d-Ribulose 1,5-Diphosphate Carboxylase from Rhodospirillum rubrum

Ribulose 1 ,5-diphosphate carboxylase has been purified IS-fold from derepressed butyrate-grown Rhodospirillum rubrum to a specific activity of 1.76 pmoles of ribulose 1,5diphosphate carboxylated per min per mg of protein at pH 8.0 and 30".The enzyme, which is recovered in 30 to 35% yield, is homogeneous by the criterion of electrophoresis in gels polymerized from several concentrations of acrylamide.Immunochemical procedures were also used to establish homogeneity.The carboxylase is strongly activated by EDTA and dithiothreitol after storage if Mg2+ is present.There is an absolute requirement of Mg2+ for catalysis as only 0.05 % activity is detected in its absence.Other divalent cations cannot be substituted for magnesium.Preincubation with Mg2+ plus bicarbonate and initiation of catalysis with ribulose 1,5diphosphate is required to achieve maximal rates.Homogeneous preparations could be stored without activity loss in the frozen state or at 2' in either phosphate or Tris buffers.Ribulose 1)5-&phosphate carboxylase (3-phospho-n-glycerate carboxy-lyase (tlimerizing), EC 4.1.1.39) is the key catalyst of the reductive pentose phosphate pathway in photosynthetic and chemosynthetic organisms.The enzyme has been isolated in homogeneous form from several higher plants (I-10) and green algae (11,12).To date the only bacterial ribulose 1 ,5-tliphosphate carbosylases that have been isolated in homogeneous form are from two species of photosynthetic purple sulfur bacteria (13, 14) and from the chemosynthctic hydrogen bacteria (15).With the exception of enzymes from the latter source, all of these car-