phosphorus-radioisotopes and Carcinoma--Krebs-2

Krebs II ascite cells suspended in Eagle medium were incubated at 37 C for up to 6 hr in the presence of [3H] glycerol or [32P] orthophosphate. After extraction, their lipids were treated with guinea pig phospholipase A1 under conditions where all diacyl-phospholipids (diacyl-PL) became hydrolyzed with 55% recovery of lyso-PL. Using a bidimensional thin layer chromatography (TLC) involving exposure to HCl fumes between the two runs, it then became possible to determine at once the specific radioactivity of the three subclasses (diacyl-, alkylacyl- and alkenylacyl-) present in choline glycerophospholipids (CGP) and ethanolamineglycerophospholipids (EGP). Compared to diacyl-PL, a lower de novo synthesis of ether subclasses was evidenced in both CGP and EGP by [3H] glycerol incorporation. Although the same profile was obtained for CGP with [32P] orthophosphate, the three EGP subclasses displayed in this case the same specific radioactivity. These data indicate a higher turnover rate of the polar head group of ether-EGP compared to either-CGP. The simple methodology used in the present study might thus prove helpful in developing enzymatic studies dealing with the mechanism of this accelerated renewal.

Topics: Animals; Carcinoma, Krebs 2; Choline; Deoxycholic Acid; Ethanolamines; Glycerol; Kinetics; Mice; Phosphates; Phospholipids; Phosphorus Radioisotopes; Phosphorylcholine; Tritium

Topics: Animals; Binding Sites; Carbon Radioisotopes; Carcinoma, Krebs 2; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Hydroxyapatites; Kinetics; Mice; Molecular Weight; Organ Specificity; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Phosphorus Radioisotopes; Protein Binding; Ribosomes; Sodium Dodecyl Sulfate; Species Specificity; Spectrophotometry, Ultraviolet; Sucrose; Tritium

Polydisperse high-molecular-weight RNA of nucleated avian erythrocytes includes sequences coding for globin chains. The RNA was extracted from immature erythrocytes of ducks and fractionated under denaturing conditions by sucrose density gradient centrifugation in 99% dimethylsulfoxide. The RNA sedimenting faster than 45 S was able to direct the synthesis of duck globins in the Krebs II ascites cell-free protein-synthesizing system. The newly synthesized globin molecules have been identified by their characteristic electrophoretic properties in polyacrylamide gels containing either urea or sodium dodecyl sulfate, and by immunoprecipitation of the released globin chains by rabbit antibodies against duck hemoglobin. In order to rule out the possibility of a contamination of the high-molecular-weight RNA with duck-globin messenger RNA tailing from the 9-10S region, rabbit-globin messenger RNA was added to duck RNA as an internal control. No rabbit-globin messenger RNA activity could be detected in the RNA fractions sedimenting faster than 45 S. It is concluded that high-molecular-weight RNAs in the nucleated erythroid cell contain sequences of globin messenger RNAs covalently attached to larger polynucleotide chains. These results support the view that polydisperse nuclear RNA is the precursor of the cytoplasmic messenger RNA fraction.

Topics: Animals; Carbon Radioisotopes; Carcinoma, Krebs 2; Cell-Free System; Centrifugation, Density Gradient; Ducks; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Globins; Immunoassay; Leucine; Molecular Weight; Phosphorus Radioisotopes; Protein Biosynthesis; RNA; RNA, Messenger; Tritium

Topics: Adenine Nucleotides; Animals; Carcinoma, Krebs 2; Centrifugation, Density Gradient; Chromatography; Dactinomycin; Ethidium; Hydroxyapatites; Mitochondria; Mitochondria, Liver; Phosphorus Radioisotopes; Rats; Ribonucleases; RNA; Tritium; Ultrafiltration