Permeability of Phospholipid Vesicles to Amino Acids

Preliminary experiments indicated a change in the distribution of molecular aggregates possessing phospholipase activity when the supernatant had been treated with colchicine before density-gradient sedimentation. To examine the possible association between microtubular protein and the phospholipase, rat brain supernatant was labelled with [3H]colchicine before ion-exchange chromatography. Enzyme activity in the peak I position was retained, but peak I1 activity was abolished, to be replaced by a new peak (111) that was eluted from the resin together with protein-bound [3H]colchicine at higher salt concentration. Density-gradient sedimentation of each peak indicated that the smaller-molecular-weight component (molecular weight 36000) was lost from peak I. Peak I11 sedimented in three main bands corresponding to molecular weights of approx. 14OOO0, 88000 and 43 000. Polyacrylamide-gel electrophoresis of peak I11 material in calibrated sodium dodecyl sulphate-urea gels showed only three protein bands, the aand p-subunits of microtubular protein (molecular weights 56000 and 52000 respectively) and a single band of molecular weight 38000. Phospholipase activities of peak I11 sedimenting as higher-molecular-weight proteins must have arisen from an association between the enzyme and subunits of microtubular protein. The maintenance of organized microtubular structures in a variety of cell types has been consistently demonstrated as a prerequisite for the co-ordination of their specialized functions. The precise mechanism by which they do this is a matter of conjecture at the present time. However, it has been proposed that microtubules perform a contractile function in the secretory process (Malaisse et al., 1971). The evidence given in the present communication suggests an additional role for microtubules in regulating phosphatidylinositol degradation in membranes. This work was supported by part by U.S. Public Health Service Grants AM 10699 and AM 05071.