PLANT PHYSIOLOGY , Vol 107, Issue 2 393-393, Copyright © 1995 by American Society of Plant Biologists
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GENE REGULATION AND MOLECULAR GENETICS |
Purification and cDNA Isolation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii
M. Kitayama and R. K. Togasaki
Department of Biology, Indiana University, Bloomington, Indiana 47405
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.
