regulation of binding of phosphofructokinase to ... - Semantic Scholar

/. exp. Biol. 137, 13-27 (1988)

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REGULATION OF BINDING OF PHOSPHOFRUCTOKINASE TO MYOFIBRILS IN THE RED AND WHITE MUSCLE OF THE BARRED SAND BASS, PARALABRAX NEBUL1FER (SERRANIDAE) BY SUSAN J. ROBERTS*, MARY SUE LOWERY AND GEORGE N. SOMERO Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093, USA Accepted 8 February 1988 Summary The binding of phosphofructokinase (PFK) to myofibrils from the white muscle of the fish Paralabrax nebulifer (Girard, 1854) is sensitive to factors known to be allosteric regulators of PFK activity. PFK in Triton-X-100-extracted muscle remains bound to myofibrils at pH 7-0 and is fully solubilized by increasing the pH to 8-0. The curve describing the pH-dependence of PFK binding to myofibrils is similar in its steepness to pH versus activity curves of PFK at low temperature. Nucleotides are also potent modulators, preventing the association of PFK with myofibrils at concentrations between 20 and 60jtimol I"1 of ATP, ADP, MgATP or GTP, listed in order of effectiveness. PFKs in the red and white muscle extracts differ in their pH-dependence of binding to myofibrils, and their kinetic and regulatory properties (response to citrate, pH and fructose-2,6-bisphosphate). Reversible binding of PFK to myofibrils may be important in the control of glycolysis, especially in the highly glycolytic white muscle fibres.

Introduction Regulation of enzymatic activity may entail the reversible association of enzymes with other cellular structures as well as the adjustment of catalytic rates through interactions between enzymes and ailosteric modulators. For example, several enzymes of the glycolytic pathway bind reversibly to muscle thin filaments, with concomitant alterations in some of the kinetic properties of the enzymes. These binding equilibria are affected by such physiologically important factors as the level of muscle stimulation (Clarke, Shaw & Morton, 1980), pH (Roberts & Somero, 1987), enzyme phosphorylation state (Kuo, Malencik, Liou & Anderson, * Present address: Department of Molecular Biology, 229 Stanley Hall, University of California, Berkeley, CA 94720, USA. Key words: compartmentalization, Paralabrax nebulifer, phosphofructokinase, red muscle, white muscle.

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S. J. ROBERTS, M. S. LOWERY AND G. N. SOMERO

1986; Luther & Lee, 1986) and the nutritional condition of the organism (Lowery, Roberts & Somero, 1987). In this study we have examined the binding of the glycolytic enzyme phosphofructokinase (PFK: EC2.7.1.11) to myofibrillar fractions of white and red muscle from the teleost fish Paralabrax nebulifer. Our first objective was to examine the binding of PFK to native myofibrils rather than purified actin or reconstituted thin filaments. The discovery that rabbit muscle PFK has a higher affinity for binding to F-actin than to reconstituted thin filaments (Roberts, 1986) indicates that the properties of PFK binding to the myofibrillar lattice may be dependent on more than one component of the myofibrillar system. Thus, to understand the nature of PFK-myofibril binding in vivo, it is important to examine myofibrillar systems containing the full complement of myofibrillar proteins. The second objective was to determine whether PFK binding is different in muscle types with different metabolic properties. One advantage of studying possible regulatory roles of PFK's association with myofibrils in fish is the clear separation of glycolytic (white) and oxidative (red) tissue in fish musculature compared with the mixed fibre types found in mammals. Fish white muscle corresponds to the fast twitch muscle fibres of mammalian muscle and is recruited for high-speed swimming and for burst swimming. Energy for muscle contraction in burst swimming comes almost exclusively from anaerobic glycolysis, and the levels of glycolytic enzymes are correspondingly high (Johnston, Davison & Goldspink, 1977; Johnston & Moon, 1980; Johnston & Salamonski, 1984). Red muscle in fish resembles the slow tonic fibres of mammals; it functions in slowspeed swimming and is fuelled by the oxidation of fatty acids. Levels of lipolytic enzymes and enzymes involved in oxidative metabolism are several times higher in red than white muscle (Love, 1970), and different isozymes of some enzymes are found in the red muscle. Red muscle of the mirror carp contains myosin light chain kinase typical of slow twitch mammalian muscle, whereas carp white and pink muscle contain myosin light chains resembling fast twitch muscle (Johnston et al. 1977). Also, the A 4 isozyme of lactate dehydrogenase (LDH) predominates in white muscle, but red muscle contains a higher proportion of the LDH B 4 isozyme. This distribution of isozymes has been correlated with the aerobic capacity of the tissue. In addition, LDH isozymes have different affinities for the paniculate fractions of muscle homogenates. The A 4 isozyme binds more tightly than B 4 to particulate fractions and this association has been postulated to serve a role in switching between aerobic and anaerobic metabolism in muscle (Nadal-Ginard & Markert, 1975; Hultin, 1975). Isozymes of other glycolytic enzymes, including PFK, may have similar patterns of subcellular distribution, but this remains to be demonstrated. Therefore, we have examined the properties of PFK in white and red muscle with regard both to differences in kinetics and to differences in binding to myofibrils. Our results indicate that PFK binds reversibly to myofibrils in preparations from fish white muscle. Binding of PFK is modulated by several physiologically important regulators of PFK activity, including adenylates and protons. We

Phosphofructokinase binding to myofibrils

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present functional and structural evidence for the existence of different isozymes of PFK in white and red muscle which differ significantly in their kinetic characteristics and in their interactions with the myofibrillar lattice. Materials and methods

Animals Barred sand bass, Paralabrax nebulifer, 15-24 cm in length (mean = 21 cm) and 82-323 g in mass (mean = 240 g) were captured with a seine net in Mission Bay, San Diego, CA, USA. Fish were maintained in large circulating aerated seawater tanks. Experiments were conducted in April to June and in January (water temperature, 15-20°C). No seasonal effects were detected in the data. Fish were fed a diet of anchovies and mackerel to satiety every other day. Assay of phosphofructokinase activity Two assays for phosphofructokinase activity were used. For measuring optimal activity, PFK was assayed using the fructose-6-phosphate (F-6-P) coupled assay of Bock & Frieden (1974). The medium contained: 1-7 ml of reaction buffer (42mmoir 1 Tris-acetate, pH8-0 at 20°C, Slmmoll" 1 KC1, 5-lmmoll" 1 NH4C1), 0-2 ml of substrate mixture (20mmoll~ l ATP, 20mmoll~' fructose-6-phosphate, l-6mmoll~' NADH, 20mmoll~' magnesium acetate, 0-1 moll" 1 Tris-acetate, 0 - l m m o i r 1 EDTA, pH7-7 at 20°C) and 0-1 ml of coupling enzymes (50 units of glycerol-3-phosphate dehydrogenase, 500 units of triose phosphate isomerase and 40units of aldolase in 0-1 moll" 1 Tris-acetate, O-lmmolT 1 EDTA, pH8-0 at 20 °C). The effects of pH, F-6-P, fructose-2,6-bisphosphate (F-2,6-P2) and citrate on PFK activity were determined using the following assay system designed to enhance the allosteric properties of the enzyme: Hepes reaction buffer ( 5 0 m m o i r l Hepes-KOH, pH7-0, lSmmolT 1 MgCl2, 1-Ommoll"1 EDTA); coupling enzymes (5 units of glyceraldehyde-3-phosphate dehydrogenase, GAPDH, 50 units of triose phosphate isomerase, and 4units of aldolase) and substrates (lmmoll" 1 ATP, l-Smmoll" 1 MgCl2 and 0-16mmoU"1 NADH). Coupling enzymes were obtained as ammonium sulphate precipitates which were pelleted by centrifugation and resuspended in buffer. For assays on PFK's regulatory properties the enzymes were dialysed overnight in the Hepes reaction buffer to remove traces of (NH 4 ) 2 SO 4 . Dilutions of F-6-P were prepared so that the same volume (0-1 ml/1-0 ml assay) was added for each final concentration of F-6-P. A sample of PFK was added to initiate the reaction. All substrates and buffers were purchased from the Sigma Chemical Co. and coupling enzymes were purchased from either Calbiochem or Boehringer-Mannheim. Preparation of PFK and myofibrils A pellet of myofibrils was obtained after three successive extractions of minced muscle with Triton extraction buffer (TEB) [lOOmmoll"1 potassium phosphate,

