STUDIES ON ASSOCIATIONS OF GLYCOLYTIC AND GLUTAMINOLYTIC ENZYMES IN MCF-7 CELLS - ROLE OF P36

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phos phoglycerate kinase, enolase, and pyruvate kinase. This complex betwee n the glycolytic enzymes is sensitive to RNase. p36 could not be detec ted within this association of glycolytic enzymes; however an associat ion of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by iso electric focusing: the mitochondrial form with an isoelectric point be tween 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associate d form with pI 7.8. The mitochondrial form comprises the mature mitoch ondrial isoenzyme (pI 9.5) and its precursor form (pI 8.9). Refocusing of the pI 7.8 form of malate dehydrogenase also gave rise to the mito chondrial isoenzyme. Thus, the pI 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the as sociation with p36. Addition of fructose 1,6-bisphosphate to the initi al focusing column induced a quantitative shift of the pI 7.8 form of malate dehydrogenase to the mitochondrial forms (pI 8.9 and 9.5). In M CF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pI 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD --> NADH direct ion (malate --> oxaloacetate direction). The association of p36 with t he mitochondrial isoenzyme may favor the flow of hydrogen from the cyt osol into the mitochondria. Inhibition of cell proliferation by AMP wh ich leads to an inhibition of glycolysis has no effect on complex form ation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP tre atment leads to an activation of malate dehydrogenase, which correlate s with the increase of pyruvate and the decrease of lactate levels, bu t has no effect on the distribution of the various malate dehydrogenas e forms. (C) 1996 Wiley-Liss, Inc.