Biosynthesis of rat-liver pI-5.0 esterases in cell-free systems and in cultured hepatocytes.
Rat liver esterases focusing at pH 5.0 (referred to below as pI-5.0 esterases) are structurally related glycoproteins which differ slightly in their mobility in sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). They reside in the lumen of the endoplasmic reticulum. We have studied their biosynthesis in cell-free systems programmed by total liver RNA, using sheep and rabbit antibodies to isolate the translation products related to these enzymes. Our results show that they are assembled as a precursor polypeptide chain (62 kDa) larger than the mature proteins. The pI-5.0 esterase mRNA could be extracted from bound but not free polysomes. Reticulocyte lysates supplemented with dog pancreas microsomes produced four esterase-related components in segregated form (61, 60, 58 and 56 kDa). The largest three correspond in electrophoretic mobility to the mature enzymes. They are glycoproteins that bind to concanavalin A, and can be reduced to the size of the shortest component by endo-beta-N-acetylglucosaminidase H (endo-H). Immunoprecipitation after biosynthetic labeling of the proteins in cultured hepatocytes also gave three glycosylated components that had the same mobility in SDS-PAGE as the mature enzymes. When tunicamycin was present in the culture medium, a single immunoprecipitable form was observed. Its apparent Mr was similar to that of the unglycosylated pI 5.0 esterase form synthesized in vitro in the presence of dog pancreas microsomes. Thus the biosynthesis of these esterases has characteristics in common with that of numerous secretory proteins, except for the rather large difference in size (approximately equal to 6 kDa) resulting from the proteolytic processing of their in-vitro-synthesized precursor.
D000118:Acetylglucosaminidase / Q000494:pharmacology; D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000096:biosynthesis*; D005260:Female; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats
D004950:Esterases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase