©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inositol 1,4,5-Trisphosphate Binding to Porcine Tracheal Smooth Muscle Aldolase (*)

(Received for publication, March 9, 1995; and in revised form, May 23, 1995 )

Carl B. Baron (1)(§) Shoichiro Ozaki (3) Yutaka Watanabe (3) Masato Hirata (4) Edward F. LaBelle (1) (2) Ronald F. Coburn (1)

From the  (1)Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the (2)Bockus Research Institute, Philadelphia, Pennsylvania 19146, the (3)Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790, Japan, and the (4)Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka 812, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(3)) 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(4)) content was 34.5 pmol/100 nmol lipid P(i). Ins(1,4,5)P(3)) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P(3) affinity chromatography. Ins(1,4,5)P(3) bound to purified aldolase (at 0 °C) in a dose-dependent manner over the range [Ins(1,4,5)P(3)] 20 nM to 20 µM, with maximal binding of 1 mol of Ins(1,4,5)P(3)/mol aldolase(4) and a K of 12-14 µM. Fru(1,6)P(2) and Fru(2,6)P(2) displaced bound Ins(1,4,5)P(3)) with a 50% inhibition at 30 and 170 µM, respectively. Ins(1,3,4)P(3) (20 µM) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P(3) binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 µM each). Aldolase-bound Ins(1,4,5)P(3) may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P(3)].


INTRODUCTION

Many reports have demonstrated the importance of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3))(^1)-evoked Ca release from endoplasmic reticulum (ER) in various non-muscle cells(1, 2) . Although a role of Ins(1,4,5)P(3) and sarcoplasmic reticulum (SR) Ca release is established for smooth muscle(3, 4, 5) , it is not proven that Ins(1,4,5)P(3) can be formed rapidly enough from phosphatidylinositol 4,5-bisphosphate (PIP(2)) 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(3)] 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(3) 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(3) by-products(6) . In other smooth muscles, transient increases in Ins(1,4,5)P(3) 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(3) content occur within 200-300 ms after stretch to L(max)(10) .

In a previous study(11) , we obtained evidence that there may be a bound or sequestered Ins(1,4,5)P(3) store in PTSM. The Ins(1,4,5)P(3) 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(3) contents have been reported for other tissues, as well(14, 15, 16) .) Ins(1,4,5)P(3) 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(3) can bind to membranes via Ins(1,4,5)P(3)-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(3) content found in PTSM. Ins(1,4,5)P(3) 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(2)), can reduce the affinity of aldolase A and B for Ins(1,4,5)P(3)(18) . Hirata and Kanematsu (19, 20, 21) have determined that in rat brain, Ins(1,4,5)P(3) binds to cytosolic phospholipase C and an unidentified 130-kDa protein. The total bound Ins(1,4,5)P(3) 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(3) 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(3) 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(3).


EXPERIMENTAL PROCEDURES

Tissue Preparation

Porcine tracheal smooth muscle strips were prepared as described previously(22) . Unstimulated strips were incubated in Krebs solution at 37 °C for 1 h and then freeze-clamped with liquid N(2)-cooled tongs. The frozen tissue was ground to a fine powder in a liquid N(2)-cooled mortar and pestle and stored at -70 °C until used.

Fractionation of Tissue

The method described below was adapted from that described by Jones et al.(23) , Caroni and Carafoli (24) and Popescu and Ignat(25) ; this preparation was originally used to prepare muscle sarcolemma.

Preparation of Ghosts and Cytosol

Frozen, powdered tissue was thawed and dispersed by slow addition to 6 ml of homogenization buffer: 20 mM HEPES-Na, pH 7.3, 250 mM sucrose, 2 mM EGTA, 0.5 mM MgCl(2) (0.45 mM free), 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µM calpain inhibitor peptide, and 5 µg/ml each of aproptinin, leupeptin, and pepstatin A. All steps in this preparation were performed at 0 °C. The suspension was homogenized in a 7-ml Tenbroeck homogenizer (Wheaton, Wheaton, NJ) and centrifuged for 5 min at 200 g. The supernatant was collected, the pellet resuspended in homogenization buffer and the above procedure repeated five more times. The pooled supernatant from multiple washes was centrifuged two times (1,000 g for 20 min; 100,000 g for 1 h). The 100,000 g supernatant is the ``cytosolic'' fraction. The pellet obtained from the 1,000 g centrifugation was added to the 200 g pellet (resulting in a 10% increase in total aldolase activity). This fraction has been termed the ``ghost'' fraction, so named because of its microscopic appearance(25) .

