Studies on the control of lactate dehydrogenase activity in mammalian cells

BIOCHIMICA ET BIOPHYSICA ACTA

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BBA 26669

S T U D I E S ON T H E CONTROL OF LACTATE D E H Y D R O G E N A S E ACTIVITY IN MAMMALIAN CELLS II. D E M O N S T R A T I O N OF R A P I D CONTINUOUS V A R I A T I O N S IN LACTATE D E H Y D R O G E N A S E ACTIVITY IN HUMAN E R Y T H R O C Y T E S AND T H E I R R E L A T I O N TO A BIOLOGICAL ACTIVATOR AND AN I N H I B I T O R OF LACTATE D E H Y D R O G E N A S E

P. D U F F Y AND J. C. S A N D E R S O N

Department of Pathology, Chermside Hospital, Rode Road, Chermside 4032, Queensland (Australia) (Received March 29th, I97 I)

SUMMARY

I. Continuous variations in the activity of lactate dehydrogenase (L(+)lactate:NAD + oxidoreductase, EC 1.1.1.27) and in the concentration of pyruvate in washed human erythrocytes in constantly stirred suspensions, have been observed. 2. Variations were also observed in the activity of an activation and inhibition effect from lactate dehydrogenase as evidenced b y variations in the effects of the erythrocyte suspension extracts on crystalline rabbit muscle lactate dehydrogenase.

INTRODUCTION

To investigate whether the novel mechanisms controlling the activity of lactate dehydrogenase (L(+)-lactate : NAD+ oxidoreductase, EC 1.1.1.27) demonstrated in the previous paper I were of general application as a cellular control process, extension of these studies to human erythocrytes was made. These cells suggested themselves, because of their occurrence in vivo in free suspension, their lack of a nucleus and general protein synthesis, and their ready availability. This paper reports that the lactate dehydrogenase activity of washed human erythrocytes in stirred suspension in vitro varies in a rapid fashion similar to that for the tissue culture cells, and that these variations appear associated with variations in the activity of an activator and inhibitor for lactate dehydrogenase. Rapid continuous variations have also been demonstrated in the pyruvate concentrations of the erythrocyte suspensions. METHODS

5-2o ml fresh blood were obtained by venous puncture from antecubital fossa of human volunteers. Blood was diluted 8-fold with Tris-citrate buffered salt solution 2 Biochim. Biophys. Acta, 244 (1971) 613 617

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P. DUFFY AND J. C. SANDERSON

and centrifuged using a bench centrifuge. Ceils were washed with Tris-citrate buffered salt solution a further 2 times and the washed erythrocytes were then taken up in 25o ml Tris-citrate buffered salt solution in a 25 ° ml erlenmeyer flask, suspended in a water bath at 37 °. The cell suspension was constantly stirred by means of a magnetic follower and stirrer. After 3 ° min, sampling was performed at the times indicated using a I-ml pipetting sampler (Oxford). Timing was done with a stop-watch. After removal, samples were immediately frozen in a solid COs-acetone bath, and then thawed in a water bath at 37 °. Samples were frozen and thawed to a total of 3 times and stored at 4 ° until assay within the following 6 h. Lactate dehydrogenase was assayed according to WROBLEWSKIAND LA DUE 3 using a recording spectrophotometer (Unicam SP 8ooo B), equipped with an automatic cell changer through the cell holder of which was circulated running tap water at ambient temperature (27-29°). Phosphate buffer was maintained at this same temperature by means of a water-jacketed vessel. Correction for temperature changes to 25 ° was made 4. Quality control of assays was maintained by using assayed lyophilised control serum of constant batch number (Hyland). Results were expressed in I.U./ml of suspension. For pyruvate assay o.5 ml of cell suspension was rapidly removed from a stirred suspension at the times indicated using an o.5-ml pipetting sampler (Oxford) and rapidly ejected into 3.o ml of 6% HC104 and pyruvate assayed enzymically according to B0CHER el al.% using phenol red as indicator, in place of methyl orange. To demonstrate the lactate dehydrogenase activator and inhibitor activity, o.5 ml of cell extract were mixed with a constant amount of rabbit muscle lactate dehydrogenase (Sigma), of assayed activity. The activity of the mixture was measured after 3o min at ambient temperature (27°)and the percentage change in rabbit nmscle enzyme activity calculated on the basis of unaltered cell extract lactate dehydrogenase activity.

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Biochim. Biophys. Acta, 244 (1971) 613-617

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LACTATE DEHYDROGENASE VARIATIONS 15

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Figs. I and 2 show the patterns of change in lactate dehydrogenase activity in the erythrocyte suspension with duplicate samples being taken at 5-min intervals. The lactate dehydrogenase activity of the suspensions is seen to be varying with an apparent period approximately that of mouse fibroblast (LS) cellsL though the amplitude is much less. Close approximation of the activities of the duplicate samples is observed, standard deviation being 1.2% for Fig. I, and 1.6% for Fig. 2.

