Troponin from Akazara scallop striated adductor muscles.

T Ojima,K Nishita

doi:10.1016/s0021-9258(18)66629-3

Abstract

Troponin was isolated from striated adductor muscles of the "Akazara" scallop (Chlamys nipponensis akazara), and purified in an active form by DEAE-cellulose (Whatman DE52) column chromatography and subsequent gel filtration on Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:1:1. The three components were separated from each other by column chromatography in the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAE-cellulose. The Mr 20,000 component was regarded as troponin C according to the Ca2+-binding properties, which was found to bind 0.7 mol of Ca2+/mol at 0.1 mM Ca2+. The association constant of Ca2+ to troponin C was estimated to be 5 X 10(5) M-1, and was not affected by the addition of 2 mM MgCl2. The Mr 52,000 component appeared to be troponin I, since it inhibited, together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin reconstituted from rabbit myosin and actin, and the inhibition of the ATPase activity was diminished by the addition of Akazara troponin C. Finally, the Mr 40,000 component appeared to be troponin T, since it co-precipitated with actin-tropomyosin filament and was indispensable with Akazara troponin C and the Mr 52,000 component (troponin I) for conferring the Ca2+ sensitivity to reconstituted actomyosin.

Highlights

The three componentswere separated from each other by columnchromatographyin the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAEcellulose
Component appeared to be troponin T, since it co-pre- Mg-ATPase activity was measured at 15 “C in a reaction medium cipitated with actin-tropomyosin filament and was in- containing 30 mM KCl, 20 mM Tris maleate, 2 mM MgC12, dispensable with Akazara troponin C and the M, 1mM ATP, and either 0.2 mM EGTA o0r
In the early 1970s, it was believed that molluscan muscles Preparation of Akazara Troponin and Tropomyosin-Akapossess only a myosin-linked regulatory system [5].zara troponin and tropomyosinwere prepared essentially by the isolation of troponin from squid mantlemuscles [11]and the methods of Ebashi et al [22]

Summary

Takao Ojima and Kiyoyoshi Nishita

From the Departmentof Chemistry, Faculty of Fisheries, Hokkaido University, Hakwlate, Hokkaido, Japan. Troponin was isolated from striated adductor mus- troponin fromscallopmuscles has not yet been successful cles of the “Akazara” scallop (Chlamys nipponensis (althoughthinfilamentsthat conferCa2+dependence on akazara), and purified in an active formbyDEAEcellulose (Whatman DE52) column chromatography and subsequent gel filtrationon Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:l:l. The M , 52,000 comvertebrate myosin ATPase were obtained[14]),andlittle information isavailable concerning the subunit structure and actual functionof molluscan troponin; thephysiological relationship between myosin-linked and actin-linked regulations in molluscan muscles remains obscure. In this paper,we report the isolation of troponin from the striated adductormuscles of Akazara scallop and the characponent appeared to be troponin I, since it inhibited, terization of its components. Together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin recon-

MATERIALS AND METHODS

RESULTS

Fraction Number

40KT rn'

DISCUSSION

Akazara from Troponin