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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034029-04
Application #
3284420
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1984-07-01
Project End
1988-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Wasco, W M; Kincaid, R L; Orr, G A (1989) Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella. J Biol Chem 264:5104-11
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Goldin, B F; Voulalas, P J; Orr, G A (1989) Characterization of the maturation-associated galactose oxidase-sensitive glycoproteins of rat caudal sperm plasma membrane and epididymal fluid. Arch Biochem Biophys 270:208-18
Lieberman, S J; Wasco, W; MacLeod, J et al. (1988) Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella. J Cell Biol 107:1809-16
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Fudem-Goldin, B; Voulalas, P; Orr, G A (1988) Synthesis and rapid purification of UDP-[6-3H]galactose. J Biochem Biophys Methods 17:199-202
Zeheb, R; Orr, G A (1986) Use of avidin-iminobiotin complexes for purifying plasma membrane proteins. Methods Enzymol 122:87-94
Orr, G A; Heney, G C; Zeheb, R (1986) Purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B. Methods Enzymol 122:83-7