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Calcium-dependent binding to phospholipid membranes is closely associated with annexin functional properties. In these studies, 31P- and 1H-nuclear magnetic resonance (NMR) experiments have been performed to study the effects of binding of recombinant rat annexin V to sonicated small unilamellar vesicles (SUVs). High-resolution 31P-NMR spectra of SUVs containing mixtures of synthetic phosphatidic acid (PA) and phosphatidylcholine (PC) show resolvable resonances corresponding to the inner-leaflet PA, outer-leaflet PA, and PC phosphoryl groups. When annexin binding occurs, the outer-leaflet PA 31P resonance shifts while that of PC is unaffected, consistent with selective binding of the protein to the phosphoryl moiety of the PA component. Further, annexin V binding to membrane outer-leaflet phospholipids has a measurable effect on inner-leaflet phospholipids of intact vesicles. 1H-NMR T1 relaxation measurements of SUVs containing acyl-chain-perdeuterated PC show no effects on the PA hydrocarbon-chain segmental motions upon annexin binding. Circular dichroism measurements indicate that the protein does not undergo a significant conformational change upon binding to the vesicles. The observed NMR changes do not correspond to proton or calcium gradients, nor to lateral segregation of extended patches of homogeneous phospholipids. The combined evidence suggests that selective, peripheral annexin-membrane interactions influence the environment of the inner vesicular surface. The mechanism proposed is a protein-induced change in vesicle morphology that corresponds to reduced curvature.

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.