Biochimica et Biophysica Acta 828 (1985) 383-386 383 Elsevier BBA 30093 BBA Report Pressure-induced dissociation of aggregates of myelin proteolipid protein Alexander Gow, Donald J. Winzor and Ross Smith * Department of Biochemistry, Unioersity of Queensland, St. Lucia, Queensland 4067 (,4ustralia) (ReceivedDecember14th, 1984) Key words: Myelin proteolipidprotein; Proteinaggregation; Molecular weight determination; Pressureeffect; Membraneprotein Sedimentation velocity and equilibrium experiments have revealed an extremely pressure-sensitive aggrega- tion of myelin proteolipid protein in the presence of Triton X-100, dissociation of the protein aggregate being observed at pressures that are several orders of magnitude lower than those effecting disaggregation of many other proteins. These results highlight the need to employ a range of angular velocities in sedimentation studies of intrinsic membrane protein. The characterization of intrinsic membrane pro- teins poses problems in that it requires the adop- tion of techniques that differ from those employed for water-soluble proteins. Specifically, molecular weight measurements must be performed either in non-aqueous solvents, or in aqueous solvents con- taining detergents, in order to maintain protein solubility. Because of the usefulness of detergents in the extraction and purification of intrinsic membrane proteins, sedimentation methods have been extended in recent years to allow molecular weights to be deduced from such experiments per- formed in the presence of amphiphiles [1,2]. How- ever, several assumptions are implicit in the deduc- tion of molecular weights from such results. (i) Frequently, the apparent partial specific volume, q/, of the protein is not measured directly, but is calculated from the amino acid composition of the polypeptide, the partial specific volumes of the amino acids and the detergent, and the amount of detergent bound per unit weight of protein. (ii) Even in instances where ~#' is measured directly, the molecular weights are deduced on the basis * To whom correspondence shouldbe addressed. that the partial specific volume and the extent of detergent binding pertain under the conditions operative during ultracentrifugation. There is thus a general anticipation that the modest pressures encountered in sedimentation equilibrium experi- ments have little influence on either the aggrega- tion behaviour of the protein or on its detergent- binding characteristics. However, a need for cau- tion in these regards is emphasized by the present investigation of the central nervous system proteo- lipid protein, which is shown to exhibit extreme pressure-sensitivity of its self-association be- haviour in 0.02 M phosphate buffer (pH 7.4) con- taining 0.05% Triton X-IO0. Proteolipid protein was isolated from bovine white matter by Triton X-100 in accordance with the procedure described previously [3], the purity of the preparation being verified by polyacryl- amide gel electrophoresis in the presence of sodium dodecyl sulphate. Prior to centrifugation, solutions were dialyzed at 4°C for 24-60 h against 20 mM phosphate buffer (pH 7.4) containing 0.05% (0.79 mM) Triton X-100, or against the same buffer made 0.1 M with respect to sodium chloride. Sedi- mentation experiments were carded out at 5°C in 0167-4838/85/$03.30 © 1985 ElsevierSciencePublishersB.V.(Biomedical Division)