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Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica

Chiu, A. Y. and Hunkapiller, M. W. and Heller, E. and Stuart, D. K. and Hood, L. E. and Strumwasser, F. (1979) Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica. Proceedings of the National Academy of Sciences of the United States of America, 76 (12). pp. 6656-6660. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:CHIpnas79

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Abstract

Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In the present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a Mr of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln- Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with [35S]methionine or [3H]leucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent Mr of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20°C.


Item Type:Article
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http://www.pnas.org/content/76/12/6656.abstractPublisherUNSPECIFIED
Additional Information:© 1979 by the National Academy of Sciences. Communicated by Theodore H. Bullock, August 15, 1979. We thank Dr. L.K. Kaczmarek for comments on the manuscript and Mr. Vince Farnsworth, Mr. Dan Viele, and Ms. Annette Yuen for technical assistance. The work was funded by National Institutes of Health Grants NS-07071 and NS-15183 to F.S., Grant GM-06965 to L.E.H., and Biomedical Research Support Grant RR 07003 and by the B.W. Foundation. Predoctoral support came from the Lawrence A. Hanson Foundation Fellowship, Spencer Foundation Fellowship (A.Y.C.), National Institutes of Health Training Grant GM-0086, and the Arthur McCallum Fund (D.K.S.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
National Institutes of HealthNS-07071
National Institutes of HealthNS-15183
National Institutes of HealthGM-06965
National Institutes of HealthRR 07003
B. W. FoundationUNSPECIFIED
Lawrence A.Hanson FoundationUNSPECIFIED
Spencer FoundationUNSPECIFIED
National Institute of HealthGM-0086
Arthur McCallum FundUNSPECIFIED
Subject Keywords:bag cells; amino acid sequence; neurosecretory peptide; molluskan neuropeptide; microsequence analysis
Record Number:CaltechAUTHORS:CHIpnas79
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CHIpnas79
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11460
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:22 Aug 2008 04:08
Last Modified:26 Dec 2012 10:14

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