Mode of Interaction of Phosphofructokinase with the Erythrocyte ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 01985 by The American Society of Biological Chemists, Inc.

Vol. 260,No. 19, Issue of September 5, pp. 10426-10433, 1985 Printed in U S A .

Mode of Interaction of Phosphofructokinase with the Erythrocyte Membrane* (Received for publication, January 4, 1985)

Jean D. Jenkins$, FerencJ. Kezdy, and Theodore L. SteckP From the Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates theenzyme in vitroby relievingtheallostericinhibition imposed by ATP (Karadsheh, N. S., and Uyeda, K. (1977) J. Biol. Chern. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme’s adenine nucleotide activation site,are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane amino vesicles and a 23-kDa fragment containing the terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. Thedependence of the residualphosphofructokinase activityon phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramersto inactivedimers. Dimers of phosphofructokinase associate withthe membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimersof phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.

from the membrane (Higashi et at., 1979). The binding site for these enzymes as well as for hemoglobin lies in the highly acidic amino-terminal portion of band 3 (Murthy et al., 1981; Tsai et al., 1982; Kaul et al., 1983; Salhany, 1983; Murthy et al., 1984; Walder et al., 1984). Phosphofructokinase also interacts with thin filaments of muscle (Liou and Anderson, 1980) and with calmodulin (Mayr andHeilmeyer, 1983). Mammalian phosphofructokinase exists ina pH- and concentration-dependent association equilibrium. The smallest active form is the tetramer (Pavelich and Hammes, 1973). Tetramers aggregate at alkaline pH andhigh enzyme concentration (Wenzel et al., 1978; Hesterberg and Lee, 1981) and dissociate to inactive dimers at high dilution in acidic media (Aaronson and Frieden, 1972; Pavelich and Hammes, 1973). The rate and extentof inactivation increase at low temperature, a property known as cold lability (Bock and Frieden, 1976a; Wenzel et al., 1978). Allosteric phosphofructokinase inhibitors, suchas ATP and citrate,also increase inactivation while allosteric activators, suchas fructose-1,6-P2 and AMP, decrease inactivation (Bock and Frieden, 1976b). In this study, we show that the bindingof phosphofructokinase to the human erythrocyte membraneprobably occurs at its adenine nucleotide activation site. An excess of band 3 leads to phosphofructokinase inactivation through the avid binding of band 3 to phosphofructokinasedimers. A preliminaryaccount of this work has been published previously (Jenkins and Steck, 1983).

Phosphofructokinase is complex a regulatory enzyme (Uyeda, 1979). Binding of phosphofructokinase to the erythrocyte membrane in uitro relieves allosteric inhibitionby ATP and 2,3-bisphosphoglycerate, converting the sigmoidal fructose-6-P saturation curve to a hyperbolic form (Karadsheh and Uyeda, 1977). Band 3, the predominant integral protein and anion transporterof the erythrocyte membrane, has been implicated as the membrane-binding site since glyceraldehyde-3-phosphate dehydrogenase and fructose bisphosphate aldolase, both of which bind to band 3 (Yu and Steck, 1975; StrapazonandSteck, 1977),displace phosphofructokinase

orMCBReagents. Purifiedrabbitliverphosphofructokinase was generously provided by Lawrence Foe ofthe Chicago Medical School. The purified 23-kDa amino-terminal fragmentof band 3 was generated by the S-cyanylation of alkali-stripped ghosts (Murthy et al., 1981) and generously provided by S. N. Prasanna Murthy of Northwestern University. Human Phosphofructokinase-Phosphofructokinase was purified from outdated human red cells according thetomethod of Karadsheh et al. (1977) and stored at -70 “C. Protein was quantified according to Bradford (1976) or by a modified Lowry procedure(Peterson, 1977) using bovine serum albumin as a standard. The enzyme was nearly homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide Portions of this paper (including part of “Experimental Procedures” and Scheme I) are presented in miniprint at the end of this paper. The abbreviation used is: PFK, phosphofructokinase. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-0016, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

* This work was supported by American Cancer SocietyGrant BC95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “c&ertisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact. $ Supported by Medical Scientist Training Program Grant PHS 5 T32 GM07281. § To whom correspondence should be addressed: 920 E. 58th St., Chicago, IL 60637.

EXPERIMENTAL PROCEDURES~

Materia/+”utdated human blood, obtained from the University of Chicago Blood Bank orUnited Blood Services, was usedwithin 57 weeks after being drawn. Biochemical reagents and enzymes were fromSigma.Otherchemicals were of reagentgradefromFisher, Mallinckrodt Chemical Works, J. T. Baker Chemical Co., Bio-Rad,

