Distribution of metabolic fluxes towards glycerol phosphate and l -lactate from fructose 1,6-biphosphate in vitro: Effect of glycerol phosphate dehydrogenase

Int. J. Biochem. Vol. 18, No. 9, pp. 853 856, 1986

0020-711X/86 $3.00+0.00 Pergamon Journals Ltd

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DISTRIBUTION OF METABOLIC FLUXES TOWARDS GLYCEROL PHOSPHATE A N D r-LACTATE FROM FRUCTOSE 1,6-BIPHOSPHATE IN VITRO: EFFECT OF GLYCEROL PHOSPHATE D E H Y D R O G E N A S E Jos~ M. RIOL-CIMAS and ENRIQUEMELI~NDEZ-HEVIA* Departamento de Bioquimiea, Facultad de Biologia, Universidad de La Lagnna, 38000 Tenerife, Canary Islands, Spain (Received 17 December 1985)

Al~tract--l. A metabolic system in vitro, which converts fructose 1,6-biphosphate into the two alternative products, lactate and glycerol phosphate, was designed to study the distribution of metabolic fluxes and, specifically, the control of glycerol phosphate production rate in rat muscle extract. 2. Experiments were carried out at several protein concentrations by dilution of rat muscle extract, showing non-linear behaviours of flux versus protein concentration. These were hyperbolic for glycerol phosphate and logarithmic for L-lactate. 3. The influence of the flux towards any alternate product on the rate giving the other was studied by stimulation of each. Results obtained show that in this system, flux towards glycerol phosphate is not affected by lactate production and the same occurs for the contrary ease. 4. Glycerol phosphate dehydrogenase seems to be the only enzyme in this system whose activity controls the flux towards glycerol phosphate.

work we have designed an experimental system for this study, which converts fructose 1,6-biphosphate into glycerol phosphate and L-lactate with rat muscle soluble extract. Fructose 1,6-biphosphate was used as first substrate of the system as the intermediary product of glycolysis just before the branching, in order to simplify the metabolic system. Results obtained on flux distribution and regulation are discussed.

INTRODUCTION In non-lipogenic tissues, such as muscle, glycerol phosphate production from glycolytic pathway is necessary for regeneration of cytoplasmic N A D ÷ by means of glycerol phosphate shuttle (see, e.g. Dawson, 1979). It has been shown that glycerol phosphate is produced in vivo and in vitro in several arganisms and tissues at rates similar to lactate (Mackenzie et al., 1983; Melrndez-Hevia et aL, 1984). In rat muscle and liver, previous experiments in our laboratory (Melrndez-Hevia et aL, 1984) have shown that flux towards glycerol phosphate from glucose 6-phosphate occurs at a rate similar to that of L-lactate in vitro. Recently (Siverio et aL, 1985) we have described changes in distribution of fluxes in vitro towards glycerol phosphate and lactate from glucose 6-phosphate by rat liver soluble extracts during liver regeneration after partial hepatectomy. Glycerol phosphate production rate, as well as distribution of glycolytic flux among glycerol phosphate and lactate are a subject of control of metabolic fluxes, whose theory has been studied by Kacser and Burns (1973, 1979), Heinrich and Rapoport (1974), Rapoport et aL (1976) and Savageau (1971, 1972) among others (see reviews in Groen et al., 1982; Westerhoff et al., 1984), who have described nonlinear behaviour in ratios of flux and individual enzyme activities. The distribution of metabolic flux between lactate and glycerol phosphate is, thus, an interesting experimental model to study the distribution of flux in branched metabolic pathways, which as an experimental system model, it can be studied in vitro from any point before branching. In the present

*Author to whom correspondence should be addressed.

