PALMITOYLATION OF BRAIN CAPILLARY PROTEINS

Palmitoylation is a reversible posttranslational modification which is involved in the regulation of several membrane proteins such as beta( 2)-adrenergic receptor, p21(ras) and trimeric G-protein alpha-subunits . This covalent modification could be involved in the regulation of th e numerous membrane proteins present in the blood-brain barrier capill aries. The palmitoylation activity present in brain capillaries was ch aracterized using [H-3]palmitate labeling followed by chloroform metha nol precipitation. Palmitate solubilizing agents such as detergents an d bovine serum albumin (BSA), were used for optimizing activity. Some palmitoylated substrates were identified using [H-3]palmitate labeling followed by immunoprecipitation with specific antibodies. Two optimal palmitate solubilization conditions were found, one involves cell per meabilization (Triton X-100) and the other represents a more physiolog ical condition where membrane integrity is conserved (BSA). Sensitivit y to the cysteine modifier N-ethylmaleimide and to hydrolysis, using h ydroxylamine or alkaline methanolysis, indicated that palmitic acid wa s bound to the proteins by a thioester bond. Maximal palmitate incorpo ration was reached after 30 or 60 min of incubation in the presence of Triton or BSA, respectively. Depalmitoylation was observed in the pre sence of BSA, but not with detergents. The palmitoylation reaction was optimal at pH 8 or 9 in the presence of Triton or BSA, respectively, but palmitoylated substrates were detectable over a wide range of pH v alues. In the presence of Triton X-100, the addition of ATP, CoA and M g2+ to the incubation medium increased palmitoylation by up to 80-fold . Two palmitoylated substrates were identified, a 42 kDa G-protein alp ha subunit and p21(ras). The study shows that the utilization of palmi tate solubilizing agents is essential to measure in vitro palmitoylati on in brain capillaries. Several palmitoylated proteins are present in the blood-brain barrier including five major substrates of 12, 21, 35 , 42 and 55 kDa. It is suggested that palmitoylation could play a cruc ial role in the regulation of brain capillary function, since the two substrates identified in this study are known to be involved in signal transduction, vesicular transport and cell differentiation.