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6th Internet World Congress for Biomedical Sciences

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ADP-RIBOSYLATION OF FILAMENTOUS ACTIN INDUCES ITS DEPOLYMERIZATION - THE ROLE OF ADP-RIBOSYLATION IN CYTOSKELETAL REORGANIZATION

Masaharu Terashima(1), Makoto Shimoyama(2), Mikako Tsuchiya(3)
(1)(2)(3)Department of Biochemistry. Shimane Medical University - Izumo. Japan

[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS AND DISCUSSION] [FIGURES] [ACKNOWLEDGEMENTS] [REFERENCES] [Discussion Board]
INTRODUCTION Previous: Testosterone modifies arylamidase activities in serum and different tissues of mice: an in vivo study. Previous: Intrinsic Membrane Properties and Synaptic Inputs Regulating The Firing Activity of the Dopamine Neurons. RESULTS AND DISCUSSION
[Biochemistry]
Next: An extractable phosphatase inhibitor is present in the CSF of subarachnoid hemorrhage patients with vasospasm 		[Cell Biology & Cytology]
Next: Upregulation of phase II enzymes decreases the oxidative stress induced cell death in astrocytes.

MATERIAL & METHODS

[adenylate-32P]NAD (29.6 TBq/mmol) was purchased from New England Nuclear. ADPRT (ADP-ribosyltransferase) was purified from the chicken heterophils as described previously (2). G-actin was prepared from chicken breast muscle by polymerization-depolymerization cycles and an anion exchange column chromatography according to Spudich and Watt and further purified by the gel filtration on a Sephadex G-150 column (3). Purity of the actin was more than 95% determined by SDS/PAGE, and the preparation should not contain ADP-ribosylated G-actin because the modified actin can not polymerize (3,4). G-actin was incubated with 50 mM KCl and 2 mM MgCl2 at 30C for 2 hr, and centrifugated at 105, 000xg for 1 hr. Resultant precipitate was resolved in the buffer containing 10 mM imidazole, pH 7.5, 0.2 mM CaCl2, 0.75 mM 2-mercaptoethanol, 50 mM KCl and 2 mM MgCl2 and used as F-actin fraction (4).

Purified actin (5 µg) was incubated with or without purified heterophil ADPRT (10 ng) in the reaction mixture containing 50 mM Tris/HCl, pH 9.0, 5 mM dithiothreitol and 0.1 mM [32P]NAD (7.4 kBq/nmol) at 25C for 15 min, and analyzed by SDS/PAGE and autoradiography. In some cases, radioactivity in the acid-insoluble fraction of the reaction mixture was measured using scintillation counter.

Increase in the viscosity of actin solution was used to estimate the increase in F-actin contents of the solution. The viscosity of the solution was measured at 25C as a flow time required for the solution to pass through a glass capillary tube, Cannon-Fenske viscometer. Relative viscosity, hr, was determined as the ratio of the flow time of actin solutions incubated with MgCl2 to that without MgCl2. Specific viscosity, hs, was calculated by the following equation; hs = hr-1. Contents of G- and F-actins in the solution were also determined with ultracentrifugation at 105,000 xg for 1 hr followed by measurement of protein amounts in the resultant supernatant and precipitate fractions, respectively.

Discussion Board
Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS AND DISCUSSION] [FIGURES] [ACKNOWLEDGEMENTS] [REFERENCES] [Discussion Board]
INTRODUCTION Previous: Testosterone modifies arylamidase activities in serum and different tissues of mice: an in vivo study. Previous: Intrinsic Membrane Properties and Synaptic Inputs Regulating The Firing Activity of the Dopamine Neurons. RESULTS AND DISCUSSION
[Biochemistry]
Next: An extractable phosphatase inhibitor is present in the CSF of subarachnoid hemorrhage patients with vasospasm 		[Cell Biology & Cytology]
Next: Upregulation of phase II enzymes decreases the oxidative stress induced cell death in astrocytes.
Masaharu Terashima, Makoto Shimoyama, Mikako Tsuchiya
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Last update: 5/01/00