Under unisite conditions (ratio of ATP to chloroplast coupling factor
(CF0CF1), approximately 1:2.8), spinach thylakoid ATPase depends on pr
ior reductive activation of CF1, just as multisite ATPase does, and is
sensitive to removal of CF1, by EDTA, Faster rates in room light than
in semidarkness and up to 80% inhibition by uncouplers only in room l
ight indicate a strong effect of protonmotive force, which can be prov
ided by room light. In addition, unisite ATPase is inhibited by azide
as long as some ADP is bound to the CF1. Several differences were foun
d between unisite and multisite ATPase, 1) The unisite activities of b
oth membrane-bound and free enzyme were stimulated up to 3-fold by 4 m
M free MgCl2 (a strong inhibitor of multisite ATPase). 2) Thylakoid un
isite ATPase was inhibited by sulfite (50% inhibition at 5 mM), a powe
rful activator of multisite ATPase. This inhibition is attributed to a
nonspecific ionic strength effect. 3) Unisite ATPase was inhibited by
trypsin treatment, which increases multisite ATPase severalfold. 4) T
he pH profile of thylakoid unisite ATPase is somewhat different from t
hat of mul tisite. 5) Alkylation of Cys-89 of the gamma subunit by N-e
thylmaleimide did not affect the unisite activity, but inhibited multi
site activity more than 90%.