| Popis: |
A new polyclonal antiserum against spinach CF1 subunit delta was produced in rabbits. It decorates only one band at 21 kDa in Western immunoblots of thylakoid proteins and does not react in ELISA with delta-free four subunit CF1(-delta); therefore it is regarded monospecific. The polypeptide used as immunogen had been purified by HPLC. Earlier antisera against CF1 delta cross-react with CF1 subunit beta. The new antiserum 306 contains different antibodies; some can be absorbed with thylakoids, i.e. by delta within the assembled CF0CF1 complex on the membrane, others still react in ELISA with isolated CF1. The former antibodies agglutinate thylakoids and inhibit PMS cyclic photophosphorylation. Therefore we conclude that part of the surface of CF1 subunit delta is exposed within the quaternary structure of the ATP-synthase complex of photosynthetically active thylakoids, but part of the surface of delta is shielded. Trypsination of isolated delta occurs at several sites, but this protease does not attack delta in situ, nor does aminopeptidase. Staphylococcus aureus protease V8 digests CF1 delta after isolation at residues Asp53, Glu61, Glu95 and Glu106, but has no access to these residues of delta in situ. Thus CF1 subunit delta seems to be shielded within the CF0CF1 complex to a large degree. Direct agglutination of thylakoids by anti delta serum 306 was weak and could be improved tenfold by a Coombs serum (goat anti rabbit gammaglobulin), whereas an anti beta serum agglutinated directly. From this we conclude that delta is not accessible at the top of the enzyme; the exposed part is extending below the large subunits alpha and beta and oriented towards the membrane. |
