Preparation and Characterization of Monoclonal Antibodies Specific for Lauroylated Isoform of Bovine Transducin α‐Subunit: Immunohistochemical Analysis of Bovine Retinas

Abstract

The photoreceptor G protein transducin [a-and /3y-subunits (TaIT/3y)] plays a central role in the visual transduction process. The amino-terminus of bovine Ta is modified by one of four distinct fatty acidslaurate (C12:0), myristate (014:0), C14:1 (5-cis), and 014:2 (5-cis, 8-cis)-but the biological significance and the localization of the four isoforms of Ta are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0-, 014:0-, C14:1-, or 014:2-nonapeptide corresponding to the N-terminal region of Ta. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the 012:0-peptide as well as purified Ta but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both 012:0 and the Nterminal amino acids of Ta. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of 012:0-Ta in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of Ta was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of Ta in a single rod cell may contribute simultaneously to a fine tuning of the photon-signal transduction process. Abbreviations used: ELISA, enzyme-linked immunosorbent assay; 0 protein, guanine nucleotide-binding regulatory protein; KLH, keyhole limpet hemocyanin; OVA, ovalbumin; PBS, phosphate-buffered saline; STI, soybean trypsin inhibitor; TBS, Trisbuffered saline; Ta and Toy, transducin a-and /3y-subunit, respectively.