MATERIALS AND METHODS Male Wistar albino rats (250-300 g) fed on a standard laboratory diet were used in all experiments. Skeletal muscles (biceps femoris) were obtained under ether anesthesia, cooled, chopped and homogenized at lg/4 ml in 0.1 M potassium phosphate buffer, pH 7.4, containing 5 mM NaC1 and 2.5 mM MgCI 2 by using a Potter-Elvejeim homogenizer with Teflon pestle in an ice-cold bath. Homogenates were clarified by centrifugation at 27,000 g in a Sorvall RC-5B centrifuge at 3--4°C for 20 min. The resulting supernatants were used immediately to assay enzyme activities according to Bergmeyer (1974) and for kinetic experiments after elimination of low molecular weight metabolites. This was achieved by chromatography using Sephadex G-25 in a column of 1 x 10 cm applying 1 ml of supernatant and with the same buffer, during 15 min to obtain 12 ml of diluted sample, where protein concentration was assayed according to Lowry et al. (1951). For kinetic experiments I mg/ml chicken egg white trypsin inhibitor (Sigma, type 11) was added to the sample. Kinetic experiments were carried out by incubation of tissue extracts and substrates diluted in the same buffer with a total volume of 5 ml in a shaker bath at 30°C during 40 min, taking 0.5 ml aliquots every 5 min to assay L-lactate and glycerol phosphate. Reactions were started with 5mM NAD + and 5mM fructose 1,6-biphosphate in the incubation mixture and the appropriate volume of tissue extract to obtain 0.08q3.5 protein mg per ml, according to the experiment. At 20 min of reaction time ADP diluted in the same buffer to obtain a concen-

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Fig. l. Results of a typical kinetic experiment with rat muscle extract showing L-lactate (Q) and glycerol phosphate (©) production in vitro from fructose 1,6-biphosphate, with 5 mM NAD ÷. Glycerol phosphate is produced linearly from starting time and lactate production was started at 20 rain of reaction time by adding 5 mM ADP (arrow), its production also being linear during the time of the experiment. Fluxes towards glycerol phosphate and lactate were obtained from slopes of these results. Note that the starting of flux towards lactate does not alter the slope of glycerol phosphate production.

tration of 5 mM in the incubation mixture was added and reactions were maintained in these conditions until conclusion of kinetic assay, taking 0.5 ml aliquots every 5 rain, as described above. All the aliquots were deprotenized immediately after being taken out by addition of 0.5 ml of 1M HC104, neutralized after 10 rain with 0.05 ml of 5 M K2CO 3 (to pH 6-7) and clarified by centrifugation at 1500g for 10 rain. L-Lactate and glycerol phosphate were assayed in these extracts according to Bergrneyer (1974) by a continuous recording UV absorbance variation of NADH at 340nm to end point with a Hitachi 100-80A Speetrophotometer at 25°C. All experiments were carried out three times using a different animal for each, giving their results a coefficient of variation, (a/.~). 100 = 3.2. All biochemical reagents, including substi'ates and auxiliary enzymes to assay L-lactate and glycerol phosphate, and to assay glycolytic enzyme activities, were obtained from Sigma Chem. Co. (St Louis, Mo, U.S.A.). All other reagents were of analytical grade, obtained from E. Merck (Darmstadt, F.R.G.). In kinetic experiments to study the role of individual enzymes on glycerol phosphate production rate, different amounts of each enzyme were added at 20 rain of reaction time, according to the experiment, at the same time of ADP addition, as described above. RESULTS AND D I S C U S S I O N

Figure 1 shows results of a typical kinetic experiment from which fluxes for L-lactate and glycerol phosphate were obtained. In all experiments, under described conditions, a good linearity for both lactate and glycerol phosphate (showing the system to be at constant flux) were obtained. These results show that after the first 20 min, addition of ADP, and consequently, the start of flux towards lactate, does not alter the glycerol phosphate production rate. This

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fact, observed in all experiments, shows that the second part of glycolysis (after glyceraldehyde 3-phosphate dehydrogenase) does not influence the flux toward glycerol phosphate on this system in vitro, suggesting glycerol phosphate production rate is not under control of this part of the system. However, the total protein concentration in the system is an important factor to determine the ratio between these fluxes. As can be seen in Fig. 2, distribution of fluxes is different at different total protein concentrations of the system, the individual relationships among enzymes remaining constant. Furthermore, neither activity for L-lactate production nor glycerol phosphate shows a linear relationship versus protein concentration. In enzyme kinetic assays, enzyme activity is expected to be linear versus protein concentration; however, in this system, where a metabolic pathway involving several enzymes exists, there is no linearity, activity for lactate being logarithmic and activity for glycerol phosphate being hyperbolic, as is shown in the insets of Fig. 2. Kacser and Burns (1979, 1981) have described hyperbolic relationships between flux and the concentration of an individual enzyme of a metabolic system, with a consistent theoretical framework, This hyperbolic relationship has also been described in vitro for the glycerol phosphate production by rat liver extracts (Torres et al., 1986). These results, however, are referred to an hyperbolic behaviour under total pro-

