Isoelectric focusing of pigment‐protein complexes solubilized from non‐irradiated and irradiated prolamellar bodies

Abstract

Preparative isoelectric focusing was employed to compare the association of protochlorophyllide and chlorophyllide with the enzyme NADPH‐protochlorophyllide oxidoreductase (PCR; EC 1.3.1.33). Photoactive protochlorophyllide‐PCR complexes were solubilized with 1‐O‐n‐octyl‐β‐d‐glucopyranoside from non‐irradiated prolamellar bodies of wheat (Triticum aestivum). Also, chlorophyllide‐PCR complexes were solubilized from prolamellar bodies irradiated under conditions either preventing or favouring a spectral shift of chlorophyllide to shorter wavelengths. Independently of the treatment prior to the solubilization, the pigments and the PCR focused together at pHs of 4 to 5. The results indicate that protochlorophyllide‐PCR complexes are conformationally similar to chlorophyllide‐PCR complexes. The results support the hypothesis that the spectral shift, referred to as the Shibata shift, reflects a breaking‐up of large chlorophyllide‐PCR aggregates to smaller chlorophyllide‐PCR units, rather than a dissociation of the chlorophyllide from the enzyme protein.

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH:protochlorophyllide oxidoreductase from Avena sativa L

“…An absorption maximum at 641 nm has been observed earlier by the treatment of a PLB preparation with 10 mM OG without glycerol [11]. The authors considered this to be due to the short-wavelength form of photoactive Pchlide, especially since photoconversion yielded only the short-wavelength Chlide form, and furthermore discussed that such short-wavelength forms which they considered as non-aggregated pigment^protein complexes are always obtained by solubilization with detergents.…”

Photoreduction of Zinc Protopheophorbide b with NADPH-Protochlorophyllide Oxidoreductase from Etiolated Wheat (Triticum aestivum L.)

“…To 0.5 ml of the prolamellar body preparation, 15 pl 30 mM NADPH was added and the suspension was irradiated with white light (approximately 3.5 pE m-' s-l) for 15 min with gentle shaking to phototransform the endogenous Pchlide. To break up the Chlide-pchlide-reductase aggregates (Wiktorsson et al 1992), and to optimize the quantitative evaluation of the enzyme assay (Griffiths 1980), 50 p1 10 mM Triton X-100 was added and the mixture was shaken for a further 10 min in the dark. To assay the phototransformation of exogenous substrate, 1-2 pl pigment dissolved in dimethylsulfoxide (Me,SO ; final concentration approximately 1 mM) was added.…”

Characterization of Serracin P, a Phage-Tail-Like Bacteriocin, and Its Activity against Erwinia amylovora , the Fire Blight Pathogen

“…The purity of serracin P was confirmed by isoelectrofocusing and ultracentrifugation on a sucrose gradient. For isoelectrofocusing analysis, 100 g of purified bacteriocin was electrofocused according to Wiktorsson et al (43) in a Rotofor apparatus (Bio-Rad Laboratories, Hercules, Calif.). For purity analysis by ultracentrifugation, 600 g of purified serracin P was subjected to overnight ultracentrifugation on a sucrose gradient (10% to 40%) at 170,000 ϫ g and 4°C.…”