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Polyphosphoinositide synthesis in rabbit erythrocyte membranes
ARCHIVES Vol. 219,
OF BIOCHEMISTRY No. 1, November,
AND BIOPHYSICS pp. 58-64, 1982
Polyphosphoinositide
Synthesis
in Rabbit
EUGENE Lkpartment
of Pharmacology, Received
April
12,
1982,
Membranes
E. QUIST
North Medicine,
Osteopathic
Erythrocyte
Texas State Fort Worth, and
in revised
Uniuersity/Texa.s Texas 76107 form
July
13,
College
of
1982
Incubation of rabbit erythrocyte ghosts at 25°C with 1 mM [T-~‘P]ATP and MgCl, results in incorporation of 32P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol 32P/mg/h, respectively. Incorporation of 32P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with 32P, did not undergo significant breakdown when incubated at 25°C for 15 to 20 min. Turnover of 32P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl, and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.
Hokin and Hokin (1) were the first to report that 32P is incorporated into DPI’ and TPI from [Y-~*P]ATP in human erythrocyte ghosts. Similar results have been found in erythrocyte ghosts prepared from other mammalian species (2, 3). The kinase activity(s) involved in the synthesis of DPI and TPI from phosphatidylinositol is membrane bound, MgC12 dependent, and located on the cytoplasmic surface of the erythrocyte membrane (l-4). Because 32P turns over rapidly in DPI and TPI in
intact erythrocytes (5), polyphosphoinositides have been thought to have an important functional role in erythrocyte membranes (3-5). Among these, polyphosphoinositides have been implicated in the regulation of energy charge (6, 7), opening and closing erythrocyte pores (S), and in ATP-dependent pinocytosis (9,lO). Recently, Quist and Reece (11) reported that there is a strong correlation between 32P incorporation into DPI from Mg-ly3”P]ATP and Mg-ATP-dependent decreases in the viscosity of rabbit erythrocyte ghosts. The increase in viscosity is associated with an echinocyticcdiscocytic shape transformation of the ghosts and possibly with an increase in membrane deformability (11). Therefore, Mg-ATP may induce viscosity or shape changes in
1 Abbreviations used: UPI, dipbosphoinositide or phosphatidylinositol I-phosphate; TPI, triphosphoinositide or phosphatidylinositol 4,5-bisphosphate; EGTA, ethylene glycol bis(&aminoethyl ether). N,N ‘-tetraacetic acid; HPLC, high-pressure liquid chromatography. 00n3-9X~1/82/13005X-07$02.00/0 Copyright 0 1982 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
58
RABBIT
ERYTHROCYTE
ghost membranes by increasing membrane DPI concentration by stimulating a Mg2+dependent phosphatidylinositol kinase activity. Neomycin was also shown to inhibit Mg-ATP-dependent viscosity and shape changes in ghosts and to decrease the concentration of 32P-labeled diphosphoinositide (11). This study was initiated to determine if 32P incorporated into phosphoinositide from [y-“2]ATP was a measure of synthesis or turnover of DPI in these ghost membranes. Conditions were identical to those used in the previous study in which the effect of Mg-ATP on shape and viscosity was determined (11). The mechanism by which neomycin decreases 32P-labeled DPI was also investigated. MATERIALS
AND
59
POLYPHOSPHOINOSITIDES
METHODS
Materials. [y-“*P]ATP was obtained from New England Nuclear. Neomycin sulfate, Triton X-100, and phospholipid standards were obtained from Sigma Chemical Company. Solvents used for phospholipid analysis and chromatography were HPLC grade. Preparation of ghosts. Fresh heparinized blood was obtained by cardiac puncture from New Zealand white rabbits and the erythrocytes were washed three times with isotonic saline to remove plasma and white cells. Ghosts were usually prepared by washing packed erythrocytes two times with 10 vol of 20 mM TrisHCI, pH 7.6, at 20,OOOg for 15 min at 5°C. Ghosts were used within 1 h of preparation. 32P incorporation into ghost phospholipids. Ghosts were usually incubated in a final volume of 0.5 ml in 25 mM imidazole HCl, pH 7.0, 5 mM MgCl,, 1 mM [y-“‘P]ATP, 1 mM EGTA, and 180 pg of membrane phospholipid (0.1 ml of packed ghosts) at 25°C. In some experiments neomycin was included in the incubation medium. The reaction was stopped by the addition of 3 ml of cold 5% trichloroacetic acid. The tubes were then centrifuged at 2000g for 10 min at 5°C and the pellet was further washed with 3 ml of cold H,O. Lipids were extracted from the pelleted ghosts at 5°C by resuspension in 2.0 ml of CHC13:CH,0H:HC1 (20:40:1), containing 0.1% butylated hydroxytoluene. After 20 min on ice, 0.75 ml of CHCl,j and H,O were consecutively added to the tubes with vortexing and the tubes were centrifuged at 2OOOg for 10 min at 5°C. The upper phase was removed by aspiration and 1.0 ml of the CHCll phase was transferred to 2.0.ml glass-stoppered tubes and dried under a stream of N,. The dried lipid extract was resuspended with 30 ~1 of CHClz:CHBOH:CH1 (6::i:O.l) and 10.~1 aliquots were spotted on silica gel
