Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities.

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 A resolution, comprises beta-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.

Cloning of a cDNA coding for an amino acid carrier from Ricinus communis (RcAAP1) by functional complementation in yeast: kinetic analysis, inhibitor sensitivity and substrate specificity.

A cDNA for the amino acid permease gene RcAAP1 has been isolated from Ricinus communis by yeast complementation and subjected to a detailed kinetic analysis. RcAAP1 cDNA is 1.5 kb with an open reading frame that codes for a protein with 486 amino acids and a calculated molecular mass of 53.1 kDa. RcAAP1-mediated histidine uptake was pH dependent with highest transport rates at acidic pH; it was sensitive to protonophores and uncouplers and the Km for histidine uptake was 96 microM. The substrate specificity was investigated by measuring the levels of inhibition of histidine uptake by a range of amino acids. The basic amino acids (histidine, lysine and arginine) showed strongest inhibition of uptake whereas acidic amino acids competed less effectively. Alanine was the most efficient competitor of the neutral amino acids. Glutamine, serine, asparagine, methionine and cysteine showed moderate inhibition whereas threonine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan showed only low levels of inhibition. Glycine, proline and citrulline caused slight stimulation. More detailed competition kinetics indicated that both lysine and arginine showed simple competitive inhibition of histidine uptake. When direct uptake measurements were carried out, both lysine and arginine were found to be effective substrates for RcAAP1.

Effects of Prolonged Washing on Primary and Secondary Transport Processes at the Plasma Membrane in Red Beet Storage Tissue.

Changing patterns of enzyme activity and solute transport in response to washing were investigated in red beet (Beta vulgaris L.) storage tissue. Washing had a pronounced effect on the plasma membrane (PM) H+-ATPase with an increase in both hydrolytic and proton-pumping activities. Immunoblotting indicated that this may be due, in part, to a higher amount of this enzyme in the PM of washed tissue. Activities of the tonoplast (V)H+-ATPase and pyrophosphatase fluctuated during a 4-d washing period, but overall showed no marked change in activity. In tissue discs sucrose (Suc), glucose (Glc), and fructose uptakes increased significantly in response to washing. Cycloheximide, cordycepin, and tunicamycin inhibited both Glc- and Suc-inducible uptake. Monensin also strongly inhibited inducible Glc uptake, but the effect on Suc was less marked. N-Ethylmaleimide inhibited both Suc and Glc uptake, with its effects being more pronounced in fresh tissue. Other protein-modifying reagents showed no significant difference in their level of inhibition between fresh and washed tissue. Transport studies, carried out using energized PM vesicles from fresh and washed tissue, indicated that there was no rise in Suc and Glc uptake rates in response to washing. Results with a range of inhibitors indicated that there was no marked change in transporter sensitivity in vesicles isolated from fresh and washed tissue. The results indicate that the well-described enhancement of solute transport in washed storage tissue may be due to an increased PM H+-ATPase activity rather than to changes in PM carrier activity or to changes in metabolism such as invertase activity.