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Turnover of esters produced from inosine in human erythrocyte ghosts
ARCHIVES
OF
BIOCHEMISTRY
Turnover
AND
BIOPHYSICS
103, 15-23 (1963)
of Esters Produced
from
Erythrocyte FABIAN From the Department
J. LIONETTI
AND
lnosine
in Human
Ghosts’ NORMAND
L. FORTIER
of Biochemistry, Boston University School of Medicine, Medical Center, Boston, Massachusetts Received
February
Boston
University
28, 1963
The kinetic variation of glucose-B-P,2 fructose-6-P, fructose diphosphate (FDP), mixed triosephosphates (G-3-P and dihydroxyacetone-P), xylulose-5-P, and ribulose5-P was studied in ghosts incubated with inosine. Extracts were made with metaphosphoric or perchloric acid and assayed eneymatically. A synthesis of all esters occurred, dependent on inosine and Pi reaching a maximum in 4 hr. and diminishing to low values in 6 hr. Approximately 1 mmole of inosine was metabolized in 4 hr., of which the esters accounted for 75yn of the inosine pentose carbon. Glucose-6-P together with F-6-P was 45’%. At the maximum, the amounts (pmoles/lOi* cells) were : G-6-P (274)) TrP (150)) Xu-5-P (115)) F-6-P (95)) Ru-5-P (57)) and FDP (42). Triosephosphate synthesis paralleled that of HMP, the ratio of the two being constant for 4 hr. and dependent on inosine concentration. Xylulose-5-P accumulated most rapidly and diminished, consistent with metabolism to HMP and TrP. In contrast, Ru-5-P appeared to be diverted to other pathways as it was low or not detectable in less than 2 hr. In 3-4 hr. of incubation, however, it was found to be equilibrated with R-5-P and Xu-5-P. Ribose-5-P metabolism by ghosts differed from inosine in that Xu-5-P and Ru-5-P equilibrated rapidly.
molarity of 0.30 prior to incubation with physiological buffers as cell suspensions. With the exception of 2,3-diphosphoglycerate, the ghosts contained little of the endogenous ester or cofactor composition of intact erythrocytes. They possessed, nevertheless, sufficient soluble and structural enzymes to facilitate net increases in phosphorylated intermediates of the order of 400-500 pmoles of esters per 1012ghosts in 2-3 hr. of incubation with inosine. The participation of the enzymes transaldolase and transketolase in the metabolism of nucleosides and R-5-P by hemolysates has been demonstrated by Dische (2, 3). Phosphopentoisomerase and epimerase also have been shown (4). Further metabolic studies of ester synthesis from R-5-P indicate that erythrocytes contain the major components of a pentose phosphate pathway (.5-7). Our previous study had
INTRODUCTION
A previous study revealed a synthesis by human erythrocyte ghosts of several hexose- and triosephosphates from the pentose of inosine (1). The ghosts were made by hemolysis of erythrocytes with water followed by restoration to an os1 These studies were supported by a grant to Boston University (HE-05353) by the National Heart Institute of the Department of Health, Education, and Welfare, and by a contract (DA49-007-MD-542) between the Research and Development Division Office of the Surgeon General, Department of the Army and Boston University. 2 Abbreviations used: P, phosphate; glucose-Bphosphate, G-6-P; fructose-6-phosphate, F-G-P; hexosemonophosphate, HMP; fructose diphosphate, FDP; triosephosphate, TrP; ribose-5-phosR-5-P; xylulose-5-phosphate, Xu-5-P; phate, ribulose-5-phosphate, Ru-5-P; inorganic phosphate, Pi; adenosinetriphosphate, ATP; and glyceraldehyde-3-P, G-3-P. 15
I6
LIONETTI
AND
suggested the production of ketopentosephosphates as products of inosine metabolism. A fraction of the incubation medium separated by chromatography on Dowex-1 (Cl-), different from F-6-P, was alkali labile as measured with orcinol, and had an absorption maximum in the cysteine carbazole reaction of 560 rnp. The present study shows that ketopentosephosphates are indeed produced in ghosts and that Xu-5-P accumulates rapidly, equilibrates eventually with R-5-P, and diminishes to low values. Hexose- and triosephosphates meanwhile steadily accumulate up to 4 hr., depending on the concentration in the medium of inosine and inorganic phosphate. The distribution and kinetic variation of esters measured can be reconciled with the presence in ghosts of the above mentioned enzymes of the pentose phosphate pathway, whose combined activity accounts for approximately 75 % of the inosine pentose carbon metabolized. EXPERIMENTAL Erythrocyte ghosts were prepared and incubated at 37” with substrates as described previously (8), except that ghosts were suspended in a medium composed of physiological buffer instead of homologous plasma in quantities sufficient to contain 5 X lo6 cells per mm.3 of suspension. Other conditions were the same. The average hemoglobin concentration of the ghosts in all experiments was 13% of that found in an equivalent number of intact cells. Extracts from the incubation mixtures of cells and medium were made with 10% metaphosphoric acid (1: 10) and immediately brought to pH 6.8 with NaOH and assayed. In a few instances, extracts were made with 507, perchloric acid (I: lo), neutralized with KOH, and the KCIOC removed after an overnight interval at 4°C. The extracts were assayed for G-6-P, F-6-P, FDP, R-5-P, Xu-5-P, Ru&P, and TrP (DHAP plus G-3-P) as described below. The major analytical reaction was the assay of TrP (G-3-P + DHAP), which was carried out according to the procedure of Cooper et al. (9)) using a mixture of a-glycero-phosphate dehydrogenase and triosephosphate isomerase. Combinations with appropriate enzymes enabled us to assay FDP, Xu-5-P, and Ru-5-P according to the summary given below: 1. G-3-P
