Enzymatic method for quantitating creatine kinase M and B isoenzyme composition in cardiac tissue: consideration for utilization in studies of developing and adult rat heart.
Creatine kinase (CK) is a cardiac enzyme of interest due to its involvement in both mitochondrial and contractile protein aspects of heart function. Studies on the structure, function, and developmental expression of myofibrillar CK led to the identification of specific isoenzymes whose synthesis can be mediated by changes in expression at two primary gene loci. Experimental interventions which alter gene expression and protein synthesis in other cardiac enzymes have been attempted to pinpoint underlying genetic and molecular CK control mechanisms. CK isoenzymes can be separated electrophoretically and then quantitated by densitometry; however, these methods are time consuming and require specialized instruments. Enzyme specific activity can be assessed spectrophotometrically, but this method, alone, does not provide isoenzyme quantitation. The present work establishes a rapid, simple spectrophotometric enzyme assay procedure based on relative thermal lability to quantitate tissue CK isoenzyme composition. Applicability to cardiac tissue having (a) heterogeneous muscle/nonmuscle cells and (b) heterogeneous subcellular fractions within a given cell population was evaluated. Results show that cardiac tissue from newborn, weanling, and adult rats undergoes dramatic and progressive augmentation of overall CK enzyme activity that may be mediated, at least in part, by altered CK gene expression. However, based on adult rat heart analyses, it is also apparent that cell makeup and fractional composition of subcellular constituents require consideration. The described activation energy method is simple, rapid, and reliable for initial screening for potential changes in CK isoenzyme expression which can then be verified by more detailed, albeit more complex, methodology involving mRNA and/or cDNA analyses.
Dowell
R T
RT
Department of Physiology & Biophysics, University of Kentucky, Lexington, USA.
Fu
M C
MC
D000375:Aging / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D000833:Animals, Suckling; D002458:Cell Fractionation; D003402:Creatine Kinase / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism; D003720:Densitometry; D004587:Electrophoresis, Agar Gel; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006321:Heart / Q000254:growth & development; D007527:Isoenzymes; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology; D009929:Organ Size / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D014599:Uterus / Q000201:enzymology
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D003402:Creatine Kinase