In vitro translation of human prostatic acid phosphatase mRNA and processing of the translation products by microsomal membranes and endoglycosidase H.
Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000135:Acid Phosphatase / Q000235:genetics*; D000818:Animals; D004285:Dogs; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000378:metabolism; D011467:Prostate / Q000201:enzymology*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger; D000135:Acid Phosphatase; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase