Barry S. Rothman

A single gene encodes multiple neuropeptides mediating a stereotyped behavior

Egg laying in Aplysia is characterized by a stereotyped behavioral array which is mediated by several neuroactive peptides. We have sequenced two genes encoding the A and B peptides thought to initiate the egg-laying process, as well as a gene encoding egg-laying hormone (ELH) which directly mediates the behavioral array. The three genes share 90% sequence homology and are representatives of a small multigene family. Each gene encodes a protein precursor in which the active peptides are flanked by internal cleavage sites providing the potential to generate multiple small peptides. Each of the three genes consists of sequences homologous to A or B peptide as well as ELH. Although these genes share significant nucleotide homology, they have diverged such that different member genes express functionally related but nonoverlapping sets of neuroactive peptides in different tissues.

Egg-laying hormone: Direct action on the ovotestis of Aplysia

The bag cells are a group of neuroendocrine cells located in the abdominal ganglion of Aplysia californica. These cells induce egg laying in the animal through the release of neurohormone(s). Previous experiments established that an extract of bag cells releases eggs from isolated ovotestis fragments in a dose-dependent manner. (F. E. Dudek and S. S. Tobe, 1978, Gen. Comp. Endocrinol. 36, 618-627.) Experiments presented here were conducted to purify and identify the component(s) of bag cell extract with the egg-releasing activity. Bag cell extracts were fractionated by means of gel-filtration chromatography followed by cation-exchange chromatography. Column eluates were assayed for egg-release activity on isolated ovotestis fragments by the method of Dudek and Tobe (1978). Only one component purified from the crude extract had egg-releasing activity. This component was identified as egg-laying hormone (ELH) based on its purification characteristics, effects on neuronal activity, and migration on thin-layer chromatography. In dose-response studies egg release increased with the concentration of ELH and had a threshold of 8 X 10(-10) M or less. ELH had the same dose-response relationship in egg release assays when present in purified form or as a component of bag cell extract. These data show that ELH acts directly on the ovotestis and that ELH is the only component of bag cell extract with egg-release activity. Taken with the results of other studies (W. D. Branton, S. Arch, T. Smock, and E. Mayeri, 1978. Proc. Nat. Acad. Sci. USA 75, 5732-5736. B. S. Rothman, P. Brownell, and E. Mayeri, 1979. Soc. Neurosci. Abstr. 5, 260. E. Mayeri and B. S. Rothman, 1982.(ABSTRACT TRUNCATED AT 250 WORDS)

A single gene encodes multiple peptide-transmitter candidates involved in a stereotyped behavior

Abstract A multidisciplinary approach is being used to study the roles of peptides in neuronal function and the regulation of behavior in Aplysia . Recent data indicate that a group of neuroendocrine cells, the bag cells, utilize two or more peptide neurotransmitters (and/or neurohormones) that are enzymatically cleaved from a common precursor protein. The gene for this precursor is a member of a small gene family that encodes several different precursor proteins for neuroactive peptides. The combined effects of the bag-cell peptides may serve to regulate the various neural and physiological events underlying a stereotyped pattern of behavior during egg laying.

Coexistence of egg-laying hormone and a-bag cell peptide in bag cell neurons of Aplysia indicates that they are a peptidergic multitransmitter system

Double-label immunocytochemistry reveals that immunoreactivity for two putative peptide transmitters, egg-laying hormone and alpha-bag cell peptide, co-exist in most bag cell somata and processes in the abdominal ganglion of the marine mollusc Aplysia. Together with previous physiological and biochemical data these findings indicate that the neuroendocrine bag cells are a multitransmitter system which utilizes two or more peptides derived from a common precursor.

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R3-R14, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R3-R11 have been postulated to play a role in cardiovascular control, peptide II was without effect at less than or equal to 10(-4) M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.

Routine fast atom bombardment mass spectral analysis of high molecular weight peptides--atrial gland peptides from Aplysia californica.

Three peptides isolated from the atrial glands of Aplysia californica were analysed by Fast Atom Bombardment Mass Spectrometry. Survey scans over the mass range 1650 to 7500 at 500 resolution were used to locate signals for the protonated molecular ion and two subunits which result from cleavage of a single disulfide bond. A more accurate mass determination was made by accumulating scans over a narrow mass range. The amounts of sample used for each measurement ranged between 10 and 30 pmoles. Measured mass values are within 0.5 amu of calculated average molecular weights. Results illustrate the utility of the technique for accurate molecular weight determinations on limited quantities of high molecular weight peptides.

Presence of immunoreactive α-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.