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First published online November 30, 2007
Journal of Experimental Biology 210, 4465-4470 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.012088

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Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila

R. Elwyn Isaac^1,*, Erik C. Johnson², Neil Audsley³ and Alan D. Shirras⁴

¹ Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK
² Department of Biology, Wake Forest University, NC, USA
³ Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK
⁴ Department of Biological Sciences, University of Lancaster, LA1 4YQ, UK

View larger version (5K): [in this window] [in a new window]   Fig. 1. Rates of hydrolysis of insect peptides by neprilysin. Rates were determined using HPLC to measure the linear decline in the peptide substrate during incubation with human neprilysin, as described in Materials and methods. Values are the means ± s.e.m. of at least three determinations. A unit of activity is 1 nmole of peptide hydrolysed per ng of neprilysin.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Hydrolysis of PDF by recombinant human neprilysin and D. melanogaster head membranes. Peptide fragments generated after incubation of PDF with either (A) neprilysin (1.25 ng) or (B,C) D. melanogaster head membranes (1 µg protein) were separated by HPLC with the UV-monitor set at 214 nm (0.2, full scale absorbance units). The identities of the metabolites PDF1-7 and PDF8-18 were established by mass spectrometry as described in Materials and methods. (C) Pre-incubation of the membranes with 10 µmol l–1 phosphoramidon abolished the PDF-degrading activity (I, inhibitor peaks).

View larger version (6K): [in this window] [in a new window]   Fig. 3. Inhibition of PDF-degrading activity of D. melanogaster head membranes by phosphoramidon and thiorphan. Inhibition curves for phosphoramidon (filled triangles, IC50=0.15 µmol l–1) and thiorphan (open squares, IC50=1.2 µmol l–1) were generated by measuring the formation of PDF8-18 from PDF in the presence of different concentrations of inhibitors, as described in Materials and methods. Data are expressed relative to uninhibited activity and IC50 values were calculated using FigP (Biosoft).

View larger version (6K): [in this window] [in a new window]   Fig. 4. Dose–response curve for the activation of the PDF receptor of D. melanogaster by PDF, PDF1-7 and PDF8-18. The ability of peptides to increase intracellular cAMP levels was determined using HEK293 cells co-transfected with the PDF receptor gene (CG13758) and a firefly luciferase reporter gene construct, as described in Materials and methods. Results are expressed as the mean percentage increase in luciferase activity above basal levels from three replicate wells from three independent transfections (s.e.m.=5–20%). The effects of PDF1-7 and PDF8-18 on the activity of intact PDF were tested by varying the dose of PDF whilst keeping the concentration of each PDF fragment constant at 10–5 mol l–1. PDF, solid circle; PDF1-7, open circle; PDF8-18, closed triangle; PDF+PDF1-7, open triangles; PDF+PDF8-18, open square.