Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

Abstract

The photosystem II core complex (TSF-IIa) is composed of pobpeptides of molecular weight 54-, 47-, 42-, and 30 kiodaltons (kD) and cytochrome b-559. After treatment with trypin or a-chymotrypsin for 20 hours, the TSF-IIa particles stfll retained their photochemical activity and the Lighti d cytochrome b-559 siga, althugh all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex fom 250,000 to 100,000 daltons as determined by gel filtratdon, and also decreased the protein to chloophyll ratio by 40%. Chlorophyll a appeared to be assocated with the 47-and 42 kD pobpeptides. Proteolysis of the complex produced a single chlophyll a band with a slightly higher electrophoretic mobility. This band was not eqivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(Y,-4khlorophenyl)-1,1-dmethylurea (DCMU), but did not completely abolish it.It was conchlded that altbough the polypeptides of the photosystem II core complex were cleaved by the proteases, many of the peptide fragments do not diciate from the complex, particularly the chlorophyll a-containing portions, leaving the complex photochemically active. This supports the concept that chlorophyll has a structural function in chlorophyll-protein complexes.Proteolytic enzymes have been used extensively to study the localization of thylakoid membrane proteins, particularly the Chlprotein complexes (3,5,18,(21)(22)(23) tochemical properties altered. These authors suggest that the Chl present in the complexes stabilizes the structure of the complex. Unfortunately, their observations were not extended to include the effects of proteolysis on PSII. The present study describes the effects of trypsin and a-chymotrypsin on the photochemical activity and structure of the isolated PSII core complex. At low concentrations of either protease, the photochemical activity ofthe complex was not inhibited; however, the component polypeptides were extensively degraded. This suggests that the reaction center polypeptide of PSII was cleaved during proteolysis but the peptide fragments, possibly held in place by their associated Chl, did not dissociate from the complex.MATERIALS AND METHODS Preparation of TSF-IIa Particles. The initial purification procedures followed those of Vernon et al. (26) with some modifications. Spinach thylakoids prepared in 50 mm Tris-Cl (pH 7.6) containing 350 mm sucrose, were twice treated with 2.0 M NaBr as described by Nelson (12) to remove coupling factor, and then washed with the above buffer. The lamellae were resuspended in 50 mm Tris-Cl (pH 8.0) and 0.5 M sucrose at a concentration of 2.5 mg Chl/ml. Triton X-100 was added to give a final ratio of 40 mg Chl:l g Triton X-100. The solution was stirred on ice for 1 h after which it was subjected to a low speed centrifugation (5,000g, 5 min.) to remove debris. The supernatant was centrifuged at 144,000g for 1 h (2 C) and the resultant supernatan...

Effect of Trypsin Treatment of Photosystem I Particles on the Electron Donation to P700

“…However, extensive proteolysis did lead to breakdown of the pigment protein complexes (6,14). In some cases, the fragmented polypeptides remained attached to the complex until the Chl molecules were removed (14,16) indicating a structural role for the Chl molecules.…”