Preparation of K-released Proteins

Ghost suspensions were homogenized in 6 ml of release buffer: 20 mM HEPES-Na, pH 7.3, and 0.6 M KCl, 2 mM EGTA, and centrifuged for 20 min at 1,000 g. The pellet was rehomogenized and centrifuged. The resulting pellet is the sarcolemmal fraction studied previously (24, 25) but not used extensively in the present study. Pooled supernatants were centrifuged at 100,000 g for 1 h to removal all membrane particles and then dialyzed against 50 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, and 1 mM HEDTA (standard buffer). Following dialysis and reduction of the ionic strength, the solution became cloudy and was clarified by centrifugation (15 min at 15,000 g) which removed proteins consisting mainly of myosin and actin. The supernatant is the ``K-released'' fraction used in this study.

Purification of Aldolase

K-released proteins were applied to a DE32 (microgranular DEAE-cellulose (Whatman, Clifton, NJ), 5 ml bed volume, and eluted with standard buffer. Aldolase-containing fractions of the DEAE wash-through were applied to a 1-ml column of no. 204 resin (26) (2-O-[4-(5-aminoethyl-2-hydroxyphenylazo) benzoyl]-1,4,5-tri-O-phosphono-myo-inositol-Sepharose 4B) (Ins(1,4,5)P(3) affinity column). This column was washed with 24 ml of 10 mM HEPES-Na, pH 7.3, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM NaN(3) (buffer A) and then with buffer A containing the indicated [NaCl]: 26 ml of 0.15 M, 30 ml gradient of 0.15-0.65 M and 8 ml of 2 M.

Aldolase Activity Assay

Aldolase was assayed by coupling the reaction to the oxidation of glyceraldehyde 3-phosphate (GAP) to 1,3-diphosphoglyceric acid by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and following the reduction of NAD (2 mol/mol Fru(1,6)P(2) hydrolyzed) at 340 nm. The assay was modified by using arsenate (27) instead of phosphate as the product, 1-phospho-3-arsenoglyceric acid, spontaneously hydrolyzes. Thus, there is no need for additional enzymes to remove products. Early assays were performed at pH 7.6; the reaction mixture contained 200 mM triethanolamine, pH 7.6, 10 mM EDTA, 2 mM Na(2)AsO(4), 1 mM Fru(1,6)P(2), and 1 unit of rabbit skeletal muscle GAPDH in 1 ml (addition of triose phosphate isomerase did not increase the rate and so was not included). Later experiments contained 100 mM HEPES-Tris, pH 7.0, 1 mM EDTA, 100 mM KCl, 2 mM Na(2)AsO(4), 1 unit of GAPDH, and 2.5-80 µM Fru(1,6)P(2). When aldolase was assayed at different pH values, 0.1 M HEPES-Tris (i.e. [HEPES] + [Tris] = 0.1 M) was used instead of triethanolamine. Mixtures were incubated at 37 °C, and base-line NADH formation was determined. The reaction was started by addition of enzyme. 1 unit of aldolase was taken to equal to 1 µmol of Fru(1,6)P(2) hydrolyzed/min at 37 °C.

Aldolase Isoforms Present in PTSM

This was determined using antibodies to aldolase A, B, and C, and by determining preferential substrates (Fru(1,6)P(2)versus Fru(1)P) for PTSM aldolase(28) .

