Control of Glycolysis in the Human Red Blood Cell*

THE JOURNAL OP B~oxuxx~ CHPMI~TRY Vol. 241, No. 21, Issue of November 10, PP. 48484854, 1966 Printed in U.S.A. Control of Glycolysis in the Human Red Blood Cell* (Received for publication, April 22, 1966) IRWIN A. ROSE AND JESSIE V. B. WARMS From the Department of Biochemistry, The Institute for Cancer Research, Philadelphia, Pennsylvania 19111 SUMMARY Red are known to use glucose and produce lactate at an increased rate in the presence of increased inorganic phosphate. The stimulation by inorganic phosphate of hexokinase and phosphofructokinase is recognized, but the cause of the necessary rate increase of pyruvate kinase is not. Kinetic studies with this enzyme from normal and human mutant and steady state studies with whole glycolyzing support the conclusion that pyruvate kinase is normally operating far below saturation with respect to phosphoenolpyruvate. The increase in the rate of formation of phosphoenolpyruvate is primarily responsible for the increase in its rate of reaction in the kinase step. Pyruvate kinase deficient with about 10% as much enzyme activity reach much higher and even saturating levels of phosphoenolpyruvate in order to utilize a larger proportion of their enzyme. This is necessary to achieve the normal glycolytic rates observed. The explanation for the accumulation of fructose diphosphate and dihydroxyacetone phosphate at high glycolytic rates in normal and deficient is that glyceraldehyde phosphate dehydrogenase becomes ratelimiting as a result of an increased ratio of reduced to oxidized nicotinamide adenine dinucleotide and increased 1,3diphosphoglyceric acid. These factors increase at high glycolytic rate because the increased phosphoenolpyruvate plus phosphoglyceric acids leave an amount of reduced nicotinamide adenine dinucleotide unoxidized and because the phosphoglycerate kinase reaction is probably at equilibrium. The large pool of 2,3diphosphoglyceric acid in many mammalian red is seen as a possible source of the reduced nicotinamide adenine dmucleotide oxidant, pyruvate, necessary to regulate this effect which, in the limit, would exhaust the cell s adenosine triphosphate supply. Stimulation of glucose utilization (1) and of lactate formation by inorganic phosphate (2, 3) in the human red cell have been reported. The increased glucose consumption has been explained by the effect of Pi in counteracting the inhibitors of the first two irreversible steps; glucose gphosphate inhibition of hexokinase (1) and adenosine triphosphate inhibition of phosphofructokinase (2, 3). The means by which the rate of the last irreversible step of glycolysis, pyruvate kinase, is increased * This investigation n~as supported by Public Health Service Research Grants CA07818, CA07819, and CA06927. in order to increase lactate formation remain to be clarified. This has now been done through studies of the isolated enzymes and of steady state glycolysis as a function of Pi with normal and pyruvate kinasedeficient red. In addition, these studies make apparent a relation between the level of monophosphoglyceric acids and the regulation of glycolysis at the glyceraldehydep dehydrogenase stage. MATERIALS AND METHODS Human red were prepared from blood freshly drawn in sodium citrate or heparin and washed with 0.9% NaCl with removal of white by aspiration. They were suspended in two volumes of medium containing: TrisCl, 0.05 M; KCl, 0.01 M; NaCl, 0.11 M; and 3 mm Pi. The ph was maintained at 7.4 with NaH2P04 during a 30min period in the cold. The were then centrifuged and suspended at 30% concentration in the above medium at ph 7.4 modified to contain sodium phosphate at 3, 20, or 50 mm Pi and suitably less NaCl to give a total ion concentration of 0.32 M. The incubations at 35 were initiated immediately with W4Cglucose, 4 to 6 mm. Samples were taken from the gently shaken flasks immediately prior to the addition of glucose or at noted periods afterward and placed in 2096 of the volume of 1 N HClO,, cooled, and centrifuged, and then the precipitate was washed twice with a like volume of 0.3 N HC104. The combined extract was adjusted to ph 6 or 7 with cold 1 N KOH and left overnight at 4. The neutral supernatant was reduced in volume at room temperature in a vacuum and assayed for glucose utilization by determining those counts which adhered to anion exchange resin (4). The amounts of various compounds were determined within the week as follows: ATP by yeast hexokinase and glucose6p dehydrogenase; ADP by pyruvate