10426

Phosphofructokinase Interaction gel electrophoresis according to Fairbanks et al. (1971). The specific activity of several preparations was 63-67 units/mg protein, about two-thirds of that previously reported (Layzer, 1975; Karadsheh et al., 1977). Phosphofructokinase was dialyzed before use against 1000 volumes of ice-cold buffer containing 5 mM KPi or 10 mM imidazole acetate of defined pH, 5 mM 2-mercaptoethanol, and 0.1 mM EDTA. Membranes-Red blood cells and their ghosts were prepared as described (Fairbanks et al., 1971) except that ghosts were washed once in 40 volumes of 150 mM NH,HCOs to elute endogenous glycolytic enzymes from the membrane (Kant and Steck, 1973) and then twice in 5 mM NaPi (pH 8). Vesicles of inside-out orientation were prepared as described (Steck and Kant,1974).Briefly, the membranes were incubated overnight at 0 'C in 40 volumes of 1 mM NaPi (pH 8) and then sedimented a t 39,000 X g for 20 min, and thepellets were homogenized by five passages through a27-gauge needle. The vesicles were washed in 5 mM KPi or 10 mM imidazole acetate of defined pH. Vesicles were stored in thecold and used within 5 days. Acetylcholinesterase activity of the vesicles and ghosts was assayed spectrophotometrically (Steck and Kant, 1974). From the fractional accessibility of acetylcholinesterase in the presence and absence of 0.1% Triton X-100, we determined that the vesicle preparations were 50-70% inside-out. Ghosts were counted ona model ZB Coulter Counter, with correction for background and coincidence. The concentration of band 3with its cytoplasmic pole exposed to themedium was calculated for each vesicle preparation by assuming that there are 1.2 X 10' band 3 monomers/ghost (Fairbanks et al., 1971) and that the specific content of acetylcholinesterase and band 3 was the same in ghosts and vesicles (Steck, 1974). Enzyme Assay-Maximal phosphofructokinase activity was measured spectrophotometrically a t room temperature in a mixture containing 50 mM Tris-C1, 10 mM NHIC1, 5 mM MgCl,, 3 mM Napi, 2 mM fructose-6-P, 0.4 mM ATP, 0.2 mM EDTA, 0.1 mM NADH, 25 units/ml aldolase, 25 units/ml triose phosphate isomerase, and 2.5 units/ml glycerol-3-phosphate dehydrogenase. The final pH was 8.5. The reaction was initiated by the addition of fructose-6-P, and absorbance was recorded at 340 nm during the second minute of reaction after an initial lag (Reinhart andLardy, 1980a). Enzyme Inactiuation-Dialyzed phosphofructokinase was diluted in the following inactivation buffer: 5 mM KPi, 1 mM dithiothreitol, 0.1% bovine serum albumin, and 10 p~ fructose-1,6-Pz. Because inactivation was highly pH dependent, the pHelectrode, buffer standards, and buffer were pre-equilibrated at the same temperature for titrations. Vesicles were added to the enzyme solution to start the reaction. Aliquots were withdrawn a t intervals, and the enzyme was diluted to a concentrationof 6 X 10"' M in the assay buffer described above. The increase in pH and ionic strength was sufficient to elute the enzyme from the membrane and prevent further inactivation, while the dilution of enzyme subunits prevented reassociation. Enzyme Reactivation-Reactivation was initiated by sedimenting the vesicles with bound inactive phosphofructokinase at 39,000 X g for 10 min at 0 "C and resuspending the vesicles in the following activation buffer: 50 mM KPi (pH 8), 2 mM fructose-1,6-Pz, 1 mM dithiothreitol, and 0.1% bovine serum albumin. At intervals, aliquots were assayed for phosphofructokinase activity. Enzyme-Membrane Interactions-Phosphofructokinase binding to vesicles was assessed by two methods. In the centrifugation assay, enzyme and vesicles were mixed in buffer containing 15 mM Mg acetate, 10 mM imidazole acetate, 1 mM dithiothreitol, 0.1% bovine serum albumin, 10 p~ fructose-1,6-P2, and additional ligands at pH 7.5. The mixture was allowed to equilibrate a t 0 "C and then centrifuged a t 39,000 X g for 10 min at 0 "C.The supernatantwas removed and thepellet resuspended in the same volume of buffer. Aliquots of the supernatant and resuspended pellet were assayed for phosphofructokinase activity. In the filtration assay, the enzyme was diluted in inactivation buffer and its activity measured. Then vesicles were added, the suspension was mixed rapidly, incubated a t 0 "C for 15 s, and the supernatantseparated from the vesicles using a 0.2-pm pore polycarbonate membrane (Nucleopore) and a D39 prefilter which had been pretreated with polethylene glycol 20,000 to eliminate enzyme adsorption. Control experiments established that this filter combination removed 97% of the vesicles from the filtrate. Aliquots of the filtrate were assayed and compared to the total activity prior to vesicle addition. RESULTS

We have confirmed that phosphofructokinase from both humanerythrocytesandrabbit muscle binds to the cyto-

with Erythrocyte Membranes

10427

plasmic face of the human erythrocytemembrane (Karadsheh and Uyeda, 1977). Because of the limited permeability of ghosts to proteins (Lieber and Steck, 1982), their binding of exogenous glycolytic enzymes often required over an hour to reach equilibrium (Jenkins, 1983). As with saponin-treated red cell membranes (Jenkins et al., 1984), inside-out vesicles circumvented this diffusion barrier andallowed rapid binding. Most of our studies were, therefore, carried out with these vesicles. Phosphofructokinase binding to red cell membranes was greatest in low ionic strength, mildly acidic buffers, and was rapidly and completely reversed at p 2 0.1 and at pH 8 (see also Higashi et al., 1979).The binding isotherm was a rectangular hyperbola (Fig. 1). From the linear Scatchard plot (inset), a dissociation constant of Kt = 2 x M was estimated. This value has little intrinsic significance,however, since binding affinity varied over several orders of magnitude with pH, ionic strength, and buffer species. Phosphofructokinase binding to red cell membranes resembled that of two other glycolytic enzymes and hemoglobin in these respects. Effect of Ligands on Phosphofructokinase Binding-In the presence of M P ,a variety of anionic ligands displaced phosphofructokinase from the membranes (Fig. 2). Their order of potency was ADP > ATP > NADH > fructose-6-P > ITP > fructose 1,6-Pz> 2,3-bisphosphoglycerate > SO:-. Inactivation of Phosphofructokinase-In the absence of magnesium, inside-out vesicles promoted the slow inactivation of phosphofructokinase (Fig. 3A). A similar effect was observed with the purified 23-kDa amino-terminal fragment (Fig. 3B), establishing that the cytoplasmic pole of band 3 was responsible. Note (a) that theinactivation did not go to completion but rather reached a plateau after about an hour and ( b ) that the kinetics were notfirst order butrather biphasic, with an initial rapid phase followedby a slower phase. Thesefeaturesare of mechanistic significance, as demonstrated below. The determinants of the plateau activity were analyzed by incubating a varying amount of phosphofructokinase with a constant concentration of band 3 which was present in large excess (Fig. 4). The plateau activity decreased with decreasing phosphofructokinase concentration, from 29% residual activity with a phosphofructokinase concentration of 63 nM to 7% residual activity with a phosphofructokinase concentration of 6 nM. This suggests that inactivation represented the revers-

