Using a PCR approach, we have cloned DNA encoding a catalytic subunit isoform (SnRK1-α1) of SNF1-related protein kinase-1 from spinach leaf. The predicted amino acid sequence falls into the SnRK1a sub-family, and is closely related to SnRK1a sequences expressed in cucumber, Arabidopsis thaliana, tobacco and potato. We have generated two affinity-purified antipeptide antibodies (anti-RASS and anti-AEF) based on the predicted amino acid sequence of spinach SnRK1-α1. They were used to analyse multiple forms of SNF1-related kinase (HRK-A, -C, -D) that were previously identified by biochemical criteria in extracts of spinach leaf (Sugden et al., Plant Physiol. 120 (1999), 257–274). Anti-AEF appears to be specific for the SnRK1-α1 isoform, whereas anti-RASS is a `pan-α' antibody that precipitates all isoforms present in spinach leaf extracts. The activities of HRK-A and HRK-C can be entirely accounted for by the SnRK1-α1 catalytic subunit. By contrast, only a small proportion of HRK-D activity (ca. 20%) can be accounted for by SnRK1-α1, with the remainder presumably being due to other isoforms (SnRK1-α2?) that are currently poorly defined. A 35 kDa polypeptide recognized by an antibody against the putative Arabidopsis β2 subunit co-precipitates with HRK-C, but not HRK-A or D.
Plant Molecular Biology – Springer Journals
Published: Oct 3, 2004
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