Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.).
Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.
Spano
A J
AJ
Department of Biology, University of Virginia, Charlottesville 22901.
Timko
M P
MP
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D005296:Ferrous Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D012689:Sequence Homology, Nucleic Acid
D002413:Cations, Divalent; D005296:Ferrous Compounds; D011163:Hydroxymethylbilane Synthase