AMINO ACID ACTIVATING ENZYMES IN RED BLOOD CELLS OF NORMAL, ANEMIC AND POLYCYTHEMIC SUBJECTS

G. Izak, … , J. Mager, A. Karshai

J Clin Invest. 1960;39(12):1763-1770. https://doi.org/10.1172/JCI104200.

Research Article

Find the latest version: https://jci.me/104200/pdf AMINO ACID ACTIVATING ENZYMES IN RED BLOOD CELLS OF NORMAL, ANEMIC AND POLYCYTHEMIC SUBJECTS *

By G. IZAK, T. WILNER AND J. MAGER WITH THE TECHNICAL ASSISTA.NCE OF A. KARSHAI (From the Department of Medicine B, and the Research Laboratory of Hadassah University Hospital and from the Department of Biochemistry, Hebreze' University-Hadassah Medical School, Jerusalem, Israel) (Submitted for publication May 18, 1960; accepted June 9, 1960)

Following the discovery by Hoagland (1) that solution was carefully decanted and served as the source the soluble cytoplasmic fraction of rat liver ho- of amino acid activating enzymes. The hemolysate could be stored at - 150 C for at least 2 weeks without ap- mogenates catalyzes an amino acid-dependent py- preciable loss of activity with respect to amino acid ac- rophosphate (PP) exchange with adenosine tri- tivation. This preparation was routinely used and no phosphate (ATP), similar systems have been de- attempt was made to exclude platelets and leukocytes, scribed in a variety of other tissues and organisms since the contribution of these cells to the over-all ac- (2-4). These so-called "amino acid activating tivity of the lysate ,was found to be negligible (see Re- sults). enzymes" (AAAE) serve, according to the cur- In some experiments the washed blood cells suspended rently prevailing opinion (5), to convert free in 0.25 M sucrose were disrupted by sonication in an ice amino acids into the corresponding aminoacyl water-cooled 10 KC-Raytheon oscillator. adenylates, thus initiating the chain of reactions Chemicals. All chemicals were of analytic or reagent mediating their incorporation into the protein grade. The radioactive pyrophosphate was prepared by pyrolysis of PU-labeled orthophosphate at 2800 C for 20 molecule. to 30 hours. The product usually contained less than The present communication provides evidence 0.1 per cent orthophosphate. for the occurrence of AAAE in human red blood Standard reaction mixture and assail procedure. Un- cells (RBC). In addition, the paper presents less otherwise stated, the following reaction mixture was quantitative data indicating the existence of some used for assaying amino acid activation: Tris buffer, pH normal level of 7.4, 50 /,moles; MgCl2, 5 Amoles, ATP, 2 &moles; PP', significant deviations from the 2 ,umoles; amino acid mixture as indicated in the legend activity of these enzymes in certain well defined to Table II; NaF, 20 /Amoles; hemolysate, 0.3 ml. The blood disorders. volume was made up to 1 ml with distilled water and the mixture was incubated for 20 minutes at 370 C. The re- MATERIALS AND METHODS action was stopped by the addition of 5 ml of 5 per cent acid. The was re- venipuncture trichloroacetic precipitated protein Blood samples. Blood was obtained by moved by centrifugation, and 2 ml samples of the clear from healthy donors and anemic or polycythemic pa- supernates were each treated with 1 ml of a 5 per cent tients. The salient hematological data are recorded in suspension of acid-washed charcoal ("Norit A") in or- Table I. The charcoal was washed freshly der to adsorb the ATP (8). Preparation of hemolysates. The cells from 5 times with pH 4.5 acetate buffer containing 10 ,Amoles drawn heparinized blood samples were sedimented by PP per ml, once with distilled water, and was then sus- with 2 centrifugation, washed 3 times vol of physiological in an alcohol: ether mixture 1 vol/vol) and and counted before pended (3: saline buffered with veronal, pH 7.3, transferred to planchets. The radioactivity were quantitatively the last washing. All the subsequent operations was measured with an end-window Geiger-Mueller carried out in the cold. counter and the activity was calculated by dividing the Two ml of the packed blood cells were hemolyzed by total counts per minute incorporated into the ATP by dilution with 1.6 vol of cold distilled water. Following the specific activity of the pyrophosphate added and ex- 10 minutes of stirring, the isotonicity of the hemolysate pressed in terms of micromoles of PP exchanged per mil- sucrose solution was restored by adding concentrated ligram protein of the homolysate or per 10' RBC. Un- and the volume was to a final concentration of 0.25 M, der the specified conditions the reaction closely followed and were removed made up to 10 ml. Ghosts particles a zero order course. 30 minutes at G in by spinning the hemolysate for 15,000 Total protein was determined The clear Analytical procedure. the Servall centrifuge. resulting supernatant by the method of Lowry, Rosebrough, Farr and Randall * This work was supported by a research grant from (9) and was estimated by the cyanmethemo- the Hadassah Medical Organization. globin procedure (10). Inorganic phosphate was deter- 1763 1764 G. IZAK, T. WILNER AND J. MAGER

