Effects of site-specific acetylation on omega-conotoxin GVIA binding and function.
Chemical modification of omega-conotoxin GVIA (omega-CgTXGVIA) was performed using nonsaturating concentrations of acetic anhydride to generate seven distinct derivatives. Following separation of these peptides using reverse-phase HPLC (RP-HPLC), their individual molecular weights were determined using fast bombardment mass spectrometry (FAB-MS). Three peptides contained a single acetylated amino group, three possessed two acetylated amino groups, and the last contained three acetylations. For each peptide, the specific site of acetylation was confirmed using a scheme of tryptic digestion, under nonreducing conditions, followed by RP-HPLC and FAB-MS. Biological profiles for each peptide were obtained by analyzing their capacity to displace native 125I-omega-CgTx GVIA binding to rat hippocampal membranes and to block K(+)-stimulated 45Ca2+ influx into chick brain synaptosomes. The data indicate that successive additions of acetyl moieties to omega-CgTx GVIA lead to a loss of both binding affinity and Ca2+ influx inhibitory potency. Within the monoacetylated series, acetylation of the amino terminal of Cys-1, as compared to the epsilon-amino group of either Lys-2 or Lys-24, leads to the greatest shift in potency. In summary, these results indicate that basic (i.e., primary amino) groups, which are brought into close proximity as a result of disulfide bridging, are important in the functional blockade of neuronal Ca2+ channels by omega-CgTx GVIA.
Lampe
R A
RA
Department of CNS Pharmacology, ICI Pharmaceuticals Group, A Business Unit of ZENZCA Inc., Wilmington, Delaware 19897.
Lo
M M
MM
Keith
R A
RA
Horn
M B
MB
McLane
M W
MW
Herman
J L
JL
Spreen
R C
RC
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000737:chemistry*; D002645:Chickens; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013574:Synaptosomes; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D008978:Mollusk Venoms; D020866:omega-Conotoxin GVIA; D002118:Calcium