The neuropeptide proctolin induces phosphorylation of a 30 kDa protein associated with the thin filament in crustacean muscle
Berit Brüstle1,
Sabine Kreissl1,*,
Donald L. Mykles2 and
Werner Rathmayer1
1 Department of Biology, University of Konstanz, D-78457 Konstanz, Germany, and
2 Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
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Fig.1. Proctolin immunoreactivity on extensor muscles and in pereion ganglion 4 of I. emarginata. (A) Immunostained varicose axons extend along the inner layer of long fibres in pereion segment 7 and pleon segment 1. Numbers indicate identified muscle fibres. Anterior is up in all parts of the figure. Scale bar, 200µm. (B) Proctolin immunostaining reveals a symmetric pair of neurones (arrowheads) in the anterior part of the ganglion in pereion segment 4. (C) Somata of efferent neurones projecting through N3 in the same ganglion, stained by retrograde labelling with Lucifer Yellow (arrowhead indicates the anterior motoneurone). (D) Double exposure of the specimen reveals the double labelled proctolin-ir motoneurone (arrowhead). Scale bar, 100µm.
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Fig.2. Effect of proctolin on caffeine-induced contractures. (A) Potentiation of caffeine (10mmoll-1) contracture by 1µmoll-1 proctolin. Two controls are shown, before and after the exposure to proctolin. (Caff, caffeine; Proc, proctolin). (B) Averages of normalised tensions obtained from 4 experiments. Control represents the average of three caffeine contractures per experiment prior to the proctolin tests. Values are means ± S.D., *P<0.05, Student’s t-test.
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Fig.3. (A) Electrophoretic separation of I. emarginata extensor muscle fibres on a 15% SDS gel. The positions of molecular mass standards (kDa) are indicated. (B) Effect of proctolin (1µmoll-1) on serine phosphorylation of I. emarginata extensor muscle proteins. The 30kDa protein band of the proctolin-treated sample (arrow) shows an increased phosphorylation signal compared to the 30kDa protein band of the untreated sample. Molecular mass standards are indicated by arrowheads. Although differences in phosphorylation of a 31kDa, 35kDa and a 36kDa protein are seen in this experiment, these effects do not occur consistently as an effect of proctolin incubation. (C) Densitometric analysis of B. The 30kDa protein band is marked (arrow).
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Fig.4. Changes in relative extinction of the 30kDa protein and a 70kDa protein (for comparison) upon treatment with 1µmoll-1 proctolin. Summary of 11 experiments. The relative changes in the extinctions of the 30kDa and the 70kDa band of proctolin-treated samples and untreated samples were compared in each experiment. The extinctions result from immunoreactivity of the anti-phosphoserine antibody. Values are means ± S.D., *P<0.05, Student’s t-test.
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Fig.5. Electrophoretic separation and western blot analysis of I. emarginata myosin, actomyosin and thin filament on 15% SDS gels. The positions of molecular mass standards (kDa) are indicated. (A) In the myosin extract three myosin light chains (myosin LC) were identified (asterisks). In myosin LC extracts obtained by guanidine–HCl precipitation two of these light chains (14kDa and 18.5kDa) are present. (B) The actomyosin and thin filament extracts contain a protein with the apparent molecular mass of 30kDa (arrow), whereas no such protein is present in the myosin extract. (C) Western blot analysis with a phosphoserine antibody reveals a number of high molecular mass proteins and two proteins at 30kDa and 31kDa in thin filament preparations. (D) In I. emarginata muscle homogenates and thin filament preparations, a major band at 30kDa (arrow) and a minor band at 31kDa are stained in western blot analysis with antiserum against lobster troponin I3.
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© The Company of Biologists Ltd 2001