TABLE I Summary of hematological data in the various groups of patients (range)

No. of Clinical diagnosis patients Hemoglobin RBC* Hematocrit Reticulocytes Serum iront Vitamin Bi4

gm % 106/cu mm % % pg % ;spg/ml serum Normal 30 13.4-15.2 4.6-5.2 44-49 0.1-0.6 79-108 180-420 Macrocytic§ hyperchromic 20 6.5-9.2 1.5-3.2 21-36 1.2-3.1 67-110 60-190 Microcytic hypochromic anemia 16 4.5-7.2 2.7-3.9 19-34 0.8-2.8 0-38 200-450 vera 10 15.9-18.0 5.7-7.5 53-70 0.1-1.1 70-115 270-390

* RBC = red blood cells. t Determined according to the method of Davies, Levin and Oberholzer (6). Determined according to the method of Grossowicz, Aronovitch and Rachmilewitz (7). § No patients with pernicious anemia (lack of intrinsic factor) or have been included in this group. mined by the method of Fiske and Subbarow (11). All in the rate of exchange to one-fourth to one-half the experiments were run in duplicate. of that observed with the complete system. It appeared likely that the considerable "background" RESULTS activity obtained without the addition of amino Conditions governing PP32-A TP exchange in acids was due to the appreciable amounts of amino hemolysates. As shown in Table II, hemolysates acids released from the RBC in the course of lysis prepared from normal RBC catalyzed the incorpo- ( 12), especially since low substrate saturation levels ration of P32-labeled pyrophosphate into ATP. are characteristic of the amino acid activating en- Omission of amino acids from the otherwise zymes (2, 3). The validity of this assumption is complete reaction mixture resulted in a decrease strengthened by the finding that the ratio between

TABLE II TABLE III PP32-A TP exchange in a normal blood hemolysate; Precipitation of the AAAE at pH 5.0 in the presence of requirements of the reaction * sodium ribonucleate *

pmoles pmoles Complete ATP3 ATP32 amino No acid amino 100 mg Treatment of mixture acid Ratio No protein 101" RBCt hemolysatet A B A/B ATP

