title: Characterisation and purification of ribulose-bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei.
abstract: The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.
authors.affiliation: Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Bombay, India.
authors.forename: R
authors.initials: R
authors.lastname: Rajagopalan
authors.forename: W
authors.initials: W
authors.lastname: Altekar
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006216:Halobacteriaceae / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D015636:Magnesium Chloride; D011189:Potassium Chloride; D012273:Ribulose-Bisphosphate Carboxylase