Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

In the present study, we followed glucose utilization in R. sylvatica erythrocytes as a function of temperature. Our data revealed a profound glycolytic block at 4°C. Identification of accumulated glycolytic intermediates suggests that PFK is the affected enzyme. This identification was made based on the pattern of glycolytic intermediate accumulation (Figure 4) and on the absence of a control on glucose accumulation by erythrocytes (Figure 2, 3). It is possible to quantify the glycolytic intermediate build-up by integrating the NMR peaks. These calculations showed that approximately 3.6 mM fructose was present in the NMR tube after 34 h at 4°C. Since fructose can equilibrate across the erythrocyte membrane it is not possible to calculate the absolute cellular concentration. However, one can also calculate that approximately 6 mM G6P and 8 mM F6P were present in these cells. This shows a profound blockage at the PFK enzyme locus leading to a high accumulation of PFK substrates. In support of this co nclusion, measurement of radioactivity equilibration across the erythrocyte membrane showed no apparent effect of temperature. This latter result agrees with the direct measurement of glucose transport across human erythrocyte membranes which indicated that the reaction proceeded in great excess when compared to the maximal glycolytic activity (4).

PFK is a prime candidate for glycolytic regulation since it has already been identified as a rate-modifying locus in many different animal models. The enzyme is complex, comprising 4 kinetically interactive subunits that may polymerize at low pH or higher temperatures. In R. sylvatica erythrocytes at 4°C, the pH was increased relative to that at 17°C (7.3 versus 7.0) suggesting that the R. sylvatica PFK enzyme is particularly sensitive to changes in temperature only. This heightened temperature sensitivity resulted in a complete blockage of glycolysis as shown by a rate of -0.08 x 0.04 mol/h/10¹⁵ cells at 4°C (Figure 1) and the accumulation of approximately 8 mM F6P (Figure 5). The physiological implications of such a glycolytic blockage are important. R. sylvatica rapidly export glucose into their circulatory system in response to a freezing stimulus: blood glucose levels increase from 5 mM to 16 mM by 4 min. post ice nucleation and reach 200 mM glucose 3 days after freezin g (1,5). The glucose is derived from liver glycogen via activation of glycogen phosphorylase (1) and export from the liver is aided by high liver glucose transporter numbers (6). High glucose concentrations are required to prevent internal cellular contents from freezing during exposure to cold while allowing controlled freezing of extacellular water (7). In order to maintain high extracellular glucose concentrations, the glycolytic activity of all cells must be profoundly regulated to prevent oxidation or fermentation (during anoxic conditions). In order to respond to the rapid rise in glucose concentrations, the mechanisms regulating glycolytic activity must also be rapid to permit a coordinated metabolic response to a freezing event. In addition, they must be readily reversible to respond to a rise in temperature. The mechanisms for regulating glycolytic enzyme activity in R. sylvatica are largely unknown (7). To date, only the liver mechanism has been identified. In liver, the concentrations of the potent PFK activator, fructose 2,6-bisphosphate decrease rapidly during freezing, probably as a function of hormonal control over 6-phosphofructo-2-kinase activity (3).

The present paper, therefore, presents novel mechanism for regulating erythrocyte glycolytic activity: temperature sensitivity of key regulatory enzymes. The advantages of this stratagem are numerous including: rapid implementation and reversibility of the control mechanism, considerable energy savings in terms of implementation and maintenance of the altered activity state and, as shown in this paper, complete inhibition at the enzyme locus. Phosphofructokinase from R. sylvatica, therefore, may be similar to that of the freeze tolerant insect, Eurosta solidaginis which is also inhibited at low temperatures to support sorbitol production from glycogen during exposure to cold temperatures. Kinetic analysis of E. solidaginis PFK revealed that the maximal velocity was extremely temperature sensitive (Q₁₀ = 3.6) . In addition, the E. solidaginis PFK affinity for its substrate, fructose 6-phosphate, was greatly reduced in the cold as was the effectiveness of allosteric activators such as AMP and fructose 2,6-bisphosphate. Similar changes may underlie the control of R. sylvatica erythrocyte PFK activity.

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