Vol. 131, No. 3 Printed in' U.S.A.
JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 1020-1022 Copyright C 1977 American Society for Microbiology
Characterization of Antiserum Directed Against Form II Ribulose 1,5-Bisphosphate Carboxylase from Rhodopseudomonas sphaeroides JANET L. GIBSON AND F. ROBERT TABITA* Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 23 June 1977
Antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides showed no cross-reactivity towards the form I enzyme as evidenced by a lack of immunoprecipitation. In addition, this antiserum failed to inhibit form I enzymatic activity.
We have reported the isolation of two differ- chloride (pH 7.5). Antibody titration of enzyme ent molecular forms of ribulose 1,5-bisphos- activity was conducted by incubating control phate carboxylase from Rhodopseudomonas (preimmune) serum or antiserum with the ensphaeroides (2). The two enzymes, designated zyme for 30 min at 300C, pH 7.2, prior to assay. peak I (form I) and peak II (form II), were The ribulose 1,5-bisphosphate carboxylase asshown to differ significantly in structural, cata- say has been described (4). Figure 1 illustrates results obtained in an lytic, and immunological properties. Form I, like the large ribulose 1,5-biphosphate carbox- Ouchterlony double-diffusion experiment in ylases typical of eucaryotes and some procar- which anti-form II serum was reacted against yotes (3), has a native molecular weight of crude extract, homogeneous form I, and homo550,000 and is composed of two types of sub- geneous form II carboxylase. A single precipitin units: a large subunit of 52,000 daltons, and a band is observed in the reaction between antismall subunit of 11,000 daltons (2). Form II, serum and crude extract or between antiserum exhibiting a molecular weight of approximately and homologous antigen. No cross-reactivity is 360,000, is composed of large subunits only, and evident between antiserum and form I carboxis probably a hexamer of such subunits (2). The ylase (Fig. 1). Moreover, anti-form II serum did two enzymes differ in response both to pH and not inhibit the activity of form I carboxylase the effector 6-phosphogluconate, a potent inhib- (Fig. 2), whereas the form II enzyme was signifitor of the large carboxylases (1, 5). It was fur- icantly inactivated by these antibodies. These results strengthen previous findings ther found that antiserum directed against the form I enzyme cross-reacts with crude extracts that suggested the distinctness of the two ribuof R. sphaeroides and the purified form I en- lose 1,5-bisphosphate carboxylases isolated from zyme, but shows no reactivity with the form II R. sphaeroides (2). The lack of any demonstracarboxylase. We report here the reactivity of tion of cross-reactivity either by immunodiffuantiserum directed against the form II enzyme. sion or by enzyme inhibition indicates differElectrophoretically pure form II carboxylase ences between the two enzymes. However, it is was obtained from photoheterotrophically possible that the lack of cross-reactivity by angrown cells of R. sphaeroides 2.4.1. Ga as previ- tiserum directed against one protein toward the ously described (2). Antiserum was raised in a other may be a function of small subunits inNew Zealand white female rabbit injected sub- ducing a conformational change in the form I cutaneously with 100 jig of enzyme emulsified enzyme so as to bury antigenic sites shared in in Freund complete adjuvant. Two additional common with the form II protein. Alternainjections were administered at 14-day inter- tively, the lack of small subunits in the form II vals, and the rabbit was bled 8 days after the protein, and its apparent hexameric structure, third injection. The blood was allowed to clot for may be a sufficient structural constraint to alseveral hours at room temperature, after which ter the immunological properties of this proit was refrigerated overnight. The clot was re- tein. The answer to these questions awaits furmoved by centrifugation, and the supernatant ther detailed chemical analyses of the form I fraction was used for further studies. Immuno- and form II carboxylases. It will thus be essendiffusion was carried out in 1% agarose contain- tial to compare peptide maps of tryptic or chying 50 mM tris(hydroxymethyl)aminomethane- motryptic digests of isolated large subunits of 1020
NOTES
VOL. 131, 1977
1021
FIG. 1. Ouchterlony double-diffusion analysis of form I and form H R. sphaeroides ribulose 1,5-bisphosphate carboxykwe. Immunodiffusion was carried out for 24 h. The center well contained 10 yd of anti-form II serum. Other wells contained: a, crude extract (200 pg); b, form I enzyme (30 jg); c, form II enzyme (7.5 pg).
*
*
FIG. 2. Antibody titration ofform I and form II R.
I4
sphaeroides
ribulose
1,5-bisphosphate carboxylase ac-
tivity. Portions of anti-form II serum were incubated for 30 min with form I (-) and form II (0) carboxyl-
\
ase as described in the text and
assayed as described (4). FORM I
l 10i
20
30
ANTISERUM EpJ
40
50
(2)
and
subsequently
1022
J. BACTERIOL.
NOTES
both enzymes to confirm whether these proteins are indeed different gene products. In addition, further immunological analyses using antisera directed againbt the isolated subunits may prove fruitful. The two enzymes isolated from R. sphaeroides will provide excellent experimental tools for the study of structure-function relationships in ribulose 1,5-bisphosphate carboxylase. This investigation was supported by National Science Foundation grant PCM 7410297.
LITERATURE CITED 1. Chu, D. K., and J. A. Bmham. 1972. Inhibition of
ribulose-1,5-diphosphate carboxylase by 6-phosphogluconate. Plant Physiol. 50:224-227. 2. Gibson, J. L., and F. R. Tabita. 1977. Different molecular forms of ribulose 1,5-bisphosphate carboxylase fromRhodopseudomonas ephaeroides. J. Biol. Chem. 252:943-949.
McFadden, B. A., and F. R. Tabita. 1974. D-ribulose1,5-diphosphate carboxylase and the evolution of autotrophy. Biosystems 6:93-112. 4. McFadden, B. A., F. R. Tabita, and G. D. Kuehn. 1975. Ribulose diphosphate carboxylase from the hydrogen bacteria and Rhodspirillum rubrum. Methods Enzymol. 42:461-472. 5. Tabita, F. R., and B. A. McFadden. 1972. Regulation of ribulowe 1,5-diphosphate carboxylase by 6-phospho-Dgluconate. Biochem. Biophys. Res. Commun. 48:1153-1160. 3.