Biomedical Engineering Reference
In-Depth Information
cortical vesicles that fuse with the plasma membrane are tightly associated
with its cytoplasmic face in the mature egg before fertilization. Fragments
of egg cortex that contain the cortical vesicles and the plasma membrane
can be utilized as a cell-free system for studying Ca
2+
-induced exocytosis
(Vogel
et al.,
1991). The cortical vesicles and plasma membranes can also
be isolated from one another and then recombined to reconstitute Ca
2+
-
riggered exocytosis (Vogel
et al.,
1991).
N
-ethylmaleimide treatments
inhibit the fusion step in both the egg-cortex-fragment and reconstituted
systems (Jackson and Modern, 1990). The
N
-ethylmaleimide-sensitive pro-
teins appeared to be firmly associated with the membranes (Jackson and
Modern, 1990).
Interestingly, reconstitution of exocytosis could be achieved by
recombination of
N
-ethylmaleimide-treated plasma membrane with
untreated cortical vesicles or of
N
-ethylmaleimide-treated cortical vesicles
with untreated plasma membrane, whereas the combination of
N-
ethylmaleimide-treated plasma membrane and
N
-ethylmaleimide-treated
cortical vesicles was inactive (Jackson and Modern, 1990). These data sug-
gested that the
N
-ethylmaleimide-sensitive protein(s) resided in both the
plasma membrane and cortical vesicles and could be supplied by either one
(Jackson and Modern, 1990).
It has been demonstrated that the cortical granules will themselves fuse
in the presence of Ca
2+
in a reaction that is inhibited by
N
-ethylmaleimide
(Vogel
et al.,
1992; Vogel and Zimmerberg, 1992). Untreated vesicles will
fuse with
N
-ethylmaleimide-treated vesicles, thereby demonstrating that
the
N
-ethylmaleimide-sensitive proteins that promote fusion need be on
only one of the fusing membranes (Vogel
et al.,
1992). There appears to be
no requirement for cytoplasmic proteins in the fusion reaction. In addition,
the cortical granules fuse with liposomes in an
N
-ethylmaleimide-inhibit-
able fashion (Vogel
et al.,
1992).
Additionally, it has been demonstrated that treatment of the mem-
branes with 3-(2-pyridyldithio)propionate coupled to dextran (dextran-
PDP) inhibited granule exocytosis in a reaction that was reversible by
subsequent addition of dithiothreitol (Whalley and Sokoloff, 1994).
The dextran-PDP appeared to react with the same thiols as the
N -
ethylmaleimide, because it prevented the irreversible inhibition of fusion
by
N
-ethylmaleimide. These data suggested that the functionally significant
thiols are readily exposed on the membranes.
2.2.6. Microsome Fusion
Homotypic fusion of microsomes derived from disrupted rat liver
endoplasmic reticular membranes is dependent on GTP and inhibited by
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