Purification and some properties of ribulose 1,5-bisphosphate carboxylases/oxygenases from Acinetobacter sp. strain JC1 and Hydrogenophaga pseudoflava.

Ribulose 1,5-bisphosphate carboxylases/oxygenases (RuBisCOs) of two carboxydobacteria, Acinetobacter sp. strain JC1 and Hydrogenophaga pseudoflava, grown on carbon monoxide were purified and partially characterized. RuBisCO of Acinetobacter sp. JC1 was purified 5-fold in eight steps to homogeneity, with a yield of 1.6%. The final specific activity of the purified enzyme was 39.5 nmol CO2 incorporated per min per mg protein. The molecular weight of the native enzyme was determined to be 520,000. Sodium dodecyl sulfate-gel electrophoresis revealed two nonidentical subunits of molecular weights 53,500 and 15,000. The Km and Vmax for CO2 were 36.7 microM and 296.1 nmol per min per mg protein, respectively, and those for ribulose 1,5-bisphosphate were 3.7 microM and 770 nmol per min per mg protein, respectively. The enzyme of H. pseudoflava was purified 55-fold in eight steps to homogeneity, with a yield of 3.6%. The final specific activity was 304.3 nmol CO2 incorporated per min per mg protein. The molecular weight of the enzyme was estimated to be 505,000. The enzyme was found to have two kinds of nonidentical subunits of molecular weights 51,500 and 14,000. The Km and Vmax for CO2 were found to be 16.4 microM and 777.8 nmol per min per mg protein, respectively, and those for ribulose 1,5-bisphosphate were 0.1 microM and 436.2 nmol per min per mg protein, respectively. The N-terminal amino acid sequences of the large and small subunits of Acinetobacter sp. JC1 enzyme were Ala-Asp-Arg-Trp-Asn-Ala-Gly-Val-IIe-Pro-Tyr-Ala-Glu-Met-Gly and Met-Arg-Ile-Thr-Glu-Gly-Thr-Phe-Ser-Tyr-Leu-Pro-Asp-Phe-Thr, respectively. The sequences of the H. pseudoflava enzyme were Ala-Thr-Lys-Thr-Tyr-Asu-Ala-Gly-Val-Lys-Glu-Tyr-Trp-Ser-Thr and Met-Ser-Met-Gln-Asp-Tyr-His-Ser-Arg-Leu-Ser-Asp-Pro-Ala-Ile, respectively. The peptide map of RuBisCO from Acinetobacter sp. JC1 grown on carbon monoxide was different from that of the bacterium grown on methanol. The two RuBisCOs, however, were found to be identical in N-terminal residue and antigenic property. The RuBisCO of Acinetobacter sp. JC1 was found to share no immunological properties with those of H. pseudoflava, Oligotropha carboxidovorans and Pseudomonas carboxydohydrogena.