Hazen S L, Gross R W
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1993 May 5;268(13):9892-900.
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.
我们之前已经证明，心肌胞质钙非依赖性磷脂酶A2是一种40 kDa的多肽，受配体调节的蛋白质-蛋白质相互作用调控（哈曾，S.L.，和格罗斯，R.W.（1991年）《生物化学杂志》266，14526 - 14534）。我们现在证明，一种85 kDa的多肽，它与磷酸果糖激酶（PFK）具有序列同源性以及化学、物理、免疫和色谱相似性，能特异性地与40 kDa的磷脂酶A2催化亚基相互作用，并且代表了在之前工作中鉴定出的假定蛋白质调节元件。多条独立的证据证明了85 kDa的磷酸果糖激酶同工型与40 kDa的心肌胞质钙非依赖性磷脂酶A2催化多肽之间的关联，包括：1）85 kDa和40 kDa多肽在凝胶过滤色谱中以400 kDa复合物形式共洗脱；2）85 kDa和40 kDa多肽之间的化学计量关系对应于由四聚体PFK同工型和40 kDa磷脂酶A2催化多肽组成的复合物；3）证明85 kDa的磷酸果糖激酶同工型作为心肌胞质磷脂酶A2催化活性的特异性和可逆亲和吸附剂；4）利用鸡抗兔骨骼肌PFK IgG对心肌胞质磷脂酶A2活性进行免疫沉淀；5）在由磷酸果糖激酶变构调节剂形成三元复合物后，磷脂酶A2从ATP - 琼脂糖上特异性释放；6）PFK对纯化的均一钙非依赖性胞质磷脂酶A2变性的选择性减弱。总体而言，这些结果证明了一种磷酸果糖激酶同工型与心肌胞质钙非依赖性磷脂酶A2的高度特异性关联，并提示了一种新的生化机制，该机制是之前在心肌和其他哺乳动物组织中观察到的磷脂分解和糖酵解协同调节的基础。
