Purification and cDNA isolation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii.
Chloroplastic phosphoglycerate kinase (PGK) was purified to homogeneity from a soluble fraction of chloroplasts of a cell-wall-deficient mutant strain of Chlamydomonas reinhardtii (cw-15) using ammonium sulfate fractionation, Reactive Blue-72 column chromatography, and native polyacrylamide gel electrophoresis. PGK activity was attributed to a single polypeptide with a molecular mass of 42 kD. Relative purity and identity of the isolated enzyme was confirmed by N-terminal amino acid sequence determination. Antiserum against this enzyme was raised and a western blot analysis of whole-cell lysate from cw-15 cells using this anti-chloroplastic PGK serum detected a single polypeptide with a molecular mass of 42 kD. The cDNA clone corresponding to the Chlamydomonas chloroplastic PGK was isolated from a Chlamydomonas cDNA expression library using the anti-PGK serum. The cDNA sequence was determined and apparently codes for the entire precursor peptide, which consists of 461 codons. The results from Southern and northern blot analyses suggest that the chloroplastic PGK gene exists as a single copy in the nuclear genome of C. reinhardtii and is expressed as a 1.8-kb transcript. The C. reinhardtii chloroplastic PGK cDNA has 71 and 66% homology with wheat chloroplastic PGK and spinach chloroplastic PGK, respectively. Based on the deduced amino acid sequence, the chloroplastic PGK of C. reinhardtii has more similarity to plant PGKs than to other PGKs, having both prokaryotic and eukaryotic features.
Kitayama
M
M
Department of Biology, Indiana University, Bloomington 47405.
Togasaki
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002458:Cell Fractionation; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000302:isolation & purification*; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010735:Phosphoglycerate Kinase