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Phycocyanobilin synthesis in the unicellular rhodophyte Cyanidium caldarium.

ABSTRACT: Light is required for synthesis of the accessory photosynethetic pigment phycocyanin in cells of the unicellular rhodophyte Cyanidium caldarium. Phycocyanin is a conjugated protein composed of polypeptide subunits to which the light-absorbing bile pigment chromophore phycocyanobilin is covalently attached. Dark-grown cells of C. caldarium are unable to make phycocyanin, but when incubated in the dark with 5-aminolaevulinate the cells synthesize and excrete a protein-free phycobilin (algal bile pigment) into the suspending medium. The electronic absorption spectrum, electron impact mass spectrum, chromatographic properties and imide products obtained after chronic acid degradation of the excreted phycobilin were identical with those of phycocyanobilin cleaved from phycocyanin in boiling methanol. This establishes the structural identity between the excreted phycobilin, which is the end product of bile-pigment synthesis in vivo, and the chromophore cleaved from phycocyanin in boiling methanol. The significance of the structure of the excreted phycobilin with respect to the events surrounding the assembly of the phycocyanin molecule in vivo is discussed.

SUBMITTER: Troxler RF

PROVIDER: S-EPMC1185732 | biostudies-other | 1978 Jun

REPOSITORIES: biostudies-other

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Project description:Allophycocyanin from the unicellular rhodophyte Cyanidium caldarium was purified by (NH4)2SO4 fractionation and ion-exchange chromatography on brushite (calcium phosphate) columns and on DEAE-Sephadex A-25 columns. The specific absorption coefficient (A0.1%1cm) at 650nm of purified allophycocyanin was 6.35 in 0.05M-potassium phosphate buffer, pH7.0. Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350 and 275 nm. Circular-dichroic spectra displayed positive-ellipticity bands at 658 and 630 nm and a major negative-ellipticity band at 340nm. Computer analysis of the circular-dichroic spectrum of allophycocyanin from 207 to 243 nm indicated 42% alpha-helix and 58% beta-form. The estimated molecular weight of purified allophycocyanin on calibrated Sephadex G-200 columns at pH7.0. was 196000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulphate/polyacrylamide gels revealed a single band with apparent mol.wt. 16000. The presence of two polypeptide subunits, with nearly the same molecular weight, was revealed on polyacrylamide gels by using a modified electrophoresis buffer. Spectral analysis of the allophycocyanin subunits resolved by ion-exchange chromatography on Bio-Rex 70 columns indicated that a single phycocyanobilin chromophore was present on each polypeptide chain. Treatment of allophycocyanin with 8M-urea (pH3.0) and subsequent removal of urea by dialysis against water yielded a derivative phycobiliprotein with spectroscopic characteristics similar to those of phycocyanin. The original allophycocyanin spectrum was regenerated after incubation in phosphate buffer, pH7.0. Automated sequences analysis of the N-terminus of allophycocyanin showed that (a) the sequences of the two subunits were different from one another and were different from the subunits of phycocyanin from the same alga, (b) the subunits occurred in a molar ratio of 1:1 and (c) the sequences homology at the N-terminus among alpha- and beta-subunits of allophycocyanin from blue-green and red algae approached 90%.

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Phycocyanobilin synthesis in the unicellular rhodophyte Cyanidium caldarium.

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