Abstract
It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.
Highlights
From the Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 227 and the $Department of Biology, Yokohama City University, Seto, Yokohama 236, Japan
The extract contained another polypeptide of which the M, was 14,000, and these two polypeptides comprised a complex with approximate M, 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose- 1,5-bisphosphate carboxylase
For the analysis of the 55- and 14-kDa polypeptides, the purified complex was subjected to SDS-polyacrylamide gel electrophoresis, and protein bands were blotted to a polyvinylidene difluoride membrane
Summary
F,, a water-soluble part of H+-ATP synthase; CF,, F1 from chloroplasts; TF,, F1 from a thermophilic bacterium PS3; octyl glucoside, l-O-n-octyl-P-D-glucopyranoside; ribulose-P, carboxylase, ribulose-l,&bisphosphate carboxylase; SDS, sodium dodecyl sulfate; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography; dansyl, 5-dimethylaminonaphthalene-l-sulfonyl; PTH, phenylthiohydantoin. Frasch et al [5] washed spinach chloroplast thylakoid membranes with 1 M LiCl and found that the major constituent of the washed extract was a polypeptide that has the same mobility as that of the ,8 subunit of CF1 in polyacrylamide gel electrophoresis in the presence of SDS They proved by Western immunoblotting that this protein (55-kDa protein) cross-reacted with anti-CF1 p subunit antibody. From the results of gel permeation chromatography using a Bio-Gel P-150 column, they suggested that the 55-kDa protein existed as a NO-kDa complex, a trimer of the p subunit This complex had rather strong ATPase activity, more than 10% of that of CF1, in the presence of 40 mM octyl glucoside. Since the finding of the ATPase-active /33 complex appears to be an important contribution for studies of F1, we attempted to obtain the & complex according to the procedures of Frasch et al [5]
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