzyme which con ordinately with cell multiplication. So far nothing definite verts dCMP to dUMP (13, 19), is present in mammalian is known about this regulation. Nevertheless, attempts tissues and manifests a high activity in such rapidly pro have been made to explain this phenomenon from data on liferating tissues as embryonic tissue or tumors (3, 13, 15, the conditions affecting the lability and stabilization of 19). In tissues with a low mitotic index, the activity of dCMP-deaminase in vitro. In the absence of substrate, the enzyme is quite low. Thus dCMP-deaminase activity this enzyme was reported to be inactivated by dialysis is low in adult rat liver but increases when cell proliferation and thermal denaturation (6) even by physiologic temper occurs in the organ, as for example after hepatectomy (14, atures (18, 20). The nature of this lability, however, is 17) or during carcinogenesis (3, 5). These circumstances not yet clear. Whereas one group of investigators con suggest that dCMP-deaminase plays an important role in sidered that the lability was caused by thermal denatur the chemical mechanism of cell proliferation and that the ation of the enzyme and contested the possibility that intracellular activity of this enzyme is regulated co dCMP-deaminase could be reactivated (20), another group reported a reactivation of inactive dCMP-deaminase 1 This work was presented, in part, at the Third Annual Meet ings of the American Society for Cell Biology, New York, N. Y., preparations (16). The finding that dCTP protected November, 1963. dCMP-deaminase against spontaneous inactivation in 2 This work was supported by grant-in-aid GB-754 from the vitro (16, 19, 20) while dTTP inhibited the enzyme led to National Science Foundation and by grant 07 171-01 from the the hypothesis of an enzymatic feed-back mechanism, National Cancer Institute. 3 The abbreviations used are : dCMP, deoxycytidylic acid; whereby these deoxynucleoside tniphosphates regulate dCTP, deoxycytidine triphosphate; dTTP, deoxythymidine tn dCMP-deaminase activity (16, 20, 21) by binding to an phosphate; dUMP, deoxyunidylic acid; P1, orthophosphate; ATP, allosteric site on the enzyme molecule (21). adenosine triphosphate ; GSH, reduced glutathione ; GSSG, oxi Despite these interesting data concerning the consti dized glutathione ; TMP, thymidylic acid ; GTP, guanosine tn phosphate. tution and properties of the enzyme, it seems clear that Received for publication October 5, 1964; revised March 1, the problem of in vivo regulation of dCMP-deaminase 1965. activity cannot be elucidated by observations on purified 922 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1965 American Association for Cancer Research. FIALA AND FIALA—dCMP Deaminase in Ehrlich Ascites Tumor Cells 923 enzyme preparations alone. The problem must also be glycyiglycine or 0.1 M Tnis buffer for 5—10mm at pH 7.0— approached by obtaining data of a more physiologic 7.2, we could solubilize all the dCMP-deaminase after 2 nature; that is, by studying the factors in homogenates extractions. In order to speed up the process, however, and tissue extracts which influence dCMP-deaminase ac only 1 extraction with 10 volumes of buffer was per tivity and correlating the levels of dCMP-deaminase formed. One milliliter of extract contained 20 mg pro activity under various physiologic conditions. Led by tein. During all the procedures prior to an experiment such considerations in our earlier work (7), we emphasized the material was kept in an ice-water bath. The “active the inverse relationship between the activities of dCMP extract― was used immediately or was stored at —15°Cfor deaminase and dCMP-phosphatase. The activity of several weeks without loss of activity. The advantage of dCMP-deaminase appeared to be proportional to the using acetone powders was that a fresh “activeextract― mitotic index of the tissue, while the activity of dCMP could be obtained at any time. On the other hand, only phosphatase seemed to parallel the generation time of the about one-third of the activity present in the intact cells cell (unpublished observations). We also reported earlier could be accounted for in the extracts, the remainder (6) thatmicrosomesfromvarioussourcescontaineda probably being lost during acetone homogenization of the factor, apparently a protein, which inactivated dCMP tissue. The extract made from 10 mg of powder con deaminase in the absence of substrate. In the present verted about 8 @imolesdCMP in 1 hr at 37°C. The work we have utilized homogenates and extracts of Ehrlich conditions of enzyme saturation were fulfilled when 0.1 ascites tumor cells to obtain more information about this ml of the extract (approximately 2 mg protein) was factor and about other aspects of the intracellular behavior incubation with 2 Mmoles dCMP. At this concentration of dCMP-deaminase. The main questions we have con the deamination of dCMP proceeded linearly for 30 mm sidered are the following: Under what conditions is dCMP at 37°Cand the amount of dCMP converted was propor deaminase stable and when is it labile? Which factors tional to the enzyme concentration. In a number of increase the stability and which increase the lability of the experiments we prepared a “heatedextract― and used it as enzyme? Finally, what conclusions can be drawn from a diluting medium for dCMP-deaminase. Such an extract these observations concerning the in vivo regulation of was prepared by heating acetone powdered cells in boiling dCMP-deaminase activity? water for 4 mm or, in later experiments, by heating to 70°C for 10 mm followed by centrifugation for 10 mm at 17,300 MATERIALS AND METHODS x g. TheheatedextractdidnowshowanydCMP Preparations of extracts from Ehrlich ascites carcinoma deaminase activity but contained nucleoside di- and cells.—Ehrlich ascites tumors obtained through the triphosphates as shown by paper electrophoresis. Deoxy courtesy of Dr. Franz A. Schmid (The Sloan Kettering nucleotides and nucleosides were purchased from Sigma Institute for Cancer Research, Rye, N. Y.), were trans Chemical Company, St. Louis, Mo. Reaction products mitted serially in Swiss albino mice (average weight 30 were followed by paper electrophoresis of KOH neutralized gm). Tumors were harvested 8—10days after inoculation, perchloric acid extracts in the model R Spinco electro yielding on the average 2.5 X 1O@cellsfrom a single mouse. phoresis cell (Durrum type) at constant voltage (200 The cell number counted in a Petroff-Hauser counter was volts) and current (4—7ma). Samples of 10—100 @lwere correlated with the optical densities measured at 520 m@s placed on Whatman No. 3MM paper strips and the eleetro (1 cm optical path) inaBeckmanmodel DUspectrophotom phoresis was performed at room temperature for 8 hr in eter. An optical density reading of 0.100 corresponded 0.5 M citrate buffer at pH 3.5. to 2.6 X 10@cells/mi, the cell number being a linear func Preparaiion of dCMP-deaminw@e inactivating factors tion of optical density in the range 0—20X 10@cells/mi. from ascitic fluid and microsomes.—The supernatant ob The cells were collected and centrifuged at 42 X g for 10 tamed after centrifuging the ascitic fluid at 3000 rpm was mm (0°C)followed by 2 resedimentations in 5—6volumes recentnifuged at the same speed for 10 mm and kept at of saline at 480 X g and 1085 X g. The resedimentation — 15°C. It was dialyzed before use in cellulose tubing for at 1085 X g was usually repeated to insure that the cells 24 hr at 2—4°Cagainst a large volume of distilled water. were completely washed. Bloody suspensions rarely The precipitate which formed was discarded. All the puri occurred and were rejected. For preparation of homog fication steps were performed at 0°C. For partial purl enates the cells were disrupted by ultrasonic vibration fication, the dialyzed material was brought to pH 5.0 with (20 kc, 10 sec at approximately 150 watts) with the diluted acetic acid and the precipitate, consisting mostly piezoelectric Sonifier 5-110 of Branson Instruments, Inc., of nucleic acids, was discarded.