Purification and characterization of non-chloroplastic α-1,4-glucan phosphorylases from leaves of Digitaria eriantha Stent

Abstract

Summary We have previously identified four major α-glucan Phosphorylase (GP) enzymes in crude leaf extracts from pangolagrass ( Digitaria eriantha Stent.). One co-isolates with the chloroplasts (cGP) while the other three are non-chloroplastic (nGP). In this report we present further characterization 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 mesophyll cells. Ion exchange column chromatography separated the leaf GPs into three peaks: A, B and C. Peak A was the most active and contained 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-purified 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 dinucleotide sugars. The co-localization of the leaf GP enzymes in both bundle sheath and mesophyll cells, and the separation of the non-chloroplastic GPs into two pools (peaks A and B) during ion exchange chromatography are unique characteristics not previously described for plant leaf GPs.