Triose-P
Isomerase
+ DHAP
FORTIER
2. DHAP
+ DPNH
+ H+
cu-Glycerol-P Dehydrogenase a-Glycerol-P
3. FDP .
4. Xu-5-P
Aldolase
+ R-5-P
>
+ DPN+
G-3-P + DHAP
Transketolase
, S-7-P + G-3-P
5. Ru-5-P
Ru-5-P
3 epimerase
) Xu-5-P
Glyceraldehyde-3-P in the extract was isomerized to DHAP (reaction 1) and reduced with DPNH to a-glycerol-P (reaction 2). The DPNH oxidized was a measure of the total TrP. Glyceraldehyde-3-P was also assayed with crystalline G-3-P dehydrogenase in the presence of arsenate (9), giving amounts of G-3-P approximately 4.5% of the total TrP and in agreement with those in an equilibrated mixture of G-3-P and DHAP. Fructose disphosphate was determined as the increment in TrP produced when purified muscle aldolase was added to the cuvette at the conclusion of the TrP assay. Since aldolase is not specific for FDP, we have referred to the results as the FDP fraction. Good agreement was obtained, however, between the total ketohexoses determined in the cysteine sulfuric reaction (10) and the sum of F-6-P and FDP determined enzymatically. Xylulose-5-P was assayed in the presence of an excess of R-5-P by the addition of transketolase (reaction 4). The G-3-P liberated was then determined as in reactions 1 and 2. When the reaction for the Xu-5-P assay had reached completion (5-10 min.), Ru-5-P was determined as the increment of DPNH oxidation obtained when Ru5-P 3 epimerase was added to the reaction medium (reaction 5). In a few instances, R-5-P was assayed with an excess of Xu-5-P and transketolase (11). In these cases the assays for Ru-5-P were confirmed alternately by the addition of R-5-P isomerase to the cuvettes. Data on the reliability of the assays for pentose phosphates are given below. Glucose-6-P was assayed with G-6-P dehydrogenase (9), as was F-6-P in the same reaction tube after the addition of phosphoglucoisomerase. Inosine and hypoxanthine were determined as described previously (12). Phosphorylated carbohydrates and some enzymes were obtained or prepared as detailed elsewhere (1). The R-5-P used in the assay for ketopentosephosphates was prepared fresh from the
ESTERS
FROM
INOSINE
barium salt. Barium R-5-P was treated with dilute NaOH to remove ketopentosephosphates prior to use (5) ; the barium ion was removed with Dowex50 (Hf) resin. Transketolase of very high specific activity, Xu-5-P (calcium salt), Xu-5-P epimerase, phosphoriboseisomerase, and several samples of enzymatically prepared mixtures of Xu-5-P, Ru5-P, and R-5-P were the very generous gifts of Dr. Ephraim Racker. Transketolase free of :DPNH oxidases, aldolases, lactic acid dehydrogenase, and epimerase was also prepared from yeast (13). Xylulosed-P epimerase was made from rabbit muscle (14). Preparations of the epimerase (Ru5-P 3 epimerase) yielding the best results were obt,ained from yeast (15). Glucose-6-P dehydrogenase and phosphoglucoisomerase were obtained from Boehringer and Soehne Co., Mannheim, Germany. The former was found free of TPNH oxidases, 6-phosphogluconic dehydrogenases, and phosphoglucomutase. The best preparations of phosphoglucoisomerase contained small amounts of transketolase whose effects could be made negligible by dilution of the preparation. The muscle aldolase contained small amounts of triosephosphateisomerase but no DPNH oxidases or lactic acid dehydrogenases. Pyruvate, a potential interfering substance when lactic acid dehydrogenase contaminates enzyme preparations, was also sought and found missing. Control experiments with standard samples of phosphate esters added to extracts of ghosts without inosine gave recoveries of the order of 957,.
IN
RBC
17
GHOSTS
ASSAY OF PENTOSE PHOSPHATES IN AN ENZYME EQUILIBRATED MIXTURE Enzymes used analytically for pentose phosphates were checked by analyzing a mixture of pentose phosphates of known composition (Table I). One of these was assayed by Dr. Asoke Datta and contained Xu-5-P, Ru-5-P, and R-5-P in proportions 40:20:40. Weighed samples of the dry powder were analyzed by us, and aliquots were added periodically to ghosts suspensions as a check on the ketopentosephosphate analysis under experimental conditions. Twenty-five mg. of the desiccated barium esters were dissolved in approximately 1.0 ml. of distilled water acidified with one drop of dilute HCl and passed over a 5.0 ml. column of Dowex-50 (Na+). The column was washed with water, the pH adjusted to 6.8, and the final volume made to 5.0 ml., giving a 0.57, solution of the mixture. The total phosphate in the prepared solution was 7.7 rmoles per ml., and the orcinol chromophore 6.1 pmoles per ml. Aliquots were chromatographed on paper according to the procedure of Runeckles and Krotkov (16) and gave three spots. Two dense spots were obtained at Rf’s relative to inorganic phosphate indicative of Xu5-P (0.90; obs. 0.87) and R-5-P (0.79; obs. 0.79). A less dense spot for Ru-5-P (0.94; obs. 0.96) overlapping that for Xu-5-P was found. Only traces of inorganic phosphate were observed. The solution was assayed for Xu-5-P and Ru-5-P wit,h an assay system similar to that given by Cooper et al. (9).
TABLE I ASSAY OF PENTOSE PHOSPHATES IN A STANDARD MIXTURE
Mixture Component~assayed Additionsto sample
xu-5-P
R-5-P
Excess R5P + Transketolase + Epimerase Excess Xu-5-P + Transketolase + R-5-P isomerase
Pentose phosphates (,moles/ml.) XII-s-Pa
R-S-P’”
3.1*3%
-
-
-
-
3.2f3yo
-
-
a The mean of seven assays in duplicate. b The mean of two determinations in duplicate. Characteristics of the mixture of pentose phosphates
are given in the text.
Ru-j-P*
K”~fl;~l)S“
1.6 =t 7%
4.7
1.7 f
14%
4.9
18
LIONETTI
AND
In some instances the determination of G-3-P after transketolase activity was carried out with crystalline G-3-P dehydrogenase and DPN+ in the presence of arsenate. However, due to the presence of much greater amounts of DHAP in ghosts extracts, it was more convenient and reliable to measure the total TrP (G-3-P + DHAP) with DPNH and ol-glycerophosphate dehydrogenase containing triosephosphate isomerase. The latter reaction goes nearly to completion in 10 min. in the presence of excess R-5-P, and indicates with good accuracy the increment of G-3-P produced when Ru-5-P epimerase is added to the cuvette. In the assay of Xu-5-P with transketolase, linearity of DPNH oxidized with volumes of standard mixture was obtained in the Zeiss spectrophotometer at 340 rnp over the range of .Ol pmoles (absorbancy, 0.062) to .07 rmoles. Aliquots of sample up to 0.2 ml. were used to adjust samples for maximum absorbancy changes. Controls with all components except pentose phosphate mixture, but with an excess of R-5-P, revealed a slow rate of DPNH oxidation due to small amounts of ketopentose-P in the R-5-P. This was subtracted from the absorbancy changes in duplicate cuvettes. Readings were taken before and after the addition of transketolase and followed until the reactions ceased. In extracts of ghosts made with metaphosphoric acid, reliability of the assays for Ru-5-P was 5-1070 less, as these depended on a second increment of DPNH oxidation after the addition of epimerase. In the Ru-5-P assay, the requirement is also critical that transketolase be free of epimerase. The R-5-P solutions usually developed 2-3% of Xu-5-P on standing, as found in controls with transketolase from which samples were omitted. Treatment with barium hydroxide (4) decreased this usually to 06% or less. The Xu-5-P as provided by Dr. Racker contained approximately 5% of R-5-P, 370 of Ru-5-P, and a trace of G-3-P, as judged by absorbancy changes in controls. The assays of pentose phosphates in the standard mixture shown in Table I agree with the per cent composition of esters as provided by Dr. Datta. It is seen that Xu-5-P equals the R-5-P, which is twice the amount of Ru-5-P. Two other different small samples of enzyme equilibrated ketopentosephosphates gave values of Xu-5-P to Ru-5-P close to 3:l. Despite the ratio of Xu-5-P to Ru-5-P of 2: 1 in the sample characterized, sufficient quantities were available for routine use as a standard mixture. The total pmoles of pentose phosphates determined enzymatically (8.0 rmoles per ml.) agrees reasonably well with the total phosphate (7.7 pmoles per ml.), and the orcinol chromophore (6.1 pmoles per ml., expected 6.6 rmoles per ml .) . In Table II data are given on the suitability of
FORTIER TABLE II RECOVERY OF PENTOSE PHOSPHATES FROM METAPHOSPHORIC ACIO EXTRACTS OF GHOSTS Ketopentose-P (pmoles/ntl.)
Ghost estract (0.1 ml.)
