Differential Regulation of GTPase Activity by Mastoparan and Galparan

The chimeric peptide galparan, composed of galanin(1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin(1-13) was found in this tissue. EC50of 12.1 ± 2.1 μM and Hill coefficient of 2.1 ± 0.6 was calculated for galparan from a dose–response curve andKiof 19.1 ± 0.3 μM was obtained by fitting the experimental data to the Michaelis–Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose–response curve for galparan inhibition. The rate of GTPγS binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel β-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of α-helix. Critical micelle concentration of galparan in buffer solution of 22 μM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.