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Metabolism and Enzymology

Isozymes of -N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019

Four isozymes of -N-acetylhexosaminidase (-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated -NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl--D-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. -NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl--D-glucosaminide and p-nitrophenyl-N-acetyl--D-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to -NAHAs from other plant sources.

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				H. B. Lin, S. M. Harley, J. M. Butler, and L. Beevers Multiplicity of clathrin light-chain-like polypeptides from developing pea (Pisum sativum L.) cotyledons J. Cell Sci., December 1, 1992; 103(4): 1127 - 1137. [Abstract] [PDF]