Molecular characterization of phosphoribulokinase and rubisco activase from Chlamydomonas reinhardtii
Roesler, Keith Robert
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https://hdl.handle.net/2142/20627
Description
Title
Molecular characterization of phosphoribulokinase and rubisco activase from Chlamydomonas reinhardtii
Author(s)
Roesler, Keith Robert
Issue Date
1990
Doctoral Committee Chair(s)
Ogren, William L.
Department of Study
Crop Sciences
Discipline
Biology, Botany
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Agronomy
Biology, Botany
Biology, Plant Physiology
Language
eng
Abstract
Chlamydomonas reinhardtii phosphoribulokinase was purified to apparent homogeneity and polyclonal antibodies were prepared. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Immunoblots also revealed substantial amounts of phosphoribulokinase protein in Chalmydomonas mutant strain 12-2B, but none in mutant strain F-60. Chlamydomonas and spinach cDNA clones containing the entire phosphoribulokinase coding region were isolated and sequenced. The Chlamydomonas and spinach mature proteins possessed 75% amino acid sequence identity and similar kinetic properties. The transit peptides possessed almost no amino acid sequence identity. Genomic DNA blot analysis suggested the presence of a single Chlamydomonas phosphoribulokinase gene. The pH optimum of the wild-type Chlamydomonas enzyme was 8.0, while that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km (Ru5P) value nearly 1000-fold greater than the wild-type value of 56 $\mu$M. Similar Km (ATP) values of less than 100 $\mu$M were observed with both wild-type and mutant enzymes. The wild-type Vmax was 450 $\mu$mol min$\sp{-1}$ mg$\sp{-1}$, while values for the 12-2B enzyme were 140 $\mu$mol min$\sp{-1}$ mg$\sp{-1}$ at pH 6.5 and 36 $\mu$mol min$\sp{-1}$ mg$\sp{-1}$ at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition which created an arginine to cysteine change at position 64 of mature phosphoribulokinase. This arginine residue is conserved in the enzyme of vascular plants, algae, and photosynthetic bacteria and appears to function in binding the phosphoribulose substrate. Immunoblots and DNA sequence analysis indicated the presence of a single Chlamydomonas rubisco activase polypeptide which possessed 60 to 65% amino acid sequence identity with the higher plant polypeptides. Chlamydomonas rubisco was less effectively activated by spinach rubisco activase than was spinach rubisco. Genomic DNA blot analysis suggested the presence of a single copy Chlamydomonas rubisco activase gene.
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