MULTIPLE FORMS OF WHEAT LEAF N-ACETYL-BETA-D-HEXOSAMINIDASE

We have identified two distinct forms of wheat leaf N-acetyl-beta-D-he xosaminidase, N-Hex-A and N-Hex-B, which preferentially hydrolyse pNP- GlcNAc and (GlcNAc)(n), respectively. Both forms were detected in all wheat cultivars examined. In leaves of increasing maturity, the specif ic activity of N-Hex-A gradually declined, whilst the specific activit y of N-Hex-B remained relatively constant. Affinity chromatography on Con A-Sepharose resulted in a 772-fold purification of N-Hex-A and a 4 41-fold purification of N-Hex-B. N-Hex-A and N-Hex-B had similar nativ e molecular weights (167 +/- 23 kDa) as estimated by gel filtration. A near-baseline separation of N-Hex-A and N-Hex-B was achieved by anion exchange chromatography. Polyclonal antibody, raised against homologo us N-Hex-A, also showed a strong cross-reaction with N-Hex-B. Chitinas e-free, semi-purified extracts, containing both N-Hex-A and N-Hex-B ac tivities, preferentially hydrolysed (GlcNAc)(3-5) in comparison to (Gl cNAc)(2). Two compounds, PUGNAC and PUCIB had no inhibitory effect on N-Hex-A activity but specifically reduced N-Hex-B activity (PUGNAC, K- i = 1.45 x 10(-7)M; PUCIB, K-i = 6.5 x 10(-6) M).