Lactate favours the dissociation of skeletal muscle 6-phosphofructo-1-kinase tetramers down-regulating the enzyme and muscle glycolysis

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure

Biochemical, biophysical and haemorheological effects of dimethylsulphoxide on human erythrocyte calcium loading

© 2002, Published by Elsevier Science Ltd.The studies using dimethylsulphoxide (DMSO) and/or the 4-bromo-calcium ionophore A23187 (Br-A23187)often neglect the precise knowledge of some of their biochemical, biophysical and haemorheological effects. The aim of the present study was to evaluate these effects on erythrocytes after whole blood incubations with DMSO or Br-A23187 dissolved in DMSO. There were no significant differences between the different aliquots in the values of P50, pH,erythrocyte deformability, erythrocyte membrane fluidity, haemoglobin and intracellular Ca2+ concentrations ([Ca2+]i). Aliquots with DMSO (independently of the presence of Br-A23187 or added Ca2+) had lower erythrocyte aggregation indexes and higher plasma concentrations of K+, Na+ and Ca2+ than the aliquots without DMSO independently of the presence of added Ca2+). Aliquots with added calcium (without the presence of Br-A23187 in DMSO) had a significantly higher erythrocyte acetylcholinesterase activity. Our data shows that calcium loading, the usual objective of Br-A23187 incubations, cannot be fulfilled with the studied experimental conditions. The coherence between our results and those obtained by other authors with different biological systems and different modulators of the rise on [Ca2+]i suggests a non-specific effect of DMSO, disabling the action of the modulator. It can be reasoned that the decreased erythrocyte aggregation (without significant changes on the deformability or membrane fluidity) can result either from the decrease of the hydrogen bonding contribution to erythrocyte aggregation or the increased ionic strength influence on the erythrocyte membrane surface.This work was supported by the Fundação para a Ciência e Tecnologia of the Portuguese Ministry of Science and Technology, and by the Calouste Gulbenkian Foundation (Portugal