60 plates (Merck) and assayed for phospholipid content by the method of Bartlett (12). Thin-layer plates were developed in CHC13:CH30H:H20:30% NH3 (25:35:7.5:2.5). Radiolabeled phospholipids were located overnight by autoradiography using Kodak OMAT R X-ray film. Labeled phospholipids were scraped from the plates and counted in 8 ml of tritisol. Recovery was 90%. Results are expressed as nanomoles of 32P incorporated per milligram of total ghost phospholipid. Effect of ATP and neomycin on 32P-labeled polyphosphoinositides. Ghosts were incubated for 20 min at 25’C in 25 mM imidazole, pH 7.0, 5 mM MgC12, 1 mM EGTA, and 1 mM [y-32P]ATP in a final volume of 0.5 ml. The reaction was stopped with 2.0 ml of cold 25 mM imidazole HCl, pH 7.0, 5 mM MgCl*, and 1 mM EGTA. The tubes were centrifuged at 20,OOOg for 15 min and the pellets were resuspended with 3 ml of the same solution and recentrifuged. Finally, the pellet was resuspended in the same medium. Where indicated, 1 mM ATP (disodium salt), neomycin sulfate, or Triton X-100 were added to the tubes. The reaction was usually stopped with 3 ml of cold 5% trichloroacetic acid and the phospholipids were extracted, separated, and counted as above. To study the effect of Triton X-100 it was necessary to stop the reaction with 2.0 ml CHCls:CHBOH:HCl (20:40:1). After 20 min on ice, 0.75 ml of CHC& and 0.25 ml of Hz0 were added. The other steps in the procedure were the same as above except that the CHCl, phase was washed twice with 2 ml of 0.1 N HCL. Measurement of Mg” -ATPase actiuity. Mg*+ATPase activity was determined in a final volume of 1 ml in 25 mM imidazole HCl, pH 7.0, 5 mM MgCl*, 1 mM EGTA, 1 mM ATP, and 0.2 ml of ghost membranes. The tubes were incubated 30 min at 25°C and the reaction was stopped with 1.0 ml of 5% sodium dodecyl sulfate and inorganic phosphate was determined by the method of Peterson (21) using K,HPO, as the standard. The M$+-ATPase activity was corrected for absorbance due to ghosts and nonenzymatic hydrolysis of ATP. RESULTS
Time
Course
of 32P Incorporation
In this study, the time courses of “‘P incorporation into DPI and TPI were determined under conditions in which MgATP decreases the viscosity and induces echinocytic-discocytic shape transformations in rabbit ghosts (11). The ionic strength was maintained hypotonic at approximately 40 mM to prevent resealing and only freshly prepared ghosts were
60
EUGENE
used. Under standard phosphorylation conditions (see Materials and Methods), 32P was incorporated into DPI with an initial rate of 15.6 nmol/h/mg of total phospholipid and incorporation plateaued after 15 to 20 min (Fig. 1). Similar curves of 32P incorporation into DPI from [y-32P]ATP have been reported in human and swine erythrocyte ghosts (1, 2). The reason for plateauing at 20 min is unclear. In rabbit ghosts, M$+-ATPase activity is 552 nmol PJmg/h and, therefore, it can be calculated that only 6% of the initial ATP would be hydrolyzed after 20 min. Additionally, membrane phosphatidylinositol concentration decreases 24% after 20 min under these conditions (Quist, unpublished observations). Therefore, substrate depletion does not appear to be the reason for plateauing unless only a part of the phosphatidylinositol pool is available for phosphorylation to DPI. Alternatively, an increase in membrane DPI may inhibit phosphatidylinositol kinase activity. Incorporation of 32P into TPI was linear for 20 min and plateaued after approximately 80 min at 25°C (Fig. 1). The initial rate of 32P incorporation into TPI was 3.6 nmol/mg/h. Correction for two phosphates incorporated per molecule of TPI would yield an actual rate 1.8 nmol/mg/h. The specific activity of 32P incorporation into polyphosphoinositides in different ghost preparations did not vary by more than 25%. This contrasts to previous studies in which very wide variations in specific activity were found (1, 2). This difference could be attributed to the use of only freshly prepared ghosts in this study compared to the use of freeze-thawed ghosts or ghosts stored overnight at 5°C (1, 2). Hydrolysis and Turnover Polyphosphoinositides
of
One of the aims of this study was to determine if 32P incorporation into polyphosphoinositides represents net synthesis, turnover, or both within 20 min of incubation at 25°C. To approach this problem, polyphosphoinositides were labeled with 32P by incubating the ghosts for 20 min with 1 mM [Y-“~P]ATP. The ghosts
E. QUIST
20
40
Time FIG. 1. Time phosphoinositide
so
80
100
(min.)
courses of 32P incorporation (0) and triphosphoinositide
into di(A).
were then washed free of [T-~*]ATP as described under Materials and Methods and the ghosts were further incubated under standard conditions +l mM cold ATP (Fig. 2). After 15 min incubation, there was a slight decrease in labeled DPI, which varied from 0 to 5% in ghost preparations. This slight loss could be due to phosphatase activity or instability of DPI. In these studies 1 mM EGTA was always present in the incubation and washing medium and EGTA should effectively chelate endogenous Ca*+ and Ca*+ often found in commercial MgC12 thus preventing hydrolysis of polyphosphoinositides by phosphodiesterase activity (13). The addition of 1 mM ATP had no effect on the concentration of prelabeled DPI after 15 min incubation. Similarly, 32P-labeled TPI did not change in the presence or absence of ATP during these incubations. Therefore, both of these phospholipids are relatively stable under these conditions and do not turnover significantly in the presence of ATP.
