Inhibition of lactate transport in Ehrlich ascites tumor cells and human erythrocytes by a synthetic anhydride of lactic acid

Abstract

The synthesis and some of the physical and biological characteristics of a new inhibitor of lactate transport are described. The inhibitor is isobutylcarbonyl lactayl anhydride (iBCLA). It is formed by the condensation of lactic acid and isobutylchloroformate. It inhibits lactate transport 50% at 0.5 microgram/mg of protein in both Ehrlich ascites tumor cells and human erythrocytes. In contrast, 15 microgram of iBCLA/mg of protein is required for 50% inhibition of phosphate transport in erythrocytes, and phosphate transport in Ehrlich ascites tumor cells is unaffected at levels as high as 50 microgram of iBCLA/mg of protein. A time-dependent and concentration-dependent reversal of lactate transport inhibition took place on exposure of iBCLA-treated Ehrlich ascites cells to hydroxylamine or dithiothreitol. These data, along with the observed sensitivity of the lactate transporter to sulfhydryl reagents [Spencer, T. L., & Lehninger, A. L. (1976) Biochem. J. 154, 405-414], suggest that iBCLA acylates an essential sulfhydryl group on the transporter. When glycolyzing Ehrlich ascites tumor cells were treated with concentrations of iBCLA sufficient for complete inhibition of lactate transport, intracellular lactate levels increased, intracellular pH and extra-cellular lactate levels decreased, and overall lactate production was inhibited.

Increases in Na+,K+-ATPase activity of erythrocytes and skeletal muscle after chronic ethanol consumption: evidence for reduced efficiency of the enzyme.

“…Details of the analysis of lactate including a slight modification in the neutralization procedure are published elsewhere (12 where appropriate, glucose plus 'Rb (1.6 Bq/nmol of K+).…”

Monocarboxylate transport in erythrocytes

“…Information on the molecular nature of the carrier and its environment in the membrane comes from [28,46] and a lactic anhydride derivative [59] suggest the involvement of amino groups. In view of their inhibitory influence on inorganic anion exchange [28,59,67] and of the rather uncharacteristic binding pattern of such reagents [93,109], they will probably not help to identify the transport protein.…”

“…In view of their inhibitory influence on inorganic anion exchange [28,59,67] and of the rather uncharacteristic binding pattern of such reagents [93,109], they will probably not help to identify the transport protein. Mercurials are more promising tools in view of their high affinity for the monocarboxylate carrier [28].…”

Spontaneous reversal of polarity of the voltage across LLC‐PK1 renal epithelial cell sheets

“…We considered that if lactate effluxes into the basolateral compartment faster than it ef'fluxes into the apical, an apical-positive diffusion PD could establish. The uptake of L-lactate by these cells was observed to be Na+-independent, but it was observed to be inhibited by the synthetic anhydride iBCLA (isobutylcarbonyl lactyl anhydride) (see Johnson et al, 1980), the bioflavanoid quercetin, and L-lactate itself (unpublished results). Thus lactate emux in LLC-PK cells is likely carrier-mediated.…”