80

l-

%PFK

bound

60

40

20 0

"

0

50

100 II( IOV

150

200

FIG. 1. Binding of human erythrocyte phosphofructokinase ( P F K ) to inside-out vesicles ( I O U . Phosphofructokinase (6 nM) was incubated with increasing amounts of vesicles for 15 s in 1.0 ml of inactivation buffer at pH 7 and 0 "C before its binding to the membranes was determined by filtration. We calculated that 100 pl of inside-out vesicles contained 930 pmol of band 3. The inset shows aScatchard plot; T - B represents enzyme-band 3 complex and B represents band 3. We assumed that band 3 was in such excess that a negligible fraction was bound to enzyme.

Phosphofructokinase Interaction with Erythrocyte Membranes

10428

6C

\ 40

0'

1

I

-00

-5

I

z-

X

I

-3 -2 Log ligand concentration, M -4

L

a 0

FIG. 2. Effect of various ligands on the dissociation of phosphofructokinase (PFK) from inside-out vesicles. Phosphofructokinase (6nM) was incubated with 100 pl of vesicles in 1ml of buffer containing 15 mM Mg acetate, 10 mM imidazole acetate, 1 mM dithiothreitol, 0.1% bovine serum albumin, 10 pM fructose-1,6-Pz, and the indicated ligands at pH7.5 and 0 "C for 5 min. Binding was measured following centrifugation. m, ADP 0, ATP; A, NADH; 0, fructose-6-P; X, ITP; A, fructose-1,6-Pz; 'I, 2,3-bisphosphoglycerate; U, Na2S04.

.h

t 2c a

Q

k k k 20 Time,minutes

OO

40

6

FIG. 4. Effect of phosphofructokinase (PFK) concentration on inactivation. Phosphofructokinase (6-63 nM) was mixed with 100 p1 of vesicles (equivalent to 700 pmol of band 3) in 1 mlof inactivation buffer at pH 7 and incubated at 0 "C. Aliquots of the suspension were assayed for phosphofructokinase activity at the indicated times. Phosphofructokinase concentrations: 0,6 nM; U, 16 nM; A, 31 nM; 0, 63 nM. solid lines are theoretical curves calculated as described in the Miniprint Supplement.

2o

t

"9 60

100 O

m

% lnltid

PFK Activity 40

20

l20 B " 0

0' 0

I

I

30

I

I

I

60

I

90

I

1 120

Tim, mhutos 10

20

3 0 4 0

n-.

5

0

6

0

n11~t.8

FIG. 3. Inactivation of phosphofructokinase (PFK) by inside-out vesicles and the 23-kDa fragment of band 3. A, phosphofructokinase (8nM) was mixed with vesicles in 1ml of inactivation buffer at pH 7 and 0 "C. Aliquots were assayed for enzyme activity a t the indicated times. 0, control; +, 10 pl of inside-out vesicles; 0, 100 pl of inside-out vesicles. (100 pl of vesicles wasequivalent to 650 pmol of band 3.) B, phosphofructokinase (6 nM) was mixed with the 23kDa fragment of band 3 (170 nM) in inactivation buffer at pH 6 and 0 "C. Aliquots were assayed at theindicated times. 0, control; 0 , 2 3 kDa fragment.

ible dissociation of active to inactive species. Centrifugation of phosphofructokinase-vesiclemixtures revealed that almost all of the phosphofructokinase,active and inactive, was bound to themembrane under these conditions. Reversibility of inactivation was demonstrated by incubating vesicles bearing inactive phosphofructokinase in a buffer

FIG. 5. Reactivation of phosphofructokinase (PFK). Phosphofructokinase (31 nM) was incubated with 100 pl/ml vesicles in inactivation buffer at pH 6 and 0 "C for 1 h, a t which time 14% of the initial activity remained. Vesicles weresedimented, and thepellets were resuspended in activation buffer and incubated a t 21 "C a t various phosphofructokinase concentrations. Aliquots of the suspension were assayed for phosphofructokinase activity at the indicated times. Phosphofructokinase concentrations (nM): 0,6; +, 15; 0, 31; 0.62.

containing 50 mM KPi and 2 mM fructose-1,6-P2at pH 8. These conditions eluted all of the phosphofructokinase from the membrane and restored its catalytic activity (Fig. 5). The rate and extentof reactivation increased with the concentration of phosphofructokinase in the reactivation buffer, in keeping with a reassociation reaction. In several experiments, the reactivated enzyme regainedall of its original activity. Since the conditions for reactivation are those which promote phosphofructokinase subunit reassociation to tetramers

10429

Phosphofructokinase Interaction Erythrocyte with Membranes (Bock and Frieden, 1976b; Wenzel et al., 1978), we analyzed the plateau enzyme activities from the experiment shown in Fig. 4 to determine whether they reflected an equilibrium mixture of active tetramers andinactive dimers or monomers. We plotted the logarithm of the tetramer concentration at equilibrium against the logarithm of the initial minus the equilibrium tetramer concentration, since the slope of such a plot should give the number of subunits intowhich the enzyme dissociated (see legend for Fig. 6). As shown in Fig. 6, the plot was linear and hada slope of 2. We conclude that inactivation proceeded by the dissociation of phosphofructokinase tetramers to dimers. To further analyze the kinetics of inactivation, membrane concentration was varied, and the initialrates of enzyme inactivation were correlated with phosphofructokinase bind3 2 1