RABBIT
ERYTHROCYTE
61
POLYPHOSPHOINOSITIDES
not undergoing rapid turnover within the first 20 min of incubation in these ghosts. Effect of Neomycin Turnover
il--I----0
6
10 Time
16
(min.)
FIG. 2. The effect of cold ATP on 32P-labeled diphosphoinositide (@) and triphosphoinositide (A). Diphosphoinositide (0) and triphosphoinositide (A) in the absence of ATP. Other conditions: 25 mM imidazole-HCl, pH 7.0, 5 mM MgC12, and 1 mM EGTA at 25°C. Data are representative of five separate experiments.
It was considered that a loss of Mg2+ kinase activity(s) during the washing procedure could account for the apparent lack of turnover. To examine this possibility, ghosts were incubated for 5 min with a relatively low concentration of [y-32P]ATP (0.1 mM) to label DPI and TPI. After 5 min, 2 mM cold ATP was added to the tubes and the tubes were further incubated for various times at 25°C (Fig. 3). The cold ATP was intended to dilute the hot ATP and thus suppress further 32P incorporation into the polyphosphoinositides. Under these conditions further synthesis of polyphosphoinositides from cold ATP could occur. After 30 min incubation there was not any appreciable change in the initial concentration of 32P-labeled DPI or TPI. Therefore, these data provide further evidence that volvnhosnhoinositides are A “. I
on Synthesis
and
In a previous study, it was shown that low concentrations of neomycin inhibit Mg-ATP-dependent shape and viscosity changes and 32P incorporatio n into DPI (11). However, the concentration curve for the effect of neomycin on 32P incorporation was not determined. In this study, neomycin was found to maximally decrease 32P incorporation into DPI at 0.1 mM in the presence of 1 mM [Y-~~P]ATP (Fig. 4). Incorporation of 32P into TPI was also maximally stimulated by this concentration of neomycin. In other studies, the polyphosphoinositides were prelabeled with 1 mM [y32P]ATP for 15 min at 25°C. The ghosts were washed free of [T-~~P]ATP and resuspended in the standard incubation medium. Incubation of the prelabeled ghosts for 15 min with 1 mM ATP, 5 mM MgC12, and 0.3 or 0.5 mM neomycin decreased 32P-
P i
i A- ' : ,' I ,I .2-j ' I /I
.
10
.
.
16
Time(min.) FIG. 3. Absence of turnover of diphosphoinositide (0) and triphosphoinositide (A) in rabbit ghosts. Data are representative of two separate experiments.
62
EUGENE
E. QUIST
labeled DPI approximately 2 nmol/mg and increased 32P-labeled TPI by exactly the same amount (Fig. 5). In the absence of MgCl* or ATP, neomycin did not have any effect on DPI or TPI concentration. It was shown in Fig. 2 that the concentration of 32P-labeled DPI or TPI does not change during incubation of the ghosts at 25°C. Because ATP and MgClz are required in addition to neomycin to increase labeled TPI at the expense of DPI neomycin may act by stimulating a kinase-mediated reaction which phosphorylates DPI to TPI. Effect of Freeze-Thawing X-l 00 on 32P-Labeled Polyphosphoinositides
and Triton
The effect of perturbing the membrane by freeze-thawing or by the addition of Triton X-100 on the turnover of DPI and TPI was examined. Ghosts were phosphorylated with Mg-[y-32P]ATP and washed free of the isotope by centrifugation as described under Materials and Methods. In some experiments, the labeled ghosts were freeze-thawed twice at -20°C. Incubation of these ghosts with 1 mM MgATP did not result in any change in the concentration of labeled DPI or TPI after 15 min at 25°C (not shown). Freeze-thawing also did not affect the initial concentrations of 3!P-labeled DPI or TPI ob-
0
0.1
0.2
[Neomycin]
0.3
0.4
0.5
mM
FIG. 5. The effect of neomycin on 32P-labeled diphosphoinositide (0) and triphosphoinositide (A) in the presence of 1 mM cold ATP. Effect of neomycin on 32P-labeled diphosphoinositide (0) and triphosphoinositide (A) in the absence of ATP. Data are representative of three separate experiments.
tained in prefrozen ghosts. Ghosts were incubated with 1 mM [Y-~~P]ATP and 0.05% (v/v) Triton X-100. This concentration of Triton X-100 was chosen because higher concentrations are highly inhibitory to 32P incorporations into TPI from [T-~‘P]ATP (Quist, unpublished observation). The concentration of 32P-labeled DPI and TPI also did not change after 15 min incubation with this detergent (not shown). DISCUSSION .;0
[Neomycin
SO,] mM
FIG. 4. The effect of neomycin on 32P incorporation into diphosphoinositide (0) and triphosphoinositide (0) from [y-=P]ATP.
It was recently hypothesized that MgATP initiates echinocytic-disco&c shape transformations and decreases the viscosity of rabbit erythrocyte ghosts by increasing membrane DPI concentration (11).
