Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii.
Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.
Toguri
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Muto
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Miyachi
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D001120:Arginine / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000096:biosynthesis*; D002696:Chlamydomonas / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D004790:Enzyme Induction; D007526:Isoelectric Point; D008861:Microsomes / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D010455:Peptides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism
D009844:Oligosaccharides; D010455:Peptides; D012333:RNA, Messenger; D011061:Poly A; D001120:Arginine; D002256:Carbonic Anhydrases