brT

ing (Fig. 7). Binding was assessed by filtration of samples 15 s after the addition of vesicles, beforea significant amount of inactivationhad occurred. The initialrate of inactivation continued to increase with increasing membrane concentration even after the binding of phosphofructokinase had reached a plateau. The inset to Fig. 7 dramatizes this result, showing that the initial rate of inactivation was not directly proportional to thefraction of phosphofructokinase bound to the membrane. This behavior rules out amechanism in which membrane-binding directly inactivated the enzyme. Therefore, we postulated that tetramers on the membrane did not readily dissociate to dimers butratherthat tetramers in solution dissociated to dimers which then bound to themembrane. To demonstrate the existence of dimers in solution, phosphofructokinase was incubated alone at various concentrations in inactivation buffer prior to assay under standard conditions. Enzyme activity fell by up to15% with increasing dilution. The dissociation rate was rapid in that the decline in phosphofructokinase activity was complete within 15 s of dilution. We used these data to determine the dissociation constant for the tetramer (2')-dimer (D)equilibrium: K, = (D)'/(T). K, can be related to the fraction of total enzyme which is active by the expression

0

EIA = C

+0.5TC.l/fi

(1)

where E is the total phosphofructokinase present, A is the measured enzyme activity at equilibrium, and C is the proportionality constant relating tetramer concentration to measured activity (Jenkins, 1983). A plot of our data on the loss -2 2 3 4 of phosphofructokinase activity with dilution according to III (E -T) Equation 1 is shown in Fig. 8. It yielded a value for the FIG. 6. Analysis of the subunit size of inactive phosphofruc- dissociation constant for the tetramer-dimer equilibrium of tokinase. The 60-min values for enzyme activity inFig. 4 were taken K. = 6 X 10"' M, albeit with only moderate precision. to reflect the equilibrium tetramer concentration ( T ) while the initial Quantitative Analysis of the Phosphofructokinase-Memvalues were taken to reflect total enzyme concentration ( E ) . If T brane Equilibrium Mixture-The findings presented above dissociates into n subunits of concentration ( X ) , (E) = ( T ) + (X)/n. are all compatible with a mechanism of phosphofructokinase At equilibrium, Q = (X)"/(T), where Q is a pseudo-dissociation constant which disregards membrane binding. It can be shown that inactivation in which the enzyme exists as an equilibrium -1

ln(T) = n ln(E - )'2 + n Inn - 1nQ (Jenkins, 1983). ln(T) was, therefore, plotted againstln(E T).The least-squares fit of the slope of the plot yielded n = 1.996, with a correlation coefficient of 0.995. From the intercept, Q = 4.8 X lo" M.

-

320

300 E - 1 A

% PFK

nY U

-80

280

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+

20 0' 0

I

50

I

100

I

150

'

' 0 200

VI Kw

FIG. 7. Correlation of phosphofructokinase (PFK) binding to vesicles with initial rates of inactivation. Phosphofructokinase (6 nM) was incubated with increasing amounts of vesicles in 1 ml of inactivation buffer at pH 7 and 0 'C (100 pl of vesicles = 930 pmol of band 3). Binding was assessed by filtration after 15 s. Initial rates of inactivation ( VO)were determined in parallel experiments from measurements of enzyme activity. For this purpose, the inactivation time course was stopped after 1,15,30, and60 s of incubation by dilution into assay buffer (see "Experimental Procedures"). 0,per cent phosphofructokinase bound; +, initial rate of inactivation, V,. The inset shows a direct comparison of initial rate of inactivation with per cent of phosphofructokinase bound. ZOV, inside-out vesicles.

260 0

I

I

I

2

4

6

8

f FIG.8. Determination of the dissociation constant,K,,for the phosphofructokinase tetramer-dimer equilibrium in solution. Phosphofructokinase was incubated a t several concentrations between 3 and 160 nM in inactivation buffer at pH 7 and 0 "C for 2 min. Aliquots of the solution were diluted in enzyme assay buffer, and phosphofructokinase activity was measured. E , the total (input) phosphofructokinase concentration, divided by A, the measured enzyme activity at equilibrium, was plottedagainst 1/a K. was determined from the slo e of the line according to theequation, E / A = C + 0.5-.1/d where C is a constant relating tetramer concentration to measured enzyme activity. U represents units of phosphofructokinase activity (see Jenkins, 1983).


10430

mixture of active tetramers andinactive dimers, each of which is in association equilibrium with band 3, as shown in Scheme 1. Here T and D are free phosphofructokinase tetramers and dimers and B is their band 3-binding site. Of the four &sociation constants, Kt was determined above to be 2 X lo-? M (Fig. 1) and K, to be 6 X 10"' M (Fig. 8). The dissociation constant describing the relationship between membrane-bound tetramers and dimers is defined as Kb = (D - B)'/(T - B)(B). Recall that in the analysis presented in Fig. 6, we determined that (D - B)'/(T - B ) = Q = 4.8 X lo-' M. Hence, Kb = 4.8 X lO-'/(B). Since the concentration of band 3 in Fig. 4 was always in large excess over phosphofructokinase, we can approximate the free band 3 concentration with the total, i.e. ( B )= 7 X IO-? M. Therefore, K b = (4.8 X 10-7)/(7 x lo-') = 0.69. K d was calculated as K t .KJKb = 1.3 X lo-' M '. Note that affinity for the membrane is approximately 15 times greater for dimers than tetramers. Thus, the inactivation of phosphofructokinase appears to occur through the preferential binding of dimeric subunits by an excess of membrane sites. To demonstrate that the mechanism shown in Scheme 1 accounts for the kinetics of inactivation, a second-order differential equationwas derived from this model (see Miniprint Supplement). Numerical integration yielded biphasic theoretical curves which closely fit the experimental data (Fig. 4). Numerous other models failed to conform to the qualitative and quantitative features of our results. Thus, the proposed mechanism not only fully agrees with all of our results butis the only model to do so. Additional experiments supported thismodel. The rate and extent of membrane-dependent inactivation increased as pH was decreased from 7.5 to 6.0 (Fig. 9A), similar to the inactivation of phosphofructokinase in simple solution (Bock and Frieden, 1976a). However, the rate and extentof membranedependent inactivation increased with temperature between 0-37 "C (Fig. 9B), which is opposite to thecold lability of the pure enzyme (Bock and Frieden, 1976a; Wenzel et al., 1978). This unexpected temperature effect emphasizes that the initial rate of inactivation is dependent both on the equilibrium between tetramers and dimers in solution and on the rate of association of tetramers anddimers with the membrane. Hybridization of Phosphofructokinase Isozymes-To confirm that red cell membranes stabilize dimeric subunits of phosphofructokinase, we tested whether membrane vesicles would promote the formation of hybrid tetramers of rabbit muscle and liver isozymes (Tsai and Kemp, 1972). Rabbit muscle and liver phosphofructokinase were incubated separately with vesicles in inactivation buffer. At the end of an hour, 40% of the liver phosphofructokinase and 95% of the muscle phosphofructokinase were inactive, and essentially all of the active and inactive forms were membrane-bound. The vesicles with bound enzyme were sedimented and then resuspended in activation/elution buffer for 2 h.The vesicles were removed by centrifugation, the supernatants dialyzed, and

