Discrimination of porcine glycogen debranching enzyme isozymes by the ratios of their 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities.

Glycogen debranching enzyme (GDE) is a single-chain protein containing distinct active sites that exhibit 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. The ratios of these two activities in porcine liver and muscle GDEs were compared using a set of homologous fluorogenic branched dextrins. For quantifying 4-alpha-glucanotransferase activity, 6(3)-O-alpha-maltotetraosyl-PA-maltooctaose (B3/84), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84), 6(5)-O-alpha-maltotetraosyl-PA-maltooctaose (B5/84) and 6(6)-O-alpha-maltotetraosyl-PA-maltooctaose (B6/84) were used as substrates and maltohexaose (G6) as the acceptor. The substrate for amylo-alpha-1,6-glucosidase activity was 6(3)-O-alpha-glucosyl-PA-maltotetraose (B3/41). HPLC analysis of the fluorogenic branched dextrin digests in the presence of G6 revealed that GDE 4-alpha-glucanotransferases produce the corresponding 6-O-alpha-glucosyl-PA-maltooctaose (GG8PA) and maltononaose (G9). The ratios of the 4-alpha-glucanotransferase activity to amylo-alpha-1,6-glucosidase activity, for the liver and muscle enzymes were respectively 0.240 and 0.0840 for B3/84, 0.204 and 0.0788 for B4/84, 0.145 and 0.0592 for B5/84, and 0.109 and 0.0458 for B6/84. These data clearly indicate that porcine liver and muscle GDEs are different from each other. The ratios of porcine brain GDE were 0.155, 0.131, 0.0990 and 0.0745 for B3/84, B4/84, B5/84 and B6/84, respectively. These results indicate that porcine brain GDE is also unique from liver and muscle enzymes, suggesting that it is either a third enzyme, or a mixture of 45% liver and 55% muscle GDEs.