Additions
to extract
XU-
Ru-5-P
5-P
I-
xu-5-P
Excess R5P + Transketolase + Pentose-P mixture + Ru-5-P epimerase
xu-5-P
Excess R5P + Transketolase + Ru-5-P epimerase + Pentose-P mixture
_;-
I-
0.0
4.6 I-
R-5-P
Excess Xu-5-P + Transketolase + R-5-P isomerase + Pentose-P mixture
0.0
1 -
(1.8)”
i
4 Values in brackets equal total ketopentose-P less the Xu-5-P in the mixture. Ribulose-5-P produced metabolically from inosine was assayed directly as done in Table I. the enzyme assay procedures of metaphosphoric acid extracts of ghosts. The possibility of contaminants occurring in the enzymes or inhibitors in the extract was checked. No endogenous esters were present, nor was there any reaction when either transketolase or Ru-5-P epimerase was added to the extract. When the standard mixture of esters was added to the cuvettes with transketolase, the expected amounts of Xu-5-P (3.1 rmoles per ml.) were obtained. The addition of Ru-5-P epimerase gave a value of 1.7 pmoles per ml. of Ru-5-P. The addition of the mixture to cuvettes containing both transketolase and epimerase gave the expected value for the sum of the ketopentosephosphates (4.6 pmoles per ml.) from which Ru-5-P was estimated by difference to be 1.5 pmoles per ml. (values for Ru-5-P by direct assay (Table I) were 1.6-1.7 pmoles per ml.). Likewise when excess Xu-
ESTERS
FROM
INOSINE
i
i
IN
RBC
GHOSTS
4
5
i
19
6
TIME (HOURS) FIG. 1. Phosphate esters from inosine and Pi. Erythrocyte ghosts were incubated in physiological buffer with inosine (10.0 mM) and Pi (4.0 mM) at a cell count of 4.0 X 10-S - 5.0 X 106 cells per mm.3 of suspension. The average pH was 7.2. At the intervals shown extracts were prepared and assayed as described in Experimental. The values are corrected to a cell count of 5 X 106 per mm.3 The bars indicate the positive deviations from the mean of four experiments on different samples of blood. 5-P was used and R-5-P was assayed no reaction was experienced with R-5-P isomerase. The addition of the pentose phosphate mixture to CUvettes containing transketolase and isomerase gave expected values for the total ketopentose-P (4.9 pmoles per ml.). RESULTS
AND
DISCUSSION
Phosphorylated esters synthesized in ghosts from the ribose of inosine are given in the bar graph (Fig. 1). The values represent averages from four experiments done weekly with ghosts from four different blood donors. Inosine was rapidly metabolized by nucleoside phosphorolysis as indicated by a rapid production of hypoxanthine amounting to 200-250 pmoles per hr. per 1012cells for 4 hr. Glucose, however, remained low and constant throughout the 6-hr. interval in which large variations in ester synthesis were found. The bars represent the total hexosemonophosketopentosephosphates, phates, and triosephosphates. The kinetic variation of phosphorylated esters revealed that all except FDP were found to increase to a maximum and diminish to lower values during 6 hr. The hexosemonophosphate
fractions showed the greatest change as both G-6-P and F-6-P increased linearly for 4 hr. Based on the assumption that hypoxanthine synthesis in 4 hr. equaled the ribose carbon metabolized, the HMP fraction accounted for 45 % of the inosine metabolized, while the total recovery of ester carbon in the group of esters shown amounted to approximately 75 %. Glucose-6-P was evidently equilibrated with F-6-P through the phosphoglucoisomerase reaction in ghosts, as the ratio of the two rapidly reached 72 f 2 % of G-6-P in the HMP and remained constant throughout the incubation. The triosephosphate fraction (TrP) showed a large increase, reaching a maximum production in 4 hr. In this, G-3-P assayed independently gave a G-3-P to DHAP ratio of 1: 22, which is in accord with the K,, for TrP equilibrated in the triosephosphate isomerase reaction (17). Of interest is the ratio of the HMP to TrP fraction, which reached a value of 2.2 in 1 hr. and remained the same (average 2.4) for 6 hr. Dische (2) has made a similar observation of a slightly smaller magnitude in studies with adenosine in hemolysates containing fluoride. He assumed for his
20
LIONETTI
AND
experimental conditions that F-6-P could form only G-6-P and that G-3-P could form DHAP and FDP. The accumulation of TrP in the suspension suggests the glyceraldehyde phosphate dehydrogenase is a rate-limiting enzyme in ghosts. The FDP fraction is shown superimposed on the TrP fraction in the bar graph. Higher concentrations of FDP relative to TrP would be expected from the equilibrium constant for aldolase. Further investigations of the low and constant magnitude of FDP revealed it to be intracellularly located, along with 2,3-DPG, within ghosts throughout the incubation interval. Monophosphates, on the other hand, were found to diffuse from cells to medium rapidly, In Fig. 1 note the relatively high endogenous value of FDP in ghosts at time zero. In experiments in which ghosts were centrifuged from the incubation medium and separated from supernatant buffers quickly (5 min.), it was found that HMP and TrP were distributed equally (50%) between ghosts and buffers. This occurred within $1 hr. and remained so distributed up to 5 hr. Intracellular FDP, however, was concentrated in ghosts up to 5 hr., 95 % being in the cells and 5 % in the supernatant. Ghosts do contain, however, sufficient aldolase to permit much greater concentrations of FDP, considering the relative high TrP present. Assays revealed ghosts to contain 30-35 % of the enzyme contained in an equal number of frozen thawed erythrocytes. In these assays FDP was added to the medium of ghosts and the TrP produced was assayed. The observaCon suggests that aldolase is peripherally located on the erythrocyte surface and is isolated from intracellular FDP. During the first hour a large synthesis of Xu-5-P occurred. A maximum amount of 115 pmoles per 1Ol2 cells was reached at 2 hr.; a diminution then occurred which was consistent with a turnover to other esters that accumulated simultaneously. Ribulose5-P was present in much lower quantities than Xu-5-P and was barely detectable at the first hour of incubation. Unlike the G-6-P to F-6-P or the HMP to TrP ratios which remained constant throughout, the Xu-5-P to Ru-5-P ratio did not indicate the ketopentosephosphates to be in equilib-
FORTIER
rium until the third and fourth hour. The rate at which Ru-5-P attained a constant proportion of the total ketopentose phosphate varied with the concentration of inosine and demonstrated a much lowel rate of synthesis than found when R-5-F’ was metabolized by ghosts. Table III describes the influence of inosine on ketopentosephosphates which accumulated kinetically in ghosts. For comparative purposes the values for inosine (10 @I) and Pi (4 &I) from Fig. 1 are tabulated. Included also are results from an experiment in which ketopentosephosphates were measured in ghosts incubated with equal concentrations of R-5-P and Pi. It is apparent that Xu-5-P is synthesized rapidly and reaches a maximum in 2-3 hr. with inosine. The magnitude of Xu-5-P which accumulated increased from the low to high inosine concentration and tended to remain high with high substrate. Ribulose-5-P was absent for the first 2 hr. for all three concentrations of inosine studied. In the 24 hr.-interval, however, Ru--5-I’ accumulated and eventually reached a relatively constant proportion of 33 % of the ketopentosephosphate. This equilibration was clearly dependent on available inosine as the value reached 33 % at 3 hr., while for the lowest inosine the Ru-5-P could not be detected until 3 hr.; at 4 hr. it had not yet reached equilibrium. In this instance, the low values obtained in the assay with epimerase were confirmed by assay with transketolase and ribose-5-P isomerase. Ribose-5-P, impossible to assay with orcinol in the presence of inosine, was assayed in this experiment with an excess of Xu-5-P and transketolase (11). Ribose-5-P was found to be synthesized from inosine and exhibited similar turnover characteristics to Xu-5-P but at a somewhat smaller order of magnitude (see legend to Table III). In 4 hr., the ratio of ketopentose phosphates to R-5-P was 1.4 for the experiment at lowest inosine concentration, which agrees with values (1.5) observed by others for hemolysates (5, 19). In the later stages of the incubations, at the higher inosine concentrations, the Xu-5-P to Ru-5-P ratios equilibrated at a value of 2.0. This value is consistent with equilibrated pentose phosphates in which the proportions of Xu-5-l’,
ESTERS
FROM
INOSINE
IN
TABLE KETOPENTOSE
I
III FROM
-
INOSINE
AND
2
Pi
34
75
0
0 0
0 -
0
--__ 6
1 4 -
10
I
4
R-S-P
10
R-5-P”
Ketopentose-P
4
4
21
GHOSTS
Time (hr.)
