ABSTRACT
Protein phosphorylation is a major mode of regulation of metabolism, gene expression and cell architecture. In chloroplasts, reversible phosphorylation of proteins is known to regulate a number of prominent processes, for instance photosynthesis, gene expression and starch metabolism. The complements of the involved chloroplast protein kinases (cpPKs) and phosphatases (cpPPs) are largely unknown, except 6 proteins (4 cpPKs and 2 cpPPs) which have been experimentally identified so far. We employed combinations of programs predicting N-terminal chloroplast transit peptides (cTPs) to identify 45 tentative cpPKs and 21 tentative cpPPs. However, test sets of 9 tentative cpPKs and 13 tentative cpPPs contain only 2 and 7 genuine cpPKs and cpPPs, respectively, based on experimental subcellular localization of their N-termini fused to the reporter protein RFP. Taken together, the set of enzymes known to be involved in the reversible phosphorylation of chloroplast proteins in A. thaliana comprises altogether now 6 cpPKs and 9 cpPPs, the function of which needs to be determined in future by functional genomics approaches. This includes the calcium-regulated PK CIPK13 which we found to be located in the chloroplast, indicating that calcium-dependent signal transduction pathways also operate in this organelle.
ABSTRACT
An enzyme-linked immunosorbent assay (ELISA) on microplates and a HPLC coupled-column switching method were compared for the determination of the nitroarene compound 1-nitropyrene in airborne particulate organic matter collected at a busy intersection over a period of 2 months. After purification of the sample extract with silica, in the multidimensional chromatographic method nitroarenes were separated on a RP18 precolumn from matrix constituents followed by on-line reduction to corresponding aminoarenes with a Pt catalyst on alumina and a further separation of 1-aminopyrene on a second RP18 column. Methanol-water (70:30, v/v) was the mobile phase used. With ELISA, a six-fold overestimation was obtained for untreated samples. After clean-up it was lowered to approximately 1.6-fold overestimation which was mainly caused by cross-reaction of 2-nitropyrene and 2-nitrofluoranthene.
