Elsevier

Peptides

Volume 19, Issue 4, April 1998, Pages 619-626
Peptides

Original Articles
Isolation and Identification of a Second Diuretic Hormone from Tenebrio molitor 1

https://doi.org/10.1016/S0196-9781(97)00475-0Get rights and content

Abstract

A diuretic hormone (DH) of unusual structure was isolated from extracts of heads of Tenebrio molitor. The hormone is a 47 amino acid peptide, Mr = 5,029.9, with the sequence AGALGESGASLSIVNSLDVLRNRLLLEIARKKAKEGANRNRQILLSL. This peptide increases cyclic AMP production in Malpighian tubules of T. molitor. We recently identified a smaller DH from T. molitor with 37 amino acids; these peptides have only 15 identical amino acids when aligned to maximize similarity to other members of the insect DH family. This family has sequence similarity to the corticotropin-releasing factor superfamily of vertebrate peptides.

Section snippets

Experimental Animals

T. molitor were reared as described earlier [14]. Heads of pupae were collected and stored at −80°C as described [14].

Bioassay

DH activity in fractions from all purification steps was detected by the elevation of cAMP in isolated Mt dissected from newly emerged adult T. molitor as described earlier [14]. Preparation of aliquots of fractions for bioassay was also performed as already described [14].

Extraction and Preliminary Purification (Step 1)

Collected heads (fresh weight ≈103 g) from ≈20,000 T. molitor pupae were homogenized in cold methylene

Extraction and Preliminary Purification (Step 1)

The preliminary purification used a Vydac C4 reversed-phase cartridge as already described for identifying T. molitor DH37 [14]. Fractions were assayed for the presence of a DH based on their ability to stimulate cAMP production in Mt of T. molitor. Strong diuretic activity was found in the fraction that eluted from the cartridge with 45% acetonitrile.

Liquid Chromatographic Purification (Steps 2 to 9)

The 45% CH3CN fraction from above was diluted with 12 volumes of 0.1% TFA and then loaded onto a Vydac C4 semipreparative column (Step 2)

Discussion

We report the identity of a second DH in head extracts of the coleopteran insect T. molitor. We named this hormone T. molitor DH47; the subscripted number signifies that it is a 47 amino acid peptide. Earlier, we identified a smaller DH from this species that we now designate T. molitor DH37 for consistency (rather than Tem-DH1). Both of these DH are members of a family of peptides that have sequence similarity to the sauvagine-CRF-urotensin I family of peptides from vertebrates (Fig. 2).

Acknowledgements

We thank Hewlett–Packard for sequence analysis of the N-terminal 40 residues of T. molitor DH47, Chris Grimley of Applied Biosystems for amino acid sequencing of the C-terminal fragment, Dr. Arnold Falick of PerSeptive Biosystems for the MALDI MS analysis of the C-terminal fragment, and Dr. George C. J. Fernandez for statistical analysis of the dose/response data. This work was supported by NIH #GM48172, and is a contribution of the Nevada Agriculture Experiment Station. We thank Dr. Houle Wang

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    1

    Taken in part from a paper presented at a Joint Meeting of the European Neuropeptide Club-Winter Discussion and the Winter Neuropeptide Conference February 8–11, 1997, Kitzbühel, Austria.

    2

    Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.

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