Ins(1,4,5)P(3) Binding Assay

We used two different assays: protein precipitation with polyethylene glycol (PEG) and equilibrium dialysis. The PEG assay was adapted from the method of Kanematsu et al.(19) and utilized 50 mM HEPES-Tris, pH 7.3, 1 mM EDTA, 100 mM KCl, 0.6 mg/ml -globulins, and 3 nM [^3H]-Ins(1,4,5)P(3) in a volume of 50 µl. Assays were performed at 0 °C. The sample was added and the mixture incubated for 20 min. 50 µl of 30% PEG (average M(r) 8,000) was added, vortexed, and left on ice for an additional 20 min. The mixture was centrifuged two times (first horizontally at 15,000 g for 15 min with about 95% of the supernatant removed and second in an angled rotor at 15,000 g for 5 min with the remaining supernatant removed). The pellet was dissolved in 0.2 ml of 0.1 N NaOH, transferred to a counting vial, and the tube rinsed with 0.4 ml of H(2)O which was added to the vial. Vials were counted in a scintillation counter. An aliquot of the mixture was taken prior to the addition of sample to determine the [^3H]Ins(1,4,5)P(3) radioactivity in the assay. PEG did not completely precipitate aldolase, as determined by measuring aldolase activity in the pellet (obtained by dissolving the pellet in 20 mM HEPES-Na, pH 7.3); this factor (0.753 ± 0.009) was used to correct for the amount of aldolase in the PEG precipitation assay. Specific binding was determined by the addition of unlabeled Ins(1,4,5)P(3). Complete displacement of radioactivity was achieved with 200 µM unlabeled Ins(1,4,5)P(3) and was the same value as -globulin alone (with or without 200 µM Ins(1,4,5)P(3)). When binding was assayed at different pH values, the same HEPES-Tris buffers were used as described in the aldolase activity assay.

The equilibrium dialysis assay conditions were the same as in the PEG assay except that aldolase and [^3H]Ins(1,4,5)P(3) were added on opposite sides; aliquots of each side were counted after 3 days of equilibration at 4 °C.

Binding of Other Inositol Phosphates and Aldolase Substrates and Products of Aldolase

Binding of these compounds was assessed indirectly by determining if they displaced bound Ins(1,4,5)P(3).

Calculation of [Ca] in Assayed Fractions

The computer program MAXC, v6.50 (29) was used.

SDS-Gel Electrophoresis

Proteins were dialyzed against water, SDS and dithiothreitol added to 1.1% and 20 mM, respectively, placed in a boiling water bath for 5 min, and 10% glycerol added. Electrophoresis was carried out in 0.4 M Tris-Cl, pH 8.8, in an 8% acrylamide running gel with a 4% acrylamide stacking gel at 45 milliamps/gel (30) . Gels were fixed and stained with 0.05% Coomassie Brilliant Blue R in methanol/water/acetic acid (20:73:7, by volume). When gels were silver stained(31) , they were first destained in methanol/water/acetic acid and then rinsed with water to remove all methanol and acetic acid. Densitometric traces (Shimadzu CS-930) of the gels were performed at 555 nm for Coomassie Brilliant Blue R and at 474 nm for silver.

Western Blots

Proteins were transferred to nitrocellulose as described previously(32) . Densitometric traces (Shimadzu CS-930) of the blots were performed at 552 nm.

Protein and Total Phospholipid Measurements

Protein was measured using the Micro BCA Protein Reagent Kit (Pierce) (prior precipitation with 7% trichloroacetic acid was used to remove interfering substances). We measured total phospholipid P(i) by the method of Ames(33) .

5`-Nucleotidase Assay

5`-Nucleotidase activity was measured by incubating aliquots of tissue extracts with 5 mM AMP, 5 mM MgCl(2), and 50 mM Tris-Cl, pH 7.4, for 30 min at 37 °C. The reaction was stopped by the addition of 10% trichloroacetic acid, the samples centrifuged for 5 min at 1,000 g, and the supernatants analyzed for released phosphate. Phosphate detected with 0.41% (NH(4))(6)Mo(7)Obullet4O, 0.375 M H(2)SO(4), and 1.1% FeSO(4) at 660 nm.

Curve Fitting

Constants and standard deviations were obtained by using non-linear curve fitting with the computer program SYSTAT (ver. 3.0). Sigmoidal curves were fitted to the general equation y = (a-c)/(1 + (x/k)) + c(34) .

