Distribution and characterization of pedal peptide immunoreactivity in Aplysia.

Pedal peptide (Pep) is a very abundant neuropeptide in Aplysia. A radioimmunoassay (RIA) was developed to quantify Pep-like immunoreactivity (IR-Pep) in tissue extracts. IR-Pep was present in very high concentrations in the central nervous system (CNS) and two peripheral tissues: the large hermaphroditic duct (LHD) and the foot. RIA of fractions from high-pressure liquid chromatography (HPLC) indicated that Pep itself was the predominant immunoreactive species in each of these tissues. Lower concentrations of Pep were found in a number of other peripheral tissues. Incorporation of labelled amino acid indicated that Pep was synthesized in the LHD, whereas Pep in the foot was synthesized primarily in central neurons and transported to the foot. IR-Pep was further localized by immunocytology. All peripheral IR-Pep appeared to be associated with neuronal fibers, most commonly varicose axons. Immunoreactive innervation of the LHD and foot was particularly dense but positive staining was also observed in other tissues including tegument, gill, gut, and heart, IR-Pep innervation in all tissues including the LHD appeared to be localized predominantly in muscular portions of the tissue. Spontaneous contractions of isolated LHD were accelerated by the application of Pep. Pep appears to act as a transmitter or neuromodulator at a number of different sites in Aplysia.

Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation.

In both vertebrates and invertebrates, long-term memory differs from short-term in requiring protein synthesis during training. Studies of the gill and siphon withdrawal reflex in Aplysia indicate that similar requirements can be demonstrated at the level of sensory and motor neurons which may participate in memory storage. A single application of serotonin, a transmitter that mediates sensitization, to individual sensory and motor cells in dissociated cell cultures leads to enhanced transmitter release from the sensory neurons that is independent of new macromolecular synthesis. Five applications of serotonin cause a long-term enhancement, lasting one or more days, which requires translation and transcription. Prolonged application or intracellular injection into the sensory neuron of cyclic AMP, a second messenger for the action of serotonin, also produce long-term increases in synaptic strength, suggesting that some of the gene products important for long-term facilitation are cAMP-inducible. In eukaryotic cells, most cAMP-inducible genes so far studied are activated by the cAMP-dependent protein kinase (A kinase), which phosphorylates transcription factors that bind the cAMP-responsive element TGACGTCA. The cAMP-responsive element (CRE) binds a protein dimer of relative molecular mass 43,000, the CRE-binding protein (CREBP), which has been purified and shown to increase transcription when phosphorylated by the A kinase. Here we show that extracts of the Aplysia central nervous system and extracts of sensory neurons contain a set of proteins, including one with properties similar to mammalian CREBPs, that specifically bind the mammalian CRE sequence. Microinjection of the CRE sequence into the nucleus of a sensory neuron selectively blocks the serotonin-induced long-term increase in synaptic strength, without affecting short-term facilitation. Taken together, these observations suggest that one or more CREB-like transcriptional activators are required for long-term facilitation.

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai.

A phosphonoglycosphingolipid, designated as FGL-IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by permethylation studies to be attached as a ketal to O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb. By sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3(Fuc alpha 1----2) (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide. Its major aliphatic components were palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. This is the first report of the occurrence of pyruvylated galactose as a constituent of animal sphingolipid.

Biopolymers from marine invertebrates. X. Mode of action of an antibacterial glycoprotein, aplysianin E, from eggs of a sea hare, Aplysia kurodai.

An antibacterial factor, aplysianin E, was purified from the eggs of a sea hare, Aplysia kurodai. Purified aplysianin E was a glycoprotein of 250 kilo daltons consisting of 3 subunits, and showed both antibacterial and antineoplastic activities. The two activities were lost in parallel on heating and at low and high pH. This factor was half-maximally active for gram-positive and -negative bacteria at 0.12-3.3 micrograms/ml and its action was not bactericidal but bacteriostatic. Aplysianin E did not induce morphological elongation of bacteria or their release of adenosine triphosphate (ATP), but it completely inhibited the syntheses of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by E. coli within 10 min. These results suggest that aplysianin E, found in an invertebrate, the sea hare, is a new antibacterial protein and that it exerts its action by inhibiting nucleic acid synthesis, as a DNA-inhibiting chemotherapeutic drug does.