kinase and lactic dehydrogenase; AMP by prior addition of ATP and myokinase, acidification, and determination as ADP; glucose6p by glucose6p dehydrogenase; dihydroxyacetonep, glyceraldehyde3p, and fructose 1,6diphosphate by aglycerolp dehydrogenase, triosep isomerase, and aldolase addition, in that order; pyruvate, phosphoenolpyruvate, 2phosphoglyceric acid, and 3phosphoglyceric acid by addition of lactic dehydrogenase, pyruvate kinase, enolase, and phosphoglycerate mutase, in that order; lactate, cuglycerolp, and glycerol were determined with lactic dehydrogenase or crglycerolp dehydrogenase plus glycerol kinase in the presence of hydrazine (5). The changes in reduced pyridine nucleotide upon which these assays depend were measured spectrophotometrically or fluorometrically depending on the concentration of material present. NAD+ and NADH were determined on separate samples by the recycling method of Lowry et al. (6) with suitable controls taken to correct for 4848

Issue of November 10, 1966 I. A. Rose and J. V. B. Warms 4849 pyruvate present in the sample. For the determination of intracellular Pi, 0.2ml samples of incubation were diluted 25 fold with cold 0.9% NaCl and centrifuged, and the were briefly washed once with cold 0.9% NaCl before making the acid extract. Pyruvate kinase from normal and mutant was prepared from small samples of by the DEAEcellulose column method of Koler et al. (7). The kinetic studies on the enzyme were conducted at 28 in the following incubation mixture: 0.15 rnru ADP, 25 mm KCl, 5 mm MgCl,, 50 mm imidazolecl buffer (ph 7.4), 0.1 mm NADH, and 1 i.u. of lactic dehydrogenase (Boehringer) free of pyruvate kinase. Penolpyruvate and ATP were varied in the incubation as required. The initial rate of reaction was measured either by absorbance or fluorescence change following the addition of the red cell enzyme. Pyruvate kinasedeficient blood was obtained through Dr. H. S. Bowman of the Harrisburg General Hospital from an llyearold female (L. Y.) previously described by Bowman and Procopio (8). The second sample was obtained from a 65yearold woman through the cooperation of Dr. D. G. Nathan of the Peter Bent Brigham Hospital, Boston. We are greatly indebted to Drs. Bowman and Nathan for their effective help. RESULTS The kinetic behavior of pyruvate kinase from red has been previously examined by Campos, Koler, and Bigley (9) who found that contrary to the muscle enzyme (10) the KPenorpyruvste and Kmp are dependent on ADP and Penolpyruvate concentrations, respectively. These data were obtained at ph 7.4 with 16 mm Mg++. The unusually high KPeno~pyruvate that was obtained (1 to 4 mm, depending on the ADP concentration in the range 0.5 to 1.25 mm) was confirmed in this laboratory but found to be a consequence of the high concentration of Mg++ used. In addition, the Michaelis constant for Penolpyruvate is increased by monovalent cations, such as triethanolamine, also used in that study. ATP was reported to be a competitive inhibitor of Penolpyruvate (9). This result was confirmed at 5 mm MgClz in imidazole buffer in which KPenolpyruvate is much lower than reported (9) (Fig. 1). With ADP at 0.15 mm the inhibition is competitive at 0.3 mm and 1 mm ATP (KPmenolpyruvate goes from 0.065 mm to 0.23 and 0.4 mm, respectively), but perhaps not at 2 mm ATP. Regardless of the nature of the inhibition, it is clear that under conditions of high ATP and low ADP such as found in the human red blood cell, the pyruvate kinase is likely to be operating in the first order region of Penolpyruvate concentration dependence. To test the possibility that other metabolites might modify the ATPinhibited state of the enzyme, the effects of fructose diphosphate (2 RIM), AMP (0.3 MM), and Pi (10 mm) on the Kpenolpyruvote in the presence of 1 mm ATP and 0.15 mm ADP were studied. All three reagents were, in fact, mild inhibitors competitive with Penolpyruvate. Thus, unlike pyruvate kinase of yeast, which is strongly activated by fructose diphosphate (ll), the red cell enzyme has characteristics that suggest that its steady state velocity in the cell will be controlled by the concentration of Penolpyruvate, ADP, and ATP, and not by allosteric effecters. In the first whole cell experiment, from a normal male were incubated at three levels of medium Pi and the rates of glucose utilization and lactate formation measured at 30 mm, 60 min, and 120 min of incubation. As seen in Fig. 2, both rates are constant during the course of the sampling which would i 1,,,,,, 2 4 6 8 IO 1 2 /PEP, (ITIM) FIG. 