Restrictions on rotational and translational diffusion of pigment in the membranes of a rhabdomeric photoreceptor.

Abstract

Individual, isolated rhabdoms from dark-adapted crayfish (Orconectes and Procambarus) were studied with a laterally incident microbeam that could be placed in single stacks of microvilli. Concentration gradients of metarhodopsin along the lengths of microvilli were produced by local bleaches, accomplished by irradiation with small spots of orange light at pH 9 in the presence of glutaraldehyde or formaldehyde. No subsequent redistribution of pigment was observed in the dark, indicating an absence of translational diffusion. On the basis of comparison with other systems, glutaraldehyde, but not formaldehyde (0.75%), would be expected to prevent diffusion of protein in the membrane. Under the same conditions photodichroism is observed, indicating an absence of free Brownian rotation. Photodichroism is larger in glutaraldehyde than in formaldehyde, suggesting that the bifunctional reagent quiets some molecular motion present after treatment with formaldehyde. Quantitative comparison of photodichroism with mathematical models indicates that the pigment absorption vectors are aligned within .+-. 50.degree. of the microvillar axes and are tilted into the surface of the membrane at an average value of .apprx. 20.degree.. The photoconversion of rhodopsin to metarhodopsin is accompanied by an increase in molar extinction of .apprx. 20% at the .lambda.max and a reorientation of the absorption vector by several degrees. The transition moment either tilts further into the membrane or loses some of its axial orientation, or both. The change in orientation is 3.5 times larger in formaldehyde than glutaraldehyde.