Confirmation of D-aspartic acid in the novel dipeptide beta-aspartylglycine isolated from tissue extract of Aplysia kurodai.

A novel o-phthalaldehyde-reactive compound was found in the h.p.l.c. chromatogram of Aplysia kurodai extract. This compound was isolated by ion-exchange chromatography and preparative high-voltage paper electrophoresis. It was shown by optical-rotatory-dispersion spectrum and optical-resolution h.p.l.c. analysis that this compound consisted of equimolar amounts of D-aspartic acid and glycine. This compound resisted cleavage in the Edman reaction. This peptide was inferred to be beta-D-aspartylglycine, and this was confirmed by synthesis. beta-D-Aspartylglycine was detected in all tissues of Aplysia kurodai, with especially high concentrations in body wall (skin and muscle) and gill.

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla.

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.

Structural characterization of alpha-bungarotoxin-binding proteins from Aplysia californica.

Structural features of alpha-bungarotoxin-binding proteins from the marine mollusc Aplysia californica have been examined as a first step toward delineating their potential role in cholinergic neurotransmission. Protein blotting with 125I-alpha-bungarotoxin was used to identify binding proteins in membranes prepared from Aplysia muscle and nervous tissue. Binding proteins from both tissues exhibited similar physical characteristics, which distinguish them from the prototypical alpha-bungarotoxin-binding protein, the nicotinic acetylcholine receptor obtained from Torpedo californica electric organ membranes. Aplysia binding activities migrate with an apparent molecular weight of 250 kDa on sodium dodecyl sulfate (SDS) gels in the presence of reducing agents. Binding of alpha-bungarotoxin to blots of Aplysia membranes is abolished by exposure of samples to heat or to low pH but is unaffected by reduction-alkylation treatment. In contrast, the alpha-bungarotoxin-binding subunit of the acetylcholine receptor from Torpedo membranes migrates on SDS gels at 40 kDa. It retains binding activity following exposure to heat or to low pH, but binding is substantially diminished by reduction-alkylation treatments. Another distinguishing characteristic of the Aplysia binding activities is revealed by examining recovery of membrane alpha-bungarotoxin-binding on protein blots; the high recovery of Aplysia binding contrasts sharply with the low recovery of Torpedo binding activity. The high apparent molecular weight of the Aplysia alpha-bungarotoxin-binding activities, their most distinguishing feature, is similar to an alpha-bungarotoxin-binding activity recently identified in lower vertebrate brain. Covalent cross-linking with 125I-alpha-bungarotoxin demonstrates, however, that the mobility of both Aplysia binding activities is due to a multimeric protein that is unusually resistant to dissociation in SDS. The covalently radiolabeled Aplysia alpha-bungarotoxin-binding activity migrates at approximately 260 kDa on SDS gels when solubilized at room temperature. When it was boiled before electrophoresis, the mobility of the radiolabeled protein shifts to approximately 70 kDa. Resistance to dissociation in the absence of boiling may explain both the high recovery of activity on blots and the insensitivity to reductive alkylation. Conversely, dissociation of the multimeric complex upon boiling may explain the observed loss of binding activity. Our results demonstrate structural similarities and differences between Aplysia alpha-bungarotoxin-binding proteins and the Torpedo acetylcholine receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].

Axial coordination of ferric Aplysia myoglobin.

