A soluble calcium-binding protein from the terrestrial annelidLumbricus terrestris L. |
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Authors: | R Huch J D'Haese Ch Gerday |
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Institution: | (1) Lehrstuhl für Morphologie und Zellbiologie, Institut für Zoologie, Universitätsstrasse 1, D-4000 Düsseldorf 1, Federal Republic of Germany;(2) Laboratoire de Biochimie musculaire, Université de Liège, Sart Tilman B 6, B-4000 Liège 1, Belgium |
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Abstract: | Summary Soluble calcium-binding proteins (SCBP) considerably different from calmodulin were purified from the body wall muscle of the earthwormLumbricus terrestris. Three isoforms were obtained with similar UV absorption spectra and amino acid compositions and an apparent molecular weight close to 20 kDa. They can be distinguished by their histidine and proline content and by their peptide maps. The tissue content, as determined by quantitative ELISA varies individually from 0.1 to 0.3 mmol kg–1. The calcium-binding property can be demonstrated by Ca2+-dependent electrophoretic mobility shift and45Ca2+ autoradiography on nitrocellulose sheets. The apparentK
D values for the SCBP-Ca2+ complex is approximately 10–7 mol l–1 as revealed by euquilibrium and flow dialysis experiments. In the presence of 1 mmol l–1 MgCl2 the maximum binding capacity of SCBP was determined to be either 2 mol Ca2+ mol–1 protein (SCBP2) or 3 mol Ca2+ mol–1 protein (SCBP3). Preliminary studies concerning the functional role of SCBP indicate that it facilitates the diffusion of Ca2+ ions by a factor of 2 and is capable of inhibiting the ATPase of isolated body wall muscle actomyosin. The results reveal that earthworm SCBP are similar to vertebrate parvalbumin and to SCBP characterized from aquatic invertebrates.Abbreviations
ABTS
2,2 -azino-di-(3-ethyl)-benzothiazolinsulfonate
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CN-PDE
3 :5 -cyclic nucleotide-phosphodiesterase
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DEAE
diethylaminoethyl
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EGTA
ethyleneglycol-bis( -aminoethyl ether)-N,N,N ,N -tetraacetic acid
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ELISA
enzyme linked immuno sorbent assay
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HEPES
N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid
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HPLC
high performance liquid chromatography
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HRP
horseradish peroxydase
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PAGE
polyacrylamide gel electrophoresis
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PBS
phosphate buffered saline
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P
i
inorganic phosphate
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PMSF
phenylmethylsulfonyl fluoride
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SCBP
soluble calcium-binding protein
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SDS
sodium dodecyl sulphate
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SPDP
N-succininydyl-3-(2-pyridyldithio)propionate
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SR
sarcoplasmic reticulum
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Tris
tris(hydroxymethyl)-aminomethane
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UV
ultraviolet |
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