In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays.

Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO fixation during C photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity.

Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide.

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography.

Immunoreactive adrenomedullin in human plasma.

A specific and sensitive radioimmunoassay for adrenomedullin has been developed. Half-maximal inhibition of binding of radioiodinated adrenomedullin was observed at 4 fmol/tube. The radioimmunoassay recognized the entire adrenomedullin molecule and has little crossreactivity with adrenomedullin fragment peptides.

Solution synthesis of Na+,K(+)-ATPase inhibitor-1 (SPAI-1).

SPAI-1, a 49-amino acid peptide including eight Cys residues with Na+,K(+)-ATPase inhibitory activity, was synthesized by the solution procedure. Protecting groups, including the formyl group on the Trp residue, were cleaved simultaneously by HF treatment in the presence of a sufficient amount of thiol compound. After removal of the Acm group on the Cys residue, the resulting octa SH peptide was subjected to an oxidative folding reaction in the presence or absence of redox reagents and/or denaturant.