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Biol Bull 142: 446-460. (June 1972)
© 1972 Marine Biological Laboratory
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ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF A PEPTIDE WHICH STIMULATES THE HINDGUT OF THE COCKROACH, LEUCOPHAEA MADERAE (FABR.)

G. MARK HOLMAN 1 and BENJAMIN J. COOK 1

1 Metabolism and Radiation Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Fargo, North Dakota 58102

1. Ethanol extracts of hindguts from L. maderae contained a material that stimulated the myogenic activity of the isolated organ. Ion-exchange chromatography of the ethanol extracts resulted in a 2000-fold purification of the active residue, and thin-layer chromatography demonstrated that the activity was associated with a single ninhydrin-positive spot.

2. The biological activity of the extracted substance was not affected by incubation for 3 hours with chymotrypsin. However, pronase completely destroyed the activity within 30 minutes, which suggests that the active material is a peptide. Gel filtration experiments indicated that the molecular weight is in the 400-600 range.

3. The peptide was isolated from extracts of terminal ganglia, proctodaeal nerves, and heads of the cockroach but not from the foregut. Interestingly, the myogenic activity of this organ was not affected by exposure to extracts from the hindgut. The peptide was found in the two cockroaches, L. maderae and P. americana, and the grasshopper, S. vaga. It was not found in the head of the house fly, M. domestica nor in fifth-instar larvae of the tobacco hornworm, M. sexta.

4. The characteristic response of the hindgut to this peptide was a sustained increase in the tonus, frequency, and amplitude of contractions. An excitation threshold was obtained with only 0.004 hindgut equivalents per milliliter or 13 ng of dry residue per milliliter. Both the isolated rectum and colon responded to the peptide, and although tetrodotoxin (10-6 g/ml) blocked neurally evoked contractions, the visceral muscle of the hindgut showed no change in sensitivity to the extracted peptide.

5. Cockroach innervated heart preparations showed a noticeable increase in frequency, amplitude, and coordination of contractile events when exposed to 0.3 µg of dry residue. If 1.0 µg or more of the dry residue was added to the heart, activity was greatly depressed.

6. Both histological and cytological evidence of neurosecretion were obtained from the proctodaeal nerve. Preliminary centrifugation experiments suggest that the extracted peptide is associated with the particulate fraction at 20,000 g. Finally, a number of reasons for considering our extracted peptide as a neurohormone are discussed.






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Copyright © 1972 by the Marine Biological Laboratory.