Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements.
Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability.
Rogers
S W
SW
Department of Biology, University of Utah, Salt Lake City 84132.
Rechsteiner
M
M
D001345:Autoradiography; D006207:Half-Life; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008845:Microinjections; D011506:Proteins / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014961:X-Ray Diffraction
D007457:Iodine Radioisotopes; D011506:Proteins