Purification of RNA associated and unassociated forms of 1,4-alpha-glucan branching enzyme from rat liver.
Two molecular forms (BE-I and -II) of 1,4-alpha-glucan branching enzyme were purified from rat liver by affinity chromatography on glycogen-adipoyldihydrazide-Sepharose 4B and glycogen-ethylenediamine-Sepharose 4B, respectively, after hydrophobic chromatography on hexylamine-Sepharose 4B, as single proteins with the same molecular weight of 82,000 on SDS-polyacrylamide gel electrophoresis. By comparing their UV spectra and by RNA detection on polyacrylamide gels after electrophoresis, BE-I and -II were identified to be RNA associated and unassociated forms, respectively. The molecular weights of BE-I and -II estimated by Sephadex G-200 gel filtration were 91,000 and 98,000, respectively, indicating that both forms consist of a monomer. As Be-II had about half the specific activity of BE-I, the RNA component was not essential for the activity of the rat liver branching enzyme, and it was also dissociable from the protein component (BE-II) on polyacrylamide gel electrophoresis at pH 7.3, whereby BE-II showed a heterogeneity of at least three microspecies as revealed by protein staining as well as by activity staining of the gels. Using the antibody against BE-I, BE-I and -II were indistinguishable on immunodiffusion, and the activity inhibition rate of BE-II by the antibody was almost the same as that of BE-I if their specific activities are considered, indicating that the RNA component may have no effect on the enzyme-antibody reaction. Immunochemically no isoenzyme was detected for this enzyme in rat tissues.
D015061:1,4-alpha-Glucan Branching Enzyme / Q000302:isolation & purification*; D000818:Animals; D000906:Antibodies / Q000032:analysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification*; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D012313:RNA / Q000302:isolation & purification*; D011817:Rabbits / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet
D000906:Antibodies; D012313:RNA; D005964:Glucosyltransferases; D015061:1,4-alpha-Glucan Branching Enzyme