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pH7-0, l m m o i r 1 EGTA, 2 m o i r 1 glycerol, lmmoll" 1 MgCl2, 0-05% Triton X-100, O-Smmoir 1 dithiothreitol (DTT) and 20 J umoir 1 phenylmethylsulphonyl fluoride (PMSF)]. 10 g of muscle mince was stirred for 5min on ice in three volumes (30 ml) of TEB. The Triton extraction buffer was based on a buffer used by Lockwood, Trivette & Pendergast (1981) for preparing cytoskeleton from whole cells. The muscle mince suspension was centrifuged at 1000 g for lOmin. The pellet was re-extracted twice in TEB. Fibrils were resuspended in five volumes of myofibril buffer (lOmmoir 1 potassium phosphate, pH8-0, 3 0 m m o i r ' potassium fluoride, 2 m m o i r L EDTA, lmoll" 1 glycerol, 0-5mmoll~J DTT and 20^moll~' PMSF) by homogenizing for 30s in a Waring blender. The pH of the slurry after homogenization was approximately 7-0, and it contained about 60 % of the PFK activity present in whole muscle. This preparation is referred to as PFKmyofibrils no. 1. For some experiments, PFK was separated from the myofibrils by washing them at a higher pH. PFK-myofibrils no. 1 was centrifuged at 6000 g for 20min, the supernatant was decanted, assayed for residual PFK activity and, if activity was negligible, discarded. Pellets, containing most of the PFK activity, were resuspended in five volumes of myofibril buffer and homogenized for 30 s in a Waring blender. The pH was adjusted to 8-0 with 0-5moll" 1 KOH and stirred for l h at 4°C. The myofibril suspension was centrifuged for 20min at 6000 g and the supernatant fraction, containing 90-100 % of the PFK activity, was decanted and used as the source of PFK (MYB-PFK) for studying the effects of ligands on binding. The myofibril pellets were stirred into five volumes of myofibril buffer, p H 8 0 , and passed through a cheesecloth mesh to remove tendons and produce a homogeneous suspension. Myofibrils were collected by centrifugation at 6000g for 20min and used in subsequent experiments. pH effects on binding Samples of the PFK-myofibrils no. 1 suspension described above were adjusted to the appropriate pH values with dilute KOH or phosphoric acid and incubated for 15 min at 20°C. 1 ml of each sample was centrifuged for 5 min at 13 000g in a Fisher microcentrifuge. Supernatants were assayed for soluble PFK activity. Previous control experiments had shown that, even at the lowest pH, 100 % of the pre-centrifugation activity could be recovered in the supernatant and pellet fractions. Effects of ligands on the binding of PFK Suspensions of myofibrils and MYB-PFK were prepared in the ratio of 0-4 g of myofibrils (wet mass) per ml of MYB-PFK and adjusted to pH7-2. lOmmoll" 1 stock solutions of ATP, MgATP, ADP, GTP, MgCl2 and CaCl2 were made up in myofibril buffer and adjusted to pH7-0. Small volumes of these stock solutions were diluted into MYB-PFK before addition of myofibrils to obtain a range of concentrations. After a 45-min incubation at 20°C, the myofibril suspensions weref assayed for PFK activity and then centrifuged at 13 000 g for 5 min in the


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microcentrifuge. Supernatants were decanted and assayed for PFK activity. Percentage bound activity was calculated from the ratio of activity in the supernatant to activity in the suspension. Pellets were routinely resuspended and assayed to ensure that there was no loss of activity during the experiment.

