Modification of essential carboxyl group in rabbit muscle phosphorylase by water-soluble carbodiimide.
Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N,N,N',N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and beta-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C]GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.
Takagi
S
S
Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Miyagi.
Kobayashi
M
M
Matsuda
K
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D003488:Cyanogen Bromide; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D010763:Phosphorylase b / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011817:Rabbits; D011198:Solanum tuberosum / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity
D000596:Amino Acids; D002234:Carbodiimides; D008024:Ligands; D010763:Phosphorylase b; D006005:Phosphorylases; D003488:Cyanogen Bromide