Td

2D

Kt

SCHEME 1

x Initial PFK Activny

% lnnbl

PFK Activity

0

I

I

10

20

-,

I

I

I

I

30

40

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FIG. 9. Effect of pH and temperature on phosphofructokinase (PFK) inactivation. Phosphofructokinase (6 nM) was mixed with vesicles equivalent to 700 nM ( A ) and 400 nM ( B )band 3 in 1 ml of inactivation buffer at different values of pH and temperature. Aliquots of the suspensions were assayed for phosphofructokinase activity a t the indicated times. A , the suspension was incubated at 0 'C at the following pH values: 0, 7.5; 0, 7.0; 0, 6.5; +. 6.0.B, the suspension was incubated at pH7.0 at thefollowing temperatures: 0, 0 "C; 0, 10 "C; +, 24 "C;0 , 3 7 "C.

phosphofructokinase distribution analyzed by ion-exchange chromatography. The isozyme composition of the enzyme peaks was determined in apolyacrylamide gel electrophoresis system (Laemmli, 1970) which resolved the muscle and liver subunits (Karadsheh etal. 1977). When muscle and liver phosphofructokinase were chromatographed either separately or in fresh mixtures, the muscle isozyme eluted early as a sharp peak and the liver isozyme later as a broad peak. A similar distribution was observed in control experiments in which muscle and liver isozymes were inactivated and reactivated separately, then mixed just prior to chromatography (Fig. 10B). (The phosphofructokinase activity eluting at the intermediate position contained both muscle and liver subunits and is presumed to reflect aggregates of the two isozymes.) The profile of the isozymes that were allowed to reactivate together is shown in Fig. 1OA. In this case, both the muscle and liver peaks were greatly reduced in favor of a large peak at theintermediate position of hybrids. The resolution of this chromatogram is not ideal. However, the results were reproduced in each of three other experiments. The resolution was not improvedby the use of a concave gradient (Vora et al., 1980). Isoelectric focusing was not attempted since phosphofructokinase isozyme mixtures yield only a single peak by this technique (Kaur and Layzer, 1977). The limitation on resolution of isozymes is probably a manifestation of the tendency of phosphofructokinase tetramers to aggregate under the conditions required for this experiment (Wenzel et al., 1978; Reinhart and Lardy, 1980b; Foe and Kemp, 1985).We conclude that this experiment provides strong, albeit imperfect, evidence that red cell membrane vesicles catalyze hybrid enzyme formation. Phosphofructokinase Subunits in Vivo-A simple experiment was performed to determine whether inactive dimers