Materials

[^3H]Ins(1,4,5)P(3) was purchased from DuPont NEN. Non-radioactive Ins(1,4,5)P(3) was from American Radiolabeled Chemicals, Inc., St. Louis, MO. HEPES, polyethylene glycol, rabbit skeletal muscle aldolase A, -globulins, EGTA, EDTA, HEDTA, rabbit skeletal muscle GAPDH, DL-GAP, NAD, arsenic acid (disodium salt), Fru(1,6)P(2), Fru(2,6)P(2), calpain inhibitor peptide, aprotinin, leupeptin, pepstatin A, and antibodies to vinculin, talin, and alpha-actinin were from Sigma. Octyl glucoside (OG) was from Calbiochem, La Jolla, CA. Triton X-100 was from Rohm and Haas, Philadelphia, PA. Antibodies to human aldolase A-C were kindly provided by Dr. Kanefusa Kato, Aichi Prefectural Colony, Japan. All other chemicals were reagent grade.


RESULTS

Characteristics of Ghosts and K-released Proteins

Gels prepared using SDS-solubilized ghosts (Fig. 1) showed that the major solubilized proteins are actin and myosin. Ghost suspensions contained about 50% of total cellular phospholipid and 80% of 5`-nucleotidase activity. About 21% of ghost proteins could be released from ghosts with 0.6 M KCl. After removal of particulate material by a 100,000 g centrifugation for 1 h, 16% of cellular proteins remained in the supernatant (the K-released preparation used in this study). The K-released fraction contained 11 and 17% of total cellular myosin and actin, respectively, as assessed by areas of peaks in gels. K-released proteins reacted strongly with antibodies to vinculin, talin, and alpha-actinin (Fig. 2). No phospholipid or 5`-nucleotidase activity was detected in the K-released fraction. Table 1summarizes data relevant to various components and properties of ghosts and K-released proteins. We have concluded from the above data that ghosts consist of extracellular matrix-sarcolemma-cytoskeleton complexes and that KCl-released protein fractions contain cytoskeleton proteins and no plasma membrane. The absence of lipid in this preparation suggests the absence of intact organelles, i.e. intact ER, SR, and nucleus. Small amounts of myosin are still present in this preparation. Although this could reflect myosin in the cytoskeleton, i.e. myosin motors, it is possible that some contractile proteins are still attached to the cytoskeleton.


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, alpha-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.





Aldolase Activity in Various Fractions

93% of cellular aldolase activity was recovered in ghost suspensions from either unstimulated or 55 µM carbachol-stimulated (20 s or 5 min) tissue and could be entirely released by treatment with high tonicity (0.6 M KCl) into the K-released fraction. The remainder of aldolase activity was recovered mainly in the cytoplasm. No activity was detectable in the sarcolemma.

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.

Purification of Aldolase and Ins(1,4,5)P(3)-binding Proteins

K-released proteins were passed through a DEAE column. Aldolase activity, and coincident Ins(1,4,5)P(3) binding, did not stick to the DEAE column, but passed through the column in the washes. These fractions were then passed through an Ins(1,4,5)P(3) affinity column. This revealed (Fig. 3) one major peak of aldolase activity (>80% of total aldolase activity) which was eluted between 0.2-0.4 M NaCl. Ins(1,4,5)P(3), up to 10 µM, did not elute any aldolase or Ins(1,4,5)P(3)-binding protein. GAPDH activity (an enzyme which can contaminate aldolase preparations as observed in other tissues) was not detected. SDS-gel electrophoresis of aldolase recovered from the affinity column contained only one major peak, at 35-40 kDa (Fig. 4). SDS-gel electrophoresis of rabbit skeletal muscle aldolase A showed a peak at an identical molecular weight as PTSM aldolase (not shown).


Figure 3: Chromatography of aldolase on Ins(1,4,5)P(3) affinity column. DEAE column fractions containing aldolase were applied and eluted from the Ins(1,4,5)P(3) 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(3) binding were coincident at a molecular mass of 138 kDa.

PTSM Total Aldolase Content

Table 2shows that aldolase was purified from K-released proteins with only an 11-fold purification. Note that the use of K-released proteins already resulted in a 17-fold purification of aldolase, given as activity/total cellular protein. Therefore, the total purification to apparent homogeneity was about 190-fold. Thus, aldolase contributes about 9% of the total K-released proteins and about 0.5% of total cellular protein.



