We have used the polymerase chain reaction to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis demonstrates that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion molecule DM-GRASP, SC-1, BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish.Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two are shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion molecule, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system.
The heavy and light chains of ~t~~~ A toxin were separated by anion exchange c~o~to~aphy. Their intracelltiar actions were studied using bovine adrenal chromafhn cells penneabilimd with streptolysin 0. Purified light chain inhibited the Gas +-stimulated p~o~~~ne release with a l&f-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.
1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.
We have used the polymerase chain reaction to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis demonstrates that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion molecule DM-GRASP, SC-1, BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish. Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two are shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion molecule, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system. Animuls. All goldfish (Curussius uururus) were maintained in tanks at 18" C. Optic nerves of goldfish (5-7 cm long) were cut under
Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells [(1989) FEBS Lett. 242, 245-248; J. Neurochem., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions of both neurotoxins on exocytosis were presently compared using streptolysin 0-permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca*+-stimulated catecholamine release from these cells. Addition of dithiothreitol lowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.
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