"Complete amino acid % activity 5 mixture"t 11.6 33.0 activity Amino acid mixture "A"t 11.2 31.0 None 100 33 3.0 10 Tryptophan 8.7 25.3 pH 5.Ot 95 28 3.4 7 No amino acids 5.1 13.7 pH 5.0 iSupernate 63 23 2.7 12 No ATP 2.1 4.5 No Mg++ 3.8 6.3 RNA§ fSediment 28 2.5 11.2 3 p3 Pi§ added instead of pp32 0 0 Pyrophosphatase added 0 0 * The results are expressed in percentage of the activity obtained with untreated hemolysate, the latter set at 100 * Conditions as described under Methods; see first sec- per cent. See footnotes * and I to Table II for definitions. tion of text for abbreviations. t Hemolysate: particle-free supernate prepared from f Based on the red cell count performed immediately whole blood as described under Methods. prior to lysis. t The pH of the ice-chilled hemolysate was lowered to "Complete amino acid mixture": arginine, aspartic, 5.0 by dropwise addition of 1 N acetic acid with continu- glycine, histidine, serine, isoleucine, leucine, lysine, phenyl- ous stirring. No precipitate was obtained. The pH of alanine, threonine, tryptophan, valine, glutamic, methi- the hemolysate was immediately readjusted with 1 N Tris onine, cysteine, tyrosine, proline. All amino acids were buffer to 7.2. of the L-series and were added at 5 X 10-4 M final con- § pH 5.0 + RNA: 30 mg/ml sodium ribonucleate centration. Amino acid mixture "A": arginine, glycine, [commercial yeast, RNA, purified according to the pro- histidine, serine, threonine, tryptophan, valine. Inorganic cedure of Woodward (13)] was added to the hemolysate pyrophosphatase (40 ,ug) was added and the reaction with stirring until dissolved. Thereafter the solution was mixture was pre-incubated for 5 to 10 minutes prior to the treated as above. The precipitate formed at pH 5.0 was addition of ATP. (Kindly supplied by Dr. M. Kunitz, the sedimented by centrifugation and dissolved in a measured Rockefeller Institute for Medical Research.) quantity of Tris buffer, 0.025 M, pH 7.2. The pH of the § Pi: inorganic phosphate. supernate was readjusted as above. AMINO ACID ACTIVATING ENZYMES IN MATURE ERYTHROCYTES 1765 the endogenous and the amino acid-stimulated TABLE IV PP32-ATP exchange activity increased from 1: 3 Rate of PP32-A TP exchange determined after increasing lengths of incubation in normal and polycythemic blood in the crude hemolysate to 1: 11 in a fraction of hemolysates * this hemolysate isolated by pH precipitation (Table III). Rate of exchange Incubation By examining various amino acids singly and time Normal Polycythemic

in combination, it was found that amino acid mix- min mumoles/hr/mg protein ture "A" (see legend to Table II) enhanced the 10 35.4 24.9 20 35.4 21.6 exchange rate almost to the same extent as did a 30 33.0 mixture of 18 natural amino acids, whereas 80 per 45 36.0 23.1 cent of the maximal effect was elicited by trypto- * Rate of exchange (R) was calculated according to the phan alone. following equation (14): The radioactivity yielded without added ATP X 2.3 100 R=(ATP) (PP32) lg was about 15 per cent of that obtained with the (ATP) + (Pp32) t X log 100 - % exchange complete reaction mixture. These counts could Conditions as in Table II. Amino acid mixture "A" was be almost entirely eliminated by using an isolated used. fraction of the hemolysate, indicating that this re- using 0.005 M citrate of pH 3.5 as the mobile sidual activity was due to the endogenous ATP phase. Under these conditions radioactivity was content of the hemolysate (Table III). detectable only in the ATP spot located by its Omission of Mg++ resulted in a roughly 70 per quenching of ultraviolet light. When the labeled cent drop of the reaction rate (Table II). ATP was hydrolyzed with 1 N HCl in a boiling P32-orthophosphate failed to induce any detecta- water bath for 10 minutes, the radioactivity was no ble exchange with ATP when substituted for pp32 longer adsorbable with charcoal. The latter re- in the standard reaction mixture. The specificity sult indicated, therefore, that the P32-label was of the system with respect to pyrophosphate was entirely confined to the acid-labile terminal pyro- even more by the find- convincingly demonstrated moiety of ATP. ing that crystalline inorganic pyrophosphatase phosphate Fractionation. The AAAE of the rat liver cy- completely abolished the exchange reaction fraction are referred to by Hoagland, (Table II). toplasmic Keller and Zamecnik (15) as pH 5.0 enzymes be- The pH of optimum PP32-ATP exchange ac- cause of their precipitating property at this pH as tivity was found to range from 6.1 to 7.3. By a The same varying the concentration of the amino acids in nucleoprotein complex. procedure applied to whole blood hemolysates failed to pro- the reaction mixture the maximal rate of exchange duce a precipitate without affecting, however, the was attained at levels close to 2 X 10-4 M. that the was read- The considerable adenosine triphosphatase and activity, provided pH quickly justed to neutrality. It appeared likely that this pyrophosphatase activity of the lysates, as meas- difference in behavior is attributable to the virtual ured by the release of orthophosphate from the re- substrates in the course of incubation, was spective TABLE V entirely eliminated by the fluoride included in the Distribution of amino acid activating enzymes in various reaction mixture. The stability of the assay sys- formed elements of the peripheral blood * tem was also borne out by the fact that the rate jLmoles pAmoles of exchange remained practically constant during ATP32 ATP32 % of an experimental period which lasted for at least 100 mg loll totalt 40 minutes (Table IV). protein cells activity Identification of the reaction product. At the Red blood cells 13.0 30.0 95.6 end of the reaction the labeled ATP was adsorbed White blood cells 69.0 78.0 0.4 Platelets 30.0 37.0 4.0 onto charcoal and the residual PP was removed Whole blood 13.57 31.3 100.0 by extensive washing. Thereafter the ATP was * eluted with 50 per cent ethanol containing 0.5 M Conditions as described under Methods. t The activity of the whole blood hemolysate was set at ammonia and isolated by paper electrophoresis, 100 per cent. 1 766 G. IZ.AkK, T. WILN'ER AND J. MAGER

