Site Directed Antisera to the D-2 Polypeptide Subunit of Photosystem II

R. Geiger Hoechst AG, Pharma-Synthese, Postfach 800320, D-6230 Frankfurt/M. 80, Bundesrepublik Deutschland R. J. Berzborn, B. Depka, W. Oettmeier, and A. Trebst Lehrstuhl Biochemie der Pflanzen, Ruhr-Universität Bochum, Postfach 102148, D-4630 Bochum 1, Bundesrepublik Deutschland Z. Naturforsch. 42c, 491-498 (1987); received November 18, 1986 Dedicated to Professor Helmut Simon on the occasion o f his 60th birthday Antibody, D-2 Polypeptide, Photosynthesis, QA Binding Protein, Thylakoid Membrane Three synthetic oligopeptides were used for preparation of antibodies against the D-2 polypep­ tide of thylakoid membranes. Their sequence was chosen from a model of the folding of the amino acid sequence of the D-2 polypeptide subunit through the membrane that predicted these se­ quences to be exposed at the membrane surface. For the Merrifield solid-phase method on a fully automated synthesizer the N“-amino group was protected by a fluorenyl-9-methylcarbonyl group. The oligopeptides were coupled to serum albumin by EDAC for immunizations in rabbits. Antisera with high titer were obtained for the two oligopeptides that contained the amino acid sequence of the D-2 protein from amino acid 230 to 235 and from 235 to 241. The antisera reacted with the D-2 polypeptide, separated on SDS gel and agglutinated the thylakoid membrane. It is known that certain photosystem II functions are impaired by short time trypsin treatment of the membrane. The antisera were used to show that under these conditions the D-2 polypeptide in the membrane is very sensitive. The trypsination yielded two cleavage products detected by the two antisera, a 20 kDa fragment blotted by antiserum against amino acids 230 to 235 and a 10 kDa fragment blotted by the antiserum against amino acids 235 to 241. As the polypeptide cleavage occurs between the two epitopes, the trypsin cut is therefore at arginine 234. This supports the prediction that the sequence containing this arginine is the most exposed part of the D-2 polypep­ tide on the membrane (matrix) surface. It is proposed that the high sensitivity of the D-2 polypep­ tide accounts for the known effect of membrane trypsination on Q A accessibility in photo­ system II.