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 FIALAâ€”dCMP Deaminase in Ehrlich Ascites Tumor Cells 923 enzyme preparations alone. The problem must also be glycyiglycine or 0.1 M Tnis buffer for 5â€”10mm at pH 7.0â€” 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 â€œactive the inverse relationship between the activities of dCMP extractâ€• was used immediately or was stored at â€”15Â°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 â€œactiveextractâ€• 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 37Â°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 37Â°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 â€œheatedextractâ€• 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 70Â°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.â€”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 8â€”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 (4â€”7ma). Samples of 10â€”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 0â€”20X 10@cells/mi. from ascitic fluid and microsomes.â€”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 (0Â°C)followed by 2 resedimentations in 5â€”6volumes recentnifuged at the same speed for 10 mm and kept at of saline at 480 X g and 1085 X g. The resedimentation â€” 15Â°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 2â€”4Â°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 0Â°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. Subsequently, the pH Stamford, Conn. For preparation of tissue extracts the was brought to 4.5 and the protein which precipitated, sedimented cells were collected with ice-cold (reagent mostly albumin, was discarded. The solution was grade) acetone and homogenized in a Virtis â€œ45â€•blender neutralized with NaOH and an equal volume of saturated for 90 sec. The acetone powder was prepared by the same ammonium sulfate (saturated at room temperature and methods described earlier for other tissues (7). Acetone adjusted to neutrality with ammonia) was added. The powder kept under vacuum at 0Â°Cshowed full dCMP deaminase activity for months. In fact, the enzyme precipitate was dissolved in 0.1 of the original volume of withstood 1 month's storage at room temperature without glycylglycine buffer (0.1 M) pH 7.2 and dialyzed against any detectable loss of activity. Acetone powder, 1 mg, distilled water for 24 hr. The precipitate was discarded corresponded to approximately 20 X 10@cells. By slowly and the pseudoglobulin in solution was lyophilized. stirring the acetone powder with 5 volumes of 0.1 M About 80 mg of white fluffy powder, completely soluble in

water, was obtained from 100 ml of the original ascitic activity could also be followed directly without de fluid. proteinization using 1 cm Beckman cuvets with spacers to For preparation of the inactivating factor from micro reduce the optical path to 0.002-0.003 cm. The extinction somes either unperfused mouse liver or rat liver perfused was read at 290 m@ where, at pH 7.2, uracil derivatives in &ituwith isotonic saline with the animal under Nembutal did not absorb. narcosis was used. The liver was disrupted in isotonic (0.25 M) sucrose with a Potter-Elvehjem glass homog RESULTS enizer. After centrifuging down the nuclear (10 mm at Deoxycytidylate deaminase in homogenates and extracts 600 X @i)and mitochondnial fractions (10 mm at 13,000 x from Ehrlich ascites tumors.â€”Deamimation of dCMP was g) the resulting supernatant was centrifuged for 90 mm at found not to occur with washed intact cells in the presence 59,000 X g in a Spinco model L preparative ultracentri of 2 @smolesof substrate even after 30 mm of incubation fuge. The microsomal pellet was rehomogenized in cold apparently because of the impermeability of the cell mem saline, dialyzed for 24â€”48hr against distilled water and brane to dCMP. The same suspensions after sonic lyophilized. This material, kept in a desiccator under disruption or by rapid freezing and thawing converted vacuum at 0â€”2Â°C,wasstable for a period of several months. dCMP to dUMP in an amount proportional to the original The inactivating factors from this material did not go into number of cells. Thus, 1 million cells are capable of solution. When an immediate testing of the microsomal converting approximately 0.1 Mmole dCMP in 1 hr. On factor was desired, an acetone powder was prepared from a weight basis, 1 gm of cells would convert approximately the microsomal pellet. In contrast to lyophilized un 100 j@molesof dCMP in 1 hr. This is about 5 times the treated microsomes, the inactivating factor could be activity of Novikoff tumor and almost 150 times as high as extracted easily from the microsomal acetone powder by that of adult rat liver and represents, to our knowledge, stirring for 10 mm at 0Â°Cwith 10 volumes of buffer (0.1 the highest activity to be reported for any animal tissue. M glycylglycine or 0.1 M Tris buffer, pH 7.0â€”7.2). For The main product of the reaction was dUMP, with only further purification the extract was dialyzed for 24 hr, the traces of deoxyuridine and no deoxycytidine. This pH brought to 5.0 by dilute acetic acid, and the mixture indicated that very little, if any, dCMP-phosphatase centrifuged. The supernatant was discarded and the activity was present in Ehrlich ascites tumor cells. Col sediment containing about two-thirds of the total protein onimetric determinations of P1 were also performed with material of the extract was redissolved in buffer, dialyzed fresh and aged (no active dCMP-deaminase) homogenates against distilled water for 24 hi, and lyophilized. About incubated for 15 mm at pH 5.5, the optimal pH for dCMP 40 mg of white fluffy material, easily soluble in distilled phosphatase (7). The test showed that the same amount water, was obtained from approximately 2 gm of acetone of P1 was liberated in the presence or absence of dCMP; dried microsomes. The active material henceforth will this indicated that the P1 was formed endogenously and be referred to as â€œR-factor.â€• was unrelated to the metabolism of dCMP. A similar Assays for dCMP-deaminase.â€”Unless otherwise mdi result was obtained in later work with extracts of acetone cated, 0.1â€”0.2mlextract, 0.2 ml of 0.1 M glycylglycine or powdered cells. The dCMP-deaminase-inactive extracts Tris buffer (pH 7.2), and 1.3â€”2.0 @molesdCMP in a total did not evince any dephosphorylation of dCMP in the volume of 0.5â€”1.0ml were incubated at 37Â°Cfor 15 mm. range of pH 5.5â€”7.5. P1 was liberated in the active Preincubation of the mixture was performed for various extracts ; however, only the formation of deoxyuridine time intervals at 37Â°Cin the absence of substrate. The was detected, indicating that dCMP is first deaminated to incubation was stopped by the addition of 5 ml of cold dUMP which is then dephosphorylated to deoxyuridine. 10 % perchlonic acid. After deproteinization the super The very high dCMP-deamimase activity accompanied by natants were diluted with 5 % perchloric acid and the barely detectable dCMP-phosphatase activity in rapidly activity measured by a spectrophotometnc method proliferating Ehrlich cells contrasted with the very low described earlier (6). When desirable the spectrophoto dCMP-deaminase and very high dCMP-phosphatase metric determination was complemented by paper electro activities observed in such nonproliferating tissue as phoresis and the colorimetric estimation of ammonia normal adult rat liver (5, 7). formation (nesslerization). In the spectrophotometric Preincubation of the homogenate led to a partial in determinations, the control tube containing the extract activation of dCMP-deamimase. The time course of incubated without dCMP served as the blank. The ratio inactivation in a suspension containing approximately @ (referred to as the â€œQvalueâ€•)was 2.06 for 40 X i0@cells (0.5 ml homogenate in a total volume of 2.3 dCMP and 0.33 for dUMP in 5 % perchloric acid. The ml) was perfectly linear for a period of 20-30 mm (Chart proportion of these mucleotides in a mixture should be a 1). In a more concentrated suspension there was a loss of linear function of the observed Q-values. Therefore the activity in the initial 5â€”10mm and no further change amount of dCMP converted to dUMP in time I (Ce) can during the remainder of the 30-mn period. An exponen be calculated from the equation C@= Co (1.188â€”0.58Q) or tial decrease of activity reflecting a simple protein de can be read directly from a momogram. The empirical maturation or other first order reaction was not observed. measurements were in agreement with this assumption; This indicated that the inactivation of dCMP-deaminase moreover, this assumption was also confirmed by other in homogenates was a more complex process than denatura workers using our method (22). The conversion of 1 tion of the enzyme. Similar observations were made @imoleof dCMP in 1 hr at 37Â°C/i mg protein was defined with the supernatant obtained after centrifuging down all as specific dCMP-deaminase activity. The enzymatic particulate matter at 105,000 X g for 90 mm. In order