Resonance Raman spectra of ferric Aplysia myoglobin in the ligand-free and the azide-bound forms have been studied over a wide pH range to determine the coordination states of the heme iron atom. In the hydroxide form at high pH (approximately 9) the iron is six-coordinate and is in a high/low spin equilibrium. As the pH is lowered below the acid/alkaline transition (pKa = 7.5), the heme becomes five-coordinate. When the pH is lowered even further no other changes in the resonance Raman spectrum are detected; thus, the heme remains five-coordinate down to pH 4, the lowest value studied. For ferric azide-bound Aplysia myoglobin, the iron is six-coordinate in a high/low spin equilibrium at all pH values (4.8-9). These data indicate (i) that the unusual reactivity toward azide previously observed at neutral pH is indeed related to the absence of a coordinated water molecule, and (ii) that causes other than the heme coordination are responsible for the spectral differences and the ligand-binding kinetics differences observed below pH 6.

Purification and sequencing of neuropeptides contained in neuron R15 of Aplysia californica.

R15 is a large identified neuron present in the abdominal ganglion of the mollusc Aplysia. Previous studies have indicated that this neuron may play a role in water balance and possibly renovascular functions. A peptidic factor contained in the neuron R15 has been shown to increase the water content of Aplysia. To determine the structure of the peptides contained in R15, we purified the extracts of 820 R15 cells by means of two steps of reverse-phase HPLC. The purification yielded a number of peptides, only one of which, R15 alpha 1, resulted in water uptake when injected into animals. Determination of the amino acid content and sequence analysis of the R15 alpha 1 peptide demonstrated that this peptide contains 38 residues, including two cysteines. The peptide failed to react with iodoacetate, indicating that the two cysteines are connected by a disulfide bridge. To confirm the assigned structure, the peptide was synthesized with a disulfide bridge. The chromatographic properties and bioactivity of the synthetic material were identical to those of the native peptide. Several other R15 peptides were inactive in the bioassay for water uptake. The sequence of one of these peptides (R15 beta) was determined, and it was established that the peptide contains 28 residues. Amino acid analysis of three other peaks was performed. One of these peaks contained a peptide (R15 beta f) whose amino acid composition suggests that it is a fragment of the R15 beta peptide. The other two peaks contained peptides with identical amino acid compositions, suggesting that they are variants of a single peptide (R15 gamma). The amino acid sequences of all the peptides identified in neuron R15 correspond to stretches of a polyprotein encoded by a recently sequenced R15 cDNA.

Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana.

The fetid secretion of a sea hare, Aplysia juliana, was lethal to crabs and also inhibited the growth of bacteria. When the secretion was partitioned between water and n-hexane, only the n-hexane layer, which had a nauseating odor, was lethal to crabs. The water-soluble fraction showed strong antibacterial activity and inhibited the growth of both Gram-positive and Gram-negative bacteria. Antibacterial activity of the water-soluble fraction was destroyed by heating at 50 degrees C for 15 min, but was resistant to treatment with proteolytic enzymes. The active principle, named julianin-S, was purified by gel filtration and ion exchange chromatography. The purified specimen gave a single protein showing a mol. wt of approximately 67,000, as determined by gel filtration. Julianin-S inhibited the growth of Bacillus subtilis by 50% at a concentration of 70 ng protein/ml. It was also cytotoxic to murine tumor cells and inhibited in vitro growth of L1210 cells by 50% at a concentration of 8 ng protein/ml.

Sequencing proteins from acrylamide gels.

The analytical power of high-resolution two-dimensional gel electrophoresis has been coupled with molecular cloning techniques to allow the sequencing of proteins directly from preparative gels.

Sequencing of proteins from two-dimensional gels by using in situ digestion and transfer of peptides to polyvinylidene difluoride membranes: application to proteins associated with sensitization in Aplysia.

We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the gel is stained with Coomassie blue and the protein spots of interest are cut out. The in situ protein is digested with Staphylococcus aureus V8 protease in a second polyacrylamide gel and the peptides are separated by one-dimensional polyacrylamide gel electrophoresis. The peptides are then electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Using this method, we have obtained amino acid sequence data for two proteins that are altered after long-term sensitization: actin and Aplysia protein 407. In addition, we have obtained amino acid sequence data for rat protein 425, a protein that appears to be homologous to Aplysia protein 407.

