Abstract
Abstract Evidence is presented that indicates that greater than 95% of the protein synthesized by intact rabbit reticulocytes is globin and that only globin nascent chains are found on the membrane-bound ribosomes from reticulocytes. This result conflicts with reports in the literature that approximately 14% of the protein synthesized by reticulocytes is non-globin and that 86% of the protein synthesized by membrane-bound ribosomes is non-globin (Bulova, S. I., and Burka, E. R. (1970) J. Biol. Chem. 245, 4907). Intact reticulocytes were incubated in the presence of either [3H]tyrosine or [3H]tryptophan, the cells were lysed, and the free ribosomes were sedimented. The postribosomal supernatant was precipitated with acid-acetone, and the precipitated material was chromatographed on a Sephadex G-100 column. Three main regions of protein were found. Bulova and Burka had designated the first two regions eluting from the column as non-globin protein and the third region as globin subunits. Material from each region was combined with uniformly labeled 14C-amino acid, globin that had been partially purified by passage through a DEAE-cellulose column before acid-acetone precipitation, and the mixture was digested with trypsin. Chromatography of the tryptic peptides on Dowex 50-X8 indicated that greater than 95% of the protein in each region is globin. Nascent chains on free ribosomes and membrane-bound ribosomes were prepared from intact reticulocytes incubated in the presence of [3H]tyrosine. Tryptic digestion of the nascent chains on the ribosomes yielded only globin peptides. Plots of the specific activity of tyrosine versus amino acid position in the globin chain declined linearly from the NH2-terminal to COOH-terminal ends of the chain and showed that the peptides had not arisen from free globin bound to the ribosomes. Additional experiments showed that no more than 25% of the radioactivity in the membrane-bound ribosomes could have arisen from contamination by free ribosomes.
Highlights
The reticulocytex were incubated for 30 min at 35” for studies involving products synthesized by the intact cells and for 10 min at 35” for the preparation of nascent chains on the ribosomes
Since the results obtained with tyrosine and tryptophan n-elc essentially the same, we present only those for tyrosinc iii detail
Bulova and Burka [11, 20] contend that a large of the acid-acetone-precipitable protein synthesized reticulocytes is non-globin in nature
Summary
Intact Cell Incubation-Reticulocytes were incubated according to the procedure of Godchaux [13]. The reticulocytex were incubated for 30 min at 35” for studies involving products synthesized by the intact cells and for 10 min at 35” for the preparation of nascent chains on the ribosomes. Following the incubation the cells were chilled to 5”, sedimented, resuspended, and lysed with either the hemolysate used by Bulova and Rurka [11] or with 2.4 volumes of Ruffer A: 1 mM. The membranes and cell debris were sedimented by centrifugation at 17,000 X g for 20 min. The membrane fraction (pellet) was used for the preparation of membrane-bound ribosomes, whereas the lysate (supcrnatant) was used for the preparation of synthesized product and free ribosomes
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