Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I).
The aim of this study was to isolate and to characterize actin from the carp liver cytosol and to examine its ability to polymerize and interact with bovine pancreatic DNase I. Carp liver actin was isolated by ion-exchange chromatography, followed by gel filtration and a polymerization/depolymerization cycle or by affinity chromatography using DNase I immobilized to agarose. The purified carp liver actin was a cytoplasmic beta-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle alpha-actin. Polymerization of purified carp liver actin by 2 mM MgCl(2) or CaCl(2) was only obtained after addition of phalloidin or in the presence of 1 M potassium phosphate. Carp liver actin interacted with DNase I leading to the formation of a stable complex with concomitant inhibition of the DNA degrading activity of DNase I and its ability to polymerize. The estimated binding constant (K(b)) of carp liver actin to DNase I was calculated to be 1.85x10(8) M(-1) which is about 5-fold lower than the affinity of rabbit skeletal muscle alpha-actin to DNase I.
Ciszak
L
L
Department of Cell Pathology, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148, Wroclaw, Poland.
Krawczenko
A
A
Polzar
B
B
Mannherz
H G
HG
Malicka-Blaszkiewicz
M
M
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002347:Carps; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism*; D011108:Polymers; D013050:Spectrometry, Fluorescence
D000199:Actins; D011108:Polymers; D003850:Deoxyribonuclease I