On the triple localization of creatine kinase in heart and skeletal muscle cells of the rat: evidence for the existence of myofibrillar and mitochondrial isoenzymes.

Abstract

Abstract 1. 1. In order to assess the intracellular localization of creatine kinase (EC 2.7.3.2) in muscle, a fractionation prodecure was developed in which (a) 1 mM EDTA was used to prevent the Ca2+-dependent binding of cytosolic creatine kinase to myosin A; (b) 0.2 mM dithiothreitol was used to prevent binding of the cytosolic marker enzyme pyruvate, kinase (EC 2.7.1.40) to the heavy particulate fraction; (c) the myofibrils were solubilized by ultrasonic treatment in 1 M KCI; (d) the washing of the minced tissue was omitted since it was found that this gave rise to the loss of up to 40% of pyruvate kinase. The tissues were fractionated in three fractions enriched in the myofibrillar marker enzyme myosin ATPase (EC 3.6.1.3.), the mitochondrial marker enzyme cytochrome c oxidase (EC 1.9.3.1) or the cytosolic marker enzyme pyruvate kinase. 2. 2. Creatine kinase was found to be located in the myofibrils, the mitochondria and the cytosol of both rat heart and rat masseter muscle. With the aid of the marker enzymes mentioned above, it was calculated that in heart 33% of the total cellular creatine kinase activity (measured with 25 mM creatine at 25°C) resides in the myofibrils, 19% in the mitochondria and 48% in the cytosol. For rat masseter muscle these values were 4.4, 2.3 and 93% respectively. The total creatine kinase activity was in heart 145 units/g wet weight, and in masseter muscle 432 units/g wet weight. 3. 3. The electrophoretic mobility at pH 9 on cellulose acetate was different for the three isoenzymes. The mobility towards the cathode increased in the following order:cytosolic (MM-type), myofibrillar and mitochondrial isoenzyme, extracted from mitochondria. Mitochondrial creatine kinase, when bound to the outside of the inner membrane, where it resides in situ, was not mobile. 4. 4. The apparent Km values for creatine for reactions catalyzed by rat-heart myofibrillar, mitochondrial and cytosolic subcellular fractions were 4.3, 13 and 20 mM, respectively. In the corresponding fractions of masseter muscle these values were 18, 13 and 28 mM, respectively. For creatine phosphate as the substrate the values were for heart 0.9, 1.0 and 1.8 mM, and for masseter muscle 3.0, 1.6 and 2.6 mM, respectively. 5. 5. It is proposed that the physiological function of the mitochondrial and cytosolic creatine kinases is the synthesis of creatine phosphate, when fatty acids or carbohydrates, respectively, are the main source of energy. As the function for myofibrillar creatine kinase is proposed the degradation of creatine phosphate to yield ATP for muscular contraction.