1. Effect of ATP concentration on K, of phosphoenolpyruvate (PEP). With the conditions given under Materials and Methods (ADP = 0.15 mm), ATP was varied: 0, 17; 0.3 mm, A; 1.0 mm, 0; and 2 mm, l. The respective values of ~~~~~~~~~~~~~~~ were: 0.065, 0.23, 0.4, and 0.43 mm. indicate that the steady state should have been established in all ratedetermining parameters. The concentrations of several compounds extracted from the at the stated intervals are shown in Table I. It is seen that the ADP and Penolpyruvate concentrations are approximately time independent and that they change significantly with varying Pi. A comparison of these data in the form given in Table II clearly shows that the primary cause of the increased pyruvate kinase rate is the increased steady state concentration of Penolpyruvate. The increase in Penolpyruvate is more than proportional to the increased rate of lactate formation as would be required by the fall in ADP that accompanies increased glycolysis. A reasonable explanation for this result would be that increased Pi stimulates the flow of glucose through hexokinase and phosphofructokinase and that this leads to an increase in the Penolpyruvate level thus causing increased pyruvate kinase rate which causes an increased rate of ADP phosphorylation to ATP and lactate formation via pyruvate. The Penolpyruvate concentration and the pyruvate kinase rate eventually achieve a compromise condition which is determined by the rate of inflow of material to that step, the amount of pyruvate kinase, and the K, values for ADP and Penolpyruvate under the conditions of the high ATP. It is apparent, as expected from the studies with the enzyme, that the Penolpyruvate concentration of the cell is well below the apparent K, of Penolpyruvate under these conditions. A test of this idea was made by using obtained from individuals known to suffer from nonspherocytic hemolytic anemia characterized by very low levels (about 10% of normal) of pyruvate kinase. In this case it was expected that Penolpyruvate would have to achieve much higher levels in order to maintain a comparable rate of lactate production. Whether the enzyme derived from these was normal in kinetic char

4850 Control of Glycolysis in Human Red Blood Cell Vol. 241, No. 21 7 6 5 4 3 2 I I I I I I I I I I 30 69 90 120 30 60 90 120 MINUTES FIG. 2. Rates of glucose utilization (left) and lactate production (right) of normal red as a function of medium Pi. These figures are derived from the data given in Table I for incubated in Pi: 0, 3 mm; 0, 20 mm; and A, 50 mm. MINUTES Glucose utilized.... Lactate formed. 1 TABLE I Responses of normal red to Pi.5 @MA& Of intracellular Pi per IId Of Cells 6 I.9 pllldek Of intracellular Pi per ml Of C&S 30 min 60 min 120 min 0.35 0.63 pmoles/d 0.76 1.33 1.42 2.65 30 min 60 min 120 Inin 0.69 1.19 p?noles/ml 1.33 2.30 2.51 4.26 _ 1 30 min 60 min 120 min pm&s/m1 1.30 2.35 4.37 2.00 3.51 6.55 Glucose6P. Fructose diphosphate DihydroxyacetoneP Glyceraldehyde3P. 0.067 3 0.076 4 9 0.087 7 0.066 3 5 0.032 4 8 0.037 3 7 0.031 0.041 0.046 0.121 0.175 0.259 0.275 0.353 0.415 3 0.016 0.024 ATP. ADP. 0.160 0.160 1.100 0.150 0.120 0.120 1.170 0.120 0.097 0.096 1.200 0.091 Penolpyruvate. 2Phosphoglycerate. 3Phosphoglycerate. 5 6 0.016 6 0.018 0.013 0.012 3 0.038 0.014 3 0.043 0.028 0.029 0.014 0.076 0.029 9 0.079 Ratio of Sphosphoglycerate to Penolpyruvatt 2.7 3.0 3.1 3.3 2.6 2.9 0 Value refers only to the 60min time. acter had not been established at the time of these experiments. This would contribute to a lowering of the apparent K, of The data for two such experiments are shown in Tables III and Penolpyruvate of the deficient cell. In both subjects it is IV. The two cases are surprisingly similar in the responses given. evident that the rate of pyruvate kinase as derived from the In both cases the Penolpyruvate levels required to achieve a rate of lactate formation has become independent of Penolrate of pyruvate kinase reaction (lactate formation) comparable pyruvate at the second level of medium Pi. This saturation to that of the normal cell are much higher. This is expected from of pyruvate kinase, at levels of Penolpyruvate below 0.061 to a substratecontrolled velocity operating with less enzyme. 0.083 pmole per ml of packed or per 0.65 ml of cell water, That the pyruvate kinase deficient cell is usually lower in occurs at lower concentrations than would be expected from the ATP than the normal cell has previously been reported (12). apparent K, measured with the enzyme from normal.