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(4)) given per 100 nmol phospholipid P(i) was 34.5 pmol/100 nmol lipid P(i).

Aldolase Isoforms

Aldolase in the K-released fraction and purified aldolase reacted strongly with anti-aldolase C antibody, but not with anti-aldolase A or B antibodies (Fig. 2). PTSM aldolase activity was 29 ± 3 times greater with Fru(1,6)P(2) as substrate than when Fru(1)P was used. This finding supports the conclusion that aldolase C is the major aldolase isoform in PTSM(28) . In rabbit tissue, aldolase A (skeletal muscle) and B (liver) ratios of activity with Fru(1,6)P(2)/Fru(1)P were reported to be 55, and 1.2, respectively(28) .

Effects of Fru(1,6)P(2), Ins(1,4,5)P(3), and pH on Aldolase Activity

The apparent K for Fru(1,6)P(2) (range 2.5-75 µM; 37 °C, [Ca] = 0, pH 7.0) was 18.5 ± 2.0 µM. The addition of 10 µM Ins(1,4,5)P(3) had no effect on purified aldolase activity either at [Fru(1,6)P(2)] of 2.5 or 25 µM (however, a higher [Ins(1,4,5)P(3)] (20 µM) caused a small (30.1 ± 4.7%), significant inhibition (p < 0.002)).

Fig. 5shows the effects of altering pH on purified aldolase activity. These data were obtained using [Fru(1,6)P(2)] 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(2) 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(3) binding. Aldolase activity (closed circles, solid line (Guassian fit)) and Ins(1,4,5)P(3) 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(3) binding versus pH was transposed to versus [OH] and the data fitted to the curve: y = (a-c)/(1+(x/k)) + c where y = [^3H]Ins(1,4,5)P(3) 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 10M (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.



Ins(1,4,5)P(3) Binding to Purified Aldolase

Data were obtained over the range [Ins(1,4,5)P(3)] 3 nM to 20 µM (Fig. 6). These data show concentration-dependent binding of Ins(1,4,5)P(3) to aldolase and that there was no difference with the two different methods of determining Ins(1,4,5)P(3) binding.


Figure 6: Binding of Ins(1,4,5)P(3) to aldolase. Data from eight separate determinations (total n = 68) were normalized to Ins(1,4,5)P(3) bound/aldolase(4) (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(3) binding to aldolase at 20 µM free Ins(1,4,5)P(3). At this concentration binding reached about 0.6 mol of Ins(1,4,5)P(3) bound/mol of aldolase(4). The K for binding and the total capacity of aldolase to bind Ins(1,4,5)P(3) were computed using the dissociation equation for the reaction:

where K = dissociation constant, [A] = the free concentration of aldolase(4), [I] = the free concentration of Ins(1,4,5)P(3), [AI] = the concentration of aldolase-Ins(1,4,5)P(3) complex, n = the number of Ins(1,4,5)P(3) bound/aldolase(4), [A] = the total concentration of aldolase(4) and thus, n[AI]/[A] = the total amount of Ins(1,4,5)P(3) bound/total aldolase(4) (mol/mol). Using [A] = [A] + [AI], the equation was transposed to n[AI]/[A] = n[I](f)^n/(K+[I](f)^n). 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(4)]; 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(3)] (= [Ins(1,4,5)P(3)] > 160 nM). However, using n = 0.99, as obtained above, Kcomputed by Scatchard analysis was 11.9 ± 0.3 µM.

Thus, considering the above, we conclude that the Kfor Ins(1,4,5)P(3) binding to aldolase was in the range of 11.9-13.6 µM (at 0 °C, pH 7.3, 100 mM KCl, 0 Ca).

Effects of [Fru(1,6)P(2)] and [Fru(2,6)P(2)] on Ins(1,4,5)P(3) Binding to Purified Aldolase

Fru(1,6)P(2) had a profound effect on Ins(1,4,5)P(3) binding (Fig. 7). Ins(1,4,5)P(3) binding was significantly inhibited at a [Fru(1,6)P(2)] of 8 µM (t test, p < 0.03) and progressively decreased as Fru(1,6)P(2) was increased to 1 mM. Inhibition of binding was not dependent upon the [Ins(1,4,5)P(3)] tested. The [Fru(1,6)P(2)] for half-maximal inhibition of maximal Ins(1,4,5)P(3) binding was calculated at be 30 ± 5 µM. Fru(2,6)P(2) was less effective in inhibiting Ins(1,4,5)P(3) binding. The [Fru(2,6)P(2)] for half-maximal inhibition was 170 ± 28 µM.


Figure 7: Inhibition of Ins(1,4,5)P(3) binding to aldolase by Fru(1,6)P(2) and Fru(2, 6)P. Purified PTSM aldolase was incubated with either 10 or 100 nM Ins(1,4,5)P(3) and Fru(1,6)P(2) or Fru(2,6)P(2) at indicated concentrations ([Ca] = 0 except where indicated): Fru(1,6)P(2) (open squares, n = 7-11/point except 2 mM, n = 3); Fru(1,6)P(2), [Ca] = 1 µM (, n = 3/point); Fru(2, 6)P(2), (open triangles, n = 5-6/point)). All points >8 µM Fru(1,6)P(2) were significantly different (p > 0.05) than 0 µM Fru(1,6)P(2). The data ([Ca] = 0; Fru(1,6)P(2), n = 56, solid line; Fru(2, 6)P(2), n = 16, long dashed line) were fitted to the equation: y = 100/(1 + (x/k)), where y = % Ins(1,4,5)P(3) bound (100% at [Fru(X, 6)P(2)] = 0 and 0% at [Fru(X,6)P(2)]) = , x = [Fru(X,6)P(2)], k = [Fru(X,6)P(2)] for displacement of half of the maximum amount of bound Ins(1,4,5)P(3) (Fru(1,6)P(2), 0.030 ± 0.005 mM; Fru(2,6)P(2), 0.170 ± 0.028 mM), and b = the steepness factor (Fru(1,6)P(2), 0.89 ± 0.09; Fru(2,6)P(2), 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(3) to aldolase(4). Ca (1 µM) did not cause a significant change in Ins(1,4,5)P(3) binding at different [Fru(1,6)P(2)] compared to values obtained at 0 [Ca].

Effects of Ins(1,3,4)P(3), Ins(1,4)P(2), Ins(4)P, and GAP on Ins(1,4,5)P(3) Binding to Aldolase

[^3H]Ins(1,4,5)P(3) binding was determined at 10 nM. Ins(1,3,4)P(3) (20 µM) displaced about 70% bound [^3H]Ins(1,4,5)P(3) (p < 0.04). Fru(1,6)P(2) and GAP (2 mM) displaced all bound Ins(1,4,5)P(3). Ins(1,4)P(2) and Ins(4)P (20 µM) had no effect on bound [^3H]Ins(1,4,5)P(3). Data are shown in Fig. 8.


Figure 8: Displacement of bound [^3H]Ins(1,4,5)P(3) by other compounds. [^3H]Ins(1,4,5)P(3) was bound to purified PTSM aldolase by incubation with 10 nM [^3H]Ins(1,4,5)P(3). 20 µM Ins(1,3,4)P(3), Ins(1,4)P(2) or Ins(4)P, or 2 mM GAP was added to the reaction mixture. Displacement of ^3H 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(3) and 2 mM Fru(1,6)P(2). In these experiments bound [^3H]Ins(1,4,5)P(3) was determined by subtracting total ^3H from [^3H]-globulin blank. Parentheses indicate number of data points. * indicates statistical significant displacement of bound [^3H]Ins(1,4,5)P(3) at p < 0.001.



Effects of pH on Ins(1,4,5)P(3) Binding to Aldolase

Ins(1,4,5)P(3) binding to aldolase was essentially independent of pH between values of 6.0 and 7.3 (Fig. 5). Between 7.3 and 7.5 there was a sharp drop in binding of about 80%, which then remained constant to pH 8.5. The halfway point of the transition was pH 7.41 ± 0.02.