Immunochemical and histochemical studies on a phosphonoglycosphingolipid, SGL-II, isolated from the sea gastropod Aplysia kurodai.

Antiserum was raised against 3-O-MeGal beta 1----3GalNAc alpha 1----3[6' -O-(2-aminoethylphosphonyl)Gal alpha 1----2 (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Ceramide (SGL-II) isolated from the skin of a mollusc, Aplysia kurodai. This antiserum reacted with SGL-II and other phosphonoglycosphingolipids of Aplysia such as SGL-I', F-21, and some minor glycolipids on TLC plates, but it did not react with ganglioside or globoside. The sugars recognized were 3-O-methylgalactose at the non-reducing end and galactose at the branched chain of the glycolipids. One membrane glycoprotein (Mr 280,000) reacted strongly, and some other proteins reacted weakly with the antiserum. Immunohistochemical examination of the nervous tissues revealed distinct staining in the periganglionic tissue of the ganglia, and the perineural sheath of the proximal portion of the peripheral nerves. The neuropil and satellite cells were also stained. In the skin, subcutaneous connective tissues were moderately stained, and the cytoplasm of small mononuclear cells and foamy cells was also stained. The staining patterns were essentially the same in paraffin and cryostat sections. From the findings with sections pretreated with chloroform-methanol (2 : 1, v/v), it was suggested that the periganglionic and perineural stainings were due to glycoproteins, including an SDS-soluble glycoprotein of Mr 280,000, while those of the other regions were due to SGL-II and glycolipids immunologically related to SGL-II. The stainings in the skin sections were largely due to glycoproteins.

Purification and characterization of a GTP-binding protein with a molecular weight of 20,000 in bovine brain membranes. Identification as the rho gene product.

We have determined that there are at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 in the crude membrane fraction of bovine brain and have purified one of them with a Mr of about 24,000 (24K G) to near homogeneity (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In this study, we have purified another G protein with a Mr of about 20,000 (20K G) to near homogeneity and have characterized it. 20K G bound maximally about 1.0 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 50 nM. [35S]GTP gamma S binding to 20K G was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP; it was also inhibited by pretreatment with N-ethylmaleimide. 20K G hydrolyzed GTP to liberate Pi, with a turnover number of about 0.01 min-1, and was not copurified with the beta gamma subunits of the regulatory G proteins of adenylate cyclase. 20K G was not recognized by the antibody against the ADP-ribosylation factor for the stimulatory regulatory G protein of adenylate cyclase. Peptide map analysis showed that 20K G was not a proteolytic product of 24K G. The partial amino acid sequence of 20K G was almost identical with that deduced from the rho gene. The amino acid composition of 20K G was similar to that of the rho gene product. These results suggest that 20K G is the rho gene product and that this G protein is present in bovine brain membranes.

Structural alignment and analysis of two distantly related proteins: Aplysia limacina myoglobin and sea lamprey globin.

Two new globin structures have recently been determined at high resolution: the globin from the mollusc Aplysia limacina at 1.6 A resolution and a new refinement of the structure from sea lamprey. Two amino acid sequences of these homologous molecules have only 30% residue identity in an optimal alignment. We discuss some of the problems arising in the alignment of Aplysia globin with other globins of known structure, a challenging problem because of the distant relationship. Four independent approaches were applied to the alignment of the Aplysia and lamprey globins, including those based on individual sequence comparisons, structural analysis, and the relatively new method of templates or fingerprints derived for an entire family of proteins. We also compare these two new structures with what is already known about the globin family. A detailed description of the two structures shows that the two molecules contain the main structural features common to all the globins so far studied, with several minor but interesting hitherto unobserved variations.

Coexistence of FMRFamide, met-enkephalin and serotonin in molluscan neurons.