RABBIT
ERYTHROCYTE
However, it was assumed that “P incorporation into DPI from Mg-[y-32P]ATP was a measure of synthesis rather than turnover of this phospholipid (11). The results of the present investigation show that under conditions identical to those in which Mg-ATP induces these shape and viscosity changes, 32P incorporation into DPI from Mg-[y-32P]ATP is clearly related to synthesis of polyphosphoinositides. For instance, polyphosphoinositide monoesterase or phosphodiesterase activity (13, 14) was negligible in ghosts incubated for 20 min at 25°C (Fig. 2). In these studies EGTA was present which could chelate Ca2+ and suppress esterase activities (13, 14). Other studies showed that turnover of 32P-labeled DPI and TPI was not measurable for at least 20 min at 25’C in the presence of cold ATP (Figs. 2 and 3). Although these data support the assumption that 32P incorporation into DPI is a measure of DPI synthesis, a difference was found in the time course of Mg-ATPdependent viscosity and shape transformations and the time course of 32P incorporation into DPI found here (Fig. 1). Mg-ATP-dependent viscosity and shape transformations are complete within 10 to 15 min at 25°C (ll), whereas 32P incorporation into DPI did not plateau until 15 to 20 min under identical conditions (Fig. 1). These data could suggest that these events are not related or that the membrane synthesizes more DPI than is required to complete the shape or viscosity changes. Polyphosphoinositide
Synthesis
Although few studies have been done to characterize Mg2+ kinase activitie(s) involved in the synthesis of DPI and TPI in erythrocyte membranes, it is generally assumed that the synthetic pathway for the synthesis of polyphosphoinositides in erythrocyte membranes is the same as in brain (5, 15-18). In brain, DPI and TPI are thought to be synthesized by the sequential phosphorylation of phosphatidylinositol by phosphatidylinositol and diphosphoinositide kinase activities (15-18). In brain, phosphatidylinositol kinase is in
POLYPHOSPHOINOSITIDES
63
the plasma membrane and diphosphoinositide kinase is a cytoplasmic enzyme (18). In erythrocyte membranes, the kinase activity(s) involved in DPI and TPI synthesis are both membrane bound (l-4). Using the pathway derived from research on polyphosphoinositides in brain, it would be anticipated that 32P-labeled DPI would be phosphorylated to TPI in the presence of Mg-ATP. However, the results of this study indicate that the mechanism for polyphosphoinositide synthesis is more complicated in erythrocyte membranes and cannot be adequately explained using this mechanism. For instance, “2P-labeled DPI was not phosphorylated to TPI in the presence of Mg-ATP under conditions which ensured that the kinase activities involved in DPI and TPI syntheses were active (Figs. 2 and 3). These data therefore suggest that in these membranes DPI and TPI are synthesized by separate kinase pathways and that these membranes contain more than one phosphatidylinositol kinase activity. The observations that DPI is not further phosphorylated to TPI would suggest that synthesized DPI is not available to kinase activity(s) involved in the synthesis TPI (except in the presence of neomycin, see below). The results of this study do not show how many kinase activities would be required for the synthesis of DPI and TPI but they do suggest that these kinase activities may be highly organized in the membrane. Further studies on the kinetic parameters and other properties of Mg2+ kinase activities in these membranes are being done in an attempt to determine how many M$+ kinase activities are involved in polyphosphoinositide synthesis. Effects of Neomycin on Polyphosphoinositide Synthesis Neomycin and other polycations are known to form strong complexes with highly acidic polyphosphoinositides and neomycin has been suggested to alter 32P incorporation into DPI and TPI in guinea pig inner ear (19) and kidney (20) by binding to polyphosphoinositides. In this way, the availability of these substrates to ki-
64
EUGENE
nase and phosphatase activities may be affected. Previously, Lang et al. (7) reported that neomycin increased 32P incorporation into DPI and to a lesser extent into TPI in intact human erythrocytes. In rabbit erythrocytes ghosts, low concentrations of neomycin markedly decrease 32P incorporation into DPI and greatly increase 32P incorporation into TPI from Mg-[y-32P]ATP (Fig. 4). The maximal effects of neomycin were attainable at approximately 0.1 mM in these ghosts. The difference between the affects of neomycin on 32P incorporation into DPI and TPI in ghosts and intact erythrocytes could be due to differences in membrane permeability, species variation, or to the presence of active phosphatase activities in intact cells versus ghosts. In these ghost membranes, 0.5 mM neomycin inhibits Mg-ATP-dependent viscosity and shape changes presumably by decreasing DPI concentration (11). In this study it was found that neomycin may decrease DPI concentration by stimulating Mg-ATP-dependent phosphorylation of DPI to TPI (Fig. 5). Because Mg-ATP in the absence of neomycin does not stimulate the phosphorylation of 32P-labeled DPI to TPI (Figs. 2 and 3), neomycin may produce this affect by altering the availability of DPI to a kinase activity not normally involved in DPI synthesis and therefore perturbs the normal synthetic pathways involved in DPI and TPI synthesis. In a previous section, it was suggested that DPI and TPI may be synthesized by separate pathways in these membranes. Because neomycin can bind to DPI (ll), neomycin may be able to displace DPI from its usual site in the membrane thus making DPI available to a kinase which can further phosphorylate it to TPI. However, perturbing the membrane by freeze-thawing or by the addition of Triton X-100 did not produce the same effect as neomycin which suggests that this effect of neomycin may be relatively selective.
E. QUIST ACKNOWLEDGMENTS I wish to thank Karen Kolquist, Barbara Ronnie Barker, and Pat Powell for their technical assistance, and Jeanne Hudson, her secretarial assistance. This research ported by Grant HL 28458 from NIH.
