Arch Microbiol (1981) 130:213-217 Archives of Microbiolngy @ Springer-Verlag1981 Structural and Immunoelectrophoretic Comparison of Soluble and Particulate Ribulose Bisphosphate Carboxylases from the Cyanobacterium Chlorogloeopsisfi'itschii T. Lanaras and G. A. Codd Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Great Britain Abstract. The soluble and particulate (carboxysomal) forms of ribulose a,5-bisphosphate (RuBP) carboxylase from the cyanobacterium Chlorogloeopsis fritschii have been purified separately. A molecular weight of 520,000 was found in each case. Large (L, 53,000) and small (S, 13,000) subunits were obtained after dissociation, indicating a L8S8 quaternary structure for the enzyme from both sources. The L and S subunits are identical in molecular weight to the major polypeptides present in isolated dissociated C. fritschii poly- hedral bodies (carboxysomes). Occasionally an additional polypeptide (mol. wt. 45,000) was found after dissociation of the soluble enzyme only, although the possibility that this may be due to proteolysis is not discounted. Immunochemical identity between the purified soluble and carboxysomal RuBP carboxylases was indicated by tandem-crossed and rocket immunoelectrophoresis. Key words: Chlorogloeopsis fritschii - Ribulose bisphos- phate carboxylase - Polyhedral bodies - Carboxysomes Ribulose 1,5-bisphosphate (RuBP) carboxylase, the COz- fixing enzyme of the Calvin cycle, is present in soluble and particulate cell-free extracts of several prokaryotic auto- trophs. In Thiobacillus neapolitanus (Shively et al. 1973), Nitrobacter agilis (Shively et al. 1977), Nitrosomonas sp. (Harms et al. 1981.)and Chlorogloeopsisfritschff (Lanaras and Codd 1980, 1981), the particulate enzyme is principally present in the polyhedral bodies, which may thus be termed carboxysomes (Shively 1974). The association of RuBP carboxylase with polyhedral bodies in centrifuged density gradients loaded with broken Anabaena cylindrica cells (Codd and Stewart 1976), and the coincidence of RuBP carboxylase activity and polyhedral body numbers in this cyanobacterium (Stewart and Codd 1975; Stewart 1977) and in Thiobacillus intermedius (Purohit et al. 1976), indicate that the polyhedral bodies of these organisms are also carboxysomes. Evidence for the presence of RuBP carboxylase in car- boxysomes includes electron microscopical demonstration of ring-shaped structures, with the general dimensions of RuBP carboxylase molecules inside the bodies (Shively et al. 1973, Abbreviations. PAGE = polyacrylamide gel electrophoresis; SDS = so- dium dodecyl-sulphate; RuBP = D-ribulose 1,5-bisphosphate; TCA = trichloroacetic acid; LTIB = low Tris isolation buffer; HTIB = high Tris isolation buffer; CIE = crossed immunoelectrophore- sis; TCIE = tandem-crossed immunoelectrophoresis; RIE = rocket im- munoelectrophoresis. 1977; Lanaras and Codd 1981). Polypeptides with tool. wts. 55,900 and 13,000 occur in SDS-disociated carboxysomes from N. agilis (Bock strain, see Westphal 1977; Biedermann and Westphal 1979). Although reports on the quaternary structure of RuBP carboxylase from this strain are lacking, it has been reasonably assumed that the 55,900 and 13,000 Daltons polypeptides are the L and S subunits of RuBP carboxylase (Biedermann and Westpha11979). RuBP carbox- ylase from the Delwiche strain ofN. agilis consists of 2 major components of tool. wt. 54,000 (L) and 12,000 (S) and a minor component of mot. wt. 48,000 with unspecified assignation (Harrison et al. 1979). SDS-dissociated C. fritschii carboxy- somes contain at least 8 polypeptide classes, including 52,000 and 13,000 Dalton species (Lanaras and Codd 1981). RuBP carboxylase has not been characterized hitherto from C. fritschii. Previous studies on the molecular properties of RuBP carboxylase from polyhedral body-containing prokaryotes do not permit comparison between the soluble and particulate enzyme (for literature see McFadden ~nd Purohit 1978; Akazawa 1979), or between dissociated particulate enzyme and carboxysomal polypeptides. We report here on the separate purification of soluble and particulate C. fritschii RuBP carboxylase. The enzymes are compared in terms of their mol. wt., subunit composition and some immuno- electrophoretic characteristics. Materials and Methods Organism and Growth Conditions. Chlorogloeopsis fritschii 1411/16, from the Culture Centre of Algae and Protozoa, Cambridge, GB, was grown photoautotrophically in pure culture in medium C of Kratz and Myers (1965) as detailed previously (Codd and Stewart 1973). Preparation of Cell-Free Extracts. Cells from 16 1 of late log phase culture were harvested by spinning at 1900 x g for 10 rain and used immediately. All subsequent steps were performed at 0-4~ unless stated otherwise. 10-15 g wet wt. of cells were washed, resuspended in 10 vols. oflow ionic strength Tris isolation buffer (LTIB; 10 mM Tris, 10 mM MgCIz, 50 mM NaHCO3, 1 mM EDTA, 12 mM /?-mer- captoethanol, pH to 7.8 with HC1 at room temperature) and broken by single passage through a French press at 110 MPa. Remaining whole cells and large fragments were removed by spinning at 1,500 • g for 10 min. The supernatant was spun at 40,000 x g for 1 h to yield a supernatant and pellet which were used to purify the soluble and particulate RuBP carboxylases respectively. 0302-8933/81/0530/0213/$01.00