The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the b-P of ATP to the phosphoglucan, whereas the g-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD.

Higher plants possess two types of glucan phosphorylase ( ). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230−8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567−576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81−84 % amino acid identity over most parts of the two sequences) with the exception of the N‐terminal transit peptide and the large insertion located between the N‐ and the C‐terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non‐denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti‐Pho1a antibodies, but only two of these reacted with the anti‐Pho1b antibodies. The isoform binding exclusively to the anti‐Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti‐Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti‐Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross‐linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a‐Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1‐type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

A wide range and occasionally high levels of persistent organic pollutants (POPs) are reported in Arctic regions, especially among top predators. Polar bears (Ursus maritimus), arctic foxes (Alopex lagopus) and some gull species (Larus spp.) often have high levels of these fat-soluble pollutants. Gulls deposit significant levels of these contaminants in their eggs. In northern regions, gull eggs are part of the traditional human diet. In the present study we have investigated the levels of POPs in gull eggs in order to determine the tolerable weekly intake (TWI) for humans. Concentrations of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were measured in 214 gull eggs collected in the spring of 2001-02. The eggs were collected from four gull species (herring gulls (Larus argentatus), great black-backed gulls (L. marinus), lesser black-backed gulls (L. fuscus) and glaucous gulls (L. hyperboreus)) at 12 different locations in Northern Norway, on the Faroe Islands and on Svalbard. The pollutant levels in gull eggs were found to be 65.5 +/- 26.9 pg toxic equivalent (TE) for dioxin and PCB g(-1) wet weight. Based on these findings and the TWI-value determined by the EU Scientific Committee on Food it is advised that children, young women and pregnant and nursing women should not eat gull eggs. Other people should limit their intake of eggs to an absolute minimum, considering the health risks associated with gull egg intake.