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Oxidative Phosphorylation in Homogenates Normal and Tumor Tissues of VAN R. POTTER AND GLORIA G. LYLE (McArdle Meinori@alLaboratory, University of Wiscon.nn, Madison 6, Wi.s.) Earlier studies from this laboratory (13, 18, @0) with homogenates prepared in isotonic KC1 showed that tumor tissues were essentially devoid of oxidative activity in reaction mixtures in which large quantities of oxygen could be used by ho mogenates of brain, heart, liver, and kidney. The circumstances of the oxidation were such that it might be assumed that the oxalacetate, when oxidized, proceeded via the Krebs citric acid cycle and that the process was phosphorylative in na tune. Studies with kidney (13, 18, @0)showed that the oxidations are maintained by the phosphate reservoirs which they in turn maintain, and it therefore became desirable to correlate the oxida tive rates in the various tissues with the status of the phosphate reservoir during the course of the reaction, since failure to maintain the reservoir might result in a loss of oxidative activity before measurement of it could be made. In addition, the nature of the oxidative pathway was studied by determining the levels of intermediates in the Knobs citric acid cycle. Malic and citric acids were determined, since the metabolic pools of these substances in an equilibrium system would be much greater than the amounts of the other in termediates. Oxalacetate was the substrate added, and the rate of its disappearance was followed analytically. w@eighed, and homogenized in cold isotonic KCI. All-glass homogenizers were used to avoid metal contamination and to maintain nuclei and mito chondnia as intact as possible. Mea@iurement of oxal,aceth, oxidase activity.— The enzyme activity measurements were carried out under the conditions described by Potter et al. (@0) with occasional slight deviations. In all cases 1.5 ml. of a stock solution similar to that de scnibed earlier (@0) was added to each flask to give a final concentration of 0.067 M KC1, 3.3 X 10—8 M MgC12, 6.6 X 10@ M potassium phos phate, and 1.3 X 10@ M cytochrome c at pH 7.0. Other properties of the system and precautions as to speed and cold, proper tonicity, use of alkaline KC1, and partially neutralized oxalacetic acid (13.8 @iMper flask) have been noted in earlier publications (18, @0).The ATP was added to give a final molarity of 0.001 M in a volume of 8.0 ml. The oxygen uptake was measured at 10- or @0minute intervals in duplicate flasks, and all the data are recorded in terms of the oxygen uptake per 10 minutes, plotted against time, in order to correlate the decline in oxidative enzyme activity with changes in the concentrations of primary substrate, intermediates in the Krebs cycle, in organic phosphate, and ammonia. Analytical methods.—Protein-free filtrates were prepared by adding @.0ml. of cold 17.5 per cent EXPERIMENTAL trichloroacetic acid per Warburg flask to give a MATERIALS AND METHODS final volume of 5.0 ml. and a trichioroacetic acid Preparation of tissues.—The tissues were oh concentration of 7 per cent. The flasks were re tamed from young, adult male rats weighing be moved from the manometers at various intervals tween @00 and 300 gm. The animals were obtained and placed in an ice bath immediately before add from the Holtzman Rat Company and were main ing the acid (14). Analytical methods were as fol tamed on a stock diet of mixed grains for 1—@lows : inorganic phosphate (8), citric acid (9, weeks before they were used. The rats were killed cf. @0),malic acid,' ketoacids (5, cf. @0),and am by decapitation, and the tissues were chilled in monia (1).2 ice-cold isotonic KC1. The tissues were blotted, Reagents.—Oxalacetic acid was prepared by Dr. C. Heidelberger, using an improved method 4 This work was aided by grants from the American Cancer Society on the recommendation of the NationalResearch cer Institute Service. Received Council, of the Committee on Growth and from The National ofthe National Institutes for publication December ofHealth, 1J@F. Speck, unpublished data. Can Public Health 2 We are greatly indebted to Jean H. Webster of the Pathology Drs. John Department W. Harmon and for making pre liminary determinations and for advice on the use of this pro cedure. 29, 1950. 