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Wheat endosperm ADP-glucose pyrophosphorylase has been purified approximately 70-fold by two different procedures. The enzyme is composed of four subunits of identical molecular weight (51,000). Antibodies prepared against the denatured 51 kD subunit of wheat endosperm ADP-glucose pyrophosphorylase recognise a 48 kD polypeptide in soluble protein extracts of wheat leaves, suggesting that wheat leaf and wheat endosperm ADP-glucose pyrophosphorylases may be the products of different genes. \ud \ud Two primary translation products of wheat endosperm ADP-glucose pyrophosphorylase mRNA have been Identified by immunoprecipitation of the nascent polypeptides with anti-wheat endosperm ADP-glucose pyrophosphorylase serum. The ADP-glucose pyrophosphorylase primary translation products have apparent molecular weights of 61,000 and 53,000. The 61 kD polypeptide Is found associated with membrane-bound polysomal RNA from wheat endosperm, while the smaller 53 kD polypeptide has been found associated almost exclusively with free polysomes. \ud \ud A cDNA clone (WL:AGA.l) encoding wheat leaf ADP-glucose pyrophosphorylase has been Isolated from a Xgtll expression library, by immunological screening with anti-spinach leaf ADP-glucose pyrophosphorylase serum. The WL:AGA.l cDNA Is 948 bp long and contains approximately 55% of the complete wheat leaf ADP-glucose pyrophosphorylase mRNA sequence, estimated from Northern blot experiments. A wheat endosperm cDNA library, consisting of 1.6x10s bacteriophage particles, was subsequently constructed in the Xgtll expression vector. Six clones hybridising to the cDNA insert of clone WL:AGA.l were isolated from the wheat endosperm cDNA library. The longest of these wheat endosperm ADP-glucose pyrophosphorylase cDNAs, clone WE:AGA.7, is nearly full-length (1798 bp), indicated by Northern blot analysis of wheat endosperm mRNA, coupled in vitro transcription and translation of the cDNA, and nucleotide sequence analysis. \ud \ud Southern hybridisation analysis and restriction enzyme mapping indicated that the wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs and genes are members of two distinct sub-families, expressed in an organ-specific manner. Southern blot analysis of wheat genomic DNA has been performed using the WE:AGA.7 and WL:AGA.l cDNA inserts as probes. This analysis indicated that each gene sub-family consists of approximately 2-5 genes. In addition, restriction enzyme mapping of wheat endosperm ADP-glucose pyrophosphorylase cDNAs revealed the presence of two classes of that sequence, indicating the existence of at least two wheat endosperm ADP-glucose pyrophosphorylase genes. \ud \ud Subsequent nucleotide sequence comparison of wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs Indicates that there is approximately 55% identity between them. In contrast, the wheat endosperm cDNAs are very closely related. Members of each class of endosperm cDNA, represented by clones WE:AGA.3 and WE:AGA.7, are 96% Identical. \ud \ud The derived amino acid sequences of wheat leaf and endosperm ADP-glucose pyrophosphorylases were determined from the nucleotide sequences of the corresponding cDNAs. These sequences are approximately 24% identical to the amino acid sequences of E.coli ADP-glucose pyrophosphorylase and 40% identical to the rice endosperm ADP-glucose pyrophosphorylase. The structure-function relationships pf substrate, activator, and inhibitor binding on the ADP-glucose pyrophosphorylase enzyme are discussed.\ud |
