Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius

Abstract

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 x g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and snall subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.

Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms.

“…Dissociation of the Aphanothece enzyme occurs at low ionic strength (339), another effective method for preparing large and small subunits for structure-function studies. In the chemolithoautotrophic bacteria, a number of reports have led to the general impression that RuBPC/O from all of these organisms is composed of the usual L8S8 structure (34,39,41,52,212,251,252,255,307). Past suggestions of L6S6 (181,218,343), L8 (254), or alternative structures (174, 217) have not been substantiated, and part of the inaccuracies of these studies may be due to problems similar to those delineated above for the cyanobacterial enzyme.…”

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

“…With the exception of Thiothrix, where no data is available on RuBisCO, all of the bacteria showing positive hybridization to the A. nidulans probe possess RuBisCO with both small subunits (SSU) and LSU [3,4,[9][10][11]29,[46][47][48][49][50][51]. Most have the common hexadecamer (8LSU-8SSU) but 2 (R. vannielii and P. oxalaticus) have a dodecamer structure (6LSU-6SSU).…”

Cloning and expression of the ?-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

“…The presence of both forms of the enzyme has been reported in Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopseudomonas blastica, and T. denitrificans [4,[14][15][16][17]. Bowman and Chollet [18] purified a form I RuBisCO from T. intermedius. Herein we report the isolation and expression of a form II gene from T. intermedius.…”