355 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. Cancer Research 856 of synthesis (6). Pyruvic acid was redistilled in vacuo, maintained as a 1 N solution in the cold, and jieutralized with K2CO3 before use. ATP was pre pared from rabbit muscle under the direction of Dr. G. A. LePage (cf. @4).Cytochrome was pre pared from beef heart according to the method of Keilin and Hartree (cf. @4). RESULTS A series of determinations of oxygen uptake was carried out with l@—l6identical reaction mix tures containing aliquots of the same tissue ho OXYGEN UPTAKE @6O 0 ly to show the time course of the changes in the reaction mixture and to facilitate comparisons between tissues. Instead of plotting all the changes for each tissue in one chart, the data on each reaction component that has been determined are plotted in separate graphs that show the changes when the various tissues were used. The results for each of the reaction components cor relate very satisfactorily with changes in the other reaction components for each of the tissues stud ied, and may be considered in terms of their inter relationships. Thus, the oxygen uptake data (Chart 1) are related to the data on substrate dis appearance (Chart @) and on intermediary metabolite formation (Chart 3), while the inor ganic phosphate changes (Chart 4) are related to both oxygen uptake and metabolite data, and the ammonia data (Chart 5) are secondary to the L&J z KETO @4C ACIDS Ui 0 >- x 0 (1) c@2C Ui -J 0 U @@256 0 CHART 1.—Changes in rate of oxygen uptake by whole homogenates of various rat tissues with oxalacetate as the added substrate. Conditions as described in text. The amount of tissue per flask was as follows: kidney, 30 mg.; heart, 40 mg.; and liver, brain, and tumor, 50 mg. Each curve is the average of two to four experiments with homogenates from different rats. The following four figures represent the results of analyses from experiments that included those reported in this figure. 2 @ mogenate. After 10, 30, 50, 70, 90, and 1@0 minutes, successive pairs of duplicate flasks were treated with trichloroacetic acid and analyzed for ketoacids, citric acid, malic acid, inorganic phos phate, and ammonia. In subsequent experiments which were extended to as much as 300 minutes, the flasks were taken of! at somewhat different in tervals. The experiments of long duration were continued in order to reach the plateau level of phosphate and ammonia output. Each tissue was tested several times in experiments in which all the analyses were performed on the same reaction mixtures. Numerous experiments in which only part of the analyses were done are unreported, but they support the data in Charts 1—5,in which the results of @—7 experiments on each tissue have been averaged. The data have been recorded graphical CHART2.—Disappearance of ketoacid during the oxidation of oxalacetate as shown in Chart 1. phosphate data. A typical experiment on kidney is shown in Chart 6, in which all the measure ments have been recorded in a single chart to show the correlation among the various findings. The data on oxygen uptake are given in Chart 1 and may be compared to the data on ketoacid disappearance in Chart @.Since in these experi ments oxalacetate was the only substrate added, and since the @onversionof oxalacetate to pyruvate does not involve ketoacid disappearance, Chart is a record of substrate utilization. The accumu lation of a-ketoglutarate is negligible under these conditions. It may be seen that oxalacetate was rapidly utilized by homogenates of heart, liver, Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. Po@ AND Lmi@—Phosphorykdion kidney, and brain but that in the latter the oxygen uptake ceased before the ketoacid was used up after a period of rapidly declining oxygen uptake. In the case of the Walker @56 carcinosarcoma, no period of oxygen uptake was observed, and there was a very slight disappearance of ketoacid in the first @0minutes of incubation. Identical results in Normal and Tumor T@ues 857 there was essentially no change in the level of ketoacid during the second hour of incubation. In heart homogenates the decline in oxygen uptake paralleled the decline in ketoacid, but in liver and kidney homogenates there was a considerable Ca&wr 5.—Changes in the concentration of ammonia ing the oxidation of oxalacetate as shown in Chart 1. dur 28 27 26 CHART 8.—Changes in the malate during the oxidation INORGANIC concentration of oxalacetate of citrate and as shown in Chart 1. 25 >- 24 x 0 23 __ PHOSPHATE @ z w -J 22 Ui I- 21 Ui I< @ = 1 0 I- OS a- CHART 6.—Typical showingcorrelatious CHART4.