Issue of November 10, 1966 I. A. Rose and J. V. B. Warms 4851 At 0.15 mm ADP the enzyme from the Harrisburg was observed to have a KPenorpyruvste of <O.Ol mm in the absence of ATP and 0.05 mm in the presence of 1 mm ATP. These are considerably below the values for the normal enzyme. The enzyme from the Boston was also abnormal in that it was not possible to obtain linear rates at low concentrations of P enolpyruvate. The rapid loss of activity upon dilution in the assay mixture was not prevented by EDTA, mercaptoethanol, or serum albumin, nor was it reversed by subsequent addition of high levels of Penolpyruvate. The general conclusion can be drawn from the constancy in 3phosphoglyceric acid to phosphoenolpyruvate ratios that the enzymes, enolase and monophosphoglycerate mutase, are able to maintain their reactants in equilibrium with each other. This point has previously been made for normal red by Minakami et al. (13). Thermodynamic considerations warrant the conclusion that at the observed concentrations of 3phosphoglyceric acid and at reasonable ratios of ATP:ADP and NAD+:NADH there can be very little fructose diphosphate if all of the enzymes, aldolase, triosep isomerase, glyceraldehyde 3P dehydrogenase, and phosphoglycerate kinase, are rapidly equilibrating. This situation is evident in the normal and deficient at the lower glycolytic rates. However, at the TABLE II Relation of Penolpyruvate, ADP, and ATP to rate of lactate production in normal Rate of lactate formation (relative) 1 1.9 3 T Steady state relative amount of 1 1 1 6.9 2.3 0.75 1.06 9.7 4.8 0.60 1.09 12.5 higher glycolytic rates for both normal and deficient, fructose diphosphate and the trioseps are found to accumulate. In all of the cases where fructose diphosphate exceeds 0.01 Hmole per ml of packed, the fructose diphosphate and dehydroxyacetonep concentrations are observed to rise continually during the incubation. Thus independence of glycolytic rate from changes in these concentrations is evident. Two questions arise from this observation. First is the problem of the role of fructose diphosphate as a control factor in glycolysis. It has been reported that fructose diphosphate is a very sensitive activator of phosphofructokinase in the ATPinhibited state (14, 15). It will be noted that at the lower twor Pi levels the increased glycolysis in the case of the normal (,Table I) and one deficient (Table III) is achieved with no change in glucose 6P. This effect is presumed to represent an exact coordination of the stimulatory effects of Pi on hexokinase and phosphofructokinase (1, 2). However, in all of the cases in which fructose diphosphate accumulates, the steady state level of glucose6p is seen to fall greatly. This can be visualized as due to an increment of phosphofructokinase stimulation that is in excess of the coordinated Pi effect. It has been reported that the effects of the several regulator substances for phosphofructokinase activity (15) and enzyme stabilization (16) are additive. However, it will be seen that in those cases of the fructose diphosphate stimulation, the fructose diphosphate is operating at saturating levels with respect to activation of phosphofructokinase since both glucose6p concentration and the glucose utilization rate are constant in spite of a steadily increasing fructose diphosphate concentration during the period of measurement. Thus within the rather narrow range of glycolytic rates observed, fructose diphosphate has gone from a level presumably too low to stimulate phosphofructokinase to one beyond the range of such control. The maximum effect on glucose utilization that can be attributed to fructose diphosphate is less than 2fold, and the greater fall in steady state Glucose Lactate utilized.. formed. TABLE Responses of pyruvate kinasedeficient (Harrisburg) III 1.5 flol& Of intracellular Pi per ml Of Cdl! j.9 Sol& Of intracellular Pi per ml Of Cells 13 pmolesa of intracellular Pj per ml of 30 min / mmin ( 120 min Jmoles/ml 0.79 1.57 30 min 60 min 120 min 0.47 0 :69 2.5 0.87 1.66 3.43. _ 30 min 1 60 min 1 120 min 1.38 2.75 5.10 1.38 3.36 5.58 Glucose6P. 0.108 0.108 0.101 0.085 0.098 0.100 0.017 0.019 0.025 Fructose diph0sphat.e. 8 5 0.011 0.191 0.443 0.640 Dihydroxyacet,oneP 0.248 0.519 0.585 3Phosphoglycerate. 0.050 0.052 0.072 0.115 0.167 0.177 0.249 0.329 0.462 2Phosphoglycerate. 5 0.022 0.013 0.029 0.034 0.044 0.069 0.078 0.135 Phosphoenolpyruvate 0.023 0.026 0.030 0.061 0.080 0.102 0.127 0.186 0.276 aglycerolp. Glycerol 0.064 0.116 0.165 0.274 0.042 0.042 ATP 0.617 0.642 0.684 0.605 0.645 0.640 0.540 0.578 0.550 ADP 0.123 0.126 0.135 0.103 0.113 0.101 0.180 0.179 0.186 AMP 0.044 0.044 0.044 0.033 0.035 0.033 0.056 0.062 0.070 ATP + ADP + AMP.. 0.784 0.812 0.863 0.741 0.793 0.774 0.776 0.819 0.806 (AMP) (ATP)/(ADP)2. 1.790 1.780 1.650 1.880 1.770 2.060 0.935 1.110 1.110 a Value refers only to the 60min time.