Elmquist, excellent B.A., for was sup-
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OF BIOCHEMISTRY No. 1, November,
AND BIOPHYSICS pp. 58-64, 1982
Polyphosphoinositide
Synthesis
in Rabbit
EUGENE Lkpartment
of Pharmacology, Received
April
12,
1982,
Membranes
E. QUIST
North Medicine,
Osteopathic
Erythrocyte
Texas State Fort Worth, and
in revised
Uniuersity/Texa.s Texas 76107 form
July
13,
College
of
1982
Incubation of rabbit erythrocyte ghosts at 25°C with 1 mM [T-~‘P]ATP and MgCl, results in incorporation of 32P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol 32P/mg/h, respectively. Incorporation of 32P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with 32P, did not undergo significant breakdown when incubated at 25°C for 15 to 20 min. Turnover of 32P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl, and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.
Hokin and Hokin (1) were the first to report that 32P is incorporated into DPI’ and TPI from [Y-~*P]ATP in human erythrocyte ghosts. Similar results have been found in erythrocyte ghosts prepared from other mammalian species (2, 3). The kinase activity(s) involved in the synthesis of DPI and TPI from phosphatidylinositol is membrane bound, MgC12 dependent, and located on the cytoplasmic surface of the erythrocyte membrane (l-4). Because 32P turns over rapidly in DPI and TPI in
intact erythrocytes (5), polyphosphoinositides have been thought to have an important functional role in erythrocyte membranes (3-5). Among these, polyphosphoinositides have been implicated in the regulation of energy charge (6, 7), opening and closing erythrocyte pores (S), and in ATP-dependent pinocytosis (9,lO). Recently, Quist and Reece (11) reported that there is a strong correlation between 32P incorporation into DPI from Mg-ly3”P]ATP and Mg-ATP-dependent decreases in the viscosity of rabbit erythrocyte ghosts. The increase in viscosity is associated with an echinocyticcdiscocytic shape transformation of the ghosts and possibly with an increase in membrane deformability (11). Therefore, Mg-ATP may induce viscosity or shape changes in
1 Abbreviations used: UPI, dipbosphoinositide or phosphatidylinositol I-phosphate; TPI, triphosphoinositide or phosphatidylinositol 4,5-bisphosphate; EGTA, ethylene glycol bis(&aminoethyl ether). N,N ‘-tetraacetic acid; HPLC, high-pressure liquid chromatography. 00n3-9X~1/82/13005X-07$02.00/0 Copyright 0 1982 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
58
RABBIT
ERYTHROCYTE
ghost membranes by increasing membrane DPI concentration by stimulating a Mg2+dependent phosphatidylinositol kinase activity. Neomycin was also shown to inhibit Mg-ATP-dependent viscosity and shape changes in ghosts and to decrease the concentration of 32P-labeled diphosphoinositide (11). This study was initiated to determine if 32P incorporated into phosphoinositide from [y-“2]ATP was a measure of synthesis or turnover of DPI in these ghost membranes. Conditions were identical to those used in the previous study in which the effect of Mg-ATP on shape and viscosity was determined (11). The mechanism by which neomycin decreases 32P-labeled DPI was also investigated. MATERIALS
AND
59
POLYPHOSPHOINOSITIDES
METHODS
Materials. [y-“*P]ATP was obtained from New England Nuclear. Neomycin sulfate, Triton X-100, and phospholipid standards were obtained from Sigma Chemical Company. Solvents used for phospholipid analysis and chromatography were HPLC grade. Preparation of ghosts. Fresh heparinized blood was obtained by cardiac puncture from New Zealand white rabbits and the erythrocytes were washed three times with isotonic saline to remove plasma and white cells. Ghosts were usually prepared by washing packed erythrocytes two times with 10 vol of 20 mM TrisHCI, pH 7.6, at 20,OOOg for 15 min at 5°C. Ghosts were used within 1 h of preparation. 32P incorporation into ghost phospholipids. Ghosts were usually incubated in a final volume of 0.5 ml in 25 mM imidazole HCl, pH 7.0, 5 mM MgCl,, 1 mM [y-“‘P]ATP, 1 mM EGTA, and 180 pg of membrane phospholipid (0.1 ml of packed ghosts) at 25°C. In some experiments neomycin was included in the incubation medium. The reaction was stopped by the addition of 3 ml of cold 5% trichloroacetic acid. The tubes were then centrifuged at 2000g for 10 min at 5°C and the pellet was further washed with 3 ml of cold H,O. Lipids were extracted from the pelleted ghosts at 5°C by resuspension in 2.0 ml of CHC13:CH,0H:HC1 (20:40:1), containing 0.1% butylated hydroxytoluene. After 20 min on ice, 0.75 ml of CHCl,j and H,O were consecutively added to the tubes with vortexing and the tubes were centrifuged at 2OOOg for 10 min at 5°C. The upper phase was removed by aspiration and 1.0 ml of the CHCll phase was transferred to 2.0.ml glass-stoppered tubes and dried under a stream of N,. The dried lipid extract was resuspended with 30 ~1 of CHClz:CHBOH:CH1 (6::i:O.l) and 10.~1 aliquots were spotted on silica gel