Substrate cont. (mM) Inosine
PHOSPHATES
RBC
Pi
-
i
3
66 12 12 --
0
75 15
xu-5-P Ru-5-P % Ru-5-P
0
__--
0
-
39 17 17 15 : ! 30
I-
90 46 45 23 33 -l112 180 60 ( 57 35 1 -I
17
118 25
xu-5-P Ru-5-P % Ru-5-P
4
I
18
-
36 I
-
4
33
xu-5-P Ru-5-P % Ru-5-P
340 160
250 120
280 135 32
-
33
-
/ T i 31
175 32
-
34
L Conditions are the same as given for the data of Fig. 1. For the lowest inosine concentration, R-5-P was assayed in the same extracts as described. The values for the same time intervals as the ketopentose phosphates were 0.0, 28, 56, 50, and 39 rmoles per lOI ghosts.
Ru-5-P, and R-5-P from inosine are 40, 20, and 40. However, it is significantly lower than the ratio 2.65-2.95 for Xu-5-P to Ru5P obtained by Dische and Shigeura in hemolysates (5). It would be expected from the presence of substantial quantities of Xu-5-P in ghosts and known characteristics of the Xu-5-P epimerase present in erythrocytes that significant quantities of Ru-5-P should be found in the early hours of the incubation. This unusual and interesting characteristic of nucleoside metabolism in ghosts remains to be explained. This has been observed also with other ribosides where high Xu-5-P to Ru-5-P ratios occur because of low Ru-5-P until a steady state of ester production is attained in 3-4 hr.3 Table III also reveals there is a big contrast between inosine and R-5-P metabolism in relation to ketopentosephosphate synthesis. With R-5-P, ghosts accumulated very large amounts of Xu-5-P in 0.5 hr. Ribulose-5-P was also very high and equilibrated at 33 % 3 Normand manuscript).
L. Fortier
and Fabian
J. Lionetti
(in
of the ketopentose phosphate. These remained at this value throughout the incubation. It is evident that the R-5-P isomerase and epimerase have the capacity for much greater accumulation of esters when R-5-P rather than inosine is the substrate. The dependence of hexosephosphate and triosephosphate synthesis on inosine and Pi concentration is shown in Fig. 2. An influence of both inosine an Pi is evident. It had previously been found that concentrations of 10 mM and 4 mM, respectively, were optimum for maximum ester synthesis in 2-3 hr.-incubations (18). In general, both ester groups revealed a dependence on inosine at constant Pi (4 rn1J) up to 10 mM, with the TrP being the most responsive to increases in inosine concentration. Variation of Pi at constant inosine revealed a dependence also on Pi. A larger net increase in each fraction was obtained with increasing Pi up to concentrat,ions of 6 mM. At this Pi concentration, maximum amounts of ketopentosephosphates were found at 4 hr., followed by a rapid tendency toward
22
LIONETTI
I
2 TIME
4 3 (HOURS)
5
FIG. 2A. Hexosemonophosphates from inosine and Pi. The conditions were similar to those of Fig. 1. Each point represent8 the sum of G-6-P and F-6-P assayed independently for the concentrations shown.
AND
FORTIER
diminution, while the HMP and Trl’ fractions, although accumulating rapidly, had not yet accumulated to a maximum quantity. As seen from the curves, at the higher concentrations of Pi the HMP to TrP ratios at various times remained constant (average, 1.3). Comparing this with the ratio 2.2 from Fig. 1, it is apparent that HMP and TrP are produced at linear rates for periods up to 4 hr. at Pi of 4 mM and up to 6 hr. at a Pi of 6 m&l and above. It is also evident that the availability of Pi to the system has a much greater effect on the synthesis of TrP. It is possible to reconcile the esters synthesized in ghosts with the enzymic composition of erythrocytes associated with the pentose phosphate pathway. Nucleoside phosphorylase and phosphoribomutase facilitate the synthesis from purine nucleosides of R-5-P. This, through the action of R-5-P isomerase and Ru-5-P epimerase, effects the production of Ru-5-P and Xu-Ti-1’. Transketolase and transaldolase then produce F-6-P and G-3-P which, through phosphoglucoisomerase and triosephosphate isomerase, result in the synthesis of G-6-P and DHAP, respectively. The number and magnitude of esters synthesized in ghosts (which contain negligible endogenous components and metabolize nucleosides for the ester carbon) reveal clearly that this pathway has the capacity for the synthesis in substantial quantities of esters common to the glycolytic pathway. ACKNOWLEDGMENT
;: k
We are very grateful to Dr. Ephraim Racker for generous gifts of enzymes and valuable advice, and to hi8 associate, Dr. Asoke Datta, for very generous assistance.
loo REFERENCES
I
2 TIME
3 4 (HOURS)
5
FIG. 2B. Triosephosphates from inosine and Pi. Each point represents the sum of TrP (DHAP + G-3-P) and twice the FDP fraction.
1. LIONETTI, F. J., MCLELLAN, WM. L., FORTIER, N. F., AND FOSTER, J. M., Arch. Biochem. Biophys. 94, 7 (1961). 2. DISCHE, Z., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. 1, p. 171. Johns Hopkins Press, Baltimore, Maryland, 1951. 3. DISCHE, Z., SHIGELJRA, H. T., AND LANDSBERG, E., Arch. Biochem. Biophys. 89, 123 (1960).
ESTERS
FROM
INOSINE
4. DICKENS, F., AND WILLIAMSON, D. H., Biothem. J. 64, 567 (1956). 5. DISCHE, Z., AND SHIGEURA, H. T., Biochim. Biophys. Acta 24, 87 (1957). 6. BRUNS, F. H., NOLTMANN, E., AND VAHLHAUS, E., Biochem. 2.330,483 (1958). 7. BRUNS, F. H., DUNWALD, E., AND NOLTMANN, E., Biochem. 2.330.497 (1958). 8. LIONETTI, F. J., REES, S. B., HEALEY, W. A., WALKER, B. S., AND GIBSON, J. G., J. Biol. Chem. 220, 467 (1956). 9. COOPER, J., SRERE, P. A., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 306 (1958). 10. DISCHE, Z., AND DEVI, A., Biochim. Biophys. Acta 39, 140 (1960). Il. DATTA, A. C., AND RACKER, E., J. Biol. Chem. 236, 617 (1961). 12. MCLELLAN, W. L., AND LIONETTI, F. J., J. Biol. Chem. 234, 3243 (1959).
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RBC
GHOSTS
23
13. SRERE, P. A., COOPER, J. R., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 295 (1958). 14. TABACHNICK, M., SRERE, P. A., COOPER, J. R., AND RACKER, E., Arch. Biochem. Biophys. 74, 315 (1958). 15. MCDONOUGH, M. W., AND WOOD, W. A., J. Biol. Chem. 236, 1220 (1961). 16. RUNECKLES, V. O., AND KROTKOV, G., Arch. Biochem. Biophys. 70,442 (1957). 17. BEISENHERZ, GERWIN, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, p. 387, Academic Press, New York. 18. LIONETTI, F. J., MCLELLAN, WM. L., AND WALKER, B. S., J. Biol. Chem. 229, 817 (1957). 19. BROWNSTONE, Y. S., AND DENSTEDT, 0. F., Can. J. Biochem. Physiol. 39, 527 (1961).
