Biology Reference
In-Depth Information
2. Lowering [Ca
2
þ
] to a Level Approaching 0
If one desires a medium in which the free Ca
2
þ
concentration approaches
''zero,'' one can prepare nominally calcium-free medium
16
that also contains
EGTA or BAPTA at millimolar concentrations. Again, to avoid problems of
rapid weakening of the Ca
2
þ
a
Y
nity of EGTA with decreasing pH, using
BAPTA at pH
<
7 is more convenient.
3. Setting Extracellular [Ca
2
þ
] to a Precisely Known Value
When the extracellular [Ca
2
þ
] must be known precisely, medium containing a
well-defined Ca
2
þ
bu
er system at a fixed pH must be made. The preparation of
such solutions is detailed in Chapter 9.
V
C. Divalent Cation Ionophores
1. Properties of Br-A23187 and Ionomycin
Br-A23187
17
and ionomycin are ionophores that form complexes with divalent
metal cations; the complexes are lipid-soluble and thus can cross cellular mem-
branes. These two ionophores are commonly used to increase the permeability of
biological membranes to Ca
2
þ
. Understanding the di
erences between the two
makes it possible to make a judicious choice during an experiment.
Ionomycin can lose two acidic hydrogens and, as a dianion, can form an
uncharged 1:1 complex with a divalent metal ion such as Ca
2
þ
or Mg
2
þ
. Br-
A23187 can lose a single acidic hydrogen and form an uncharged 2:1 complex
with a divalent metal ion. This di
V
V
erence makes ionomycin potentially more
V
e
ective in binding and transporting divalent cations (e.g., two molecules of Br-
A23187 are needed to bind and carry a single Ca
2
þ
whereas only one molecule of
ionomycin is su
cient).
Compared with Br-A23187, ionomycin has somewhat better selectivity for Ca
2
þ
over Mg
2
þ
; ionomycin prefers Ca
2
þ
by a factor of
Y
2, whereas Br-A23187 shows
essentially no preference for one cation over the other (
Liu and Hermann, 1978
). In
addition, these ionophores actually do not bind Ca
2
þ
very tightly [e.g., K
d
(Ca
2
þ
)
100
m
M for ionomycin; J.P.Y. Kao, unpublished results.
18
] These factors suggest
that the two ionophores would be ine
cient in mediating Ca
2
þ
transport when
relatively low Ca
2
þ
concentrations are involved (i.e., at [Ca
2
þ
]
Y
1
m
M),
because at such low Ca
2
þ
concentrations, only a minute fraction of total
K
d
, e.g.,
<
16
Because calcium is a ubiquitous ''contaminant'' in the environment, nominally calcium-free
solutions still can contain micromolar levels of Ca
2
þ
.
17
The parent compound, A23187, is fluorescent and should be avoided in fluorescence work. The
presence of the bromine atom in 4-bromo-A23187 (Br-A23187) e
V
ectively quenches the intrinsic
fluorescence of the ionophore and makes the molecule useful in fluorescence microscopy.
18
Determined by absorption spectroscopy at pH 11, at which all ionomycin in solution would be
present as the fully deprotonated dianion.