60 plates (Merck) and assayed for phospholipid content by the method of Bartlett (12). Thin-layer plates were developed in CHC13:CH30H:H20:30% NH3 (25:35:7.5:2.5). Radiolabeled phospholipids were located overnight by autoradiography using Kodak OMAT R X-ray film. Labeled phospholipids were scraped from the plates and counted in 8 ml of tritisol. Recovery was 90%. Results are expressed as nanomoles of 32P incorporated per milligram of total ghost phospholipid. Effect of ATP and neomycin on 32P-labeled polyphosphoinositides. Ghosts were incubated for 20 min at 25’C in 25 mM imidazole, pH 7.0, 5 mM MgC12, 1 mM EGTA, and 1 mM [y-32P]ATP in a final volume of 0.5 ml. The reaction was stopped with 2.0 ml of cold 25 mM imidazole HCl, pH 7.0, 5 mM MgCl*, and 1 mM EGTA. The tubes were centrifuged at 20,OOOg for 15 min and the pellets were resuspended with 3 ml of the same solution and recentrifuged. Finally, the pellet was resuspended in the same medium. Where indicated, 1 mM ATP (disodium salt), neomycin sulfate, or Triton X-100 were added to the tubes. The reaction was usually stopped with 3 ml of cold 5% trichloroacetic acid and the phospholipids were extracted, separated, and counted as above. To study the effect of Triton X-100 it was necessary to stop the reaction with 2.0 ml CHCls:CHBOH:HCl (20:40:1). After 20 min on ice, 0.75 ml of CHC& and 0.25 ml of Hz0 were added. The other steps in the procedure were the same as above except that the CHCl, phase was washed twice with 2 ml of 0.1 N HCL. Measurement of Mg” -ATPase actiuity. Mg*+ATPase activity was determined in a final volume of 1 ml in 25 mM imidazole HCl, pH 7.0, 5 mM MgCl*, 1 mM EGTA, 1 mM ATP, and 0.2 ml of ghost membranes. The tubes were incubated 30 min at 25°C and the reaction was stopped with 1.0 ml of 5% sodium dodecyl sulfate and inorganic phosphate was determined by the method of Peterson (21) using K,HPO, as the standard. The M$+-ATPase activity was corrected for absorbance due to ghosts and nonenzymatic hydrolysis of ATP. RESULTS
Time
Course
of 32P Incorporation
In this study, the time courses of “‘P incorporation into DPI and TPI were determined under conditions in which MgATP decreases the viscosity and induces echinocytic-discocytic shape transformations in rabbit ghosts (11). The ionic strength was maintained hypotonic at approximately 40 mM to prevent resealing and only freshly prepared ghosts were
60
EUGENE
used. Under standard phosphorylation conditions (see Materials and Methods), 32P was incorporated into DPI with an initial rate of 15.6 nmol/h/mg of total phospholipid and incorporation plateaued after 15 to 20 min (Fig. 1). Similar curves of 32P incorporation into DPI from [y-32P]ATP have been reported in human and swine erythrocyte ghosts (1, 2). The reason for plateauing at 20 min is unclear. In rabbit ghosts, M$+-ATPase activity is 552 nmol PJmg/h and, therefore, it can be calculated that only 6% of the initial ATP would be hydrolyzed after 20 min. Additionally, membrane phosphatidylinositol concentration decreases 24% after 20 min under these conditions (Quist, unpublished observations). Therefore, substrate depletion does not appear to be the reason for plateauing unless only a part of the phosphatidylinositol pool is available for phosphorylation to DPI. Alternatively, an increase in membrane DPI may inhibit phosphatidylinositol kinase activity. Incorporation of 32P into TPI was linear for 20 min and plateaued after approximately 80 min at 25°C (Fig. 1). The initial rate of 32P incorporation into TPI was 3.6 nmol/mg/h. Correction for two phosphates incorporated per molecule of TPI would yield an actual rate 1.8 nmol/mg/h. The specific activity of 32P incorporation into polyphosphoinositides in different ghost preparations did not vary by more than 25%. This contrasts to previous studies in which very wide variations in specific activity were found (1, 2). This difference could be attributed to the use of only freshly prepared ghosts in this study compared to the use of freeze-thawed ghosts or ghosts stored overnight at 5°C (1, 2). Hydrolysis and Turnover Polyphosphoinositides
of
One of the aims of this study was to determine if 32P incorporation into polyphosphoinositides represents net synthesis, turnover, or both within 20 min of incubation at 25°C. To approach this problem, polyphosphoinositides were labeled with 32P by incubating the ghosts for 20 min with 1 mM [Y-“~P]ATP. The ghosts
E. QUIST
20
40
Time FIG. 1. Time phosphoinositide
so
80
100
(min.)
courses of 32P incorporation (0) and triphosphoinositide
into di(A).
were then washed free of [T-~*]ATP as described under Materials and Methods and the ghosts were further incubated under standard conditions +l mM cold ATP (Fig. 2). After 15 min incubation, there was a slight decrease in labeled DPI, which varied from 0 to 5% in ghost preparations. This slight loss could be due to phosphatase activity or instability of DPI. In these studies 1 mM EGTA was always present in the incubation and washing medium and EGTA should effectively chelate endogenous Ca*+ and Ca*+ often found in commercial MgC12 thus preventing hydrolysis of polyphosphoinositides by phosphodiesterase activity (13). The addition of 1 mM ATP had no effect on the concentration of prelabeled DPI after 15 min incubation. Similarly, 32P-labeled TPI did not change in the presence or absence of ATP during these incubations. Therefore, both of these phospholipids are relatively stable under these conditions and do not turnover significantly in the presence of ATP.