1. Coexistence of FMRFamide, met-enkephalin and serotonin immunoreactivities was examined in Achatina fulica and Aplysia kurodai. 2. Coexistence of FMRFamide and serotonin was found in some neurons of the visceral, right parietal and pedal ganglia of Achatina fulica, and in the pedal ganglion of Aplysia kurodai. 3. In Achatina fulica, coexistence of FMRFamide and met-enkephalin was found in a neuron of the left parietal ganglion and that of met-enkephalin and serotonin was found in a giant neuron of the right parietal ganglion. 4. Based on these results, the biological significance of coexistence was discussed.

Purification and characterization of aplysianin E, an antitumor factor from sea hare eggs.

An antitumor factor, aplysianin E, inducing tumor lysis was purified to apparent homogeneity from the supernatant of a homogenate of eggs of the sea hare Aplysia kurodai. Purified aplysianin E was a 250-kDa glycoprotein containing three different subunits. This factor was half-maximally active at 2-114 ng protein/ml and lysed all the tumor cells tested but did not lyse normal white or red blood cells. Aplysianin E was labile on treatments with heat, low pH, urea, guanidine, sodium lauryl sulfate, and periodate, but not with proteases or organic solvents, Aplysianin E completely inhibited the syntheses of DNA, RNA, and protein by tumor cells within 2 h and caused their complete cytolysis within 15 h. Tumor lysis by aplysianin E was inhibited by N-acetylneuraminic acid, suggesting that recognition of the sugar moiety is a key step in cytolysis induced by aplysianin E. Aplysianin E also prolonged the survival of mice bearing syngeneic MM46 ascites or solid tumors. These results suggest that aplysianin E, found in an invertebrate, the sea hare, is a new antitumor factor.

Structure of a triphosphonopentaosylceramide containing 4-O-methyl-N-acetylglucosamine from the skin of the sea hare, Aplysia kurodai.

A novel phosphonoglycosphingolipid named SGL-I containing 3 mol of 2-aminoethylphosphonate residues was isolated from the skin of a sea gastropod, Aplysia kurodai. The saccharide moiety of the glycolipid was characterized as 4-O-methyl-GlcNAc alpha 1----4GalNAc alpha-1----3 [6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6)Gal beta 1----4(2-aminoethylphosphonyl----6) Glc beta 1----1-ceramide. The major aliphatic components of the ceramide portion were palmitic acid, stearic acid, octadeca-4-sphingenine, and anteisononadeca-4-sphingenine. This glycolipid is unique in containing 4-O-methyl-N-acetylglucosamine and 3 mol of 2-aminoethylphosphonate residues, one of which is attached to C-6 of glucose.

Presence of immunoreactive alpha-bag cell peptide[1-8] in bag cell neurons of Aplysia suggests novel carboxypeptidase processing of neuropeptides.

alpha-bag cell peptide (alpha BCP) is a putative neurotransmitter released from bag cell neurons of the marine mollusc Aplysia. alpha BCP is present in bag cell extracts and releasate from bag cells in two neuroactive forms: alpha BCP[1-9] and alpha BCP[1-8]. alpha BCP[1-8] is 30 times as potent as [1-9] in inhibiting target neurons, suggesting that both forms of the peptide serve as neurotransmitters. However, biochemical and molecular genetic data suggest that only alpha BCP[1-9] is originally cleaved directly from a larger precursor protein and that generation of alpha BCP[1-8] would require an unusual C-terminal leucine cleavage of alpha BCP[1-9]. To further ascertain which forms of alpha BCP are normally present in bag cells, we generated highly specific antisera to each peptide. We found intense immunostaining for both peptides in bag cell somata and nerve terminals. Moreover, both forms were stable in bag cell extract for at least 1 hr, which suggests that proteolysis in the extracts had been effectively inhibited. These results suggest that both alpha BCP[1-8] and [1-9] are normally present in bag cell somata and terminals and that a small amount of alpha BCP[1-9] is processed to alpha BCP[1-8] in vesicles before release. The results support the interpretation that the activity of an intravesicular carboxypeptidase generates alpha BCP[1-8] and thereby regulates the amount of inhibitory activity released during a bag cell discharge.