4852 Control of Glycolysis in Human Red Blood Cell Vol. 241, No. 21 TABLE Responses of pyruvatedeficient (Boston) IV I! 1.0 /AllIOlea Of ijitixcdlu1~~ Pi IYX ml Of CdlS j.3 jmmlesd of intracellular Pi per ml of 11.0 NllOleSa Of intracellular Pi per ml Of Cells 30 min 60 min 120 min 30 min 60 min / 120 min 30 min I 60 min I 120 min Glucose Lactate utilized.. formed. 0.43 0.94 0.83 1.71 pm&s/n1 1.42 0.85 1.76 3.84 1.19 2.48 5.10 3.26 1.64 3.23 6.60 1.73 3.66 7.68 Glucose6P. Fructose diphosphate DihydroxyacetoneP Glyceraldehyde3P. 0.073 0.072 0.082 0.049 0.054 0.049 0.028 0.030 0.025 0.011 0.084 0.102 0.211 0.185 0.283 0.502 0.030 0.212 0.345 0.339 0.465 0.465 0.620 0.013 0.026 0.019 0.039 0.039 0.062 3Phosphoglycerate. 2Phosphoglycerate.. Phosphoenolpyruvate @GlycerolP. 0.094 O.OiEl 0.047 0.065 0.093 0.039 0.074 6 0.034 0.211 0.260 0.348 0.250 0.332 0.438 0.029 0.033 0.040 0.022 0.039 0.050 0.083 0.120 0.139 0.097 0.139 0.181 0.048 0.075 0.146 0.069 0.136 0.288 ATP... ADP.... 0.475 0.079 0.440 0.088 0.465 0.465 0.495 0.501 0.450 0.495 0.538 0.100 0.086 0.087 0.086 0.103 0.103 0.110 a Value refers only to the 60.min time. TABLE V excess of the amount of 3phosphoglycerate found in the high Pi case of the normal cell (Table I). It is certain that the acidextracted 1,3diphosphoglycerate would have been hydrolyzed to 3phosphoglycerate by the time of assay. Furthermore, subsequent studies have failed to detect 1,3diphosphoglycerate in a comparable freshly neutralized extract. This failure to detect 1,3diphosphoglycerate is, however, to be expected if 2nd hour the dehydrogenase is slow and the kinase equilibrates its reac 1st hour No With tants. pymvate pyruvate (3m) An explanation for glyceraldehydep dehydrogenase becoming ratelimiting with additional increasing Pi must next be sought. It is clear that with the limited oxidative capacity of the mature red cell the elevation in the concentration of 3phosphoglyceric acid, 2phosphoglyceric acid, and Penolpyruvate must, to the Effect of pyruvate on glycolysis at high Pi A 30y0 suspension of normal red in 50 mm Pi medium was incubated as usual for 1 hour. Sodium pyruvate, at a final concentration of 3 mm, was added to onehalf of the suspension and both halves were incubated for a 2nd hour. Glucose utilized.. Lactate formed. Glucose6P. DihydroxyacetoneP Glyceraldehyde3P. Fructose diphosphate 3Phosphoglycerate. ATP... ADP. NADH NAD+ NADH:NAD+ ratio. 2.04 3.00 9 0.520 0.055 0.349 0.068 0.950 0.082 1.84 0.92 2.50 3.80 0.013 0.027 0.705 0.080 0.544 0.073 0.078 1.030 0.980 0.072 0.072 0.025 0.017 0.039 0.048 0.640 0.350 glucose6p seen in Tables III and IV at high Pi is probably the consequence of the additional stimulation of phosphofructokinase provided by the increases in AMP and ADP. A second problem arising from the accumulation of fructose diphosphate and trioseps is to identify the ratecontrolling step beyond phosphofructokinase that is responsible and to understand why this step has changed from a quasiequilibrium state when the further addition of Pi promotes glycolysis to a doubling of its rate. It can be logically concluded that if one step is responsible it must be the glyceraldehyde3p dehydrogenase step. If aldolase were limiting there would be no triosep. If triosep isomerase were limiting there would be no fructose diphosphate. If phosphoglycerate kinase were limiting there would be substantial amounts of 1,3diphosphoglycerate in extent that these are derived from glucose and not from the cell s 2,3diphosphoglyceric acid, be accompanied by a shift to reduced NAD. If there is a net breakdown of 2,3diphosphoglyceric acid in the presence of glucose to supply the increment of