1.5

>- I-. > I-. C-)

@ 0@5 ae

@ I I I I I 0_0 5 10 5 20 25 30 I 2 3 4 TIME IN MINUTES TIME IN HOURS

CHART 1.â€”Linear time-course of dCMP-deaminase inactivation CHART 2.â€”The time-course of dCMP-deaminase inactivation in in extracts and a homogenate. Extracts Iâ€”IVwere preincubated dependence on enzyme concentration. Specific activities of ex at 37Â°Cinduplicatetubesfor 0â€”30mm.At designatedtimein tracts: I, 40 units in 0.2 ml; II, 40 units in 1 ml; flI, 15 units in tenvals the substrate (1.3 pmole dCMP) was added to one of the 1 ml; IV, 8 units in 1.0 ml. Preincubation 0-4 hr at 37Â°Cin Tnis tubes and incubated for 15 mm. Extract I was preincubated un buffer, pH 7.1. Incubation time was 15 mm, dCMP 1.3 Mmole,in diluted (0.2 ml), extracts IIâ€”IV were diluted with Tnis buffer a final volume 1.2 ml. A unit is defined as 1 @moledCMPdeami (0.1 M, pH 7.1) to a volume of 1.0 ml. For incubation the sub natedin1 hr at 37Â°Cby1 mg protein. stnate and buffer added to a final volume of 2.0 ml. Homogenate (H) made from 40 X 10@cellsin 0.5 ml, was further diluted with Tnis buffer to 1.0 ml for preincubation. For incuba 4 hr still retained about 50 % of the original dC\IP tion the substrate and buffer added to a final volume of 2.3 ml. deamimase activity. A less concentrated extract was in activated more rapidly, while a still more dilute extract to study this process, therefore, we used only the soluble displayed a very rapid rate of inactivation. Superficially, phase obtained from the extract of acetone powdered cells. the time course of inactivation in the last case resembled The contrast between the stability of dCMP-deaminase the exponential course; however, plotting values for log in intact cells and the lability of this enzyme in extracts A 0/Agversustime did not give a straight line. suggested that the inactivation was initiated by dilution of The effect of dilution in initiating dCMP-deaminase the cell content. This conclusion was corroborated by inactivation was also studied using a stable extract diluted experiments concerning dilution, dialysis, and adsorption with buffer to different volumes. By this means the of active extracts. Thoroughly washed cells were used concentration of enzyme was varied during preincubation for preparation of these acetone powders. The extracts while the total amount of enzyme remained the same in made from cells which were not thoroughly washed lost all samples. After preincubation all samples were their activity more rapidly and showed a greater van adjusted to the same volume so that the concentration of ability in dCMP-deaminase activity than did extracts enzyme was now the same in all samples. The samples from washed cells. This was explained eventually (see were then incubated with substrate for 30 mm. Chart 3 next section) by the presence of ascitic fluid in the um shows that dilutions up to 40 % of the initi'@1concentration washed cells. The inactivation of washed extracts had little effect on the rate of inactivation ; however, showed a more uniform pattern. Concentrated extracts dilution beyond that point markedly accelerated the rate prepared with 10 volumes of buffer were completely stable of inactivation. No inactivation occurred during pre for at least 30 mm and sometimes these extracts did not incubation when the samples were diluted with a heated lose activity to any noticeable extent even after several extract instead of buffer. The protective ability of the hours at 37Â°C. However, the extracts were gradually heated extract was lost when it was treated with acid inactivated upon dilution, the process proceeding linearly washed charcoal (Norite, 20 mg/mi). Elution of the with time when observed at 5-mn intervals (Chart 1). charcoal with 50 % ethanol, 1 % NH3 and concentration Occasionally a plateau was reached without any further of the eluate by vacuum and freeze drying yielded a inactivation of the enzyme for another 1 or 2 hr. The material which protected against inactivation by the constant rate of inactivation indicated that the process â€œdilutioneffect.â€• An extract heated at 70Â°Cfor 10 mm initiated by dilution followed zero order kinetics. This protected even more strongly and, in contrast to the was no longer apparent when observations were made at extract heated at 100Â°C,did not show a rapidly moving 30- to 60-mm time intervals; however, even in this case, band in paper electrophoresis corresponding to nucleoside the effect of dilution was quite conspicuous (Chart 2). triphosphates. A similar heated extract prepared from A concentrated extract was stable for almost 1 hr and after @iormaladult rat liver which showed this band protected to

z 0 TABLE 1 PROTECTION OF dCMP-DEAMINASE BY dCTP AND THE HEATED EXTRACT AGAINST INACTIVATION BY THE DILUTION EFFECT' C-) 100 - z Volume(ml)Preincubatedâ€”0.81 â€˜U .52.5Incubated2.72.72.72.7ActivityAdditionâ€”1.160.780.460.21dCTP1.141.12 a.