r

10431

Band 3 relieves the allosteric inhibition of phosphofructokinase by ATP and 2,3-bisphosphoglycerate (Karadsheh and Uyeda, 1977) just as do other effectors which bind to its allosteric activation sites. Two of the most effective ligands which displace phosphofructokinase from the membrane, ADP and NADH, bind avidly only to the adenine nucleotide activation site(Kemp and Krebs, 1967;Gottschalk and Kemp, 1981). (Because ATP binds to three sites/phosphofructokinase protomer (Kemp and Krebs, 1967),the manner inwhich it elutes the enzyme is ambiguous.) NADH and its analogue, adenine diphosphoribose, bind to the adenine nucleotide activation site but do not activate the enzyme (Gottschalk and Kemp, 1981), implying that change to anactive conformation does not accompany their binding. That NADH is a potent elutor of phosphofructokinase, therefore, seems to signify ita direct competition with band 3 at the nucleotide activation site. Band 3 appears to inactivate phosphofructokinase in vitro by shifting the equilibrium between its subunitsto theinactive dimeric form. It does this by binding to thedimer with higher affinity than thetetramer, presumably because of interactions with the dimer that arehindered in the tetramer. This can be understood in terms of a recently proposed structure for the enzyme, wherein the adenine nucleotide activation site resides within the cleft between phosphofructokinase subunits (Poorman et al., 1984). Our results suggest an explanation for the inactivation of phosphofructokinase by other cellular proteins: (a) muscle filaments (Mansouret al., 1966; Poon and Wood, 1968; Choate and Mansour, 1983); ( b ) purified F-actin'; and ( c ) calmodulin (Mayr and Heilmeyer, 1983). In the last of these studies, Fraction inactivation by calmodulin was correlated with the dissociaFIG.10. Ion-exchange chromatography of control and hybridized muscle and liver phosphofructokinase (PFK). Rabbit tion of phosphofructokinase to a smaller, possibly dimeric muscle and liver phosphofructokinase (46 nM) were incubated sepa- form. rately with 100 pl/ml vesicles in inactivation buffer at pH 6.0 and Phosphofructokinase resembles hemoglobin in its interac0 "C for 1 h. In p a n e l A , the two inactive enzymes were mixed prior tion with band 3. The cytoplasmic domain of band 3 binds to to reactivation to allow hybridization of dissociated phosphofructo- tetrameric deoxyhemoglobin (Shaklai and Abrahami, 1980) kinase subunits. In the control (panel B ) , the isozymes were first incubated separately in activation buffer a t 20 "C for 2 h and then with a higher affinity than for oxyhemoglobin (Arnone et aL, combined. Membranes were removed by centrifugation and the su- 1983;Walder et al., 1984).In addition, the cytoplasmic portion the dissociation equilibrium of oxyhemoglobin pernatants dialyzed against 20 mM Tris base, 6 mM citric acid, 5 mM of band 3 shifts (NH&SO4, 2 mM EDTA, 1 mM dithiothreitol, 0.1 mM ATP, and 0.1 toward the dimer (Cassoly, 1983).Thus, band promotes 3 both mM fructose-1,6-P2at pH8.5 (column buffer). Each enzyme solution the deoxy conformation of the tetramer and the dimeric form was applied to a 10-ml DEAE-Sephadex A-25 column and eluted with of oxyhemoglobin (Salhany, 1983).It has recently been shown a linear gradient of 5-550 mM (NH4)2S04 incolumn buffer. by x-ray crystallography that the amino-terminal portion of band 3 bindsdeep within the cleft between the @-subunitsof accumulate in the intact erythrocyte. Fresh red cells were hemoglobin (Walder et al., 1984). lysed with 0.2% saponin in 1.5 volumes of activation buffer. It is surprising that no detectable pool of phosphofructokiEnzyme activity was then monitored periodically over time. nase dimers was found in fresh red cells, despite the higher It was reasoned that phosphofructokinase activity would in- affinity of the membrane for dimers. Indeed, the concentracrease if inactive subunits, bound to the membrane in the tion of phosphofructokinase in the red cell (calculated at 25 intact cells, were released and reassociated into tetramers in pg/ml based on activity (Karadsheh et al., 1977)) is of the uitro. However, we found that the activity 2 h after lysis (1.9 same order of magnitude as thehighest phosphofructokinase f 0.2 units/ml of cells) was the same as that found at the concentration in our inactivation experiments (20 pg/ml). time of lysis (1.9 f 0.1 units). Thus, there was no detectable This discrepancy is probably explained by the presence of pool of inactive phosphofructokinase in intact red cells, al- ligands and the high concentration of protein within the though cycling of enzyme through a small membrane-bound erythrocyte which would stabilize the tetramer and minimize dimer pool is possible. its dissociation to dimers (Bock and Frieden, 197613; Fulton, 1982). DISCUSSION Approximately half of the phosphofructokinase in the inThe present data suggest a simple mechanism for the inter- tact human erythrocyte is associated with the membrane in a action of phosphofructokinase with the erythrocyte mem- rapidly reversible fashion (Jenkins et al., 1984). I n uitro, such brane; the polyacidic amino-terminal sequence of band 3 binds association activates the enzyme (Karadshehand Uyeda, electrostatically to the polybasic adenine nucleotide-activa- 1977). Band 3 could be the major physiological activator of tion sites on the enzyme. That the consequences of binding phosphofructokinase in the human erythrocyte, since this cell are bothenzyme activation and inactivation canbe explained as follows. L. G . Foe and R. G . Kemp, personal communication. I

I

I

I

I

I

I

1

10432

Phosphofructokinase Interaction Erythrocyte with

lacks fNCtOSe-2,6-P2, the principal activator of phosphofructokinase in other cells (Hers et d.,1982; Kuwajima and Uyeda, 1982). If so, most of the active phosphofructokinase in the human red cell might be membrane-bound, since the unbound fraction would be highly inhibited by the millimolar levels of ATP and 2,3-bisphosphoglycerate in thered cell. Conversely, glyceraldehyde-3-P dehydrogenase (Brindle et al., 1982; Tsai et al., 1982) and aldolase (Strapazon and Steck, 1977; Murthy et al., 1981) are competitively inhibited when they bindto the same region of band 3. Although the physiological significance of these effects cannot be specified at present, they suggest that membrane binding could have a major impact on the activity of three successive enzymes in theglycolytic pathway. The human erythrocyte exists in circulation for approximately 4 months (Berlin and Berk, 1974), during which time its proteins cannotbe biosynthetically renewed. The maximal activity of some of the glycolytic enzymes declines substantially with cell age, and theconsequent metabolic deficit may contribute to the clearance of aged erythrocytes from the circulation (Seaman et al., 1980). We have previously suggested that the amino-terminal sequence of human band 3 may serve to stabilize glycolytic enzymes againstthermal denaturationand proteolytic degradation (Jenkins et al., 1984). That is, just asmetabolites stabilize the native conformation of enzymes, so may binding to thepolyanionic region of band 3. The enzymes in question would always tend to be liganded, equilibrating rapidly between metabolites and band 3 (Kliman and Steck, 1980; Jenkins et aZ., 1984). The stabilization of enzymes by band 3 could be more important to cellular homeostasis than its modulation of their catalytic activity. That avian and rodent red cell membranes do not appear to bind glycolytic enzymes (Jay, 1983; Ballasand Smith, 1983) may be related to the significantly shorter lifespan of these cells (530 days (Berlin and Berk, 1974)). It is likely that thedimeric subunits of phosphofructokinase would be particularly sensitive to irreversible inactivation, so that their high avidity for band 3 might be particularly beneficial to thelong-term stability of the enzyme. While we have not found a substantial pool of dimeric subunits of phosphofructokinase in the erythrocyte, such forms may arise continuously (albeit transiently) and be protected by binding to band 3 prior to their reassociation to tetramers. Acknowledgments-We wish to thank Donald P. Madden for his expert technical assistance and Dr. Lawrence G. Foe for helpful discussions and generously providing purified rabbit liver phosphofructokinase.

