PURIFICATION AND CHARACTERIZATION OF NON-CHLOROPLASTIC ALPHA-1,4-GLUCAN PHOSPHORYLASES FROM LEAVES OF DIGITARIA-ERIANTHA STENT

We have previously identified four major a-glucan phosphorylase (GP) e nzymes in crude leaf extracts from pangolagrass (Digitaria eriantha St ent.). One co-isolates with the chloroplasts (cGP) while the other thr ee are non-chloroplastic (nGP). In this report we present further char acterization and leaf cell-type localization of the non-chloroplastic enzymes. In contrast to observations in maize, all of the pangolagrass GP enzymes were present in both isolated bundle sheath strands and me sophyll cells. Ion exchange column chromatography separated the leaf G Ps into three peaks: A, B and C. Peak A was the most active and contai ned three non-chloroplastic GP (nGP), active bands separable by native polyacrylamide gel electrophoresis (NPGE). Peak B GP enzymes migrated identically to peak A enzymes during NPGE. Peak C contained a single chloroplastic GP (cGP). The two major nGPs in the peak A fraction co-p urified and migrated as a single band during SDS-PAGE, but they could be separated by IEF-column chromatography. Kinetic properties of these peak A nGP enzymes were similar to those of other plant nGP enzymes, with the exception that the pangolagrass nGP is not inhibited by dinuc leotide sugars. The co-localization of the leaf GP enzymes in both bun dle sheath and mesophyll cells, and the separation of the non-chloropl astic GPs into two pools (peaks A and B) during ion exchange chromatog raphy are unique characteristics not previously described for plant le af GPs.