In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product.
Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.
Chapdelaine
P
P
Dubé
J Y
JY
Frenette
G
G
Tremblay
R R
RR
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D002474:Cell-Free System; D004285:Dogs; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011467:Prostate / Q000201:enzymology*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases