(Received for publication, March 9, 1995; and in revised form, May 23, 1995 )
From the
A cytoskeletal fraction of porcine tracheal smooth muscle (PTSM)
was found to contain >90% of total cellular aldolase (fructose
1,6-bisphosphate aldolase, EC 4.1.2.13) activity. PTSM aldolase was
purified by DEAE and inositol 1,4,5-trisphosphate
(Ins(1,4,5)P) affinity chromatography and found to react
with an antibody directed against human aldolase C, but not
anti-aldolase A and B. The molecular mass of native aldolase was about
138 kDa (on Sephacryl S-300); SDS-denatured enzyme was 35 kDa
(comigrated with rabbit skeletal muscle aldolase). Total cellular
aldolase tetramer (aldolase
) content was 34.5 pmol/100 nmol
lipid P
. Ins(1,4,5)P
) binding activity coeluted
with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P
affinity chromatography. Ins(1,4,5)P
bound to
purified aldolase (at 0 °C) in a dose-dependent manner over the
range [Ins(1,4,5)P
] 20 nM to 20
µM, with maximal binding of 1 mol of
Ins(1,4,5)P
/mol aldolase
and a K
of 12-14 µM.
Fru(1,6)P
and Fru(2,6)P
displaced bound
Ins(1,4,5)P
) with a 50% inhibition at 30 and 170
µM, respectively. Ins(1,3,4)P
(20
µM) and glyceraldehyde 3-phosphate (2 mM) were
also potent inhibitors of Ins(1,4,5)P
binding, but not
inositol 4-phosphate or inositol 1,4-bisphosphate (20 µM each). Aldolase-bound Ins(1,4,5)P
may play a role in
phospholipase C-independent increases in free
[Ins(1,4,5)P
].
Many reports have demonstrated the importance of inositol
1,4,5-trisphosphate (Ins(1,4,5)P)(
)-evoked
Ca
release from endoplasmic reticulum (ER) in various
non-muscle cells(1, 2) . Although a role of
Ins(1,4,5)P
and sarcoplasmic reticulum (SR) Ca
release is established for smooth
muscle(3, 4, 5) , it is not proven that
Ins(1,4,5)P
can be formed rapidly enough from
phosphatidylinositol 4,5-bisphosphate (PIP
) during receptor
activation to be involved in initial increases in free
[Ca
] which drive force development. This
requires that increases in [Ins(1,4,5)P
]
to levels which release SR Ca
occur within a
few 100 ms of receptor activation of the muscle(5) . We did not
detect any rapid or sustained increases in total Ins(1,4,5)P
content in porcine tracheal smooth muscle (PTSM) during
muscarinic receptor-evoked force development. However, phospholipase C
was activated as determined by measurements of flux in inositol
phospholipids and increases in Ins(1,4,5)P
by-products(6) . In other smooth muscles, transient
increases in Ins(1,4,5)P
have been measured for a few
seconds following receptor
activation(7, 8, 9) . In a vascular smooth
muscle, large stretch-activated increases in Ins(1,4,5)P
content occur within 200-300 ms after stretch to L
(10) .
In a previous
study(11) , we obtained evidence that there may be a bound or
sequestered Ins(1,4,5)P store in PTSM. The
Ins(1,4,5)P
content in resting, unstimulated muscle
averaged 2.7 µM (assuming uniform mixing in cellular
water), a value much higher than that required to release ER and SR
Ca
(12, 13) . (High values of resting
Ins(1,4,5)P
contents have been reported for other tissues,
as well(14, 15, 16) .) Ins(1,4,5)P
content decreased during atropine-induced relaxation of
carbachol-contracted PTSM to levels about 60% of the unstimulated
level. This suggests that a portion of the sequestered store is
released during the carbachol-stimulated contraction and is not
refilled during an atropine-induced relaxation.