—Changesin the concentration of inorganic phos pilate during the oxidation were obtained of oxalacetate as shown in Chart 1. with Flexner-Jobling rat carcino mas. It appears that in brain there was an active enzyme system initially, but the system became inactivated rapidly and was no longer capable of utilizing ketoacid, while in the tumor tissue the amount of enzyme was much smaller initially, or it was inactivated even sooner. In both tissues, experiment with 30 mg. between chemicalchanges rat kidney, occurringin the reaction mixture. Phase I is shown during 0-10 minutes, phase II, 10-50 minutes, and phase Ill, 50-120 minutes. Original oxalacetate concentration was 18.3 paL. amount of oxygen taken up after the ketoacid level had fallen to less than 1 @M per flask. A partial explanation for the continuation of oxygen uptake in the liver and kidney homoge nates may be seen in Chart 3, which shows the changing levels of citrate and malate during the incubation period. There was an increase in these Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. Cancer Research 858 two metabolites during the period when ketoacid disappearance was very rapid, and after the keto acid level reached values of 1—@ j@Mper flask (or about 0.0005 M), the citrate and malate levels no longer increased, but declined somewhat in con junction with continuing utilization of oxygen notably in liver. In heart and brain there was no decline in malate or citrate after the ketoacid utilization ceased, and thus after the first hour the homogenates of these tissues were essentially inert as far as fuel combustion was concerned. The ability to reduce ketoacid concentration to very low levels and to continue to oxidize the citrate and malate is definitely correlated with the maintenance of a favorable phosphate balance as shown in Chart 4. The homogenate of tumor tissue did not oxidize ketoacid and therefore did not maintain ATP; a rapid output of inorganic phos phate was observed. In brain, kidney, and liver homogenates there was an initial output of phos phate during the period of malate accumulation, when the oxalacetate was at its highest concentra tion and was competing with oxygen for the avail able hydrogen and electrons. This was followed by an uptake of phosphate, as the oxidative phase became dominant, and finally by an output small in comparison with the total in these experi ments. The curves for ammonia formation (Chart 5) are closely correlated with the phosphate curves of Chart 4, with the exception of the brain ho mogenates. In the other tissues, ammonia is given off shortly after the phosphate is split off, but in brain the ammonia output is very slow. In kidney, the ammonia output occurs only during phases I and Ill, and the concentration of free ammonia remains constant during phase II when inorganic phosphate is being removed from the reaction. This stationary phase of the ammonia curves in kidney homogenates is much more pronounced in individual experiments, e.g., as shown in Chart 6, than in Chart 5, which represents the average of five homogenates. The data suggest that ATP, as such, cannot be deaminated, and that deamina tion occurs only after at least one phosphate has been removed from theATP molecule. The removal ofamnioniaisevidentlynotpartofthedephosphory lation reaction, because in brain the phosphate out put is not accompanied by a corresponding out put of ammonia. Furthermore, the two reactions can be dissociated by fractionating the homoge nate, as shown elsewhere (19). of phosphate as the ketoacid was used up. These three phases were noted earlier in kidney homoge nates by Potter et al. (18), but at that time the data on the oxidizable substrates and ammonia were not available. The reversals in the trend of the phosphate curve, although seen as single points in Chart 4, are not experimental errors, since experiments with closely spaced points have shown that true reversals occur as indicated (cf. Chart 5 and [18]). The failure of the oxidative mechanism is nearly simultaneous with the change in the phosphate balance from uptake to output, and comparison of Charts @—4 shows that after the phosphate output of phase III sets in, the metabolites are no longer oxidized. The case of liver is of special interest, because the period of phosphate uptake (phase II) is prolonged and because the phosphate level does not plateau dun lag phase III at the level that it does with the other tissues. It is also seen that with liver there is an absolute fall in inorganic phosphate that ap proximates the difference between the final values obtained for liver and the other tissues. It seems possible that the data may reflect the formation of inorganic pyrophosphate during phase II in liver in view of previous reports (cf. @,8). The source of the phosphate in these mixtures is ATP plus a small amount of organic phosphate present in the homogenate. Measurements of total phosphorus (18) showed that in kidney the phosphorus re leased from the homogenate is quantitatively DISCUSSION The data in the present study show that a valid assay for the oxidizing enzymes of the Krebs cycle can be obtained only during the period in which the phosphate balance is being maintained, and that in homogenates