RABBIT
ERYTHROCYTE
61
POLYPHOSPHOINOSITIDES
not undergoing rapid turnover within the first 20 min of incubation in these ghosts. Effect of Neomycin Turnover
il--I----0
6
10 Time
16
(min.)
FIG. 2. The effect of cold ATP on 32P-labeled diphosphoinositide (@) and triphosphoinositide (A). Diphosphoinositide (0) and triphosphoinositide (A) in the absence of ATP. Other conditions: 25 mM imidazole-HCl, pH 7.0, 5 mM MgC12, and 1 mM EGTA at 25°C. Data are representative of five separate experiments.
It was considered that a loss of Mg2+ kinase activity(s) during the washing procedure could account for the apparent lack of turnover. To examine this possibility, ghosts were incubated for 5 min with a relatively low concentration of [y-32P]ATP (0.1 mM) to label DPI and TPI. After 5 min, 2 mM cold ATP was added to the tubes and the tubes were further incubated for various times at 25°C (Fig. 3). The cold ATP was intended to dilute the hot ATP and thus suppress further 32P incorporation into the polyphosphoinositides. Under these conditions further synthesis of polyphosphoinositides from cold ATP could occur. After 30 min incubation there was not any appreciable change in the initial concentration of 32P-labeled DPI or TPI. Therefore, these data provide further evidence that volvnhosnhoinositides are A “. I
on Synthesis
and
In a previous study, it was shown that low concentrations of neomycin inhibit Mg-ATP-dependent shape and viscosity changes and 32P incorporatio n into DPI (11). However, the concentration curve for the effect of neomycin on 32P incorporation was not determined. In this study, neomycin was found to maximally decrease 32P incorporation into DPI at 0.1 mM in the presence of 1 mM [Y-~~P]ATP (Fig. 4). Incorporation of 32P into TPI was also maximally stimulated by this concentration of neomycin. In other studies, the polyphosphoinositides were prelabeled with 1 mM [y32P]ATP for 15 min at 25°C. The ghosts were washed free of [T-~~P]ATP and resuspended in the standard incubation medium. Incubation of the prelabeled ghosts for 15 min with 1 mM ATP, 5 mM MgC12, and 0.3 or 0.5 mM neomycin decreased 32P-
P i
i A- ' : ,' I ,I .2-j ' I /I
.
10
.
.
16
Time(min.) FIG. 3. Absence of turnover of diphosphoinositide (0) and triphosphoinositide (A) in rabbit ghosts. Data are representative of two separate experiments.
62
EUGENE
E. QUIST
labeled DPI approximately 2 nmol/mg and increased 32P-labeled TPI by exactly the same amount (Fig. 5). In the absence of MgCl* or ATP, neomycin did not have any effect on DPI or TPI concentration. It was shown in Fig. 2 that the concentration of 32P-labeled DPI or TPI does not change during incubation of the ghosts at 25°C. Because ATP and MgClz are required in addition to neomycin to increase labeled TPI at the expense of DPI neomycin may act by stimulating a kinase-mediated reaction which phosphorylates DPI to TPI. Effect of Freeze-Thawing X-l 00 on 32P-Labeled Polyphosphoinositides
and Triton
The effect of perturbing the membrane by freeze-thawing or by the addition of Triton X-100 on the turnover of DPI and TPI was examined. Ghosts were phosphorylated with Mg-[y-32P]ATP and washed free of the isotope by centrifugation as described under Materials and Methods. In some experiments, the labeled ghosts were freeze-thawed twice at -20°C. Incubation of these ghosts with 1 mM MgATP did not result in any change in the concentration of labeled DPI or TPI after 15 min at 25°C (not shown). Freeze-thawing also did not affect the initial concentrations of 3!P-labeled DPI or TPI ob-
0
0.1
0.2
[Neomycin]
0.3
0.4
0.5
mM
FIG. 5. The effect of neomycin on 32P-labeled diphosphoinositide (0) and triphosphoinositide (A) in the presence of 1 mM cold ATP. Effect of neomycin on 32P-labeled diphosphoinositide (0) and triphosphoinositide (A) in the absence of ATP. Data are representative of three separate experiments.
tained in prefrozen ghosts. Ghosts were incubated with 1 mM [Y-~~P]ATP and 0.05% (v/v) Triton X-100. This concentration of Triton X-100 was chosen because higher concentrations are highly inhibitory to 32P incorporations into TPI from [T-~‘P]ATP (Quist, unpublished observation). The concentration of 32P-labeled DPI and TPI also did not change after 15 min incubation with this detergent (not shown). DISCUSSION .;0
[Neomycin
SO,] mM
FIG. 4. The effect of neomycin on 32P incorporation into diphosphoinositide (0) and triphosphoinositide (0) from [y-=P]ATP.
It was recently hypothesized that MgATP initiates echinocytic-disco&c shape transformations and decreases the viscosity of rabbit erythrocyte ghosts by increasing membrane DPI concentration (11).