acarbon acids, it is staying remarkably within limits since there is little or no pyruvate formed in these incubations. This explanation suggests that glyceraldehydep dehydrogenase has become ratelimiting because of the substrate concentration nature of the pyruvate kinase control which causes an inhibitory accumulation of NADH. This, in turn, would cause an accumulation of fructose diphosphate that would stimulate phosphofructokinase and hence cause increased influx of material through the hexokinase step which would have been accelerated by the lowered glucose6p. To test the possibility that glyceraldehydep dehydrogenase had become ratelimiting as a result of a shift in NADH:NAD+ ratio at high medium Pi, the effect of pyruvate addition to the incubation was studied (Table V). Pyruvate would be expected to provide a good oxidant of NADH via the lactic dehydrogenase reaction of the cell. Normal, after an incubation with high Pi for 1 hour, were further incubated with or without added pyruvate and the consequences to the levels of intermediates and the glycolytic rates were determined. It will be noted that the subsequent hourly rate of glucose utilization was

Issue of November 10, 1966 I. A. Rose and J. V. B. Warms 4853 halved by the presence of pyruvate. This was accompanied by a complete depletion of the fructose diphosphate, dihydroxyacetonep, and glyceraldehyde3p pools and an increase of the glucose6p level. The observed increment in lactic acid for the 2nd hour was 1.3 pmoles greater when pyruvate was present. This corresponds very well to the 1.27 Fmoles of 3 carbon unit that could have been derived from the combined fructose diphosphate, dihydroxyacetonep, and glyceraldehyde 3P that was present at the end of the 1st hour. The amount of lactate that would be derived from the added pyruvate would be that necessary to shift the NADH:NAD+ ratio and is negligible. It will be noted that although the amount of lactate produced in the presence of pyruvate was considerably greater than in its absence, the 3phosphoglyceric acid (and hence the Penolpyruvate which was not measured) was not much higher in the former case. It is clear, however, that by the end of the hour, the accelerated rate of lactate formation must have been complete since the fructose diphosphate had disappeared, and in fact a somewhat lower level of 3phosphoglyceric acid should have been expected. The shift in NADH:NAD+ ratio was from 0.64 which is comparable to the 0.77 ratio found in whole blood (17) to 0.35 which is close to 0.40 found in incubated with 3 mm Pi in which no fructose diphosphate was present after 2 hours of incubation. Methylene blue (5 pm) also caused the disappearance of fructose diphosphate from the incubated with high Pi in similar experiments. To examine the question of whether the occurrence of fructose diphosphate reported in whole blood (18) is a consequence of control at the glyceraldehydep dehydrogenase step, freshly drawn heparinized venous blood was incubated at 35 for 15 min in the presence of varying amounts of pyruvate. The results were unambiguous in showing that the amount of fructose diphosphate plus dihydroxyacetonep of incubated blood decreased from 0.11 to 0.07 to 0.037 to 0.01 pmole per ml of whole blood as the pyruvate level of the whole blood measured at 15 min varied from 0.01 to 0.023 to 0.057 to 0.146 pmole per ml of whole blood. It should be mentioned that these levels of pyruvate were insufficient to cause stimulation of lj4cglucose oxidation to %02, which was doubled at 3 mm pyruvate but not affected at 0.3 mm. Further experiments with red in plasma will be necessary to determine what factors contribute to the ratelimiting level of NADH in Vito. Of course it is clear that any inhibition of pyruvate kinase leads to a proportional increase in the Penolpyruvate level and promotes the shift toward an increased NADH:NAD+ ratio. On the other hand, reducing compounds in blood such as cysteine and ascorbic acid may also participate in such a shift. DISCUSSION The earlier data of Minakami and Yoshikawa (2) are especially indicative of a causal relation between substrate concentration and pyruvate kinase rate in that they observed a parallel between the 3phosphoglyceric acid (and hence the Penolpyruvate) concentration