Detection and relative quantitation of mRNA for creatine kinase isoenzymes in mRNA from myogenic cell cultures and embryonic chicken tissues.
The presence of mRNA coding for creatine kinase M (Mck) and creatine kinase B (B-CK) in RNA from myogenic and fibrogenic cell cultures, embryonic muscle, and embryonic brain tissue was demonstrated by "in vitro" translation in a heterologous cell-free protein-synthesizing system from rabbit reticulocytes. The products were isolated by sensitive immunochemical methods and their identity with isolated M-CK and B-CK was shown by the following criteria: (a) the in vitro synthesized creatine kinases react with the specific antibody against these antigens; (b) the labeled peptides co-migrate with purified creatine kinase on sodium dodecyl sulfate gels in single bands; (c) the labeled peptides form homo- and heterodimers with isolated enzymatically active creatine kinase, thus behaving like authentic creatine kinases. The assay was shown to be reproducible and gave a linear response with increasing amounts of RNA, allowing relative quantitation of mRNA in polysomal RNA for the creatine kinases M and B. MRNA for M-CK was detected in polusomal RAN and total cellular RNA from myogenic cells. It is also present in polysomal RNA from enbryonic muscle and the fraction binding to oligo(dT)-cellulose. mRNA for B-CK could be found in RNA extracted from young myogenic cultures and the fraction of polysomal embryonic brain RNA binding to oligo(dT)-cellulose.
D000818:Animals; D002474:Cell-Free System; D002642:Chick Embryo; D003402:Creatine Kinase / Q000096:biosynthesis / Q000235:genetics*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D009132:Muscles / Q000196:embryology* / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D007527:Isoenzymes; D012333:RNA, Messenger; D003402:Creatine Kinase