DIFFERENTIAL REGULATION OF GTPASE ACTIVITY BY MASTOPARAN AND GALPARAN

The chimeric peptide galparan, composed of galanin(1-13) in the N-term inus and mastoparan in the C-terminus, was recently designed and synth esized. The effect of galparan on GTPase activity of rat brain cortica l membranes was studied in comparison with the effect of mastoparan an d galanin. GTPase was activated by mastoparan but it was noncompetitiv ely inhibited by galparan, while no effect of galanin and galanin(1-13 ) was found in this tissue. EC50 of 12.1 +/- 2.1 mu M and Hill coeffic ient of 2.1 +/- 0.6 was calculated for galparan from a dose-response c urve and K-i of 19.1 +/- 0.3 mu M was obtained by fitting the experime ntal data to the Michaelis-Menten equation valid in the presence of no ncompetitive 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-cataly zed ribosylation of G proteins from rat brain cortical membranes resul ted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose-response curve for galparan inhibitio n. The rate of GTP gamma S binding to G proteins from rat brain cortic al membranes was not influenced by galparan, CD spectra revealed predo minantly antiparallel beta-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid ve sicles it adopted a higher amount of alpha-helix. Critical micelle con centration of galparan in buffer solution of 22 mu M was determined. I t is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these tw o peptides on G proteins. (C) 1998 Academic Press.