and the rate of lactate formation as a function of Pi over the ph range 7 to 8. This indicates, as now pointed out, that in the normal cell the pyruvate kinase is functioning at a rate that is linearly dependent on Penolpyruvate concentration. Thus the rate of lactate formation is primarily determined by the net rate of the hexokinase plus phosphofructokinase and the pyruvate kinase keeps step simply by virtue of the increase in Penolpyruvate level. The Penolpyruvate and ADP levels seem to be responsible for determining the pyruvate kinase rate under the variety of conditions seen in the present experiments, namely varying Pi and approximately constant intracellular ATP. The accumulation of fructose diphosphate and trioseps in some of the did not alter the control characteristics of the pyruvate kinase in these. This is contrary to what would be expected for yeast where pyruvate kinase is stimulated by fructose diphosphate (11). Yeast, unlike the human red blood cell, therefore, has a device for preventing the pyruvate kinase step from being responsible for large accumulations of triosei s and fructose diphosphate. Thus, in yeast, the Penolpyruvate and fructose diphosphate levels may exercise control on each other if the elevation of Penolpyruvate brings about an accumulation of fructose diphosphate, as in the red cell. The explanation that has been suggested for the accumulation of fructose diphosphate at high glycolytic rates was that the necessary increase in Penolpyruvate requires a concomitant increase in NADH:NAD+ due to a decreased proportion of pyruvate available to act as an oxidant. This would lead to inhibition of glyceraldehydep dehydrogenase by NADH (19, 20). This explanation is supported by the effect of added oxidant in causing the rapid fall in fructose diphosphate. An alternative or contributing explanation for glyceraldehydep dehydrogenase becoming ratelimiting at high glycolytic rates is that with the increase in Penolpyruvate and the monophosphoglyceric acids must go a parallel increase in 1,3diphosphoglycerate which is known to inhibit the dehydrogenase (20). This explanation would seem inadequate in view of the stimulation by oxidants. However, Eckel et al. (21) have recently reported that pyruvate stimulates fructose diphosphate breakdown in red without the expected increase in total pyruvate plus lactate. It was found that the decrement of total triosep units due to pyruvate addition is accounted for as 2,3diphosphoglyceric acid and it was suggested that the effect of pyruvate is to stimulate the diphosphoglycerate mutase. Whether the pyruvate is acting directly or through its effect on NAD+NADH is unclear. The choice between NADH and 1,3diphosphoglyceric acid as the immediate regulator of glyceraldehydep dehydrogenase under conditions of high glycolytic rate, therefore, cannot be made at this time with certainty. However, it is significant that the control, which is presumably introduced during the short interval of transition from a low to a high glycolytic rate, is sustained for the many hours thereafter. Tsuboi and Fukunaga (3) report that fructose diphosphate and dihydroxyacetonep continue to increase over 6 hours in red glycolyzing at high Pi. Thus the controlling factor must be kinetically quite stable. This would probably better characterize the Penolpyruvate level and its directly related 1,3diphosphoglycerate than the NADH level since the former is directly related to the sustained glycolytic rate whereas the NADH produced in the transition between metabolic states is not. The combined 3carbon acids, Penolpyruvate, and the monophosphoglyceric acids were able to achieve steady state concentrations much higher than the available NAD. Assuming that these come from the increased rate of glucose metabolism rather than from the pool of 2,3diphosphoglyceric acid, an oxidant other than glucosederived pyruvate must be available. Glucosederived dihydroxyacetonep is evidently inadequate as an oxidant as shown by the insufficient amount of glycerol and