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Biochim. Biophys. Acta, 244 (1971) 6 1 3 - 6 1 7

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In Fig. 3, the patterns of change in the lactate dehydrogenase in the erythrocyte suspension with sampling at i-rain intervals, is compared with the pattern obtained by joining every fifth point starting at an arbitary origin. The I-min pattern shows a rapid fine variation which is significantly different from the random 5-min pattern. In Figs. 4 and 5, the effect of the erythrocyte suspension extracts upon added crystalline rabbit muscle lactate dehydrogenase (Sigma) are shown. The erythrocytes suspension extracts are seen to have a varying activation-inhibition effect on the rabbit muscle enzyme. The graph of this effect is seen to be the converse of the graph of the suspension lactate dehydrogenase activity. Fig. 6 shows the result of pyruvate assays of an erythrocyte suspension with samples being taken at I5-sec intervals. There is a rapid oscillation in the pyruvate levels of the cells, with a period of approximately 3 rain. DISC USSION

Tile present studies demonstrate the previously unreported existence in human erythrocyte suspensions of a rapidly varying activity of lactate dehydrogenase and suggest the presence of the novel activator and inhibitor described in the previous paper 1. Use of erythrocytes has confirmed that the variations are not related to growth synchronization and that protein synthesis is not involved. The method of sampling used in the experiments in which whole cell suspensions were frozen and thawed, confirms that the variations in the cells are not produced by passage of enzymes across the cell membrane, but rather a change in the total activity of lactate dehydrogenase in the suspension. The rapid variations in the pyruvate levels in the suspensions suggest that oscillations of glycolysis substrates are occurring in the suspensions. These have arisen spontaneously and would appear to be continuous. While oscillations of glyeolytic intermediates of cells and cell free extracts are well documented 6-~ there would appear to be no precedent for the spontaneous oscillations observed here intact in mammalian cells. For an oscillation to appear in the activity of an enzyme or amount of substrate in a cell suspension, the activity or amount in an individual cell must be oscillating

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Biochim. Biophys.. Mcta, 244 (1971) 613-617

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and there must be some form of synchronization of the cells as a whole, or of a significant proportion of them. Otherwise with the cell numbers involved (lO l° lO 11 in these experiments) oscillations in individual cells would be out of phase and unrecordable. The theory of oscillations of glycolytic substrates is well documented: lO and demonstration of such oscillations has usually followed experimental shift of glucose or other substrate concentration. Such oscillations have been self limiting. No such experimental manipulation was used in the present experiments and no continuing alteration in glucose or other substrate was made. The mechanism of initiation of the oscillations of glycolytic substrates, their propagation and synchronization in the cell suspensions in the present experiments is obscure. Oscillations of glycolytic enzymes would be expected to occur in a glycolytic system in the case of those enzymes activated or inhibited by an oscillating intermediate glycolytic substrate. Such an oscillation would be expected to have a period similar to its intermediate activator or inhibitor. Activation or inhibition of lactate dehydrogenase by glycolytic intermediates has not been described. That oscillations in glycolytic intermediates would occur with oscillations of lactate dehydrogenase activity was postulated in the previous paper 1. The observed oscillation lends credence to the hypothesis that oscillating intermediates related to the glycolytic cycle are cofactors for the activator and inhibitor of lactate dehydrogenase. The postulated cofactors would be expected to be oscillating with a period similar to that observed for lactate dehydrogenase activity and to pass across the cell membrane. i¢.EFERENCES i 2 3 4 5 6 7 8 9 IO

DUI:FY, Biochim. Biophys. Acta, 244 (I97 I) 607. PAUL, Cell and Tissue Culture, Livingstone, E d i n b u r g h and London, 4th ed., 197 o, p. 94. WROBLEWSKI AND J. S. LA DUE, Proc. Soc. Exp. Biol. Med., 9 ° (1959) 21o. VARLEY, Practical Clinical Biochemistry, H e i n e m a n n , London, 4th ed., 1967, p. 279. BUCHER, R. CYOK, W. LAMPRECHT AND E. LATZKO, in T. BERGMEYER, Methods o / E n z y m atic Analysis, Academic Press, L o n d o n and New York, 1963, p. 253. B. CHANCE,B. SCHOENER AND S. ELSAESSER, J. Biol. Chem., 240 (1965) 317o. J. HIGGINS, Ind. Eng. Chem., 59 (1967) 19. D. GARFINKEL, R. A. FRENKEL AND L. GARFINKEL, Comp. Biomed. Res., 2 (1968) 68. H. H E s s ANO A. BOITEUX, Z. Physiol. Chem., 349 (1968) 1567. B. C. GOODWlN, Adv. Enzyme Regul., 3 (1965) 425 •

P. J. F. H. T.

Biochim. Biophys. Acta, 244 (1971) 613-617