Cloning of DNA encoding a catalytic subunit of SNF1-related protein kinase-1 (SnRK1-alpha1), and immunological analysis of multiple forms of the kinase, in spinach leaf.
Using a PCR approach, we have cloned DNA encoding a catalytic subunit isoform (SnRK1-alpha1) 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-alpha1. 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-alpha1 isoform, whereas anti-RASS is a 'pan-alpha' 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-alpha1 catalytic subunit. By contrast, 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 isoforms (SnRK1-alpha2?) that are currently poorly defined. A 35 kDa polypeptide recognized by an antibody against the putative Arabidopsis beta2 subunit co-precipitates with HRK-C, but not HRK-A or D.
Crawford
R M
RM
Biochemistry Department, Dundee University, UK.
Halford
N G
NG
Hardie
D G
DG
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018744:DNA, Plant; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D018515:Plant Leaves / Q000201:enzymology; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D018724:Spinacia oleracea / Q000201:enzymology*
D018744:DNA, Plant; D007527:Isoenzymes; D010455:Peptides; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases