PHYSICAL AND BIOCHEMICAL PROPERTIES OF THE CYTOPLASMIC DOMAIN OF THE ERYTHROCYTE MEMBRANE PROTEIN, BAND 3 AND DIFFERENTIAL SCANNING CALORIMETRY OF MILK FAT GLOBULE MEMBRANES

The cytoplasmic domain of the erythrocyte membrane protein, band 3, is a major center of membrane function, linking the cytoskeleton and several cytoplasmic proteins to the plasma membrane. A 40,000-dalton cytoplasmic fragment, generated by the chymotryptic digestion of band 3, was isolated and shown to retain all of the structural as well as functional properties of the native protein. Determination of the Stokes radius, sedimentation coefficient and frictional ratio demonstrated that this polypeptide was released as a highly elongated, noncovalent dimer. Furthermore, the fragment\u27s structure was found to be extraordinarily sensitive to pH. Thus, differential scanning calorimetry, intrinsic and extrinsic fluorescence and gel filtration chromatography were used to show that the cytoplasmic fragment undergoes pH-dependent structural changes over the physiological pH range. These data indicate that the ionization of critical groups in the fragment exert significant control over the structure/stability of the cytoplasmic region of band 3. The structural interdependence of the membrane-spanning and cytoplasmic domains of band 3 was also investigated. On the basis of both calorimetric and fluorescence observations, we found that the interaction between the two structural domains of band 3 is either minimal or nonexistant. In addition, the overall similarities in the pH sensitivity of the cytoplasmic domain before and after cleavage from the membrane indicates that the pH-dependent conformational equilibrium observed for the isolated fragment also occurs on the membrane. In a separate study, we examined the calorimetric properties of the milk fat globule membrane. At least six major endotherms were observed between 10(DEGREES) and 90(DEGREES)C, corresponding to order-disorder transitions of discrete structural domains of the membrane. We present evidence that one of these, the C transition, involves lipid, while another, the D transition arises from the unfolding of the major protein, butyrophilin (band 12), of a polypeptide coat which lines the inner face of the membrane