Ins(1,4,5)P can bind to membranes via Ins(1,4,5)P
-receptors (17) and to cytosolic proteins (18, 19, 20) ; however, it is unknown if this
binding could explain the high unstimulated Ins(1,4,5)P
content found in PTSM. Ins(1,4,5)P
can bind to
aldolase (fructose 1,6-bisphosphate aldolase, EC 4.1.2.13) A (skeletal
muscle) and B (liver); the substrate for the enzyme, fructose
1,6-bisphosphate (Fru(1,6)P
), can reduce the affinity of
aldolase A and B for Ins(1,4,5)P
(18) . Hirata and
Kanematsu (19, 20, 21) have determined that
in rat brain, Ins(1,4,5)P
binds to cytosolic phospholipase
C
and an unidentified 130-kDa protein. The total bound
Ins(1,4,5)P
store in brain has not yet been determined, and
it is unknown if, in brain cells, metabolic events release bound
Ins(1,4,5)P
which could then exert effects on cellular
function.
In the present study we developed and studied a
cytoskeleton fraction in PTSM. Data were obtained which indicate that
aldolase is almost entirely associated with the cytoskeleton in this
muscle. We purified aldolase recovered from this fraction and showed
that it is aldolase C and that it binds and releases Ins(1,4,5)P under conditions which may occur physiologically. Total cellular
aldolase content was quantitated and shown to be very large suggesting
there is a potential site for binding large amounts of
Ins(1,4,5)P
.
The equilibrium dialysis assay conditions
were the same as in the PEG assay except that aldolase and
[H]Ins(1,4,5)P
were added on opposite
sides; aliquots of each side were counted after 3 days of equilibration
at 4 °C.
Figure 1:
SDS electrophoresis of K-released and
ghost proteins. Aliquots (20 µg of protein) of either K-released or
ghost proteins were separated by SDS-PAGE and the gels stained with
Coomassie Brilliant Blue. R for actin was
confirmed, using antibodies to actin, to be 0.6 corresponding to a
molecular mass of 40 kDa. The peak at R
0.06 is myosin.
Figure 2:
Western blots. A, study of
K-released proteins using to antibodies to vinculin, -actinin, and
talin. B, identity of aldolase isoforms. Lanes 1-3 contained K-released proteins (15 µg), and lane 4 contained 0.75 µg of purified PTSM aldolase. Antibodies to
aldolase A-C were applied as shown in the
figure.
Since it is possible that high tonicity could break bonds between aldolase and plasma membrane components, we solubilized ghosts using the detergents Triton X-100 (0.5%) or octyl glucoside (30 mM). This treatment did not release aldolase activity into the supernatant nor change release of aldolase evoked by a subsequent increase in KCl. We concluded that particulate aldolase was released from the cytoskeleton during 0.6 M KCl wash of ghosts and not from the plasma membrane. These data were similar to those reported in other tissues (27, 35) .
Assay of aldolase activity in K-released proteins was entirely dependent on the presence of added GAPDH. However, with ghosts aldolase activity in the entire absence of added GAPDH was as large as 60% of that in the presence of added GAPDH. This indicates that endogenous GAPDH and aldolase are associated with the intact ghosts.
Figure 3:
Chromatography of aldolase on
Ins(1,4,5)P affinity column. DEAE column fractions
containing aldolase were applied and eluted from the Ins(1,4,5)P
affinity column as described under ``Experimental
Procedures.'' The column fractions were monitored for aldolase
activity (open squares) and adsorption at 280 nm (solid
line); estimated [NaCl] is also indicated (interrupted dashed line).
Figure 4: SDS-gel electrophoresis of purified PTSM aldolase and Western blot against anti aldolase C antibody. Densitometric tracings (aligned at the front) of silver-stained aldolase (solid line) (1 µg) following SDS-PAGE and a Western blot run against anti-aldolase C (dotted line; blot is shown Fig. 2B) as described under ``Experimental Procedures.'' The upper x axis was determined from the molecular weights of standard proteins.
An estimate of the
molecular weight of native aldolase was obtained by Sephacryl S-300 HR
chromatography of K-released proteins in standard buffer containing 0.6 M KCl. Aldolase activity and Ins(1,4,5)P binding
were coincident at a molecular mass of 138 kDa.
The total aldolase content in the tissue was computed by measurement
of aldolase activity in the entire crude fraction and the specific
activity of purified smooth muscle aldolase. This assumes aldolase
specific activity is constant throughout the tissue and is the same as
the specific activity of purified aldolase. This calculation is given
under ``Appendix.'' Total aldolase content was computed to be
0.52 µg/mg cellular protein. The total content of the aldolase
tetramer (aldolase) given per 100 nmol phospholipid P
was 34.5 pmol/100 nmol lipid P
.