OF
BIOCHEMISTRY
Turnover
AND
BIOPHYSICS
103, 15-23 (1963)
of Esters Produced
from
Erythrocyte FABIAN From the Department
J. LIONETTI
AND
lnosine
in Human
Ghosts’ NORMAND
L. FORTIER
of Biochemistry, Boston University School of Medicine, Medical Center, Boston, Massachusetts Received
February
Boston
University
28, 1963
The kinetic variation of glucose-B-P,2 fructose-6-P, fructose diphosphate (FDP), mixed triosephosphates (G-3-P and dihydroxyacetone-P), xylulose-5-P, and ribulose5-P was studied in ghosts incubated with inosine. Extracts were made with metaphosphoric or perchloric acid and assayed eneymatically. A synthesis of all esters occurred, dependent on inosine and Pi reaching a maximum in 4 hr. and diminishing to low values in 6 hr. Approximately 1 mmole of inosine was metabolized in 4 hr., of which the esters accounted for 75yn of the inosine pentose carbon. Glucose-6-P together with F-6-P was 45’%. At the maximum, the amounts (pmoles/lOi* cells) were : G-6-P (274)) TrP (150)) Xu-5-P (115)) F-6-P (95)) Ru-5-P (57)) and FDP (42). Triosephosphate synthesis paralleled that of HMP, the ratio of the two being constant for 4 hr. and dependent on inosine concentration. Xylulose-5-P accumulated most rapidly and diminished, consistent with metabolism to HMP and TrP. In contrast, Ru-5-P appeared to be diverted to other pathways as it was low or not detectable in less than 2 hr. In 3-4 hr. of incubation, however, it was found to be equilibrated with R-5-P and Xu-5-P. Ribose-5-P metabolism by ghosts differed from inosine in that Xu-5-P and Ru-5-P equilibrated rapidly.
molarity of 0.30 prior to incubation with physiological buffers as cell suspensions. With the exception of 2,3-diphosphoglycerate, the ghosts contained little of the endogenous ester or cofactor composition of intact erythrocytes. They possessed, nevertheless, sufficient soluble and structural enzymes to facilitate net increases in phosphorylated intermediates of the order of 400-500 pmoles of esters per 1012ghosts in 2-3 hr. of incubation with inosine. The participation of the enzymes transaldolase and transketolase in the metabolism of nucleosides and R-5-P by hemolysates has been demonstrated by Dische (2, 3). Phosphopentoisomerase and epimerase also have been shown (4). Further metabolic studies of ester synthesis from R-5-P indicate that erythrocytes contain the major components of a pentose phosphate pathway (.5-7). Our previous study had
INTRODUCTION
A previous study revealed a synthesis by human erythrocyte ghosts of several hexose- and triosephosphates from the pentose of inosine (1). The ghosts were made by hemolysis of erythrocytes with water followed by restoration to an os1 These studies were supported by a grant to Boston University (HE-05353) by the National Heart Institute of the Department of Health, Education, and Welfare, and by a contract (DA49-007-MD-542) between the Research and Development Division Office of the Surgeon General, Department of the Army and Boston University. 2 Abbreviations used: P, phosphate; glucose-Bphosphate, G-6-P; fructose-6-phosphate, F-G-P; hexosemonophosphate, HMP; fructose diphosphate, FDP; triosephosphate, TrP; ribose-5-phosR-5-P; xylulose-5-phosphate, Xu-5-P; phate, ribulose-5-phosphate, Ru-5-P; inorganic phosphate, Pi; adenosinetriphosphate, ATP; and glyceraldehyde-3-P, G-3-P. 15
I6
LIONETTI
AND
suggested the production of ketopentosephosphates as products of inosine metabolism. A fraction of the incubation medium separated by chromatography on Dowex-1 (Cl-), different from F-6-P, was alkali labile as measured with orcinol, and had an absorption maximum in the cysteine carbazole reaction of 560 rnp. The present study shows that ketopentosephosphates are indeed produced in ghosts and that Xu-5-P accumulates rapidly, equilibrates eventually with R-5-P, and diminishes to low values. Hexose- and triosephosphates meanwhile steadily accumulate up to 4 hr., depending on the concentration in the medium of inosine and inorganic phosphate. The distribution and kinetic variation of esters measured can be reconciled with the presence in ghosts of the above mentioned enzymes of the pentose phosphate pathway, whose combined activity accounts for approximately 75 % of the inosine pentose carbon metabolized. EXPERIMENTAL Erythrocyte ghosts were prepared and incubated at 37” with substrates as described previously (8), except that ghosts were suspended in a medium composed of physiological buffer instead of homologous plasma in quantities sufficient to contain 5 X lo6 cells per mm.3 of suspension. Other conditions were the same. The average hemoglobin concentration of the ghosts in all experiments was 13% of that found in an equivalent number of intact cells. Extracts from the incubation mixtures of cells and medium were made with 10% metaphosphoric acid (1: 10) and immediately brought to pH 6.8 with NaOH and assayed. In a few instances, extracts were made with 507, perchloric acid (I: lo), neutralized with KOH, and the KCIOC removed after an overnight interval at 4°C. The extracts were assayed for G-6-P, F-6-P, FDP, R-5-P, Xu-5-P, Ru&P, and TrP (DHAP plus G-3-P) as described below. The major analytical reaction was the assay of TrP (G-3-P + DHAP), which was carried out according to the procedure of Cooper et al. (9)) using a mixture of a-glycero-phosphate dehydrogenase and triosephosphate isomerase. Combinations with appropriate enzymes enabled us to assay FDP, Xu-5-P, and Ru-5-P according to the summary given below: 1. G-3-P
Triose-P
Isomerase
+ DHAP
FORTIER
2. DHAP
+ DPNH
+ H+
cu-Glycerol-P Dehydrogenase a-Glycerol-P
3. FDP .
4. Xu-5-P
Aldolase
+ R-5-P
>
+ DPN+
G-3-P + DHAP
Transketolase
, S-7-P + G-3-P
5. Ru-5-P
Ru-5-P
3 epimerase
) Xu-5-P
Glyceraldehyde-3-P in the extract was isomerized to DHAP (reaction 1) and reduced with DPNH to a-glycerol-P (reaction 2). The DPNH oxidized was a measure of the total TrP. Glyceraldehyde-3-P was also assayed with crystalline G-3-P dehydrogenase in the presence of arsenate (9), giving amounts of G-3-P approximately 4.5% of the total TrP and in agreement with those in an equilibrated mixture of G-3-P and DHAP. Fructose disphosphate was determined as the increment in TrP produced when purified muscle aldolase was added to the cuvette at the conclusion of the TrP assay. Since aldolase is not specific for FDP, we have referred to the results as the FDP fraction. Good agreement was obtained, however, between the total ketohexoses determined in the cysteine sulfuric reaction (10) and the sum of F-6-P and FDP determined enzymatically. Xylulose-5-P was assayed in the presence of an excess of R-5-P by the addition of transketolase (reaction 4). The G-3-P liberated was then determined as in reactions 1 and 2. When the reaction for the Xu-5-P assay had reached completion (5-10 min.), Ru-5-P was determined as the increment of DPNH oxidation obtained when Ru5-P 3 epimerase was added to the reaction medium (reaction 5). In a few instances, R-5-P was assayed with an excess of Xu-5-P and transketolase (11). In these cases the assays for Ru-5-P were confirmed alternately by the addition of R-5-P isomerase to the cuvettes. Data on the reliability of the assays for pentose phosphates are given below. Glucose-6-P was assayed with G-6-P dehydrogenase (9), as was F-6-P in the same reaction tube after the addition of phosphoglucoisomerase. Inosine and hypoxanthine were determined as described previously (12). Phosphorylated carbohydrates and some enzymes were obtained or prepared as detailed elsewhere (1). The R-5-P used in the assay for ketopentosephosphates was prepared fresh from the
ESTERS
FROM
INOSINE
barium salt. Barium R-5-P was treated with dilute NaOH to remove ketopentosephosphates prior to use (5) ; the barium ion was removed with Dowex50 (Hf) resin. Transketolase of very high specific activity, Xu-5-P (calcium salt), Xu-5-P epimerase, phosphoriboseisomerase, and several samples of enzymatically prepared mixtures of Xu-5-P, Ru5-P, and R-5-P were the very generous gifts of Dr. Ephraim Racker. Transketolase free of :DPNH oxidases, aldolases, lactic acid dehydrogenase, and epimerase was also prepared from yeast (13). Xylulosed-P epimerase was made from rabbit muscle (14). Preparations of the epimerase (Ru5-P 3 epimerase) yielding the best results were obt,ained from yeast (15). Glucose-6-P dehydrogenase and phosphoglucoisomerase were obtained from Boehringer and Soehne Co., Mannheim, Germany. The former was found free of TPNH oxidases, 6-phosphogluconic dehydrogenases, and phosphoglucomutase. The best preparations of phosphoglucoisomerase contained small amounts of transketolase whose effects could be made negligible by dilution of the preparation. The muscle aldolase contained small amounts of triosephosphateisomerase but no DPNH oxidases or lactic acid dehydrogenases. Pyruvate, a potential interfering substance when lactic acid dehydrogenase contaminates enzyme preparations, was also sought and found missing. Control experiments with standard samples of phosphate esters added to extracts of ghosts without inosine gave recoveries of the order of 957,.
