Sulfhydryl Involvement in Fusion Mechanisms - Fusion of Biological Membranes and Related Problems - page 490

chaperone for other proteins besides those involved in membrane fusion
(Haas, 1998). There are two ATPase domains in NSF (Tagaya et al., 1993),
referred to as D1 and D2, which differ in affinity for ATP (Matveeva et al.,
1997).The low-affinity sites, which are on the D1 domains, appear to be crit-
ical for NSF to associate with the SNAP-SNARE complex (Matveeva et al.,
1997; Whiteheart et al., 1994).
It -phosphate

binding loop of D1 contains the thiol that reacts with N -ethylmaleimide
(Tagaya et al., 1993). Consistent with this hypothesis is the fact that the yeast
homologue of NSF, Sec18p, has a threonine residue at the corresponding
position and is resistant to inactivation by N -ethylmaleimide (Steel et al.,
1999). However, it would seem prudent if the site of modification were
determined directly; N -ethylmaleimide treatments that abolish ATPase
activity do not appear to inhibit ATP binding, as might have been expected
if the modification were in the

has been suggested that a cysteine residue in the β

β -phosphate binding loop (Matveeva et al.,

1997).

2.1.2. Calpains

It would appear that the most prominent example of the effects of
thiol-reagent treatments upon membrane fusion is a protein whose pre-
sumed modified cysteine is not, in fact, essential for function; it is the mod-
ification that is inhibitory. Potential targets of thiol-modifying reagents
that do possess active-site cysteine residues are the calpains (intracellular,
Ca 2+ -dependent thiol proteases). The possibility of the participation of cal-
pains in various membrane-fusion processes has been investigated. The
membrane mobility agent 2-(2-methoxyethoxy)ethyl- cis -8-(2-octylcyclo-
propyl)octanoate(A 2 C) promotes fusion of rat erythrocytes in the presence
of Ca 2+ . Cell fusion and the Ca 2+ -dependent membrane-protein degradation
that appears to precede it are prevented by pretreatment of the erythro-
cytes with N -ethylmaleimide or monobromobimane (Glaser and Kosower,
1986). In contrast, there was no effect when the cells were preincubated
with pepstatin, phenylmethylsulphonyl fluoride, or 1,10-phenanthroline,
which are inhibitors, respectively, of aspartyl proteases, serine proteases,
and metalloproteases. Erythrocyte ghosts undergo A 2 C and Ca 2+ -dependent
cell fusion and membrane-protein degradation solely when they
contain hemolysate. Cell fusion and membrane-protein degradation are not
detected with erythrocyte ghosts reconstituted with hemolysate treated
with N -ethylmaleimide or monobromobimane (Glaser and Kosower, 1986).
A partially purified proteolytic activity, that possessed the biochemical and
chromatographic characteristics of calpain I, when loaded with Ca 2+ into the