Fig. 5shows the
effects of altering pH on purified aldolase activity. These data were
obtained using [Fru(1,6)P] 1 mM and
standard conditions quoted above (i.e. [Ca
] = 0). Maximal activity was
recorded at pH 8.0. The low Fru(1,6)P
hydrolytic activity
observed at pH 6.0 was not due to a reduced activity of the added GAPDH
used in the coupled enzymatic assay. This was verified by increasing
the amount of GAPDH by 10-fold.
Figure 5:
Effect of pH on aldolase activity and
Ins(1,4,5)P binding. Aldolase activity (closed
circles, solid line (Guassian fit)) and Ins(1,4,5)P
binding (open squares, long dashed line (fit),
interrupted dotted line (± 1 S.D.)) were measured at different
pH values as described under ``Experimental Procedures.''
Ins(1,4,5)P
binding versus pH was transposed to versus [OH
] and the data fitted to
the curve: y = (a-c)/(1+(x/k)
) + c where y =
[
H]Ins(1,4,5)P
bound (counts/min), x = [OH
] (M), a = maximum binding = 131.2 ± 5.4 counts/min, c = minimum binding = 24.9 ± 4.8
counts/min, k = [OH
] midway
between the maximal and minimal binding transitions = 2.57
± 0.09
10
M (which occurred
at pH = 7.41 ± 0.02), and b = the
steepness factor or the number of H
-binding sites
undergoing transition = 14.6 ±
9.0.
Figure 6:
Binding
of Ins(1,4,5)P to aldolase. Data from eight separate
determinations (total n = 68) were normalized to
Ins(1,4,5)P
bound/aldolase
(mol/mol). Data were
obtained by PEG precipitation assay (
) and equilibrium dialysis
(+). The graph is presented in log-log form for easy visualization
of all data; the inset shows data in linear form. The solid line is the fit to the K
equation described in the text.
We were not able to saturate Ins(1,4,5)P binding to aldolase at 20 µM free
Ins(1,4,5)P
. At this concentration binding reached about
0.6 mol of Ins(1,4,5)P
bound/mol of aldolase
.
The K
for binding and the total capacity
of aldolase to bind Ins(1,4,5)P
were computed using the
dissociation equation for the reaction:
where K = dissociation
constant, [A]
= the free
concentration of aldolase
, [I]
= the free concentration of Ins(1,4,5)P
,
[AI
] = the concentration of
aldolase-Ins(1,4,5)P
complex, n = the
number of Ins(1,4,5)P
bound/aldolase
,
[A]
= the total concentration of
aldolase
and thus,
n[AI
]/[A]
= the total amount of Ins(1,4,5)P
bound/total aldolase
(mol/mol). Using
[A]
= [A]
+ [AI
], the equation was
transposed to
n[AI
]/[A]
=
n[I]
/(K
+[I]
).
The values obtained from the mathematical fitting (Fig. 6) of
n[AI
]/[A]
versus [I]
were 13.6
± 0.8 µM for K
and
0.99 ± 0.01 for n.
We also used a Scatchard analysis
(Klotz plot multiplied by
[aldolase]
; not shown) to compute K
and n. Using this method, the
computed n was unreasonably small (0.023), which was
apparently due to the large deviation of the fit from the points at
values <6 µM
for
[Ins(1,4,5)P
]
(=
[Ins(1,4,5)P
] > 160 nM). However,
using n = 0.99, as obtained above, K
computed by Scatchard analysis was 11.9
± 0.3 µM.
Thus, considering the above, we
conclude that the Kfor Ins(1,4,5)P
binding to aldolase was in the range of 11.9-13.6
µM (at 0 °C, pH 7.3, 100 mM KCl, 0
Ca
).
Figure 7:
Inhibition of Ins(1,4,5)P
binding to aldolase by Fru(1,6)P
and Fru(2, 6)P. Purified
PTSM aldolase was incubated with either 10 or 100 nM Ins(1,4,5)P
and Fru(1,6)P
or
Fru(2,6)P
at indicated concentrations
([Ca
] = 0 except where indicated):
Fru(1,6)P
(open squares, n =
7-11/point except 2 mM, n = 3);
Fru(1,6)P
, [Ca
] = 1
µM (
, n = 3/point); Fru(2,
6)P
, (open triangles, n =
5-6/point)). All points >8 µM Fru(1,6)P
were significantly different (p > 0.05) than 0
µM Fru(1,6)P
. The data
([Ca
] = 0; Fru(1,6)P
, n = 56, solid line; Fru(2, 6)P
, n = 16, long dashed line) were fitted to the
equation: y = 100/(1 + (x/k)
), where y = %
Ins(1,4,5)P
bound (100% at [Fru(X,
6)P
] = 0 and 0% at
[Fru(X,6)P
]) =
, x = [Fru(X,6)P
], k = [Fru(X,6)P
] for
displacement of half of the maximum amount of bound Ins(1,4,5)P
(Fru(1,6)P
, 0.030 ± 0.005 mM;
Fru(2,6)P
, 0.170 ± 0.028 mM), and b = the steepness factor (Fru(1,6)P
, 0.89
± 0.09; Fru(2,6)P
, 0.93 ±
0.13).
Fig. 7also shows results of
experiments which aimed at determining if altering
[Ca] changed the affinity for binding of
Ins(1,4,5)P
to aldolase
. Ca
(1 µM) did not cause a significant change in
Ins(1,4,5)P
binding at different
[Fru(1,6)P
] compared to values obtained at 0
[Ca
].
Figure 8:
Displacement of bound
[H]Ins(1,4,5)P
by other compounds.
[
H]Ins(1,4,5)P
was bound to purified
PTSM aldolase by incubation with 10 nM
[
H]Ins(1,4,5)P
. 20 µM Ins(1,3,4)P
, Ins(1,4)P
or Ins(4)P, or 2
mM GAP was added to the reaction mixture. Displacement of
H was determined by measuring bound counts using the PEG
assay. The graph also shows displacement (data similar to that given
above) caused by addition of 20 µM cold Ins(1,4,5)P
and 2 mM Fru(1,6)P
. In these experiments
bound [
H]Ins(1,4,5)P
was determined
by subtracting total
H from
[
H]
-globulin blank. Parentheses indicate number of data points. * indicates statistical
significant displacement of bound
[
H]Ins(1,4,5)P
at p <
0.001.
Major findings in this study relate to smooth muscle aldolase
and Ins(1,4,5)P binding to smooth muscle aldolase. The
total aldolase content of PTSM was surprisingly large, i.e. about 0.5% of total cellular protein. 93% of aldolase was found in
the K-released fraction which contains cytoskeletal proteins. On the
basis of studies on other tissues, it is likely that aldolase is bound
to cytoskeletal actin(36) . Since some contractile proteins
were present in ghost and K-released fractions, it is possible that
some or all cellular aldolase was bound to contractile proteins, as
suggested by previous workers who used skeletal or smooth
muscle(27, 37) . Our finding that aldolase was
resistant to detergent solubilization of ghosts is consistent with
aldolase binding to the cytoskeleton.
The finding that virtually all
cellular aldolase was found in the K-released fraction had practical
importance in that the enzyme was already 17-fold purified in this
fraction, when related to total cellular proteins (Table 2). The
apparent molecular mass of native aldolase was 138 kDa while
SDS-denatured enzyme was 35 kDa indicating that there are four aldolase
monomers which associate as a tetramer. The K for the substrate Fru(1,6)P
, 18.5
µM, is similar to that reported for pig skeletal muscle,
29 µM(38) , and higher than the rabbit aldolases:
muscle, 6 µM; liver, 1 µM; and brain, 2
µM(39) .
Immunoblot data indicate the presence
of an aldolase C isoform, and we could not detect reaction with
aldolase A and B antibodies. The presence of aldolase C was supported
by determining the ratio of PTSM aldolase activities driven by
Fru(1,6)P compared to Fru(1)P, an early method of
identifying aldolase isoforms. The presence of aldolase C in uterine
and gastric smooth muscle has been reported previously (40) but
in combination with aldolase A.
Strong evidence was obtained that
Ins(1,4,5)P binding to purified aldolase is specific.
Binding was displaced by cold Ins(1,4,5)P
,
Fru(1,6)P
, Ins(1,3,4)P
, and GAP, but not by
Ins(1,4)P
or Ins(4)P. The finding that Fru(2,6)P
displaced Ins(1,4,5)P
only at very high
concentrations argues for the specificity of Fru(1,6)P
displacement of Ins(1,4,5)P
.
The sharp loss of
Ins(1,4,5)P binding, at pH > 7.3 (Fig. 5) is of
interest in respect to the binding site of the highly negatively
charged Ins(1,4,5)P
to aldolase. Protein pH effects between
pH 6 and 8 may be associated with titration of positively charged
histidyl residues (41, 42, and analysis of our pH data (using the
``steepness factor'' described under ``Results'')
suggests 15 histidyl residues were titrated/aldolase
over
this pH range. Aldolase isoforms are rich in histidyl residues (i.e. A = 44, B = 36, and C = 28 per
tetramer(43) . Thus, Ins(1,4,5)P
may be bound to
aldolase C histidyl residues. These may also be the same
phosphate-binding residues which were reported to be ``capable of
immobilizing ten negative charges''(44) .
The maximal
molar binding of 1/tetramer bound in PTSM contrasts with molar binding
of Ins(1,4,5)P to skeletal muscle aldolase A of
4/tetramer(18) . The K
for
binding to PTSM aldolase, which was estimated to be in the range
11.9-13.6 µM, was higher than that reported for
skeletal muscle aldolase, 0.58 µM, and liver, 0.83
µM(18) . These differences may be explained, in
part, by different properties of aldolase C (PTSM), aldolase A (rabbit
skeletal muscle), and aldolase B (rabbit liver).
The question of
whether or not Ins(1,4,5)P binding to aldolase C can
entirely explain sequestered or bound Ins(1,4,5)P
in
unstimulated smooth muscle (11) has not been fully answered by
our study. The large content of aldolase in PTSM supports an additional
non-glycolytic role of this protein and provides a large, potential
sink for Ins(1,4,5)P
binding. In our previous
study(11) , 13.3 pmol/100 nmol lipid P
of
Ins(1,4,5)P
was sequestered or bound in unstimulated PTSM.
As indicated above, total aldolase
content determined in
the present study was 34 pmol/100 nmol lipid P
. Thus, the
potential Ins(1,4,5)P
binding to aldolase is more than
2-fold greater than the measured sequestered or bound Ins(1,4,5)P
in unstimulated muscle. The high K
for binding found in our study argues that only a fraction
of total aldolase binds Ins(1,4,5)P
under physiological
conditions. The finding that easily measurable Ins(1,4,5)P
binding to purified PTSM smooth muscle aldolase occurred even at
[Ins(1,4,5)P
] as low as 3 nM suggests a
physiological importance, if binding sites are in close proximity to
Ins(1,4,5)P
-sensitive SR. Uncertainties in projecting our
current binding data to amplification of free
[Ins(1,4,5)P
] in intact cells are related to: 1)
effects of temperature on binding (our binding studies were performed
at 0 °C), 2) whether aldolase bound to the cytoskeleton has a
different affinity for Ins(1,4,5)P
than does the soluble
form of the enzyme, 3) whether changes in Fru(1,6)P
and
associated metabolites concentration occur, and 4) other unknown
factors.
It was shown here that Ins(1,3,4)P releases
Ins(1,4,5)P
from the aldolase
Ins(1,4,5)P
complex (this is apparently the first described function for
Ins(1,3,4)P
). Further studies are required to determine if
Ins(1,3,4)P
formed from phospholipase C-derived
Ins(1,4,5)P
functions to release Ins(1,4,5)P
from aldolase-binding sites. Fru(1,6)P
and GAP also
release aldolase-bound Ins(1,4,5)P
. Further work is planned
in our laboratory to determine if the large, rapid increases in
Fru(1,6)P
content which occur in other tissues during
stimulation(45, 46, 47) , also occur in PTSM.
A steep pH-sensitive Ins(1,4,5)P
binding plot was shown in
this study. It still needs to be determined if pH increases which occur
in stimulated smooth muscle (48, 49, 50) could trigger Ins(1,4,5)P
release from the Ins(1,4,5)P
aldolase complex.
Total cellular aldolase
activity/mg of total cellular protein (Ald units/mg cellular protein) was computed as:
mg aldolase/mg cellular total protein was computed from the
specific activity of purified aldolase (SA) units/mg
purified aldolase) and Ald
(units/mg cellular
protein):