IN
RBC
17
GHOSTS
ASSAY OF PENTOSE PHOSPHATES IN AN ENZYME EQUILIBRATED MIXTURE Enzymes used analytically for pentose phosphates were checked by analyzing a mixture of pentose phosphates of known composition (Table I). One of these was assayed by Dr. Asoke Datta and contained Xu-5-P, Ru-5-P, and R-5-P in proportions 40:20:40. Weighed samples of the dry powder were analyzed by us, and aliquots were added periodically to ghosts suspensions as a check on the ketopentosephosphate analysis under experimental conditions. Twenty-five mg. of the desiccated barium esters were dissolved in approximately 1.0 ml. of distilled water acidified with one drop of dilute HCl and passed over a 5.0 ml. column of Dowex-50 (Na+). The column was washed with water, the pH adjusted to 6.8, and the final volume made to 5.0 ml., giving a 0.57, solution of the mixture. The total phosphate in the prepared solution was 7.7 rmoles per ml., and the orcinol chromophore 6.1 pmoles per ml. Aliquots were chromatographed on paper according to the procedure of Runeckles and Krotkov (16) and gave three spots. Two dense spots were obtained at Rf’s relative to inorganic phosphate indicative of Xu5-P (0.90; obs. 0.87) and R-5-P (0.79; obs. 0.79). A less dense spot for Ru-5-P (0.94; obs. 0.96) overlapping that for Xu-5-P was found. Only traces of inorganic phosphate were observed. The solution was assayed for Xu-5-P and Ru-5-P wit,h an assay system similar to that given by Cooper et al. (9).
TABLE I ASSAY OF PENTOSE PHOSPHATES IN A STANDARD MIXTURE
Mixture Component~assayed Additionsto sample
xu-5-P
R-5-P
Excess R5P + Transketolase + Epimerase Excess Xu-5-P + Transketolase + R-5-P isomerase
Pentose phosphates (,moles/ml.) XII-s-Pa
R-S-P’”
3.1*3%
-
-
-
-
3.2f3yo
-
-
a The mean of seven assays in duplicate. b The mean of two determinations in duplicate. Characteristics of the mixture of pentose phosphates
are given in the text.
Ru-j-P*
K”~fl;~l)S“
1.6 =t 7%
4.7
1.7 f
14%
4.9
18
LIONETTI
AND
In some instances the determination of G-3-P after transketolase activity was carried out with crystalline G-3-P dehydrogenase and DPN+ in the presence of arsenate. However, due to the presence of much greater amounts of DHAP in ghosts extracts, it was more convenient and reliable to measure the total TrP (G-3-P + DHAP) with DPNH and ol-glycerophosphate dehydrogenase containing triosephosphate isomerase. The latter reaction goes nearly to completion in 10 min. in the presence of excess R-5-P, and indicates with good accuracy the increment of G-3-P produced when Ru-5-P epimerase is added to the cuvette. In the assay of Xu-5-P with transketolase, linearity of DPNH oxidized with volumes of standard mixture was obtained in the Zeiss spectrophotometer at 340 rnp over the range of .Ol pmoles (absorbancy, 0.062) to .07 rmoles. Aliquots of sample up to 0.2 ml. were used to adjust samples for maximum absorbancy changes. Controls with all components except pentose phosphate mixture, but with an excess of R-5-P, revealed a slow rate of DPNH oxidation due to small amounts of ketopentose-P in the R-5-P. This was subtracted from the absorbancy changes in duplicate cuvettes. Readings were taken before and after the addition of transketolase and followed until the reactions ceased. In extracts of ghosts made with metaphosphoric acid, reliability of the assays for Ru-5-P was 5-1070 less, as these depended on a second increment of DPNH oxidation after the addition of epimerase. In the Ru-5-P assay, the requirement is also critical that transketolase be free of epimerase. The R-5-P solutions usually developed 2-3% of Xu-5-P on standing, as found in controls with transketolase from which samples were omitted. Treatment with barium hydroxide (4) decreased this usually to 06% or less. The Xu-5-P as provided by Dr. Racker contained approximately 5% of R-5-P, 370 of Ru-5-P, and a trace of G-3-P, as judged by absorbancy changes in controls. The assays of pentose phosphates in the standard mixture shown in Table I agree with the per cent composition of esters as provided by Dr. Datta. It is seen that Xu-5-P equals the R-5-P, which is twice the amount of Ru-5-P. Two other different small samples of enzyme equilibrated ketopentosephosphates gave values of Xu-5-P to Ru-5-P close to 3:l. Despite the ratio of Xu-5-P to Ru-5-P of 2: 1 in the sample characterized, sufficient quantities were available for routine use as a standard mixture. The total pmoles of pentose phosphates determined enzymatically (8.0 rmoles per ml.) agrees reasonably well with the total phosphate (7.7 pmoles per ml.), and the orcinol chromophore (6.1 pmoles per ml., expected 6.6 rmoles per ml .) . In Table II data are given on the suitability of
FORTIER TABLE II RECOVERY OF PENTOSE PHOSPHATES FROM METAPHOSPHORIC ACIO EXTRACTS OF GHOSTS Ketopentose-P (pmoles/ntl.)
Ghost estract (0.1 ml.)
