Evidence for processing of maize catalase 2 and purification of its messenger RNA aided by translation of antibody-bound polysomes.
Two-dimensional gel analysis of the in vitro and in vivo labeled catalase 2 (CAT-2) isozyme protein of Zea mays L. and western gel analysis of native CAT-2 and in vitro labeled CAT-2 indicated that the protein is processed from a precursor to a lower molecular weight form in the scutellum. The CAT-2 from each source appeared on two-dimensional gels as one major species and two to three subspecies of the same molecular weight. We have also purified the mRNA encoding CAT-2 from scutella of line R6-67 using the procedure of polysome immunoadsorption. As a midcourse check on the progress of purification, we translated a small portion of the purified Cat2 mRNA-containing polysomes while they were still complexed with CAT-2 antibodies and bound to protein A-Sepharose. This revealed the presence of highly purified Cat2 polysomes. The final mRNA could not be translated in the wheat germ system but was highly active in the reticulocyte lysate system. The translation product had a molecular weight of 56 000, compared to that of 54 000 for purified CAT-2 protein. We have also enriched for Cat2 mRNA by size selection on methylmercury-agarose gels. The Cat2 resided with and slightly above the 18S ribosomal contaminant band of the total poly(A+) mRNA. It is therefore about 1805 bases long, which is 224 bases longer than the calculated coding length of 1581 bases.
Skadsen
R W
RW
Scandalios
J G
JG
D000818:Animals; D000906:Antibodies; D002374:Catalase / Q000235:genetics* / Q000302:isolation & purification; D002458:Cell Fractionation; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011132:Polyribosomes / Q000201:enzymology* / Q000648:ultrastructure; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D000906:Antibodies; D007527:Isoenzymes; D012333:RNA, Messenger; D002374:Catalase