Cell-free translation of messenger RNAs from adult and fetal human muscle. Characterization of neosynthesized glycogen phosphorylase, phosphofructokinase and glucose phosphate isomerase.
Using a procedure of ethanol precipitation in concentrated guanidine . HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high-molecular-weight cellular RNA from human fetal and adult skeletal muscle. About 500 micrograms RNA were obtained/g of fetal muscle and 50 micrograms RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200,000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micro-method of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.
D000818:Animals; D002474:Cell-Free System; D005260:Female; D005333:Fetus; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics*; D006801:Humans; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010452:Peptide Biosynthesis; D010732:Phosphofructokinase-1 / Q000235:genetics*; D006005:Phosphorylases / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D011247:Pregnancy; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D006005:Phosphorylases; D010732:Phosphofructokinase-1; D005956:Glucose-6-Phosphate Isomerase