Additions
to extract
XU-
Ru-5-P
5-P
I-
xu-5-P
Excess R5P + Transketolase + Pentose-P mixture + Ru-5-P epimerase
xu-5-P
Excess R5P + Transketolase + Ru-5-P epimerase + Pentose-P mixture
_;-
I-
0.0
4.6 I-
R-5-P
Excess Xu-5-P + Transketolase + R-5-P isomerase + Pentose-P mixture
0.0
1 -
(1.8)”
i
4 Values in brackets equal total ketopentose-P less the Xu-5-P in the mixture. Ribulose-5-P produced metabolically from inosine was assayed directly as done in Table I. the enzyme assay procedures of metaphosphoric acid extracts of ghosts. The possibility of contaminants occurring in the enzymes or inhibitors in the extract was checked. No endogenous esters were present, nor was there any reaction when either transketolase or Ru-5-P epimerase was added to the extract. When the standard mixture of esters was added to the cuvettes with transketolase, the expected amounts of Xu-5-P (3.1 rmoles per ml.) were obtained. The addition of Ru-5-P epimerase gave a value of 1.7 pmoles per ml. of Ru-5-P. The addition of the mixture to cuvettes containing both transketolase and epimerase gave the expected value for the sum of the ketopentosephosphates (4.6 pmoles per ml.) from which Ru-5-P was estimated by difference to be 1.5 pmoles per ml. (values for Ru-5-P by direct assay (Table I) were 1.6-1.7 pmoles per ml.). Likewise when excess Xu-
ESTERS
FROM
INOSINE
i
i
IN
RBC
GHOSTS
4
5
i
19
6
TIME (HOURS) FIG. 1. Phosphate esters from inosine and Pi. Erythrocyte ghosts were incubated in physiological buffer with inosine (10.0 mM) and Pi (4.0 mM) at a cell count of 4.0 X 10-S - 5.0 X 106 cells per mm.3 of suspension. The average pH was 7.2. At the intervals shown extracts were prepared and assayed as described in Experimental. The values are corrected to a cell count of 5 X 106 per mm.3 The bars indicate the positive deviations from the mean of four experiments on different samples of blood. 5-P was used and R-5-P was assayed no reaction was experienced with R-5-P isomerase. The addition of the pentose phosphate mixture to CUvettes containing transketolase and isomerase gave expected values for the total ketopentose-P (4.9 pmoles per ml.). RESULTS
AND
DISCUSSION
Phosphorylated esters synthesized in ghosts from the ribose of inosine are given in the bar graph (Fig. 1). The values represent averages from four experiments done weekly with ghosts from four different blood donors. Inosine was rapidly metabolized by nucleoside phosphorolysis as indicated by a rapid production of hypoxanthine amounting to 200-250 pmoles per hr. per 1012cells for 4 hr. Glucose, however, remained low and constant throughout the 6-hr. interval in which large variations in ester synthesis were found. The bars represent the total hexosemonophosketopentosephosphates, phates, and triosephosphates. The kinetic variation of phosphorylated esters revealed that all except FDP were found to increase to a maximum and diminish to lower values during 6 hr. The hexosemonophosphate
fractions showed the greatest change as both G-6-P and F-6-P increased linearly for 4 hr. Based on the assumption that hypoxanthine synthesis in 4 hr. equaled the ribose carbon metabolized, the HMP fraction accounted for 45 % of the inosine metabolized, while the total recovery of ester carbon in the group of esters shown amounted to approximately 75 %. Glucose-6-P was evidently equilibrated with F-6-P through the phosphoglucoisomerase reaction in ghosts, as the ratio of the two rapidly reached 72 f 2 % of G-6-P in the HMP and remained constant throughout the incubation. The triosephosphate fraction (TrP) showed a large increase, reaching a maximum production in 4 hr. In this, G-3-P assayed independently gave a G-3-P to DHAP ratio of 1: 22, which is in accord with the K,, for TrP equilibrated in the triosephosphate isomerase reaction (17). Of interest is the ratio of the HMP to TrP fraction, which reached a value of 2.2 in 1 hr. and remained the same (average 2.4) for 6 hr. Dische (2) has made a similar observation of a slightly smaller magnitude in studies with adenosine in hemolysates containing fluoride. He assumed for his
20
LIONETTI
AND
experimental conditions that F-6-P could form only G-6-P and that G-3-P could form DHAP and FDP. The accumulation of TrP in the suspension suggests the glyceraldehyde phosphate dehydrogenase is a rate-limiting enzyme in ghosts. The FDP fraction is shown superimposed on the TrP fraction in the bar graph. Higher concentrations of FDP relative to TrP would be expected from the equilibrium constant for aldolase. Further investigations of the low and constant magnitude of FDP revealed it to be intracellularly located, along with 2,3-DPG, within ghosts throughout the incubation interval. Monophosphates, on the other hand, were found to diffuse from cells to medium rapidly, In Fig. 1 note the relatively high endogenous value of FDP in ghosts at time zero. In experiments in which ghosts were centrifuged from the incubation medium and separated from supernatant buffers quickly (5 min.), it was found that HMP and TrP were distributed equally (50%) between ghosts and buffers. This occurred within $1 hr. and remained so distributed up to 5 hr. Intracellular FDP, however, was concentrated in ghosts up to 5 hr., 95 % being in the cells and 5 % in the supernatant. Ghosts do contain, however, sufficient aldolase to permit much greater concentrations of FDP, considering the relative high TrP present. Assays revealed ghosts to contain 30-35 % of the enzyme contained in an equal number of frozen thawed erythrocytes. In these assays FDP was added to the medium of ghosts and the TrP produced was assayed. The observaCon suggests that aldolase is peripherally located on the erythrocyte surface and is isolated from intracellular FDP. During the first hour a large synthesis of Xu-5-P occurred. A maximum amount of 115 pmoles per 1Ol2 cells was reached at 2 hr.; a diminution then occurred which was consistent with a turnover to other esters that accumulated simultaneously. Ribulose5-P was present in much lower quantities than Xu-5-P and was barely detectable at the first hour of incubation. Unlike the G-6-P to F-6-P or the HMP to TrP ratios which remained constant throughout, the Xu-5-P to Ru-5-P ratio did not indicate the ketopentosephosphates to be in equilib-
FORTIER
rium until the third and fourth hour. The rate at which Ru-5-P attained a constant proportion of the total ketopentose phosphate varied with the concentration of inosine and demonstrated a much lowel rate of synthesis than found when R-5-F’ was metabolized by ghosts. Table III describes the influence of inosine on ketopentosephosphates which accumulated kinetically in ghosts. For comparative purposes the values for inosine (10 @I) and Pi (4 &I) from Fig. 1 are tabulated. Included also are results from an experiment in which ketopentosephosphates were measured in ghosts incubated with equal concentrations of R-5-P and Pi. It is apparent that Xu-5-P is synthesized rapidly and reaches a maximum in 2-3 hr. with inosine. The magnitude of Xu-5-P which accumulated increased from the low to high inosine concentration and tended to remain high with high substrate. Ribulose-5-P was absent for the first 2 hr. for all three concentrations of inosine studied. In the 24 hr.-interval, however, Ru--5-I’ accumulated and eventually reached a relatively constant proportion of 33 % of the ketopentosephosphate. This equilibration was clearly dependent on available inosine as the value reached 33 % at 3 hr., while for the lowest inosine the Ru-5-P could not be detected until 3 hr.; at 4 hr. it had not yet reached equilibrium. In this instance, the low values obtained in the assay with epimerase were confirmed by assay with transketolase and ribose-5-P isomerase. Ribose-5-P, impossible to assay with orcinol in the presence of inosine, was assayed in this experiment with an excess of Xu-5-P and transketolase (11). Ribose-5-P was found to be synthesized from inosine and exhibited similar turnover characteristics to Xu-5-P but at a somewhat smaller order of magnitude (see legend to Table III). In 4 hr., the ratio of ketopentose phosphates to R-5-P was 1.4 for the experiment at lowest inosine concentration, which agrees with values (1.5) observed by others for hemolysates (5, 19). In the later stages of the incubations, at the higher inosine concentrations, the Xu-5-P to Ru-5-P ratios equilibrated at a value of 2.0. This value is consistent with equilibrated pentose phosphates in which the proportions of Xu-5-l’,
ESTERS
FROM
INOSINE
IN
TABLE KETOPENTOSE
I
III FROM
-
INOSINE
AND
2
Pi
34
75
0
0 0
0 -
0
--__ 6
1 4 -
10
I
4
R-S-P
10
R-5-P”
Ketopentose-P
4
4
21
GHOSTS
Time (hr.)
