A new colorimetric method for determination of creatine phosphokinase

A new colorimetric method has been developed for the determination of creatine phosphokinase (CPK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with p-nitrophenylglyoxal (PNPG) under alkaline conditions to produce a colored product which absorbs maximally at 480 nm. At 25 degrees C the reaction was complete after 10 min in 0.1 M sodium pyrophosphate, pH 12, containing 0.15 M sodium ascorbate. The colored product was stable for at least 24 h and obeyed Beer's law in the range 0.005-0.05 mM creatine. The color reaction was used to determine the activity of CPK in serum and tissue extracts. The results obtained by this method agreed well with the results obtained by other available methods for CPK determination. However, the PNPG method was more rapid and more sensitive than other colorimetric methods and required a single chromogenic reagent.     

Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.     

Inhibition of cardiac creatine phosphokinase by fluorodinitrobenzene

With electrophoretic evidence for a mitochondrial isoenzyme of creatine phosphokinase (CPK), we feel that functional studies are necessary to help further elucidate the properties of this isoenzyme. As one approach, fluorodinitrobenzene (FDNB) was used to examine its effect on mitochondrial CPK. In both polarographic studies and direct enzymatic studies, 10^?5 M FDNB was shown to almost completely inhibit the enzyme activity, as has been shown in skeletal muscle. In addition it was observed that the mitochondrial CPK was just as susceptible to the inhibitory effect of FDNB as the cytoplasmic isoenzyme.     

Utilization of Barritt color reaction for studying synaptosomal creatine phosphokinase

The assay for creatine phosphokinase activity depending upon the color formation from creatine was reinvestigated. Its sensitivity was improved to 5 nmol of creatine. The method gave satisfactory Michaelis constants for both creatine phosphate and adenosine-dephosphate and was useful in detecting low creatine phosphokinase activity.     

Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase

ATPase and creatine phosphokinase (CPK) activities of isolated cardiac myofibrils were determined with ³²P γ-labeled ATP alone and with the addition of phosphorylcreatine (PC). With ATP and PC as substrates the label in the inorganic phosphate formed is greatly diluted indicating that the ATP formed by PC through CPK can reach the ATPase active site more readily than labeled ATP from the medium. The tight coupling of the ATPase and CPK activities further strengthens our view that PC serves an important role as high energy carrier between the energy producing sites (mitochondria) and the energy utilizing sites (myofibrils).     

determination of the N-terminal sequence and amino acid composition of MM- and BB-isoenzymes of the swine creatine phosphokinase

Amino-acid composition of porcine M and B creatine phosphokinase subunits and a sequence of 22 N-terminal amino acids have been determined. A comparative analysis with the structure of creatine phosphokinase from other sources made it possible to discover a sequence with 8 to 15 amino-acid residues, which is the most suitable for the preparation of polyclonal antibodies.