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Table 1. Glycolytic profile of enzyme activities of rat muscle extract used in these experiments. Means ± SD for three animals are given Enzyme UnitsO/protein Unitsa/tissue g Hexokinase 0.022 ___3.42 x 10 3 0.63 _+0.095 Phosphoglucomutase 0.98 ___0.24 31.25 ± 13.04 Phosphoglucose isomerase 6.94 _+0.75 188 + 20.25 Phosphofructokinase 0.13 ± 0.035 3.73 + 0.94 Fructose 1,6-biphosphate aldolase 1.72 ± 0.134 50.76 + 4.62 Triose phosphate isomerase 25.39 ± 0.92 768 ± 21.17 Glyceraldehyde 3-phosphate dehydrogenase 10.06 ± 4.90 275 ± 129 3-Phosphoglycerate kinase 6.90 ± 0.559 182 ± 12.9 Phosphoglycerate mutase 17,41 ± 1.46 458 ± 31.20 Enolase 3,48 ± 1.42 94,6 + 38.58 Pyruvate kinase 8.63 Z 0.43 238 ± 11.8 Lactate dehydrogenase 13.48 ± 2.19 388 + 35.0 Glycerol phosphate dehydrogenase 0.40 ± 0.08 11.2 ± 2.13 uUnits of enzyme activity are defined as ,umol of product released per rain at saturating concentrations of substrates. All assays were carried out at pH 7.4.

tein concentration (Fig. 2) in a different experimental system. Thus, the hyperbolic relationship shown in Fig. 2 for glycerol phosphate flux suggests that a particular enzyme activity may control the glycerol phosphate production rate, the concentration changes of other enzymes not being significant for the control of the system in the studied range. Thus, we have assayed the Sensitivity of the system to all enzymes of the pathway by addition of important enzyme quantities; only addition of glycerol phosphate dehydrogenase gives positive results, shown in Fig. 3. As can be seen, the increase in glycerol phosphate dehydrogenase activity enhances the flux towards glycerol phosphate, showing the system to be sensitive to changes on this enzyme activity, it there-

Acknowledgement--This work was supported by a research grant from the Comisi6n Asesora de lnvestigaci6n Cientifica y Trcnica, Re±, No. 0567/81.

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fore being an important control point of the system. In fact, in the assayed glycolytic profile (Table 1) glycerol phosphate dehydrogenase stands out as the minimal enzyme activity involved in this system. On the other hand, it can be seen, in results shown in Fig. 3 that this important increase in the flux towards glycerol phosphate does not alter the lactate production rate, this being insensitive to changes in glycerol phosphate dehydrogenase activity. This fact is in the same line as the discussed above (see Fig. 1) on the starting of flux towards lactate, which is seen not to alter the glycerol phosphate production rate. It can be concluded from these results that the two fluxes of this system are submitted to independent control, the influence of each on the other being negligible.

REFERENCES

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Fig. 3. Effect of glycerol phosphate dehydrogenase activity on flux towards glycerol phosphate. Addition of glycerol phosphate dehydrogenase to the system enhances the velocity of glycerol phosphate production showing this enzyme activity controls the flux. Additions of glycerol phosphate dehydrogenase to rat muscle extract were: none (O); 10 fold plus of the extract (1.92 Units) (A); 30 fold plus of the extract (5.76 Units) (I--1). These experiments were carried out at 0.24 protein mg per ml of total mixture. Flux towards L-lactate ( 0 ) was the same in the three conditions being not affected by glycerol phosphate dehydrogenase addition.

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