TABLE VI Mean values of PP32-A TI' exchange in blood lysates from normlal subjects and from anemic and polycythemic patients

Normal Polycythemia pmoles ATP32 ymoles ATP32 Mmoles ATP32 Jlmoles ATP32 Mmoles ATP32 Mimoles ATP32 pmoles ATP32 Amoles ATP32 100 mg 100 mg 100 mg 100 mg protein 1011 RBC protein 1011 RBC protein 101" RBC protein 1011 RBC Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Amino acid mixture "A"* 10.6 3.0 31.0 6.8 17.8 4.7 42.3 6.6 24.4 5.3 50.0 14.5 6.9 1.1 15.3 3.2 Tryptophan 8.1 1.9 24.9 6.2 14.4 4.6 35.0 7.3 20.0 5.2 40.0 10.2 7.0 2.1 15.6 4.7 No amino acids 4.5 1.4 12.3 2.7 7.5 2.1 18.3 4.4 8.0 1.5 14.1 4.1 2.9 0.4 6.7 1.3 No ATP 2.0 0.8 5.0 2.0 2.1 0.5 6.0 1.5 1.9 0.6 3.5 1.5 1.2 0.2 2.7 0.4

* Amino acid mixture "A"; see footnote: to Table II. Conditions otherwise as in Table II. absence of nucleic acids in the hemolysate. This blood cell activity resides in the RBC. On the seems to b)e confirmed by the finding that when other hand, the specific activity of the lysates cal- RNA was added to the hemolysate, a precipitate culated per unit number of cells is highest in the was obtained at pH 5.0 which contained about leukocytes and lowest in the RBC. 25 per cent of the total AAAE activity present in Leukocytes and RBC injured by a single hypo- the original hemolysate (Table III). No further tonic treatment retained about as much activity as fractionation was attempted. that released into the supernatant solution. The The distribution of AAAE in the peripheral residual activity could be solubilized by subjecting blood cells. The distribution of the AAAE in the the ghosts resedimented by centrifugation to re- various formed elements of the peripheral blood peated osmotic lysis or to sonication for 2 to 3 was studied by determining the PP32-ATP ex- minutes. On the other hand, ghosts of platelets change in lysates prepared separately from eryth- obtained after a single hypotonic lysis exhibited rocytes, leukocytes and platelets isolated by a no detectable PP32-ATP exchange. It may be modification of a conventional technique (16). pointed out in this connection that no PP32-ATP As evident from Table V, the bulk of the total exchange could be demonstrated with any of the

ATP AuM ATP AM 100mg Protein 10" RBC

- 30.0

20.0

* 10.0

0 1st 2nd 3d 4th I 2nd 3d 41 determination FIG. 1. REPEATED DETERMINATIONS OF PP52-ATP EXCHANGE AT BIWEEKLY INTERVALS. AMINO ACID ACTIVATING ENZYMES IN MATURE ERYTHROCYTES 1767

ATP puM 17.0 Hemoglobin 100 mg Prdein gm % 16.0 13.0

15.0 12 .

