THE EFFECT OF CROSS-LINKING OF THE SUBUNITS OF NADPH-PROTOCHLOROPHYLLIDE OXIDOREDUCTASE ON THE AGGREGATIONAL STATE OF PROTOCHLOROPHYLLIDE

Prolamellar bodies (PLBs) isolated from dark-grown, 6.5-d-old leaves o f wheat (Triticum aestivum L. cv. Kosack) were treated with the carbox ylic acid cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbo (EDC) or with the lysine specific cross-linker 2-iminothiolane. SDS-PAGE sho wed that the most prominenent crosslinked product was a dimer of the N ADPH-protochlorophyllide oxidoreductase (PCR), but also larger aggrega tes of the polypeptide were identified by immunological detection on e lectro-blots. A two-dimensional diagonal gel showed that much of the c ross-linking was between the PCR polypeptides. The cross-linkers induc ed a shift of the fluorescence peak to shorter wavelengths, a bandwidt h increase of the fluorescence peak, and an increase of the fluorescen ce yield. In the presence of NADPH the blue shift was reduced, but the increase in the fluorescence yield still occurred. A cross-linker tre atment of PLBs prior to solubilization with 1-O-n-octyl-beta-D-glucopy ranoside (octylglucoside) delayed, but did not prevent the spectral sh ifts from 657 to 646 nm and from 646 to 635 nm observed in non-cross-l inked detergent-treated PLBs. The cross-linking did not prevent a spec tral shift, corresponding to the Shibata shift, of Chlide. Thus the sp ectral shifts are not strictly coupled to disaggregation of the PCR po lypeptides.