DISCUSSION

Major findings in this study relate to smooth muscle aldolase and Ins(1,4,5)P(3) 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(2), 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(2) 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(3) binding to purified aldolase is specific. Binding was displaced by cold Ins(1,4,5)P(3), Fru(1,6)P(2), Ins(1,3,4)P(3), and GAP, but not by Ins(1,4)P(2) or Ins(4)P. The finding that Fru(2,6)P(2) displaced Ins(1,4,5)P(3) only at very high concentrations argues for the specificity of Fru(1,6)P(2) displacement of Ins(1,4,5)P(3).

The sharp loss of Ins(1,4,5)P(3) 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(3) 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(4) 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(3) 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(3) 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(3) binding to aldolase C can entirely explain sequestered or bound Ins(1,4,5)P(3) 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(3) binding. In our previous study(11) , 13.3 pmol/100 nmol lipid P(i) of Ins(1,4,5)P(3) was sequestered or bound in unstimulated PTSM. As indicated above, total aldolase(4) content determined in the present study was 34 pmol/100 nmol lipid P(i). Thus, the potential Ins(1,4,5)P(3) binding to aldolase is more than 2-fold greater than the measured sequestered or bound Ins(1,4,5)P(3) 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(3) under physiological conditions. The finding that easily measurable Ins(1,4,5)P(3) binding to purified PTSM smooth muscle aldolase occurred even at [Ins(1,4,5)P(3)] as low as 3 nM suggests a physiological importance, if binding sites are in close proximity to Ins(1,4,5)P(3)-sensitive SR. Uncertainties in projecting our current binding data to amplification of free [Ins(1,4,5)P(3)] 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(3) than does the soluble form of the enzyme, 3) whether changes in Fru(1,6)P(2) and associated metabolites concentration occur, and 4) other unknown factors.

It was shown here that Ins(1,3,4)P(3) releases Ins(1,4,5)P(3) from the aldolasebulletIns(1,4,5)P(3) complex (this is apparently the first described function for Ins(1,3,4)P(3)). Further studies are required to determine if Ins(1,3,4)P(3) formed from phospholipase C-derived Ins(1,4,5)P(3) functions to release Ins(1,4,5)P(3) from aldolase-binding sites. Fru(1,6)P(2) and GAP also release aldolase-bound Ins(1,4,5)P(3). Further work is planned in our laboratory to determine if the large, rapid increases in Fru(1,6)P(2) content which occur in other tissues during stimulation(45, 46, 47) , also occur in PTSM. A steep pH-sensitive Ins(1,4,5)P(3) 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(3) release from the Ins(1,4,5)P(3)bulletaldolase complex.


APPENDIX

Calculation of Total Aldolase Stores

Units of aldolase activity and total cellular proteins were converted/100 nmol of lipid P(i) giving values of 0.0165 ± 0.0005 units/100 nmol lipid P(i) and 0.91 ± 0.13 mg/100 nmol lipid P(i), respectively.

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):


FOOTNOTES

*
This work was supported by National Heart, Lung, and Blood Institute Grants HL 50532-01 and R37 H137498-08. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology/6085, University of Pennsylvania, Philadelphia, PA 19104-6085. Tel.: 215-898-8069; Fax: 215-573-5851.

(^1)
The abbreviations used are: Ins(1,4,5)P(3), inositol 1,4,5-trisphosphate; PIP(2), phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PTSM, porcine tracheal smooth muscle; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; Fru(1,6)P(2), fructose 1,6-bisphosphate; Fru(2,6)P(2), fructose 2,6-bisphosphate; HEDTA, N-hydroxyethlethylenediamine triacetic acid; GAP, D-glyceraldehyde 3-phosphate; GAPDH, D-glyceraldehyde 3-phosphate dehydrogenase; PEG, polyethylene glycol; I(4)P, inositol 4-phosphate; Ins(1,4)P(2), inositol 1,4-bisphosphate; Ins(1,3,4)P(3). inositol 1,3,4-trisphosphate; aldolase(4), aldolase tetramer.


ACKNOWLEDGEMENTS

We acknowledge, with kind appreciation, Dr. Kanefusa Kato, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya, Kasugai, Aichi 480-30, Japan for providing the aldolase anti-bodies.


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