â€˜U I-. U. . DILUTIONSWITHBUFFER I- (0.22)Heatedextract1.171.24 (0.79)1.16 (0.46)1.12 > 0 DILUTIONS WITHHEATED EXTRACT 50 0 DILUTIONS WITH HEATED EXTRACT (0.80)1.2 (0.4)1.16 (0.20) AFTER TREATMENT WITH NORITE 1 a Activity is expressed in pmoles dCMP deaminated by the U extract (dry weight 20 mg) in 30 mm at 37Â°C. Samples were pre U. U incubated for 60 mm at 37Â°C,except for those in the first column â€˜U a. which were not preincubated. The mixtures consisted of extract U) (0.2 ml), additions (1.0 @@moledCTPor 20 mg, dry weight, of heated extract) and Tnis buffer (0.1 M, pH 7.2) to make volumes of 0.8, 1.5, and 2.5 ml, as indicated above. After preincubation @ 0 I I I I II I _l t@ @I dCMP (2.0 pmoles) and Tnis buffer added to a final volume of $0 20 30 40 50 60 70 80 90 100 2.7 ml. The degree of dilution during preincubation increased the rate of dCMP-deaminase inactivation. Addition of dCTP % INITIAL CONCENTRATION or of heated extract to preincubated samples protected dCMP CHART 3.â€”Dilution effect in the inactivation of dCMP-deami deaminase against inactivation, addition after preincubation did nase.â€”The concentration of the undiluted extract (0.2 ml) was not protect (values indicated in parentheses). taken as 100% of the initial concentration. Initial activity of this extract without preincubation was 1.16 @smoles(15 mm) and was designated in the Chart as 100% specific activity. After 30 mm was only 50 % after this period even at 2-4Â°C. In 48 hr of preincubation the activity of this undiluted extract dropped to 90% of the initial activity. The increasing dilution, up to the activity was always completely lost. Even after this 10% of the initial concentration, with a buffer alone (glycylgly period, however, it was found invariably that mixing the cine, 0.2 M, pH 7.1) or with Nonite-adsonbed heated extract, led completely inactive preparation with a fresh active one to increased loss of activity during pneincubation. In contrast, considerably stimulated the activity of the mixture. The the dilution with the heated extract preserved and even increased same degree of reactivation was obtained when the heated the activity above the initial level. Volume of incubated mix tunes was 2.2 ml, including 1.3 pmoles dCMP. extract from the same source or even from adult rat liver was added to an inactive dialyzed sample; dCTP acted in a lesser extent than did heated extract from Ehrlich tumor the same way. The reactivation was always a partial cells. dCTP protected against the dilution effect (Table one, leaving no doubt that 2 processes occurred during 1), whereas ATP did not protect. However, we cannot the dialysis: the first an essentially reversible inactivation affirm at present that the physiologically active substance due to the dissociation of the enzyme; the second, a slow was dCTP because of the following observations : the 70Â°C irreversible rearrangement of the enzyme. This irrevers heated extract from Ehnlich ascites cells had a more ible process was induced by the removal of a low molecular pronounced protective effect than did 1 @moledCTP in weight compound from the extract during dialysis. protecting against the dilution effect, although no band Further data on the influence of protecting and activating corresponding to nucleoside triphosphates was visible in compounds on dCMP-deaminase inactivation were ob paper electrophoresis. This point must await further tamed by adsorbing the extracts on charcoal. A stable clarification. What should be noted, however, is that extract was treated in 1 sample with 5 mg/mi Norite, in a neither the heated extract nor dCTP could reverse the 2nd sample with 10 mg/mi Norite, in a 3rd sample with inactivation when dilution was followed by preincubation 20 mg/mi Norite. In the 1st case the initial activity was at 37Â°Cfor 30 mm (Table 1). On the other hand, dilution unchanged but a progressive inactivation was initiated. at 0Â°Cdid not lead to any appreciable loss of activity With a greater amount of adsorbent there was a loss in during the same period, thus indicating that dilution initial activity and an increase in the rate of inactivation. itself did not cause the inactivation. These observations Finally, with a large amount of adsorbent, there was a indicated that dilution probably started the inactivation further decrease in the initial activity and a considerable process by dissociating either dCMP-deaminase or a decrease in the rate of inactivation. All this suggested complex between enzyme and a protective compound or a that the rate of inactivation depended primarily on the complex between an inactivating enzyme and its inhibitor. concentration of the protective compound and only Additional information concerning these possibilities secondarily on the concentration of a complex between was obtained by dialyzing stable extracts against distilled dCMP-deaminase and some inactivating compound. water for various time intervals. In 2 hr at 0Â°Cno loss of The zero order reaction reflected a constant concentration activity was seen ; after 18 hr 85 % of the original activity of this complex for a certain period during the inactivation was lost in some cases, although in other instances the loss of dCMP-deaminase. The possibility that the inactiva

