Biology Reference
In-Depth Information
presumably with an on-rate similar to that of the other EGTA derivatives. Ca
2
þ
a
Y
nity drops sharply to
1 mM on photolysis with time constants of 14 and
ciency (0.7) and absorbance (18,400 M
1
cm
1
) are extremely
high, as is the change in Ca
2
þ
a
520
m
s. Quantum e
Y
Y
nity (10,000-fold at pH 7.2), making this a very
attractive candidate for future-caged Ca
2
þ
research.
B. Calculating Changes in [Ca
2þ
]
i
Calculating [Ca
2
þ
]
i
changes on photolysis of NP-EGTA and its congeners is
similar to that for the nitr compounds (if the pH dependence of binding constants
is ignored), since Mg
2
þ
binding is not an issue. Since the chelators' Ca
2
þ
a
nities is
similar to resting cytoplasmic [Ca
2
þ
]
i
levels, filling cells with a half-Ca
2
þ
-loaded
chelator will not disturb [Ca
2
þ
]
i
but can release substantial amounts of Ca
2
þ
(
Y
ers.
However, except for NDBF-EGTA, the low absorbance usually limits flash pho-
tolysis to at most about 20%.
Quantifying changes in [Ca
2
þ
]
i
caused by photolysis is much more di
1 mM) which will be reduced about 100-fold by the cell's endogenous bu
V
cult for
DM-nitrophen. The initial level of [Ca
2
þ
]
i
before photolysis depends upon the total
concentrations of Mg
2
þ
,Ca
2
þ
, DM-nitrophen, ATP, and native Ca
2
þ
bu
Y
V
ers,
because at least two bu
V
ers (DM-nitrophen and endogenous bu
V
ers) compete
for Ca
2
þ
, two bu
ers (ATP and DM-nitrophen) compete for Mg
2
þ
, and, after
partial photolysis, both cations also bind to the two photoproducts. Calculating
equilibrium Ca
2
þ
levels involves simultaneous solution of at least six nonlinear
bu
V
er equations (
Delaney and Zucker, 1990
), which is a tedious chore at best.
Also, the various dissociation constants depend on ionic strength, and have been
measured only at 150 mM. The high a
V
nity of DM-nitrophen for Ca
2
þ
might
Y
ering of Ca
2
þ
in cytoplasm, but this idea is misleading.
A solution of DM-nitrophen that is 50% saturated with Ca
2
þ
will hold the free
[Ca
2
þ
]
i
at 7 nM at pH 7.2; this action will be independent of the total DM-
nitrophen concentration. However, 5 mM DM-nitrophen with 2.5 mM Ca
2
þ
and
5mMMg
2
þ
will bu
V
appear to dominate the bu
er free [Ca
2
þ
]
i
to about 2
m
M; now doubling all concentra-
tions results in a final [Ca
2
þ
]
i
of around 5
m
M. Since the total [Mg
2
þ
]
i
available, as
free or weakly bound to ATP, is several millimolar, partially Ca
2
þ
-loaded DM-
nitrophen may bring the resting Ca
2
þ
level to a surprisingly high level. Because the
solution is still bu
V
ered, this [Ca
2
þ
] may be reduced only gradually by pumps and
uptake, but eventually Ca
2
þ
will be pumped o
V
the DM-nitrophen until the [Ca
2
þ
]
i
is restored to its normal level. Then photolysis may lead to only tiny jumps in
[Ca
2
þ
]
i
. However, if a large amount of Ca
2
þ
-loaded-DM-nitrophen is introduced
into a cell relative to the total [Mg
2
þ
]
i
,Ca
2
þ
can be bu
V
ered to low levels while
photolysis can release a large amount. In fact, if enough DM-nitrophen is intro-
duced into cells with no added Ca
2
þ
, it may gradually absorb Ca
2
þ
from cytoplasm
and intracellular stores and photolysis can produce a substantial jump in [Ca
2
þ
]
i
.
Therefore, both resting and the postphotolysis levels of Ca
2
þ
may vary over very
wide ranges, depending on [DM-nitrophen]
i
, [Mg
2
þ
]
i
, and cellular [Ca
2
þ
]
i
control
V
