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Journal of Biochemistry Advance Access originally published online on January 2, 2008
Journal of Biochemistry 2008 143(3):435-440; doi:10.1093/jb/mvm240
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© 2008 The Japanese Biochemical Society.

Donor Substrate Specificity of 4-{alpha}-Glucanotransferase of Porcine Liver Glycogen Debranching Enzyme and Complementary Action to Glycogen Phosphorylase on Debranching

Yumiko Watanabe, Yasushi Makino and Kaoru Omichi*

Department of Chemistry, Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Sakai, Osaka 599-8531, Japan

*To whom correspondence should be addressed. Tel: +81-72-254-9191, E-mail: komichi{at}c.s.osakafu-u.ac.jp

Received October 22, 2007; Accepted December 5, 2007


  Abstract

Glycogen debranching enzyme (GDE) has both 4-{alpha}-glucanotransferase and amylo-{alpha}-1,6-glucosidase activities. Here, we examined 4-{alpha}-glucanotransferase action of porcine liver GDE on four 64-O-{alpha}-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 64-O-{alpha}-glucosyl-PA-maltooctaose (B4/81) was liberated from 64-O-{alpha}-maltopentaosyl-PA-maltooctaose (B4/85), 64-O-{alpha}-maltotetraosyl-PA-maltooctaose (B4/84) and 64-O-{alpha}-maltotriosyl-PA-maltooctaose (B4/83), whereas 64-O-{alpha}-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-{alpha}-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-{alpha}-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-{alpha}-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-{alpha}-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the {alpha}-1,6-linked glucosyl residue is retained.

Key Words: 4-{alpha}-glucanotransferase, branched maltooligosaccharide, fluorogenic substrate, glycogen debranching enzyme, phosphorylase, substrate specificity

Abbreviations: B4/81, 64-O-{alpha}-(-glucosyl-PA-maltooctaose; B4/82, 64-O-{alpha}-(-maltosyl-PA-maltooctaose; B4/83, 64-O-{alpha}-(-maltotriosyl-PA-maltooctaose; B4/84, 64-O-{alpha}-(-maltotetraosyl-PA-maltooctaose; B4/85, 64-O-{alpha}-(-maltopentaosyl-PA-maltooctaose; GDE, glycogen debranching enzyme; GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue; G8PA, PA-maltooctaose; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; PA, pyridylamino


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