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1965 American Association for Cancer Research. FIALA AND FIALAâ€”dCMP Deaminase in Ehrlich Ascites Tumor Cells 927 tion was due to the action of an inactivating enzyme, was made improbable by these observations and was excluded when all attempts failed to increase the rate of dCMP deaminase inactivation by adding the inactivated (at 37Â°C),dialyzed and lyophilized extract material to a fresh extract. 1.1 In experiments designed to elucidate the nature of the inactivating compounds, the possible role of heavy metals 0 Ui was investigated. Neither o-phenanthroline, a,a'-di 0.9 pynidyl, 8-hydroxyquinoline nor mercaptoethylamine at Ui 1 X i0â€”@Mhad any effect on the course of inactivation. z> 0 C) The presence of heavy metals, therefore, could not be of 02 major importance in the spontaneous inactivation of a- dCMP-deaminase. The extracts placed in a closed C) polarographic vessel showed a rapid consumption of U) 0.5 Ui -i dissolved (approximately 1 X 10@ N) oxygen which was 0 almost complete within 30 mm. The inactivation of â€” / dCMP-deaminase did not seem to be connected with this 0.3 process, however, because the rate of inactivation ap peared to be unchanged whether the process proceeded in the presence or absence of oxygen. The rate of inactiva 0.1 tion as a function of pH showed a maximum at pH 8.0 when measured in the range 6.0â€”8.2. At pH 7.8 the rate 0 5 10 15 20 25 30 was about 100 % greater than at pH 6.0. A phosphatase TIME IN MINUTES which dephosphorylated dCTP was detected in extracts CHART 4.â€”Inactivation of dCMP-deaminase in presence of and also in microsomes; but sodium fluoride (5 X 10@ M), ortho-(P1) and pyrophosphate. Effect of orthophosphate on the which completely inhibited this phosphatase, had no rate of dCMP-deaminase inactivation is shown in straight lines effect on the course of dCMP-deaminase inactivation. Iâ€”V. The mixture for preincubation consisted of 0.1 ml extract, Therefore the phosphatase could not be involved in the pH 7.2, and phosphate buffer, pH 7.2 (0.1 M) added in the amounts of 0.0, 0.05, 0.1, 0.2, and 0.4 ml; volume filled up with glycylgly inactivation of dCMP-deaminase. On the other hand, the cine buffer (0.1 M, pH 7.2) to 0.5 ml. Preincubation time was 15 preparation preincubated for short intervals (5â€”15mm) and 30 mm. Incubation time was 15 mm with 1.3 jsmole dCMP at 37Â°Ccould be reactivated with heated extract or with in total volume 0.7 ml. Effect of sodium pyrophosphate com dCTP only to the extent of 10â€”20%. The inactivation pared in Curves IA (no pyrophosphate) and IIA (0.1 ml of 2 X showed a linear time course, indicating that a zero order 102 M Na4P2O@ added to 0.1 in! extract and 0.3 ml glycyiglycine buffer). Preincubation time was 15 and 30 mm. Incubation reaction determined the over-all process. Once again the time was 15 mm, dCMP 1.3 Mmole, and final volume 0.7 ml. inactivation seemed to consist of 2 separate processes, possibly performed by a single agent : the initial removal of the protective factor followed by a rather rapid (at 37Â°C) tion approaching a limit with increasing concentration of irreversible rearrangement of the enzyme molecule. orthophosphate. The pH of the added phosphate solu Further experiments, therefore, were directed toward tion was carefully matched with the pH of mixture (pH elucidating the nature of these agents which might have 7.2). dCTP counteracted the action of orthophosphate. been present or possibly formed in the extracts and which Sodium pyrophosphate was seen to be a much stronger inactivated dCMP-deaminase. inactivator of dCMP-deaminase than orthophosphate. Physiologically occurring inactivators of dCMP-de Again its inactivating effect could be counteracted but not aminase.â€”As inactivators of dCMP-deaminase we desig reversed by dCTP. This fact prompted us to investigate nate those compounds which increase the lability of this whether the inhibitory effect of dTTP, known to be enzyme so that it is gradually inactivated in the absence reversed by dCTP (16) might also be a function of time. of substrate. We use this definition in order to distin Chart 5 shows that the inactivation of dCMP-deaminase guish these compounds from simple inhibitors which by dTTP was itself time-dependent. In distinction to affect the affinity of the enzyme for the substrate but do inactivation by orthophosphate, the inactivation by dTTP not change the rate of inactivation. Among the inhibitors was completely reversed by the addition of dCTP. This of the latter variety was cupric ion which, at a concentra feature thus distinguished dTTP from the irreversible tion of 5 X i0@ M inhibited the activity of dCMP inactivators. deaminase to 40 % of the original value but was without In subsequent experiments we investigated whether am any effect on the stability of the enzyme. Similarly, intracellular factor might be present in Ehnlich ascites deoxyuridine and deoxyadenosine (1.5 Mmole)had a slight carcinoma which could inactivate dCMP-deaminase. inhibitory effect on dCMP-deaminase, but no inactivating Previously (6) we had seen that the rate of dCMP effect. In contrast, orthophosphate was found to be an deaminase inactivation in Novikoff rat tumors was en inactivator of dCMP-deaminase (Chart 4). A completely hamced by preincubation with microsomes from the same stable dCMP-deaminase extract was gradually inactiva tumor or from normal rat liver. In comparison with ted by the addition of 0.5 Mphosphate buffer, the inactiva parenchymatous organs such as liver, Ehrlich ascites

TABLE 2 THE EFFECT OF SUBCELLULAR FRACTIONS ON THE INACTIVATION OF dCMP@DEAMINASEa 1.0 5@j PREINCUBA DEAMIN (%)FractionOriginâ€”30mmâ€”â€”3.0â€”RRatliver1.84015TIONINCUBATIONADDITIONdCMP AThD (,anole)Loss

a â€˜U LU > z 0 mm30 mmâ€” U R Mouse liver 0.4 87 a- R Ehrlich car 0.5 83 U 0.5 cinoma U) â€˜U N Rat liver 2.0 30 0 (+dCTp) M Rat liver 2.4 20 R Rat liver 0.26 92 R (80Â°C, Rat liver 2.7 10 (10mm) Râ€” Rat kidney2.9 0.44 87

