Two Active Fragments of Outer Arm Dynein from Tetrahymena Cilia; Purification of Fragment-β and -γ

URI http://harp.lib.hiroshima-u.ac.jp/pu-hiroshima/metadata/12356
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Title
Two Active Fragments of Outer Arm Dynein from Tetrahymena Cilia; Purification of Fragment-β and -γ
Author
氏名 Masuyama Etsuko
ヨミ マスヤマ エツコ
別名
Abstract

When dynein obtained by dialyzing Tetrahymena ciliary axonemes against a low ionic strength buffer was digested with various amounts of thermolysin at 27 ℃for 30 min, the ATPase activity increased about four-fold and degradation of dynein heavy chains was observed by SDSPAGE. The digested dynein with thermolysin (dynein : thermolysin=10 : 1, by weight) was also analyzed by PAGE using ATPase active staining. Dynein having a large molecular mass was consistently degraded into two smaller and stable active fragment bands. Other major fragment bands were not observed in polyacrylamide gel electrophoretic patterns. Furthermore, to purify the dynein fragments, the digested dynein was chromatographed on Q-Sepharose column and two peaks possessing high ATPase activities were eluted in 0.15 M and 0.25 M NaCl in HEPES buffer (pH 7.4), respectively. Both molecular weights of the purified fragments were estimated to be approximately 400 kDa by gel filtration and both of the specific activities were 8-9 μmol Pi / mg per min per 1 mM ATP. The two fragments were composed in equal molar ratio of approximately 178 k, 89 k, and 50 kDa, and 178 k and 126 kDa, respectively. Futhermore, affinity purified
polyclonal antibodies against the two purified fragments cross-reacted with β and γ heavy chains of the outer arm dynein, respectively, on immunoblots. We concluded that the two fragments were very similar to fragment A from sea urchin with respect to the molecular mass, the polypeptide composition and the activation of ATPase.

Journal Title
県立広島大学人間文化学部紀要
Volume
11
Spage
43
Epage
54
Published Date
20160301
Publisher
県立広島大学
ISSN
2186-5590
NCID
AA12116573
Language
eng
NIIType
Departmental Bulletin Paper
Text Version
出版社版
Sort Key
002
Set
pu-hiroshima