crglycerolp formed (Tables III and IV). It seems probable that net breakdown of 2,3diphosphoglyceric acid controlled by a phosphatase provides the required oxidant, pyruvate. This role of 2,3diphosphoglycerate is shown by the fact that that have been preincubated in the absence of glucose fail to accumulate fructose diphosphate in high Pi medium following the addition of glucose. This is now readily explained since pyruvate is observed in such preincubated. The breakdown of 2,3diphosphoglycerate in red has been known to be greatly accelerated by lowering the ph a few tenths of a unit below ph 7.4 (22). This formation of NADH oxidant probably provides the explanation for the rapid disappearance of fructose diphosphate from blood that is placed at ph 7.1 in the acid citrate dextrose of the blood bank (23). In most cases of pyruvate kinase deficiency previously reported, the enzyme has a normal or somewhat elevated Kpbenol Byruvate (9, 24, 25) unlike the Harrisburg sample studied here. Because of the lower activity of the deficient cell, the enzyme will be operating nearer the level of substrate saturation than in the normal cell. The levels of Penolpyruvate, Zphosphoglycerate, and 3phosphoglycerate found with the mutant used in the present studies would be still higher in mutant with enzyme of normal or higher KPenolgyruvate. NADH and 1,3diphosphoglyceric acid would therefore tend toward higher values, and the deficient red cell would attain the state of limitation by glyceraldehydep dehydrogenase and of accumulation of fructose diphosphate more readily than the normal cell. Perhaps the greatly diminished life span of these is a result of the altered concentration of intermediates tending toward higher ADP :ATP, NADH : NAD+, fructose diphosphate, and dehydroxyacetonep, which may alter the development or survival properties. Since these four factors are returned toward normal values by the addition of small amounts of pyruvate, the possibility that administering pyruvate or another oxidant of NADH during erythropoiesis might give rise to a more stable pyruvate kinasedeficient cell should be considered. The question of whether pyruvate kinase can be considered a ratedetermining factor in lactate formation is complex. Under conditions of no fructose diphosphate accumulation, i.e. adequate glyceraldehydep dehydrogenase rate, an increase in the activity of pyruvate kinase would simply have the effect of decreasing the steady state level of Penolpyruvate without increasing the rate of lactate formation. This would be equally true of an increase in the level of ADP insofar as the pyruvate kinase step is concerned since, as shown by previous workers, KADP is rather high, 0.1 to 0.3 mm (9, 25), and is higher than this in the presence of 1 mm ATP. However, if conditions were such that glyceraldehydep dehydrogenase was ratedetermining, then increased pyruvate formation due to increased pyruvate kinase or increased ADP acting at the pyruvate kinase step would cause an increased flow through the dehydrogenase due to the consequent decreases in NADH and 1,3diphosphoglycerate concentrations. This would result in an increased rate of lactate formation as long as accumulated fructose diphosphate was the source of the lactate. However, when the fructose diphosphate pool fell below the level that stimulates phosphofructokinase there would be a decrease in the rate of glucose utilization that would be similar to the effect seen with added pyruvate (Table V). In the experiments of Whittam and Ager Control of Glycolysis in Human Red Blood Cell Vol. 241, No. 21 (26), red were preincubated in the presence of sdenosine in the cold in order to accumulate high levels of Na+ and phosphate esters, most important of which would be fructose diphasphate. It was observed that upon subsequent incubation in the presence of ouabain or in the absence of K+ the production of lactate and Pi was significantly slower than when K+ was included in the medium and active cation transport was occurring. A reasonable mechanism for the coupling of ATP utilization to glycolysis in this case would be that a decrease in ADP causes the pyruvate kinase to operate at a higher Penolpyruvate level and hence both the NADH :NAD+ ratio and 1,8diphosphoglycerate level would be increased tending to decrease the flow through the glyceraldehydep dehydrogenase step. REFERENCES 1. ROSE, I. A., WARMS, J. V. B., AND O CONNELL, E. L., Biochem. Biophys. Res. Commun., 16,33 (1964). 2. 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