in which no phase II occurs the period of valid oxygen uptake rate may be very brief as in brain, heart, and tumor homoge nates (Chart 1). In this chart, the data suggest that tumor tissue must have a relatively low level of the oxidative system, since it appears inactive at the outset. However, the data on ketoacid re moval (Chart @) show that there was a small initial period of activity in tumor homogenates during the interval before maximum phosphate output had occurred and before oxygen uptake meas urement was begun. This trartvient period of keto acid removal is in contrast to the slow but con stant rate of extra ketoacid disappearance in gly colyzing tumor homogenates in which ATP was maintained (17). In the latter, the rate was com parable to that which had been observed in tumor slices (4). The net conclusion from the data in Charts 1—5 is that in the tumor homogenates studied the breakdown of ATP is so rapid in these reaction mixtures that no oxidative activity can be ex pected. The next question is whether the reaction components might be altered so as to make condi tions more favorable. In these experiments, oxalacetate was used as the sole substrate, be Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. POTTER AND LmF'@—Phosphoryla@ion cause it could serve as a source of both oxalacetate and pyruvate, and its disappearance could readily be followed analytically. However, this compound is rapidly reduced to malate (Chart 3) during phase I, at which time the oxygen uptake in liver and kidney is submaximal and ATP breakdown occurs. This substrate thus appears to compete with oxygen, thereby lowering the yield of phos phate bond energy (cf. 7). In earlier studies with water homogenates, in which the phosphatase action is more pronounced than in KC1 homogenates (18), Potter (14) was able to convert a negative phosphate balance to a positive phosphate balance with sodium fluoride, which has frequently been used to cut down phos phatase activity in systems involving oxidative phosphorylation. However, in those experiments and in subsequent tests upon the improved kid ney system (13), fluoride decreased the rate of oxygen uptake, and no attempt was made to increase the activity of inactive tumor homoge nates by fluoride at that time. Meanwhile, other investigators, using isotopically labeled substrates with tumor slices, had shown that hepatomas could form CO,@from glucose more rapidly than could normal liver (11, @7),while pyruvate was handled equally well by both tissues (11). Al though the glucose finding may be related to a special inability of liver slices to burn glucose (10), recent studies with labeled fatty acids, including acetate (@5), have shown that labeled citrate and CO2 could be formed from these substrates in several kinds of tumor slices which were compared to liver and kidney slices. The occurrence of the “condensing enzyme,― aconitase, and isocitric dehydrogenase was also demonstrated in these these (@6), and earlier studies from the McArdle Laboratory had demonstrated significant activity for succinic and malic dehydrogenases and for hy drogen transport components. However, when slice studies with labeled acetate were begun here they showed an almost complete lack of the ability of two types of rat tumor to convert acetate to in Normal and Tumor 359 Tissues malonate as inhibitors, and the results support the experiments of Weinhouse et al. (f25, @6),in so far as they indicate the existence of a Krebs cycle in tumor tissues. These observations are in harmony with the findings in the present report in that both indicate that the homogenization process and sub sequent conditions used previously have led to the loss of Krebs cycle activity from tumor homo genates but not from liver and kidneyhomogenates; but an important point to be settled is whether or not the slice technic can be taken to indicate the relative levels of the oxidative enzyme com ponents in normal and tumor tissues, as it would seem likely that the ease with which the Krebs cycle activity is lost from the homogenate may be an indication of a low level of these enzymes as well as a high level of destructive enzymes. Sepa rate studies with liver and kidney mitochondria and nuclei (19, @) show that the level of oxidative activity is affected by the balance between phos phate breakdown and phosphate uptake, so that an inhibitor which affects this balance may stimu late in one system and inhibit in another (cf. @3). Thus, fluoride, which inhibited the kidney ho mogenate (see phosphate uptake in Chart 4), might stimulate the tumor homogenate (see phos phate output in Chart 4). Preliminary studies by Potter have shown that when a mixture of pyru vate and fumarate