14.0 11.0

13.0 10.0

12.0 9.0

11.0 8D

10.0 7.0 0 10 20 30 40 D a y s FIG. 2. GRADUAL DECREASE OF PPS-ATP EXCHANGE PARALLELING THE INCREASE IN HEMOGLOBIN VALUES IN TWO ANEMIC PATIENTS. * Amino acid activating enzyme. intact cells of the peripheral blood, apparently with macrocytic anemia were selected from a owing to their impermeability to ATP. large group of women diagnosed as anemic dur- Amino acid activation capacity of normal blood ing pregnancy and postpartum, while cases with hemolysates. Table VI summarizes the results of uncomplicated hypochromic deficiency anemia a series of determinations of PP32-ATP exchange were chosen from subjects available at the Hema- performed on blood cell hemolysates from 30 nor- tology Outpatient Department of the Hadassah mal individuals. There was a considerable varia- University Hospital. In both groups the amino tion in the values obtained in different persons. acid-dependent pyrophosphate exchange was However, when repeated determinations were markedly higher than the average values observed made on the same subject at definite time inter- in the normal group, the increase being particu- vals, the levels of activity were found to be re- larly striking in the hypochromic anemia patients markably constant (Figure 1). (Table VI). The significance of this correlation Macrocytic and microcytic . Patients between the biochemical alteration and the patho-

ATP AiM O AT P AiM 100mg Protein Hemoglobin 100 mg Protein gm% 400 ml 350 ml * Hemoglobin gm% 16.0 _ blood drawn blood drawn 18.0 I I I 0 14.0 17.0

12.0 16.0 0~~~~~~~ 10.0 I 15.0

8.0 14.0

6.0 13.0

I 4.UAA L I I ~~~~~~~I* ' ' I 1 2 3 4 5 6 7 8 9 W e e k s FIG. 3. INCREASE OF PP32-ATP EXCHANGE FOLLOWING REPEATED VENISECTIONS IN A POLYCYTHEMIC PATIENT. 1 768 G. IZAK, T. WILNER AND J. MAGER logical condition is further borne out by the fol- this respect from the behavior of some other en- low-up of six treated patients in whom progres- zymes, as described by Marks, Johnson, Hirsch- sive decline of activity down to the normal range berg and Banks (20). paralleled the improvement in the hematological status. Two representative cases are illustrated DISCUSSION in Figure 2. . In untreated patients with The occurrence of amino acid activating en- zymes mature red blood cells which are known polycythemia vera the pyrophosphate exchange in was of a considerably lower order than in normal to be incapable of protein production is surprising subjects. Addition of tryptophan alone to the re- in view of the presumed essential role of these en- action mixture elicited maximal response which zymes in the biosynthesis of protein (5). It is was not increased by further addition of other possible, of course, that the AAAE perform in amino acids (Table VI). In these patients the the mature RBC a different function which re- mains to be elucidated. It is likewise conceivable, activity rose substantially after repeated venesec- however, that the system concerned represents a gradually returning, however, to the original tions, nonfunctional rudiment carried over from the nu- low level within several weeks following the last cleated of erythropoiesis. phlebotomy (Figure 3). stages The pronounced differences in the AAAE ac- Osmnotic fragility and AAAE. The high ac- tivity observed between blood cells from normal in anemic patients suggested the pos- tivity found subjects and those from patients with various he- sibility that the elevated values were due to a matological disorders seem to reflect early disturb- "shift to the left" in the age of the red cell popu- ances occurring at somiie stage of the RBC matura- lation in view of the shortened RBC life span tion rather than a change in the type of the ma- characterizing these conditions (17, 18). This tture blood cells. The uniform distribution of this possibility was explored by examining the enzymic activity within the blood cell population, regard- activity as a function of the osmotic fragility which less of the differences in the osmotic fragility in is known to vary directly with the age of the red both normal subjects and anemic patients, indi- cells (19). cates a lack of correlation between the level of Table VII shows that the activity per unit num- AAAE and the age of RBC. This conclusion is ber of red cells lysed remained roughly constant. also in line with the observation that the reticu- The anemic cells showed an essentially similar be- locyte count did not seem to affect significantly the havior. It may be relevant to mention in this activity observed within a given group. context that there was no correlation between the The nature of the biochemical lesion responsible degree of and the PP32-ATP ex- for the observed deviations from the normal level change activity in the various groups studied. of activity is not clear. It is tempting to speculate Thus the AAAE exhibits a uniform pattern of that the enhanced AAAE activity found in the distribution in the RBC population, differing in anemic disorders described manifests an attempt of the hematopoietic system to compensate for the TABLE VII PP32-A TP exchange in relation to deficient hemoglobin synthesis. This hypothesis osmotic fragility would also account for the gradual decrease of the activity, down to normal values, occurring some Microcytic hypo- Normal chromic anemia time after a normal hemoglobin level had been at-

