Cloning of DNA encoding a catalytic subunit of SNF1-related protein kinase-1 (SnRK1-alpha 1), and immunological analysis of multiple forms of the kinase, in spinach leaf

Using a PCR approach, we have cloned DNA encoding a catalytic subunit isofo rm (SnRK1-alpha1) of SNF1-related protein kinase-1 from spinach leaf. The p redicted amino acid sequence falls into the SnRK1a sub-family, and is close ly related to SnRK1a sequences expressed in cucumber, Arabidopsis thaliana, tobacco and potato. We have generated two affinity-purified antipeptide an tibodies (anti-RASS and anti-AEF) based on the predicted amino acid sequenc e of spinach SnRK1-alpha1. They were used to analyse multiple forms of SNF1 -related kinase (HRK-A, -C, -D) that were previously identified by biochemi cal criteria in extracts of spinach leaf (Sugden et al., Plant Physiol. 120 (1999), 257-274). Anti-AEF appears to be specific for the SnRK1-alpha1 iso form, whereas anti-RASS is a `pan-alpha' antibody that precipitates all iso forms present in spinach leaf extracts. The activities of HRK-A and HRK-C c an be entirely accounted for by the SnRK1-alpha1 catalytic subunit. By cont rast, only a small proportion of HRK-D activity (ca. 20%) can be accounted for by SnRK1-alpha1, with the remainder presumably being due to other isofo rms (SnRK1-alpha2?) that are currently poorly defined. A 35 kDa polypeptide recognized by an antibody against the putative Arabidopsis beta2 subunit c o-precipitates with HRK-C, but not HRK-A or D.