A Chlamydomonas reinhardii mutant with catalytically and structurally altered ribulose-5-phosphate kinase |
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Authors: | Michael E Salvucci William L Ogren |
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Institution: | (1) Agricultural Research Service, U.S. Department of Agriculture, 1102 S. Goodwin Ave., 61801 Urbana, IL, USA |
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Abstract: | The biochemical lesion in a light-sensitive, acetate-requiring Chlamydomonas mutant was identified. This strain, designated rpk, exhibited photosynthetic rates less than 3% of the wild-type. Analysis of photosynthetic products by high-performance liquid chromatography demonstrated an accumulation of 14C label in pentose and hexose monophosphates. After 1 min of photosynthesis in 14CO2 these intermediates comprised 27.5% of the label in the mutant compared with 8% in the wild-type. The mutant pheno-type was caused by a 20-fold reduction in ribulose-5-phosphate (Ru5P)-kinase (EC 2.7.1.19) activity. The mutant exhibited wild-type levels of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1) indicating that the mutation specifically affected Ru5P kinase. In a cross of the mutant with the wild-type, tetrad progeny segregated in a Mendelian fashion (1:1) and light-sensitivity cosegregated with reduced Ru5P-kinase activity and an acetate requirement for growth. Almost normal levels of Ru5P-kinase protein were detected in the mutant by probing nitrocellulose replicas of sodium dodecylsulfate-polyacrylamide gels with anti-Ru5P-kinase antibody. The subunit size of the mutant enzyme, 42 kDa, was identical to that of the wild-type. Isoelectric focusing of the native protein determined that the mutant protein was altered, exhibiting a more acidic isoelectric point than the wild-type protein. Thus, the molecular basis for the lesion affecting Ru5P-kinase activity in mutant rpk is a charge alteration which results in a partially impaired enzyme.Abbreviations Chl
chlorophyll
- Da
dalton
- FCCP
carbonylcyanide-p-trifluorophenylhydrazone
- RuBP
ribulose-1,5-bisphosphate
- Ru5P
ribulose-5-phosphate |
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Keywords: | Calvin cycle Chlamydomonas mutant Chlorophyta Mutant (Chlamydomonas) Photosynthetic carbon metabolism Ribulose-5-phosphate kinase |
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