The specific association of a phosphofructokinase isoform with myocardial calcium-independent phospholipase A2. Implications for the coordinated regulation of phospholipolysis and glycolysis.
We have demonstrated previously that myocardial cytosolic calcium-independent phospholipase A2 is a 40-kDa polypeptide regulated by ligand-modulated protein-protein interactions (Hazen, S.L., and Gross, R.W. (1991) J. Biol. Chem. 266, 14526-14534). We now demonstrate that an 85-kDa polypeptide which possesses sequence homology to and chemical, physical, immunological, and chromatographic similarities with phosphofructokinase (PFK) specifically interacts with the 40-kDa phospholipase A2 catalytic subunit and represents the putative protein regulatory element identified in previous work. Multiple independent lines of evidence document the association between the 85-kDa phosphofructokinase isoform and the 40-kDa myocardial cytosolic calcium-independent phospholipase A2 catalytic polypeptide, including 1) the coelution of the 85- and 40-kDa polypeptides which migrate as a 400-kDa complex during gel filtration chromatography, 2) the stoichiometry between the 85- and 40-kDa polypeptides which corresponds to a complex comprised of a tetrameric PFK isoform and a 40-kDa phospholipase A2 catalytic polypeptide, 3) the demonstration that the 85-kDa phosphofructokinase isoform acts as a specific and reversible affinity adsorbent for myocardial cytosolic phospholipase A2 catalytic activity, 4) the immunoprecipitation of myocardial cytosolic phospholipase A2 activity utilizing chicken anti-rabbit skeletal muscle PFK IgG, 5) the specific release of phospholipase A2 from ATP-agarose after formation of a ternary complex comprised of allosteric modifiers of phosphofructokinase, and 6) the selective attenuation of the denaturation of purified homogeneous calcium-independent cytosolic phospholipase A2 with PFK. Collectively, these results demonstrate the highly specific association of a phosphofructokinase isoform with myocardial cytosolic calcium-independent phospholipase A2 and suggest a novel biochemical mechanism underlying the coordinated regulation of phospholipolysis and glycolysis previously observed in myocardium and in other mammalian tissues.
Hazen
S L
SL
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Gross
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006019:Glycolysis; D006706:Homeostasis; D006801:Humans; D007106:Immune Sera; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008066:Lipolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000302:isolation & purification* / Q000378:metabolism; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007106:Immune Sera; D007527:Isoenzymes; D010743:Phospholipids; D010732:Phosphofructokinase-1; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium