Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.
The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.
D000740:Anemia / Q000139:chemically induced / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism*; D005914:Globins / Q000097:blood; D007501:Iron / Q000494:pharmacology; D007930:Leucine / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010455:Peptides / Q000097:blood; D010659:Phenylhydrazines; D011691:Puromycin / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D013997:Time Factors
D001798:Blood Proteins; D010455:Peptides; D010659:Phenylhydrazines; D011691:Puromycin; D005914:Globins; D003513:Cycloheximide; D007501:Iron; D007930:Leucine