% tained. On the other hand, the decrease in the Hemo- % Hemo- Activ- globin Activ- globin AAAE level observed in patients with polycy- NaCi ity in in ity in in solutioni supernate sul)ernate supernate sulernate themia vera may reflect an opposite tendency, 0.4 30 39 14 17 aimed to restrain the excessive activity of the 0.35 90 92 60 5 1 (errytbroloietic tissule. Tt is interestino to men- 0.30 95 93 90 84 100* 100 100 100 tiOn in this colllectioln that the AA.\I activity de- termined in the IRBC of a limited number of pa- * The values obtained after lysis with distilled water are was found to arbitrarily conisidered as 100 per cenit. tients with secondary polycythemia AMINO ACID ACTIVATING ENZYMES IN MATURE ERYTHROCYTES 1769 be within the normal range. As to the transient cells and the possible mechanism underlying the increase of the PP32-ATP exchange observed in deviations from the normal level in various he- polycythemic patients following repeated venesec- matologic disorders are discussed. tions, this represents a response of the protein syn- thesizing system to the drastic reduction in the REFERENCES circulating red cell mass. 1. Hoagland, M. B. An enzymic mechanism for amino Thus the mechanisms discussed above can be acid activation in animal tissues. Biochim. bio- viewed as two facets of a homeostatic device operat- phys. Acta 1955, 16, 288. ing through a restrictive effect exerted by the red 2. DeMoss, J. A., and Novelli, G. D. An amino acid cell proteins on the formation of the enzymic sys- dependent exchange between inorganic pyrophos- phate and ATP in microbial extracts. Biochim. tem mediating their own biosynthesis. No in- biophys. Acta 1955, 18, 592. formation is available as to the nature of the regu- 3. Davies, E. W., Konigsberger, V. V., and Lipmann, F. latory factor (s) involved in this mechanism. The isolation of a tryptophan-activating enzyme Further work is required to test the validity of from pancreas. Arch. Biochem. 1956, 65, 21. this interpretation. 4. Mager, J., and Lipmann, F. Amino acid incorpora- tion and the reversion of its initial phase with cell- free tetrahymena preparations. Proc. nat. Acad. SUMMARY Sci. (Wash.) 1958, 44, 305. 5. Lipmann, F., Hulsmann, W. C., Hartmann, G., Human whole blood hemolysates were found to Boman, H. G., and Acs, G. Amino acid activation catalyze an amino acid-dependent exchange of and protein synthesis. J. cell. comp. Physiol. 1959, pyrophosphate with adenosine triphosphate. Out 54, 75. of a number of amino acids tested, tryptophan 6. Davies, G., Levin, B., and Oberholzer, V. G. 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