The Use of Avidin as a Probe for the Distribution of Mitochondrial Carboxylases in Developing Chick Retina

Abstract

During development, the inner chick retina progresses from an aerobic to an anaerobic metabolic basis because of the lack of a vascular system. To investigate this process further, we have examined the expression and distribution of mitochondrial carboxylases. Because these enzymes use covalently bound biotin as a co-enzyme, we were able to develop a new detection protocol for mitochondria using avidin as a probe for the biotin. Chemiluminescent detection of bound avidin–peroxidase was used to examine a developmental series of extracts of retinas that had been separated by electrophoresis and blotted to nitrocellulose. Avidin–peroxidase, visualized with the sensitive peroxidase substrate True Blue, permitted detection in epoxy-embedded tissue sections. In the extracts, specific bands of approximate molecular weights 130 and 70 kD were found, corresponding to biotinylated subunits of several mitochondrial carboxylases. During development, the intensity of the bands decreases, although at different rates. In tissue sections, 8-day embryonic retinas display reaction product throughout the tissue, with higher local concentrations in the vitread and sclerad regions. During further development, the reaction product becomes segregated into bands at the borders of the plexiform layers. As the photoreceptors mature, stain becomes concentrated in the developing ellipsoids and the sclerad ends of Müller cells.