RABBIT
ERYTHROCYTE
However, it was assumed that “P incorporation into DPI from Mg-[y-32P]ATP was a measure of synthesis rather than turnover of this phospholipid (11). The results of the present investigation show that under conditions identical to those in which Mg-ATP induces these shape and viscosity changes, 32P incorporation into DPI from Mg-[y-32P]ATP is clearly related to synthesis of polyphosphoinositides. For instance, polyphosphoinositide monoesterase or phosphodiesterase activity (13, 14) was negligible in ghosts incubated for 20 min at 25°C (Fig. 2). In these studies EGTA was present which could chelate Ca2+ and suppress esterase activities (13, 14). Other studies showed that turnover of 32P-labeled DPI and TPI was not measurable for at least 20 min at 25’C in the presence of cold ATP (Figs. 2 and 3). Although these data support the assumption that 32P incorporation into DPI is a measure of DPI synthesis, a difference was found in the time course of Mg-ATPdependent viscosity and shape transformations and the time course of 32P incorporation into DPI found here (Fig. 1). Mg-ATP-dependent viscosity and shape transformations are complete within 10 to 15 min at 25°C (ll), whereas 32P incorporation into DPI did not plateau until 15 to 20 min under identical conditions (Fig. 1). These data could suggest that these events are not related or that the membrane synthesizes more DPI than is required to complete the shape or viscosity changes. Polyphosphoinositide
Synthesis
Although few studies have been done to characterize Mg2+ kinase activitie(s) involved in the synthesis of DPI and TPI in erythrocyte membranes, it is generally assumed that the synthetic pathway for the synthesis of polyphosphoinositides in erythrocyte membranes is the same as in brain (5, 15-18). In brain, DPI and TPI are thought to be synthesized by the sequential phosphorylation of phosphatidylinositol by phosphatidylinositol and diphosphoinositide kinase activities (15-18). In brain, phosphatidylinositol kinase is in
POLYPHOSPHOINOSITIDES
63
the plasma membrane and diphosphoinositide kinase is a cytoplasmic enzyme (18). In erythrocyte membranes, the kinase activity(s) involved in DPI and TPI synthesis are both membrane bound (l-4). Using the pathway derived from research on polyphosphoinositides in brain, it would be anticipated that 32P-labeled DPI would be phosphorylated to TPI in the presence of Mg-ATP. However, the results of this study indicate that the mechanism for polyphosphoinositide synthesis is more complicated in erythrocyte membranes and cannot be adequately explained using this mechanism. For instance, “2P-labeled DPI was not phosphorylated to TPI in the presence of Mg-ATP under conditions which ensured that the kinase activities involved in DPI and TPI syntheses were active (Figs. 2 and 3). These data therefore suggest that in these membranes DPI and TPI are synthesized by separate kinase pathways and that these membranes contain more than one phosphatidylinositol kinase activity. The observations that DPI is not further phosphorylated to TPI would suggest that synthesized DPI is not available to kinase activity(s) involved in the synthesis TPI (except in the presence of neomycin, see below). The results of this study do not show how many kinase activities would be required for the synthesis of DPI and TPI but they do suggest that these kinase activities may be highly organized in the membrane. Further studies on the kinetic parameters and other properties of Mg2+ kinase activities in these membranes are being done in an attempt to determine how many M$+ kinase activities are involved in polyphosphoinositide synthesis. Effects of Neomycin on Polyphosphoinositide Synthesis Neomycin and other polycations are known to form strong complexes with highly acidic polyphosphoinositides and neomycin has been suggested to alter 32P incorporation into DPI and TPI in guinea pig inner ear (19) and kidney (20) by binding to polyphosphoinositides. In this way, the availability of these substrates to ki-
64
EUGENE
nase and phosphatase activities may be affected. Previously, Lang et al. (7) reported that neomycin increased 32P incorporation into DPI and to a lesser extent into TPI in intact human erythrocytes. In rabbit erythrocytes ghosts, low concentrations of neomycin markedly decrease 32P incorporation into DPI and greatly increase 32P incorporation into TPI from Mg-[y-32P]ATP (Fig. 4). The maximal effects of neomycin were attainable at approximately 0.1 mM in these ghosts. The difference between the affects of neomycin on 32P incorporation into DPI and TPI in ghosts and intact erythrocytes could be due to differences in membrane permeability, species variation, or to the presence of active phosphatase activities in intact cells versus ghosts. In these ghost membranes, 0.5 mM neomycin inhibits Mg-ATP-dependent viscosity and shape changes presumably by decreasing DPI concentration (11). In this study it was found that neomycin may decrease DPI concentration by stimulating Mg-ATP-dependent phosphorylation of DPI to TPI (Fig. 5). Because Mg-ATP in the absence of neomycin does not stimulate the phosphorylation of 32P-labeled DPI to TPI (Figs. 2 and 3), neomycin may produce this affect by altering the availability of DPI to a kinase activity not normally involved in DPI synthesis and therefore perturbs the normal synthetic pathways involved in DPI and TPI synthesis. In a previous section, it was suggested that DPI and TPI may be synthesized by separate pathways in these membranes. Because neomycin can bind to DPI (ll), neomycin may be able to displace DPI from its usual site in the membrane thus making DPI available to a kinase which can further phosphorylate it to TPI. However, perturbing the membrane by freeze-thawing or by the addition of Triton X-100 did not produce the same effect as neomycin which suggests that this effect of neomycin may be relatively selective.
E. QUIST ACKNOWLEDGMENTS I wish to thank Karen Kolquist, Barbara Ronnie Barker, and Pat Powell for their technical assistance, and Jeanne Hudson, her secretarial assistance. This research ported by Grant HL 28458 from NIH.
Elmquist, excellent B.A., for was sup-
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