Characterization and solubilization of the FMRFamide receptor of squid.

The optic lobe of squid (Loligo pealei) contains FMRFamide receptors that can bind an iodinated FMRFamide analog: [125I]-desaminoTyr-Phe- norLeu-Arg-Phe-amide ([125I]-daYFnLRFa). Radioligand binding assays revealed that squid FMRFamide receptors are specific, saturable, high affinity sites (Kd = 0.15 nM) densely concentrated in optic lobe membranes (Bmax = 237 fmole/mg protein). The receptors appeared to be coupled to Gs because guanine nucleotides inhibit receptor binding and the stimulation of adenylate cyclase by FMRFamide is GTP-dependent. Both the binding and cyclase data showed that FMRFamide, but not FMRF-OH, interacts at FMRFamide receptors; thus the C-terminal Arg-Phe-amide is critical for binding. The high binding affinity of FMRFamide (0.4 nM IC50) was specific for FMRFamide-like peptides. The structure-activity relations of many FMRFamide analogs were defined in detail and were nearly identical for both the membrane-bound and detergent-solubilized receptors. We also found that squid optic lobe contains FMRFamide-like reactivity as measured with both a radioimmunoassay and a radioreceptor assay. Moreover, we have sequenced a fragment of genomic DNA that encodes a FMRFamide precursor. Our findings in sum suggest that FMRFamide is a neurotransmitter in squid optic lobe, and that this tissue is a good source from which to purify FMRFamide receptors.

Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis.

Neurotransmitter release is triggered by Ca2+ ions binding to an unknown Ca2+ receptor within presynaptic terminals. Synaptotagmin, a Ca2(+)-binding protein of synaptic and other secretory vesicles, has been proposed to mediate vesicle-plasma membrane interactions during neurotransmitter release. Here we test this hypothesis using the giant synapse of the squid Loligo pealei, which because of its unusually large size and well established physiology is uniquely suited for dissecting presynaptic events. We find that injection of peptides from the C2 domains of synaptotagmin into squid giant presynaptic terminals rapidly and reversibly inhibits neurotransmitter release. Our data are consistent with these peptides competitively blocking release after synaptic vesicle docking and indicate that Ca2+ probably initiates neurotransmitter release by regulating the interaction of synaptotagmin with an acceptor protein.

Purification of squid synaptic vesicles and characterization of the vesicle-associated proteins synaptobrevin and Rab3A.

Two proteins associated with mammalian synaptic vesicles, the integral membrane protein synaptobrevin and the GTP-binding protein rab3A, are identified in squid nervous tissue using Western blotting and subcellular fractionation of synaptosomes. They both copurify with synaptic vesicles prepared from squid optic lobe. Synaptobrevin (18.1 kDa) is present at high levels in synaptic terminals but at very low levels in axon. Rab3A (27.5 kDa) is a member of the rab family of low-molecular weight GTP-binding proteins which regulates vesicle traffic in secretory and endocytic processes. As resolved with 2-dimensional gels, squid neurons contain at least 16 GTP-binding species (19-29 kDa), and most of these are present in both soluble and particulate fractions. The 24 kDa class of GTP-binding proteins is highly enriched in axonal transport organelles. The characterization of synaptobrevin and rab3A in squid synaptic vesicles extends their known distributions to invertebrates and points to a fundamental importance of these proteins in neurotransmitter release.

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

We find that half of the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i) association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G protein-mediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our findings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals.

Characterization of synaptophysin and G proteins in synaptic vesicles and plasma membrane of Aplysia californica.

Two components of synaptic terminals that may be involved in transmitter release are synaptophysin (p38) and G proteins. In order to study release mechanisms in Aplysia californica we have prepared subcellular fractions from nervous tissue to characterize and localize these components. We identify Aplysia synaptophysin by Western blot analysis with monoclonal antibody SY38, find that it is enriched in synaptic vesicles, and, using immunocytochemistry, show that it is localized to neuropil. These characteristics indicate that Aplysia synaptophysin is closely related to mammalian synaptophysin; it appears to be much smaller, however, having a mass of 28 kDa instead of 38 kDa. We previously determined that G protein subunits in Aplysia are enriched in neuropil and synaptosomes. We now show that within the synaptic terminal the pertussis toxin-sensitive alpha-subunit as well as the beta-subunit are associated with plasma membrane using [32P]ADP-ribosylation and Western blotting with G protein-specific antibodies.