was used as a substrate with isotonic KC1 tumor homogenates, the addition of sodium fluoride permitted the demonstration of steady rates of oxidation for @0—40 minutes in reaction systems otherwise the same as herein de scribed. The maximum rates were essentially in dependent of the fluoride concentration, but the maintenance of the rate increased with increasing fluoride concentration up to M/75. On the scale shown in Chart 1, 100-mg. quantities of Flexner Jobling tumor homogenates gave rates of l5—@0 MI. of 02 per 10 minutes, which correspond to Qo2 values of about 6—9,approximately the rate of oxygen uptake obtained with tumor slices. Space does not permit a discussion of the C02,ascomparedto liverandkidney(1@). Studies various lines of evidence that show a decreased with labeled pyruvates (carbonyl and carboxyl) amount of various oxidative enzymes in tumor were then undertaken, and it was found that in tissues as compared to brain, liver, kidney, and tumor slices pyruvate could be oxidized to CO2 at heart tissues, which in slices show Qo@values of a rates that approached the magnitude seen with magnitude similar to that seen in tumor slices. liver and kidney.3 However, in contrast to the These tissues may be differentiated from tumors tumors studied elsewhere (@5), only the hepatoma on the basis of a variety of enzyme measurements. slices were active with acetate, and the previous However, a number of other tissues, such as spleen, observations with acetate (1@) were confirmed. lung, and thymus, have amounts of these oxidative The pyruvate oxidation was carried out under a enzymes which do not differ markedly from levels variety of conditions with trans-aconitate and found in tumor. It is therefore of considerable in terest that these tissues are sharply differentiated aPotter, V.R.;Watson, L.S.;andHeidelberger, C.The from tumor tissues when tested for their ability to Oxidation of Labeled Pyruvate and Acetate in Tumor Tissue, accumulate citric acid in vivo following the injec in manuscript. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. Cancer Research 360 tion of fluoroacetate (15). We believe that the comparison between slices and optimally supple mented homogenates may be interpreted to mean that the oxidative enzymes in the tumor slice must be operating at near capacity, while the slices of normal tissues high in oxidative enzymes are not operating at capacity (cf. @1).This sug gests that the oxidative enzymes in the tumor slice may operate at higher concentrations of sub strate than the corresponding enzymes in the nor ma! tissues. Whether significant and unique al terations in metabolic patterns can result from such differences remains to be demonstrated, but the possibility is worthy of serious considera tion (16). SUMMARY 1. The oxidation of oxalacetic acid was studied in isotonic homogenates of rat kidney, brain, heart, liver, and transplantable tumors. Analytical studies were carried out to correlate the changes in the concentration of ketoacid, malate, citrate, inorganic phosphate, and ammonia with changes in the rate of oxygen uptake. @. In each tissue, the appearance of free am monia was secondary to the output of inorganic phosphate from the adenosine triphosphate reser voir, and the latter was correlated with the failure of the oxidative process. 3. The demonstration of oxidative enzyme ac tivity in the Krebs cycle in the reaction system employed was considered to depend upon a balance between the formation of high energy phosphate by the oxidative enzymes and the destruction of high energy phosphate bonds by various phos phatases. It was shown that in the Flexner-Jobling carcinoma and the Walker @56 carcinosarcoma the rate of phosphate breakdown completely over balanced the rate of oxidative phosphorylation so that no significant ketoacid removal occurred after 10 minutes. REFERENCES 1. CONWAY,B. E. J. Microdiffusion Analysis and Volumetric Error. London: Crosby LOCkWOOd& Sons, 1947. 2. Cons, C. F. Phosphorylation of Carbohydrates. In A Symposium on Respiratory Enzymes, p. 175. Madison: University of Wisconsin Press, 1942. 3. CRoss, R. J.; Taggart, J. V.; Covo, G. A.; and Green, D. E. Studies on the Cyclophorase System. VI. The Coupling of Oxidation and Phosphorylation. J. Biol. Chem., 177:65578, 1949. 4. Ei.uoi-r, K. A. 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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research. Oxidative Phosphorylation in Homogenates of Normal and Tumor Tissues Van R. Potter and Gloria G. Lyle Cancer Res 1951;11:355-360. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/11/5/355 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1951 American Association for Cancer Research.