a Isolated nuclei (N) , mitochondnia (M), and microsomal fraction (R), corresponding each to 15 mg dry weight, were resuspended in 0 5 30 45 0.1ml Tris buffer 0.1 M, pH 7.2 and addedto active extract in a final volume of 3.5 ml. TIME IN MINUTES CHART 5.â€”Time course and reversibility of dCMP-deaminase inactivation by dTTP. Extract (0.1 ml) was preincubated in GSH at 1 X i0@ M substantially lowered the effect of the absence (E) and in presence (E + dTTP) of 1 @moledTTP;glycyl R-factor, whereas GSSG at the same concentration had glycine buffer (0.1 M,pH 7.2) up to volume of 0.6 ml. To a parallel set of tubes preincubated with dTTP, 1 ,@moledCTP was added no effect. Mercaptoethylamine protected dCMP-de after 30 mm and preincubation continued for another 15 mm. aminase against added R-factor during preincubatiom but Incubation time was 15mm. dCMP 1.3 @mole,finalvolume 1.0ml. did not prevent spontaneous inactivation. This in dicated that R-factor did not play an essential role in the carcinoma showed a relative scarcity of preformed cyto inactivation of dCMP-deaminase in extracts from Ehrlich plasmic granules. For example according to our deter ascites tumor cells. minations, the protein nitrogen content of the microsomal We have observed that the amount of R-factor which fraction in Ehnlich ascites carcinoma was only 1.6 X can be extracted from acetone powdered microsomes is iO_12 g (2) in comparison to 33 X i012 g in adult rat liver. lower in liver 24 hr after hepatectomy than in the control. When the amount of microsomes in terms of protein from Also, the general depletion of the microsomal fraction in Ehnlich tumors was adjusted to that of normal liver or tumors is reflected in the less potent R-factor which can kidney a comparable inactivation was observed (Table 2). be extracted from such acetone powdered tumors as The inactivating effect of microsomes seemed to be a Novikoff rat tumor, hepatoma obtained after 200 days of general phenomenon and there seemed to be no species 3'-methyl-4-dimethylaminoazobenzeme feeding and mouse specificity since microsomes from mouse or rat liver, rat Sarcoma 180. It seems, therefore, that the potency of the kidney or mouse Sarcoma 180 produced the same effect. R-factor varies inversely with the rate of proliferation of On the other hand, the inactivating effect of other sub the tissue. In Ehrlich ascites carcinoma there is little cellular fractions was small and probably due to micro R-factor present. The R-factor is destroyed by heating somal contamination. An increased rate of inactivation to 58â€”60Â°Cfor10 mm, is nomdialyzable and can be pre or complete inactivation of a stable dCMP-deamimase in cipitated from microsomal extracts at pH 5. This the presence of microsomes was confirmed by paper material, which is obviously still a mixture of proteins, has electrophoresis, paper chromatography and the colori an absorption maximum at 270 m@i. The fact that the metric determination of ammonia liberation, as well as by R-factor is a protein and that the inactivation of stable spectrophotometric tests. Substrate protected dCMP dCMP-deaminase induced by R proceeds linearly with deaminase against inactivation by R-factor but this time (Chart 6) led us to assume (6) that R might inactivate protection was only partial in the presence of an excess of dCMP-deaminase enzymatically. However, the fact microsomal factor (Table 3). dCTP protected dCMP that orthophosphate and pyrophosphate also induce a deaminase against R-factor. Neither dCTP nor dCMP, progressive inactivation dCMP-deaminase and that the however, could reactivate dCMP-deaminase when they pH dependence of R action is similar to that for spon were added after preincubation of the extract with R taneous inactivation weakens the argument for the enzy factor. Sodium fluoride at a concentration of 1 X iO_2 matic nature of R action. Instead it indicates that R M, pH 7.2, had no effect on the R-factor, but a fresh acts similarly to P1 or pyrophosphate as an irreversible solution of 2 X 10â€”2Mmercaptoethylamine or mercapto inhibitor of dCMP-deaminase. In agreement with this ethanol at pH 7.2 completely inhibited the R-factor. interpretation is the finding that the amount of dCMP

TABLE 3 PROTECTION OF DCMP-DEAMINASE BY VARIOUS FACTORS AGAINST THE INACTIVATING EFFECT OF MICR05OMAL (R) FRACTI0Na

AcnvITY

Expazi additionsNo preincubation with RENT No. Without preincuba + tionAfter + dCTPR + + MER + GSHR Heated additionsHeated extractRR MEAR extract

1 1.17 0.26 1.1 0.0 1.01 (0.0)â€• 2 0.88 0.33 0.9 0.0 0.88 (0.0) 3 1.01 0.73 0.9 0.0 1.0 (0.0) 4 1.19 0.79 0.25 0.71 1.2 5 0.84 0.40 0.0 0.37 0.8 6 0.9 0.49 0.0 0.34 0.92 7 1.2 1.11 0.64 0.94 1.2 8 0.67 0.3 0.0 0.29 0.75

a Activity refers to @@moles dCMP deaminated in 15 mm at 37Â°C. Preincubation time 30 mm. Pre incubated mixture consisted of extract (0.2 ml), additions and Tnis buffer (0.1 M, pH 7.2) in a volume of 1.0 ml. Additions: R (4 mg), dCTP (1.0 pmole), MEA (mercaptoethylamine, 20 @imoles),ME(men captoethanol 20 Mmoles), GSH (glutathione 1 @zmole),heated extract 0.2 ml; dCMP 1.3 smole in 0.2 ml. Final volume 1.2 ml. b Numbers in parenthesis refer to activities obtained when additons were made after preincubation.

