Immunological and Biochemical Evidence that Blepharismin is not a Prosthetic Group

Abstract

A polyclonal, multispecific antiserum was raised against a whole 3[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate‐extract of nonirradiated Blepharisma japonicum cells. It was used to reveal the composition of solutions that were hypothesized to contain the photoreceptor of the ciliate. A Bio‐Gel A 1.5 m fine column chromatography of the extract allowed recovery of a single elution peak isolated by recording the 580 nm light absorbance. Fused‐rocket immunoelectrophoresis of this material revealed a large number of >300 kDa coeluted proteins. Blepharismin‐rich material with a molecular mass of approximately 50 kDa, consisting of at least nine proteins was obtained when the same extract underwent preparative isoelectric focusing before column chromatography separation. Purification of the pigment obtained from light‐exposed cells gave blepharismin‐rich material with a molecular weight of approximately 200 kDa. Comparison of the materials obtained under the same conditions, either from the dark‐kept or light‐irradiated cells, by means of pore‐gradient electrophoresis confirmed that proteins present in the two preparations were different. It revealed only a very small amount, if any, of proteins in the chromatography fractions with the highest absorbance at 600 nm. Results are discussed on the basis of the hypothesis that a specific blepharismin‐binding protein does not exist in the protozoan.

Spectroscopic study of the chromophore–protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum

“…8 A specific carrier protein for the blepharismin chromophore might even not exist at all. 9 Recent findings, obtained comparing spectroscopic properties and singlet oxygen production rate of the free pigment and of the putative pigment-protein complex suggest that oxyblepharismin is buried into the protein in a site poorly accessible to oxygen. 10 The molecular structures of these chromophores 11-13 are reported in Fig.…”

Photomodulated Azoaldolase: A Model for Light Intervention in Biological Systems?

“…A second, well-described case, is that of the multidomain photoactive yellow protein, in which the chromophore is p-hydroxy-cinnamic acid covalently linked to the apoprotein as a thioester (2,12); this soluble protein has been proposed as the prototype of various multidomain sensors (13). However, the apoprotein approach cannot cover all the possibilities in photobiological processes; sometimes the investigation's progress is in itself the demonstration that such a protein does not exist (14). The chromophore interacts with more then one single protein; in the case of blepharismine, the primary photoprocess changes from an order-disorder transition to a redox reaction (15).…”