摘要
近年来不少工作者对叶绿体偶联因子复合物的结构、功能和发生的问题颇为关注(程秋琛等 1986,Merchant等 1985,Nelson 1982,Pick等1979,Strontman等1983,Suss等 1983)。
The term chloroplast CF_0-CF_1 complex of
higher plant is used to designate a complex of
energy-transducing ATP Synthase which is
associated with a movement of proton across
thylakoid membranes. The complex can be
functionally divided into two parts: an integral
membrane sector (CF_0), which is embedded in
the lipid core of the membrane and acts as a
proton channel, and an extrinsic membrane
sector (CF_1), which emerges from the membrane
and contains the catalytic sites responeible for
ATP synthesis or hydrolysis. There exists no
simple and economical procedure for the isola-
tion of a highly active CF_0-CF_1 complex from
higher plant with the full subunit composition.
In this paper we described a procedure for
the isolation and purification of CF_0-CF_1 com-
plex from higher plant chloroplasts. The CF_0-
CF_1 complex from pea chloroplasts has been
isolated by using a newly developed detergent
CHAPS which was introduced as an alternative
to some detergents such as cholate, Triton X-100
and octylglucoside to solubize membrane pro-
tein. The procedure for the isolation of CF_0-
CF_1 complex from pea chloroplasts involves
four main steps. The first Involves washing
chloroplast membranes with 10 mmol/L Na-
PPi (pH 8.0) buffer three times. The mem-
branes which were washed thoroughly yielded
a final CF_0-CF_1 complex preparation with very
little high molecular weight contaminants.
The second involved using. CHAPS to solubilize
the membrane-bound CF_0-CF_1 complex. There
is a constant optimum contentration lying be-
tween 0.1%～1.0%. The third involved using
37%～45% (NH_4)_2 SO_4 to precipitate the CF_0-
CF_1 protein from the CHAPS extract. The
protein precipitate by 45% (NH_4)_2SO_4 showed
the activity of ATP-^(32)p exchange reaction in
proteoliposomes. Then the protein precipi-
tate was suspended in a small volume STK buf-
fer. The sample was further purified by sucrose
gardient centrifugation in the presence of 0.2%
CHAPS. The activity of ATP-^(32)p exchange
reaction of CF_0-CF_1 in different fractions of
sucrose gradient which was reconstitute in
liposomes is distributed in the fourth to eighth
fractions. The highest specific activity of ATP-
^(32)p exchange of proteinoliposomes is 107.2 nmoll
ATP mg^(-1) protein min^(-1).The subunit compo-
sition of partly purified CF_0-CF_1 complex is
shown in Fig.7.
We have founed five subunits bands of
CF_1 and two additional bands 1n the map of gel
electrophoresis. The two additional bands
have approximate molecular weights of 22 000
and 14 000 when calibrated against standard
proteins. But the protein band with mol. wt
<14 000 was not alway present in all
experiment,which was possibly related to the
proportion with the smallest subunits in the
CF_0-CF_1 complex loaded on gel.
基金
美国NIH基金和中国国家自然科学基金
关键词
洗涤剂
叶绿体
豌豆
detergent CHAPS
pea chloroplasts
CF_1-ATPase
chloroplast CF_0-CF_1 complex