REFERENCES Aaronson, R. P., and Frieden, C (1972)J. Biol. Chem. 247, 75027509 Arnone, A., Chattejee, R., Rogers, P., Musso, G. F., Kaiser, E. T., Steck, T. L., and Walder, J. (1983)Fed. Proc. 42,2196 Ballas, S. K., and Smith, E. D. (1983)Fed. Proc. 42, 2194 Berlin, N. I., and Berk, P. D. (1974)in The Red Blood Cell (Surgenor, D. MacN., ed) 2nd Ed., Vol. 2,pp. 957-1019,Academic Press, New York Bock, P. E., and Frieden, C. (1976a)J. Biol. Chem. 251,5630-5636 Bock, P. E., and Frieden, C. (1976b)J. Biol. Chem. 251,5637-5643 Bradford, M. M. (1976)Anal. Biochem. 72,248-254 Brindle, K. M., Campbell, I. D., and Simpson, R. J. (1982)Biochem. J. 208,583-592 Cassoly, R. (1983)J. Biol. Chem. 258, 3859-3864 Choate, G. L., and Mansour, T. E. (1983)Fed. Proc. 42, 2080

Membranes

Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971)Biochemistry 10,2606-2617 Foe, L. G., and Kemp, R. G. (1985)J. Biol. Chern. 260,726-730 Fulton, A. B. (1982)Cell 30,345-347 Gottschalk, M. E., and Kemp, R. G . (1981)Biochemistry 20, 22452251 Hers, H-G., Hue, L., and Van Schaftingen, E. (1982)Trends Biochem. Sci. 7,329-331 Hesterberg, L.K., and Lee, J. C. (1981)Biochemistry 20,2974-2980 Higashi, T.,Richards, C. S., and Uyeda,K. (1979)J. Biol. Chem. 254,9542-9550 Jay, D. C. (1983)J. Biol. Chem. 258,9431-9436 Jenkins, J. D. (1983)Ph.D. dissertation, University of Chicago Jenkins, J. D., and Steck, T. L. (1983)Fed. Proc. 42, 2079 Jenkins, J. D., Madden, D. P., and Steck, T. L. (1984)J. Biol. Chem. 259,9374-9378 Kant, J. A., and Steck, T. L. (1973)J. Biol. Chem. 248,8457-8464 Karadsheh, N. S., and Uyeda, K. (1977)J. Biol. Chem. 252,74187420 Karadsheh, N. S., Uyeda, K., and Oliver, R. M. (1977)J. Biol. Chem. 252,3515-3524 Kaul, R. K.,Murthy, S. N. P., Reddy, A. G., Steck, T. L., and Kohler, H. (1983)J. Biol. Chem. 258,7981-7990 Kaur, J., and Layzer, R. B. (1977)Biochem. Genet. 15,1133-1142 Kemp, R. G., and Krebs, E. G . (1967)Biochemistry 6,423-434 Kliman, H. J., and Steck, T. L. (1980)J. Biol. Chem. 255, 63146321 Kuwajima, M., and Uyeda, K. (1982)Biochem. Biophys. Res. Commun. 1 0 4 , 8 4 4 Laemmli, U. K.(1970)Nature 227, 680-685 Layzer, R. B. (1975)Methods E n z y m l . 42,110-115 Lieber, M. L., and Steck, T . L. (1982)J. Biol. Chem. 257, 1165111659

Liou, R.-S., and Anderson, S. (1980)Biochemistry 19,2684-2688 Mansour, T. E., Wakid, N., and Sprouse, H. M. (1966)J. Biol. Chem. 241,1512-1521 Mayr, G . W., and Heilmeyer, L. M. G. (1983)FEBS Lett. 159,5157 Murthy, S. N. P., Liu, T., Kaul, R. K., Kohler, H., and Steck, T. L. (1981)J. Biol. Chem. 256,11203-11208 Murthy, S. N. P., Kaul, R. K., and Kohler, H. (1984)Hoppe-Sqler's 2. Physiol. Chem. 365,9-17 Pavelich, M. J., and Hammes, G. G. (1973)Biochemistry 12, 14081414 Peterson, G . L. (1977)Anal. Biochem. 83,346-356 Poon, W. M., and Wood, T. (1968)Biochem. J. 110,792-794 Poorman, R. A., Heinrikson, R. L., Randolph, A., and Kemp, R. G. (1984)Nature 309, 467-469 Ross, S. L. (1964) Di#erentiul Equations, pp. 286-289, Blaisdell Publishing Co., New York Reinhart, G. D., and Lardy, H. A. (1980a)Biochemistry 19, 14771484 Reinhart, G. D., and Lardy, H. A. (1980b)Biochemistry 19, 14911495 Salhany, J. M. (1983)J. Cell Biochem. 23,211-222 Seaman, C., Wyss, S., and Piomelli, S. (1980)Am. J. Hematol. 8,3142 Shaklai. N.. and Abrahami.. H. (1980) . , Biochem. Biophvs. Res. Commun. 95,'1105-1112 Steck. T. L. (1974)Methods Membr. Biol. 2.245-281 Steck; T. L., and Kant, J. A. (1974)Methods Enzymol. 31, 172-180 Strapazon, E., and Steck, T. L. (1977)Biochemistry 16,2966-2971 Tsai, M. Y.,and Kemp, R. G . (1972)Arch. Biochem. Biophys. 150, 407-411 Tsai, I.-H., Murthy, S. N. P., and Steck, T. L. (1982)J. Biol. Chem. 267, 1438-1442 Uyeda, K. (1979)Adv. Enzymol. Relat. Areas Mol. Biol. 48, 193-244 Vora, S., Seaman, C., Durham, S., and Piomelli, S. (1980)Proc. Natl. Acad. Sci. U. S. A. 77, 62-66 Walder, J. A., Chatterjee, R., Steck, T. L., Low, P. S., Musso, G. F., Kaiser, E. T., Rogers, P. H., and Arnone, A. (1984)J. Bwl. Chem. 259, 10238-10246 Wenzel, K.-W., Muller, D., Blir, J., and Hofmann, E. (1978)Acta Biol. Med. Ger. 37,519-526 Yu, J., and Steck, T. L. (1975)J. Biol. Chem. 250, 9176-9184 "