I00 I.0 a I .0 â€˜U I- a â€˜U â€˜U 80 z> â€˜U S 0 z > U 0 z 1- 0 a. U 60 U a. @0 , z

U .@ Ui 0.5 - -I 0 40 â€˜U 0 0 0 20

0 2 4 6 8 10 0 5 10 15 20 25 30 35 4-0 TIME IN MINUTES R(mg) CHART 6.â€”Effect of factors from microsomes and ascitic fluid CHART 7.â€”dCMP-deaminase inactivation : Dependence on the on inactivation of dCMP-deaminase. Extracts (0.2 ml) diluted amount of microsomal inactivator (R). An extract (0.2 ml) pre with glycyiglycine buffer (0.1 M, pH 7.2) to 1.0 ml and preincu incubated in 6 parallel sets with increasing amounts of R (0.0, bated for 30 and 40 mm, (C1 and C2, respectively). In absence 2.0, 4.0, 6.0, 8.0, and 10mg) in a volume of 1.0 ml. Incubation time of R (C1) and in absence of the ascitic fluid factor (C2) both ex was 15 mm with 1.3 @@moledCMPand a final volume of 1.2 ml. tracts were stable. In presence of R (5 mg, black squares) and of the ascitic fluid factor (10 mg, C + AF) a rapid linear decrease of activity occurred during preincubation. Incubation time was 15 The fact that the spontaneous inactivation of dCMP mm; dCMP 1.3 pmole, final volume 1.2 ml. deaminase in extracts could not be ascribed to the effect or R-factor and the circumstance that in many cases the deaminase inactivated is proportional to R up to a certain rate of dCMP-deaminase inactivation was more rapid limit ; the addition of excess R does not produce any further than could be ascribed to thelevel of orthophosphate pres effect (Chart 7). In this respect the effect of R is also ent in the extracts (as illustrated in Chart 4), led us to analogous to the effect of orthophosphate. suspect that another inactivating factor might sometimes

TABLE 4 COMPARISON OF THE RATE OF DCMP-DEAMINASE INACTIVATION IN 10 INITIAL ExTRACTSa

Paxp@a&TIoN12345678910Activitywithoutpreincu

bation Activityafter30minpre 0.8 0.21 0.22 0.33 0.57 0.37 0.52 1.20 1.2 1.02 incubation Enzyme activity lost (%)1.0 200.9 771.2 820.88 630.88 351.1 661.0 481.25 41.2 01.3 22

a Activity expressed in @@moles dCMP deaminated after 30 mm incubation. Extracts (0.2 ml) made in glyclyglycine buffer, 0.1 M, pH 7.2, diluted with the same buffer to a volume of 0.8 ml. After pre incubation 1.3 @moledCMPadded to a final volume of 1.0 ml.

TABLE 5 the electrolytes by dialysis and behaved like a pseudo THE INACTIVATION OF dCMP-DEAMINASE BY THE AscITIc FLUIDâ€• globulin. Comparison of various preparations by paper - electrophoresis and scanning of bromphenol blue stained or @cni bands suggested that it migrated together with a-globulin Expazi VITY A7TZR RENT No.Exra@crAc@xvx@Loss PREINCUBA pre pre fraction. The almost identical electrophoretic pattern of incubation1 (%)WithoutincubationAfter TION the ascitic fluid with that of serum suggested that the same or a similar factor might occur in serum too. In deed, mouse serum showed a slight inactivating effect on U 0.50 0.22 56 dCMP-deaminase during preincubation and a similar 2W W 1.04 0.83 20 effect was exerted also by a-globulin subfractions IV-1 W+A.F.0.56 1.040.45 0.020 100 and IV-4 of human sera. However, the effect of serum

a Acetone powder used for extracts W and U was made either was only about 10 % that of the same volume of the from cells washed 3 times with physiologic saline (W) or from un ascitic fluid, with approximately the same protein con washed cells (U). Preincubation time 30 mm. Extracts 0.2 ml, tent. ascitic fluid (A.F.) 0.2 ml, dCMP (1.3 jzmole) and Tris buffer (0.1 DISCUSSION M, pH 7.2) to a final volume of 0.8 ml. b Activity expressed in pxnoles of dCMP deaminated in 30 mm The experiments described in this paper have demon at 37Â°C. strated that dCMP-deaminase is completely stable at 37Â°Cin tissue extracts. The enzyme becomes labile upon be present. When the extracts were made from acetone dilution or dialysis. The same lability can be effected powdered cells which were carefully washed (see also by the addition of certain factors which are either â€œMaterials and Methodsâ€•), we observed on the whole, normally present in tissue or are formed in vitro during the the same uniform pattern of dCMP-deaminase inactiva preincubation of extracts. Upon dissociation by dilution tion already described. However, extreme variations in or dialysis the enzyme becomes inactive but can be re the stability of dCMP-deaminase were observed in initial activated partially by the addition of heated extract or by extracts prepared before the importance of removing the dCTP. The partial reactivation of dCMP-deaminase ascitic fluid from the cells was realized (Table 4). This first demonstrated by Maley and Maley (16), the stimula suggested that the ascitic fluid might contain a dCMP tion of dCMP-deaminase by 6-aza-2'-deoxycytidine-5'- deaminase inactivating factor. When half of the cells phosphate reported by KÃ¡ra and @orm (ii) and the were washed 3 times prior to making the acetone powder reactivation data in this paper demonstrate that the con and the other half was not washed, there was little dif tention (21) that dCMP-deaminase is â€œeitheractive or ference in the activity of both extracts in the presence of denaturedâ€• is erroneous. There exists, in fact, an in dCMP. However, preincubatiom in the absence of active enzyme state between the states of active enzyme substrate led to a considerably greater loss of activity in and irreversibly inactivated enzyme. The intermediate the unwashed sample. The addition of ascitic fluid to state may be long-lived at low temperature (for example stable (washed) extract led to the complete loss of activity during dialysis at 0Â°C),but has a short duration at 37Â°C. after 30 mm (Table 5). Heating the ascitic fluld to 80Â°C Even at this temperature, however, it may be fixed to a for 10 mm abolished this effect. The addition of a purl long-lived state with dTTP. The linear time course of fled fraction of the ascitic fluid to a stable dCMP-de dCMP-deaminase inactivation persistently observed in aminase induced a linear time course of inactivation extracts after dilution indicates that even at 37Â°C the (Chart 6). Neither NaF nor mercaptoethylamime (5 X process is not due to a simple denaturation of the protein 10â€”sM) inhibited the ascitic factor; however, dCTP molecule. The linear time course of dCMP-deaminase protected against it. The factor was thermolabile (80Â°C, inactivation contrasts with the exponential decrease of 10 mm), withstood prolonged dialysis (72 hr) and could activity which is observed in a monomolecular process be salted out with ammonium sulfate in the globulin such as denaturation. A linear time course of dCMP fraction. The factor did not precipitate after removal of deaminase inactivation was also observed by Myers â‚¬1al.

