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Articles

Isolation of Photosystem I Complexes from Octyl Glucoside/Sodium Dodecyl Sulfate Solubilized Spinach Thylakoids ¹

Characterization and Reconstitution into Liposomes

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309

We have used the nonionic detergent octyl--D-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P₇₀₀, and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI^*, is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.

In addition, the PSI reaction center complex CPI isolated from preparative gels and PSI-B were reconstituted into lecithin liposomes for structural analysis using freeze-fracture electron microscopy. The results suggest that the native PSI complex produces 12- to 13-nanometer particles, while the PSI reaction center, depleted of LHCI and peripheral proteins, produces particles with an average diameter of 10 nanometers.

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