G proteins in Aplysia: biochemical characterization and regional and subcellular distribution.

We studied G proteins and regulation of adenylate cyclase in nervous tissue and muscle of Aplysia using bacterial toxin-catalyzed ADP-ribosylation. We identified Gs alpha, a Mr 45,000 cholera toxin substrate, Go alpha, a Mr 40,000 pertussis toxin substrate, and G beta (Mr 37,000) by Western blot analysis with antisera specific for bovine brain G protein subunits. Partial proteolysis suggests that the neuronal pertussis toxin substrates are heterogeneous. The concentration of these substrates in membranes from Aplysia ganglia is similar to that of rat, squid and Helix; in Aplysia nervous tissue, G protein subunits are most enriched in synaptosomes and neuropil. The stimulation of adenylate cyclase by serotonin (5-HT), low concentrations of GTP-gamma-S, and cholera toxin, and the inhibition by high concentrations of GTP-gamma-S that is blocked by pertussis toxin indicate that both a Gs and a Gi protein regulate the Aplysia enzyme. These results support the idea that G proteins in Aplysia are important in regulating synaptic function.

Aplysia synaptosomes. II. Release of transmitters.

A subcellular fraction (P3) from Aplysia is enriched in synaptosomes (Chin et al., 1988) and is capable of accumulating 5-HT and choline. At an external 3H-5-HT concentration of 1.8 microM, the P3 fraction took up 0.12 nmol/mg protein in 30 min. Uptake was dependent on external Na+. Electron microscopic autoradiography showed that much of the accumulated 3H-5-HT is localized to synaptosomes. At 0.5 microM 3H-choline, P3 took up 0.11 nmol/mg protein in 30 min and converted 40% to 3H-ACh. This synaptosomal fraction was also capable of releasing transmitter. After 3H-5-HT or 3H-choline was taken up, P3 released about 5% of the total radioactive transmitter in a Ca2+-dependent manner during a 30 sec exposure to a depolarizing concentration of K+ (100 mM). Identified, prelabeled synaptosomes were prepared by injecting 3H-choline into the large cholinergic neuron L10. The abdominal ganglia containing the injected cells were then fractionated, yielding synaptosomes containing radioactivity derived from L10. After this synaptosomal fraction was exposed to high K+, 2% of the radioactivity was released in a Ca2+-dependent manner. This release was completely blocked by 0.1 mM histamine, a modulatory transmitter that has previously been shown to cause presynaptic inhibition in L10.

Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions.

We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3-fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.

Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains.

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.

Adenosine 5'-O-([gamma-18O]gamma-thio)triphosphate chiral at the gamma-phosphorus: stereochemical consequences of reactions catalyzed by pyruvate kinase, glycerol kinase, and hexokinase.

The 2-[18O]phosphorothioate of D-glycerate, chiral at phosphorus, was prepared. The chiral phosphoryl group was transferred enzymically to ADP [by using enolase and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase; EC 2.7.1.40)] resulting in the synthesis of adenosine 5'-O-([gamma-18O],gamma-thio)triphosphate. This labeled ATP was used as a thiophosphoryl group donor in the reactions catalyzed by glycerol kinase (ATP:glycerol 3-phosphotransferase; EC 2.7.1.30) and by hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1). The product from the latter (glucose 6-phosphorothioate) was converted enzymically into glycerol phosphorothioate. Determination of the relative configurations and diastereoisomeric purities of the samples of glycerol phosphorothioate demonstrates that all three phosphokinases (pyruvate kinase, glycerol kinase, and hexokinase) transfer the thiophosphoryl group with complete stereospecificity, and further shows that these reactions follow an identical stereochemical course.