(17) in their study on dCMP-deaminase in regenerating of the cofactor by relatively high levels of inorganic liver; however, they did not analyze this phenomenon any phosphate, by sodium pyrophosphate and especially by further. We have concluded from our observations that the action of a protein factor from microsomes and another the linear time course of dCMP-deaminase inactivation in protein factor from ascitic fluid. The linear course of in extracts is probably due to am interaction between dCMP activation induced by these compounds means that the deaminase and some factors present in the extracts. This concentration of the complex between enzyme and in has been confirmed by the finding that orthophosphate activator is kept constant for a certain period of time. induces such a gradual inactivation of a stable dCMP It is difficult to evaluate the extent to which these deaminase preparation. Orthophosphate acts as an ir factors may participate in vivo in the regulation of dCMP reversible inhibitor. Irreversible inhibition is often deaminase activity. The R-factor may be the means by characterized by a progressively increasing inhibition with which the cell disposes of dCMP-deaminase when the time, as had been demonstrated by Aidridge (1) in the enzyme is no longer needed, whereas dTTP may regulate case of acetylcholinesterase inhibition by diethyl p-nitro the extent of its activity. These factors, dTTP and the phenyl thiophosphate. The progressively irreversible R-factor may change the activity of dCMP-deaminase inactivation of dCMP-deaminase with time in moderately within minutes. Besides such a rapid mechanism there diluted extracts may be due to orthophosphate accumu seems to be a slower regulatory mechanism that may be lated by gradual dephosphorylation of various nucleo of even greater fundamental importance. The high tides. The linear time course of dCMP-deaminase in activity of dCMP-deaminase accompanied by vanishingly activation also led us to the discovery of 2 proteins which low dCMP-phosphatase activity in Ehrlich ascites tumor inactivated dCMP-deaminase irreversibly. One of them, cells is another example of the reciprocal relationship be the pseudoglobulin present in the ascitic fluid is respon tween these enzymes. We observed (3, 7) that whenever sible for the rapid inactivation of dCMP-deaminase in all there is a change in the rate of cell proliferation in a tissue, preparations made from poorly washed cells. This factor such as during embryonic development, regeneration or whose origin is as yet unknown might be a product of carcinogenesis in rat liver, a high dCMP-deaminase is ac peritoneal cells secreted into the ascitic fluid. On the companied by a low dCMP-phosphatase activity and other hand, it might be identical with a weakly active vice versa. It is possible that both enzymes have different component from serum which accumulates in ascitic fluid. active life times in the cell so that the period of activity of The second protein, which we find in the microsomes (6), one enzyme may coincide with the period of inactivity of is present in Ehrlich ascites tumor cells in amounts too the other. This could explain the various activities of small to play a role in the inactivation of dCMP-deaminase both enzymes encountered in different tissues while the in dilute extracts. However, the observations that this concentration in the â€œactiveâ€•period might not vary too factor if present in small amounts in rapidly proliferating much. This hypothesis awaits further investigation. Ehrlich ascites tumor cells and regenerating rat liver is However this may be, the reciprocal relationship between plentiful in microsomes of such nonproliferating tissues as the activities of dCMP-deaminase and dCMP-phosphatase liver or kidney, suggests that it might play a role of indicates that the activity of these enzymes is in addi physiologic importance. In any case, the rejection by tion regulated by induction and repression through the Scarano et al. (20) of a microsomal inactivator of dCMP genetic system of the cell. deaminase simply on the basis of a priori reasoning and Finally, we might mention that the behavior of dCi\IP measurements of thermal inactivation is invalid on the deaminase in extracts, as described in this paper, bears basis of our experimental deta. The fact that all the some striking resemblances to the behavior of thymidylate irreversible inhibitors of dCMP-deaminase enumerated kinase, the enzyme which catalyses the phosphorylation of can be counteracted by the simultaneous addition of T1\'IP to thymidine diphosphate in the presence of ATP. dCTP indicates that all of these compounds together with The activity of thymidylate kinase is low in nonproliferat dTTP, may share a common site of attachment to the ing mammalian tissues but is high in rapidly proliferat dCMP-deaminase molecule. The difference between the 1mgtissues (10, 12). Hiatt and Bojarski (9) have observed irreversibility of these inactivators and the reversibility of that this enzyme is very labile in the absence of thymidy dTTP might be due to a difference in the configuration of late or thymine. However, a substance which protects the site of attachment. the labile enzyme against inactivation can be adsorbed on The foregoing experiments give thus the following charcoal from a heated extract and can be eluted. These picture of inactivation of dCMP-deaminase in tissue authors left open the question whether the lability of extract : The protection of dCMP-deaminase by dCTP or, TMP-kimase stems from the presence of an enzyme possibly, by other compounds, holds as long as the con inactivating protease or from the inherent lability of the centration of these compounds is kept at a certain level. protein. Having observed the instability of the enzyme According to the law of mass action, the dissociation of a in purified preparations, they favored the latter explana complex is facilitated by dilution or dialysis (another form tion. However, Gray et at. (8) have observed a non of dilution), with the removal of the protective cofactor dialyzable, thermolabile factor in rat liver which reduces and the production of an inactive enzyme, which is now in the activity of TMP-kinase. In view of the similarities in an intermediate reversible state still capable of reactiva the inactivations of dCMP-deaminase and TMP-kinase, tion. This intermediate state is, however, labile and at one may speculate whether TMP-kinase is really such a 37Â°Cundergoes a rapid irreversible rearrangement. The labile enzyme and whether it might not be considerably dissociation of the enzyme is also facilitated at low levels stable in the absence of the inactivating factors described

