Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Complex patterns of [125I]omega-conotoxin GVIA binding site expression during postnatal rat brain development.

In the mature CNS, N-type calcium channels regulate neurotransmitter release. The role of these channels in developing brain is less clear. Study of [125I]omega-conotoxin GVIA binding sites in developing rat brain using autoradiography reveals that putative N-type channels appear and disappear in complex temporal-spatial profiles including: (1) gradual increase to adult levels (cerebral cortex); (2) substructure differentiation (cerebellum); (3) transient expression (pons); and, (4) selective depletion (medulla). Transient expression of N-type calcium channels may influence specific neurodevelopmental processes.

A new Conus peptide ligand for Ca channel subtypes.

A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.

Localization of [125I]omega-conotoxin GVIA binding in human hippocampus and cerebellum.

The peptide toxin omega-conotoxin GVIA (omega-CgTx) has been shown to be a high affinity ligand for N-type calcium channels in the brain. We have employed [125I]omega-CgTx to localize N-type channels in human hippocampus and cerebellum using autoradiography. Ten micron thick slide-mounted tissue sections of human cerebellum and hippocampus were labeled with [125I]omega-CgTx under various conditions. Specific binding to human cerebellum was virtually irreversible and saturable. It was displaceable by the N-channel antagonist, omega-conotoxin MVIIA, but not by L- or P-channel ligands. Binding sites were heterogeneously distributed with denser binding in the molecular layer than the granule cell layer of cerebellum and with specific laminar patterns evident in the hippocampus. [125I]omega-CgTx should be a useful tool for the study of N-type calcium channels in human brain tissue.