A general biochemical approach is being developed in this laboratory for probing the composition and organization of the plasma membrane of eukaryotic cells. Exploiting the pH dependent interaction of avidin with 2-iminobiotin, we have demonstrated that it is possible to label specific membrane proteins with 2-iminobiotin and then to retreive the """"""""tagged"""""""" proteins uncontaminated by other membrane components. We propose now to extend this technology and to synthesize an arsenal of 2-iminobiotin-containing, photo-activable reagents designed specifically to interact with and label the hydrophobic domains of integral membrane proteins. Two basic structures will be synthesized and used to transport the photoactivatable group into the lipid bilayer: (a) 1-palmitoyl-2-aminoalkanoyl-snglycerol-(N-2-iminobiotinylaminoethyl)phosphat e and (b) 2-iminibiotinyldiiodotyrosine. A variety of photoactivatable groups can be attached at either the acylchain amino terminus of the phospholipid analog or the carboxyl group of the diiodotyrosyl residue. The inclusion of 2-iminobiotin as part of the hydrophobic labeling reagent offers two important advantages over currently available reagents and will increase both the scope and potential of this approach. First, it will be possible to detect """"""""tagged"""""""" compoments by transferring electrophoretically resolved polypeptides onto a suitable matrix followed by specific visualization of 2-iminobiotinylated proteins by a variety of overlay procedures. This method of detection obviates the necessity for the synthesis of radioactive labeling reagents, a laborious and often hazardous procedure. Second, the presence of the 2-iminobiotin group will facilitate subsequent structural analysis since """"""""tagged"""""""" components can be readily separated from unlabeled material. Moreover, the protein blotting/overlay technique can be used in combination with chemical or enzymatic degraditive procedures and one and two dimensional gel electrophoresis to provide information on labeling domains within a protein. The human erythrocyte will be used as a model system for evaluation of these hydrophobic labeling reagents. The following questions will be asked: 1) Is labeling restricted to intramembranous domains of integral membrane proteins? 2) Is labeling restricted to the outer segment of the lipid bilayer? and 3) Does the reagent show selectivity in labeling of integral membrane proteins? The long term goals are to determine the topographical arrangement of a 32,000 Mr maturational-associated caudal sperm membrane glycoprotein within the lipid bilayer.