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1965 American Association for Cancer Research. 932 Cancer Research Vol. 25, July 1965 in this paper. It might also be mentioned that Hoagland Biology, Vol. XXVI, The Biological Laboratory, Cold Spring et at. (10) have reported recently that microsomes contain Harbor, L.I. New York, 1961. 10. Hoagland, M. B., Scornik, 0. A., and Pfeffenkorn, L. C. As a thermolabile, nondialyzable factor which inhibits the pects of Control of Protein synthesis in Normal and Re rate of protein synthesis and might be completely counter generating Rat Liver, II. A Microsomal Inhibitor of Amino acted by GTP. The relationship, if any, between this Acid Incorporation Whose Action is Antagonized by Guano factor and the dCMP-deaminase inhibiting factor from sine Tniphosphate. Proc. Natl. Acad. Sci. U.S., 61: 1189â€”91, 1964. microsomes which can be counteracted by dCTP should 11. KÃ¡ra,J., and @orm,F.Activation of Deoxycytidylate Deami also be investigated. nase from Ehrlich Ascites Tumor Cells by 6-Aza-2'-deoxy cytidine 5'-phosphate. Biochim. Biophys. Acta, 80: 154-57, ACKNOWLEDGMENT 1964. 12. Kielley, R. K. Patterns of Synthesis of Thymidine Tniphos We wish to acknowledge the help of Mr. Harold E. Kasinsky phate and other Deoxynibonucleotide Tniphosphates in Mouse in the revision of this manuscript, and of Emerich S. Fiala, who Liver and in Mouse Ascites Hepatoma. Cancer Res., @3:801â€” determined the conditions for the spectrophotometnic assay of 10, 1963. dCMP-deaminase. 13. Maley, F., and Maley, G. F. Nucleotide Interconversions in Embryonic and Neoplastic Tissues. I. The Conversion of REFERENCES Deoxycytidylic Acid and Thymidylic Acid. J. Biol. Chem., @34:2975â€”SO,1959. 1. Aldridge, W. N. Some Properties of Specific Cholinesterase 14. . Elevation of Deoxycytidylate Deaminase and Thy with Particular Reference to the Mechanism of Inhibition by midylate Synthetase in Regenerating Rat Liver. Ibid., 255: Diethyl p-Nitrophenyl Tniphosphate (E.605) and Analogues. 2968â€”70,1960. Biochem. J., 46: 451-60, 1950. 15. . Nucleotide Interconversions. IV. Activities of Deoxy 2. Fiala, S. Fractionation, Nucleic Acid Distribution and Respi cytidylate Deaminase and Thymidylate Synthetase in Normal ration of Ehnlich Ascites Tumor Cells. Naturwissenschaften, Rat Liver and Hepatomas. Cancer Res., 21: 1421â€”26,1961. 45: 368-69, 1958. 16. . Nucleotide Interconversions. IX. The Regulatory 3. Fiala, S., Fiala, A. E., and Glinsmann, W. Proliferation of Influence of Deoxycytidine 5'-tniphosphate and Deoxythy Tumor Cells in Rat Liver and Its Relationship to the Trans midine 5'-tniphosphate on Deoxycytidylate Deaminase. J. formation of Deoxycytidylic Acid. Federation Proc., 9: 398, Biol. Chem., 257: PC3311â€”13,1962. 1960. 17. Myers, K. K., Hemphill, C. A., and Townsend, C. H. Deoxy 4. . Deoxycytidylic Deaminase in Carcinogenic Rat Liver. cytidylate Deaminase Levels in Regenerating Liver. Can. J. Naturwissenschaften, 47: 45-46, 1960. Biochem. Physiol., 59: 1043â€”54,1961. 5. . Mechanism of Carcinogenesis and Proliferation of 18. Myers, D. K. Stability of Deoxycytidylate Deaminase in Tumor Cells in Rat Liver. Pathol. Biol. Semaine Hop. 9: Vitro. Can. Ibid., 39:1656â€”58,1961. 613â€”16,1961. 19. Scanano, E. 5'-Deoxycytidylic Acid Deaminase: Enzymic 6. Fiala, S., and Fiala, A. E. Microsomal Inactivator of De Production of 5'-Deoxyunidylic Acid. Biochim. Biophys. oxycytidylic Acid Deaminase. Biochim. Biophys. Acta, 49: Acta, 29: 459â€”60,1958. 228â€”31,1961. 20. Scarano, E., Bonaduce, L., and dePetrocellis, B. The Enzy 7. Fiala, S., Fiala, A. E., Tobar, G., and McQuilla, H. : Deoxy matic Aminohydrolysis of 4-Aminopynimidone Deoxynibo nucleotidase Activity in Rat Liver and Certain Tumors. J. nucleotides. III. Purification and Properties of 2'-deoxy Natl. Cancer Inst., Â£8:1269â€”89,1962. nibosyl 4-aminopynimidone Phosphate Aminohydrolase from 8. Gray, E. D., Weissmann, W. M., Richards, J., Bell, D., Kirs, Monkey Liver. J. Biol. Chem., 257: 3742â€”51,1962. H. M., Smellie, R. S., and Davidson, J. N. Studies of the Bio 21. Scarano, E., Geraci, G., Poizella, A., and Campanile, E. The synthesis of Deoxyribonucleic Acid Extracts of Mammalian Enzymatic Aminohydrolysis of 4-Aminopyrimidone Deoxy Cells. V. Factors Interfering with Biosynthesis. Biochim. nibonucleotides. IV. On the Possibility of the Occurrence of Biophys. Acta, 45: 111â€”20,1960. an Allosteric Site on 2'-Deoxyribosyl-4-aminopyrimidone 9. Hiatt, H. H., and Bojarski, T. B. The Effects of Thyrnidine 2,5'-phosphate Aminohydrolase. Ibid., 258: PC1556â€”57,1963. Administration on Thymidylate Kinase Activity and on DNA 22. Sidler, A. J., and Holtz, M. T. Regulatory Mechanisms on the Synthesis in Mammalian Tissues. Cellular Regulatory Mocha Deoxyribonucleic Acid Metabolism of Lactobacillus R-26. nisms In: Cold Spring Harbor Symposium Quantitative Ibid., 258: 697â€”701,1963.

Silvio Fiala and Anna E. Fiala

Cancer Res 1965;25:922-932.

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