10433 (E), w a s described in termsof

The total enzymeconcentration,

SUPPLEMENTARY MATERIAL TO

tetramer as

E = T-B + T + D/2 + D-B/2 = T-B

MODE OF INTEBACnON OF PHOSPHOFRUCTOKINASE

T +*/2

t

+ D-B/2

D-B = 2E

- 2T-B -

2T

Eq. 4 w a s differentiatedto

b

- YK,T

(8)

yield:

w][dT/dt]

dS/dt = [I +%/4

Jean D. Jenkins, Perenc J. Kezdy, and Theodore 1. Steck

(7)

w a s rearranged:

Thisequation

UlTE THE ERYTHROCYTE MEMBRANE

(9)

Therefore, dT/dt =+ [%d /S4/fdi t]] / [ l

EXPERIMENTAL PROCEDURES Thereactionmechanismpostulated

(Scheme I) was it was consistent with

in thetext

analyzedmathematicallytodeterminewhether thekineticsofinactivation.

dT/dt = [kdT-B + krD-B/Z

eq. 10:

- kaBT - kfBD/21/

[l + e S / 4 f i 1

(11)

(D) (eq. 3) and (D-B) (eq. 8) weresubstitutedinto

The valuesfor

To recapitulate,

(10) (eq. 5) w a s substitutedinto

TheexpressionfordS/dt

eq. 11:

- kr)T-B + krE - (kaB + kr)T - (kr + k f B m / 2 ] +/ [f 1l S / 4 E 1

dT/dt = [(kd

To modelthetimecourse

(12)

of inactivation ofPFK

by band 3,

we developed an explicit second order differential equation

by

differentiating eq. 1: d2T-B/dt2 = kaB [dT/dt

- kd [dT-B/dt 1

1

(13)

(eq. 6) and dT/dt (eq. 12) weresubstitutedinto

ExpressionsforT eq. 13: T and D represent PFK tetramer and dimer, respectively,

while

d2T-B/dt2 = -kd(dT-B)/dt +

B represents band 3 and T-B and D-B, the enzyme-band 3 complexes. K t , Ks, Kd, and Kb aredissociationconstants; are rate constants

- [ ( k r + k f B ) ~ s / 2 ] ~ ( d T - B / d t ) / k a B + kdT-B/kaB) (14) Eq.14

Threesimplifyingassumptionswere be in a

made. First, T and Dwere

rapid association equilibrium, based

in Fig. 8. Second,since

experimentdescribed e x c e s over the enzyme,

band 3 is not significantlyaltered

on the

band 3 was in large

we assumed that the concentration

of free

by the binding of PFK.

Third,

T-B to 2 D-B transition is very slow

we assumed thatthedirect

comparedtotheotherthreereactions. (1).

provides an explicitdescription

molar concentrations in the

followingequations.)

numericallyintegratedit toobtainvalues

- kdT-B

(1) of thesolubleenzyme

as

active enzyme concentration

(Ross, 1964) The

was calculated as the sum of the free

and bound tetramerconcentrations

by substitutingvaluesfor

and d(T-B)/dt obtained from the integration

(T-B)

of eq. 1 4 into an

6:

T-B + T = T-B + (dT-B/dt)/kaB + kdT-B/kaB To make these calculations,

(15)

we assigned the values

S = T + m i 2

of thefour

as follows.

ka and kd werederived

from thehalf-time

of the binding of the tetramer to

The rate of change of thesolubleenzyme dS/dt = kdT-B + krD-B/2

w a s expressed as:

- kaBT - kfBD/2

(5)

inactive enzyme. Starting with these initial

shown in Fig. 4.

in eq. 1 4 by trial and error The best-fitkineticconstants shown in Fig. 4 were:

used togeneratethetheoreticalcurves ka = 8.33 x l o 3 M - l see-1, kd = 1.58

(6)

band 3, while

of reactivation of

toobtaincurvesbest-fittingtheexperimentallyobserveddecay enzymeactivity

T:

+ kdT-B/kaB

from therate

estimates, we variedtheparameters (4)

To

guidethecomputer-fittingprocess,initialestimates

membrane-bound eq. 2, we obtained

of the

(Ks, K t , and Kd) determined in thetext.

kr and hence k f wereobtained

D from eq. 3into

T = [dT-B/dt]/kaB

by theRunge-Kuttamethod

kineticconstantsweremade (2 )

Since K, = D ~ / T ,

Equation 1 w a s solvedfor

process in

We therefore

of d(T-B)/dt and (T-B) a t any given time.

dissociationconstants

We definedthemolarconcentration S = T + D/2

Substitutingfor

of therate

is toocomplextointegrateanalytically.

(For simplicity, the symbols for the

reactants will be used todenotetheir dT-B/dt = kaBT

questionbut

expanded form ofeq.

The rate of change of the bound tetramer was described by differential equation

- (l+kr/k,B) (dT-B/dt)

{-(l+kd/kaB)krT-B + krE

for association and dissociation of TandDwith

band 3. taken to

{kaB/[ 1+fls/4 ddT-B/dt)/kaB + kdT-B/kaBl)

ka, kf, kd and kr

sec-l, and k f = 5 x 104 M - l sec-l.

x

sec-l, kr = 5.83 x

of