Substrate cont. (mM) Inosine
PHOSPHATES
RBC
Pi
-
i
3
66 12 12 --
0
75 15
xu-5-P Ru-5-P % Ru-5-P
0
__--
0
-
39 17 17 15 : ! 30
I-
90 46 45 23 33 -l112 180 60 ( 57 35 1 -I
17
118 25
xu-5-P Ru-5-P % Ru-5-P
4
I
18
-
36 I
-
4
33
xu-5-P Ru-5-P % Ru-5-P
340 160
250 120
280 135 32
-
33
-
/ T i 31
175 32
-
34
L Conditions are the same as given for the data of Fig. 1. For the lowest inosine concentration, R-5-P was assayed in the same extracts as described. The values for the same time intervals as the ketopentose phosphates were 0.0, 28, 56, 50, and 39 rmoles per lOI ghosts.
Ru-5-P, and R-5-P from inosine are 40, 20, and 40. However, it is significantly lower than the ratio 2.65-2.95 for Xu-5-P to Ru5P obtained by Dische and Shigeura in hemolysates (5). It would be expected from the presence of substantial quantities of Xu-5-P in ghosts and known characteristics of the Xu-5-P epimerase present in erythrocytes that significant quantities of Ru-5-P should be found in the early hours of the incubation. This unusual and interesting characteristic of nucleoside metabolism in ghosts remains to be explained. This has been observed also with other ribosides where high Xu-5-P to Ru-5-P ratios occur because of low Ru-5-P until a steady state of ester production is attained in 3-4 hr.3 Table III also reveals there is a big contrast between inosine and R-5-P metabolism in relation to ketopentosephosphate synthesis. With R-5-P, ghosts accumulated very large amounts of Xu-5-P in 0.5 hr. Ribulose-5-P was also very high and equilibrated at 33 % 3 Normand manuscript).
L. Fortier
and Fabian
J. Lionetti
(in
of the ketopentose phosphate. These remained at this value throughout the incubation. It is evident that the R-5-P isomerase and epimerase have the capacity for much greater accumulation of esters when R-5-P rather than inosine is the substrate. The dependence of hexosephosphate and triosephosphate synthesis on inosine and Pi concentration is shown in Fig. 2. An influence of both inosine an Pi is evident. It had previously been found that concentrations of 10 mM and 4 mM, respectively, were optimum for maximum ester synthesis in 2-3 hr.-incubations (18). In general, both ester groups revealed a dependence on inosine at constant Pi (4 rn1J) up to 10 mM, with the TrP being the most responsive to increases in inosine concentration. Variation of Pi at constant inosine revealed a dependence also on Pi. A larger net increase in each fraction was obtained with increasing Pi up to concentrat,ions of 6 mM. At this Pi concentration, maximum amounts of ketopentosephosphates were found at 4 hr., followed by a rapid tendency toward
22
LIONETTI
I
2 TIME
4 3 (HOURS)
5
FIG. 2A. Hexosemonophosphates from inosine and Pi. The conditions were similar to those of Fig. 1. Each point represent8 the sum of G-6-P and F-6-P assayed independently for the concentrations shown.
AND
FORTIER
diminution, while the HMP and Trl’ fractions, although accumulating rapidly, had not yet accumulated to a maximum quantity. As seen from the curves, at the higher concentrations of Pi the HMP to TrP ratios at various times remained constant (average, 1.3). Comparing this with the ratio 2.2 from Fig. 1, it is apparent that HMP and TrP are produced at linear rates for periods up to 4 hr. at Pi of 4 mM and up to 6 hr. at a Pi of 6 m&l and above. It is also evident that the availability of Pi to the system has a much greater effect on the synthesis of TrP. It is possible to reconcile the esters synthesized in ghosts with the enzymic composition of erythrocytes associated with the pentose phosphate pathway. Nucleoside phosphorylase and phosphoribomutase facilitate the synthesis from purine nucleosides of R-5-P. This, through the action of R-5-P isomerase and Ru-5-P epimerase, effects the production of Ru-5-P and Xu-Ti-1’. Transketolase and transaldolase then produce F-6-P and G-3-P which, through phosphoglucoisomerase and triosephosphate isomerase, result in the synthesis of G-6-P and DHAP, respectively. The number and magnitude of esters synthesized in ghosts (which contain negligible endogenous components and metabolize nucleosides for the ester carbon) reveal clearly that this pathway has the capacity for the synthesis in substantial quantities of esters common to the glycolytic pathway. ACKNOWLEDGMENT
;: k
We are very grateful to Dr. Ephraim Racker for generous gifts of enzymes and valuable advice, and to hi8 associate, Dr. Asoke Datta, for very generous assistance.
loo REFERENCES
I
2 TIME
3 4 (HOURS)
5
FIG. 2B. Triosephosphates from inosine and Pi. Each point represents the sum of TrP (DHAP + G-3-P) and twice the FDP fraction.
1. LIONETTI, F. J., MCLELLAN, WM. L., FORTIER, N. F., AND FOSTER, J. M., Arch. Biochem. Biophys. 94, 7 (1961). 2. DISCHE, Z., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. 1, p. 171. Johns Hopkins Press, Baltimore, Maryland, 1951. 3. DISCHE, Z., SHIGELJRA, H. T., AND LANDSBERG, E., Arch. Biochem. Biophys. 89, 123 (1960).
ESTERS
FROM
INOSINE
4. DICKENS, F., AND WILLIAMSON, D. H., Biothem. J. 64, 567 (1956). 5. DISCHE, Z., AND SHIGEURA, H. T., Biochim. Biophys. Acta 24, 87 (1957). 6. BRUNS, F. H., NOLTMANN, E., AND VAHLHAUS, E., Biochem. 2.330,483 (1958). 7. BRUNS, F. H., DUNWALD, E., AND NOLTMANN, E., Biochem. 2.330.497 (1958). 8. LIONETTI, F. J., REES, S. B., HEALEY, W. A., WALKER, B. S., AND GIBSON, J. G., J. Biol. Chem. 220, 467 (1956). 9. COOPER, J., SRERE, P. A., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 306 (1958). 10. DISCHE, Z., AND DEVI, A., Biochim. Biophys. Acta 39, 140 (1960). Il. DATTA, A. C., AND RACKER, E., J. Biol. Chem. 236, 617 (1961). 12. MCLELLAN, W. L., AND LIONETTI, F. J., J. Biol. Chem. 234, 3243 (1959).
IN
RBC
GHOSTS
23
13. SRERE, P. A., COOPER, J. R., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 295 (1958). 14. TABACHNICK, M., SRERE, P. A., COOPER, J. R., AND RACKER, E., Arch. Biochem. Biophys. 74, 315 (1958). 15. MCDONOUGH, M. W., AND WOOD, W. A., J. Biol. Chem. 236, 1220 (1961). 16. RUNECKLES, V. O., AND KROTKOV, G., Arch. Biochem. Biophys. 70,442 (1957). 17. BEISENHERZ, GERWIN, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, p. 387, Academic Press, New York. 18. LIONETTI, F. J., MCLELLAN, WM. L., AND WALKER, B. S., J. Biol. Chem. 229, 817 (1957). 19. BROWNSTONE, Y. S., AND DENSTEDT, 0. F., Can. J. Biochem. Physiol. 39, 527 (1961).