Comparison of the binding of PFK in red and white muscle The designation red or white refers to preparations obtained from either the red or the white muscle of the fish. 0-3 g of either red or white myofibrils and 2-5 ml of either red or white myofibrillar supernatants were mixed in the following combinations: (1) white myofibrils plus white myofibrillar supernatant; (2) white myofibrils plus red myofibrillar supernatant; (3) red myofibrils plus white myofibrillar supernatant or (4) red myofibrils plus red myofibrillar supernatant. After a 30-min incubation at 20 °C, each sample was centrifuged for 5min at 13 000g in the microcentrifuge. The percentage of PFK bound was determined from the amount of enzyme activity in the supernatant divided by the amount of enzyme activity in the suspension before it had been centrifuged.

Kinetic studies of PFK activity in red and white muscle extracts Muscle from P. nebulifer was homogenized at 5 rnlg" 1 tissue in myofibril buffer and centrifuged for 20min at lOOOOg. Supernatants were saved and diluted to obtain equivalent levels of activity in the red and white muscle extracts. Kinetic parameters were determined using the Hepes reaction buffer system described above.

Influence of exercise on muscle pH Fish were strenuously exercised by forcing them to swim until they no longer responded to being prodded. Resting values were obtained from control fish that were kept in a small tank, allowed to acclimate, and then anaesthetized with MS 222. Fish were killed by a blow to the head, 0-5 g of the white muscle was removed and then homogenized in 10 ml of distilled deionized water that had previously been adjusted to pH7-0. The pH of the muscle homogenates was measured with a Radiometer pH electrode and pH meter.

Electrophoresis A 10% acrylamide w/v running gel (0-13% w/v bisacrylamide) with a 5 % acrylamide w/v stacking gel (0-13 % w/v bisacrylamide) was run under denaturing conditions with the discontinuous buffer system of Laemmli (1970). Gels were gained with Coomassie Blue or silver-stained using the procedure of Morrissey (1981).

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S. J. ROBERTS, M. S. LOWERY AND G. N. SOMERO Results

Effect of pH on PFK binding to myofibrils In the process of purifying PFK from P. nebulifer white muscle, we discovered that bound PFK was released from the myofibrillar fraction when the pH of the medium was raised from 7-0 to 8-0. At pH7-0 the supernatant of a centrifuged (lOOOOg, 20min) myofibrillar suspension contained only 15-30 % of the total PFK activity, but at pH 8-0 100 % of the PFK activity was in the supernatant. This effect was seen in every fish we tested (N = 18). At the same time, we also assayed for the activities of pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and aldolase. None of these enzymes showed a similar response to pH. At pH7-0, all the activity of these enzymes was in the supernatant. Electrophoretic analysis of this pH elution phenomenon revealed that PFK was the major protein in the myofibrils that exhibited pH-dependent solubility (Fig. 1A). This effect was reversible; lowering the pH of a myofibrillar suspension from 8-0 to 7-0 caused PFK to reassociate with the sedimentable filaments. The pH response of PFK binding is more thoroughly described by the curve in Fig. 2. Maximal binding was obtained below pH7-l and the apparent pK for halfmaximal binding is 7-35 at 20°C. At pH7-7, the enzyme was completely eluted from the myofibrils. To determine if P. nebulifer white muscle might experience changes in pH that could cause the changes in PFK solubility described above, we compared the pH of muscle homogenates of tissue taken from exhaustively exercised fish and fish at rest. The average pH of the exercised fish tissue was 6-45 (N= 3) and the average pH of the tissue of the fish at rest was 6-88 (N = 3). Because this technique involves dilution and disruption of intracellular compartments it probably does not yield an accurate estimate of pHi, but the relative difference in pH should reflect the pH change of the muscle caused by metabolic activity.

Effect of ligands on dissociation of bound phosphofructokinase The effects of ATP, MgATP, ADP and GTP upon the dissociation of the enzyme from myofibrils are shown in Fig. 3. The binding curves indicated that ATP ^ ADP > MgATP > GTP in effectiveness in eluting PFK from the myofibrils at pH7-2. Between 20 and 50/imolP 1 50% dissociation occurred for the four nucleotides tested. At 50 ^mol I" 1 ATP or ADP essentially all of the PFK had been eluted from the myofibrils. The results of these studies were confirmed electrophoretically with SDS-PAGE (Fig. IB). As was the case for the pH effect, PFK was the major protein that changed its distribution in response to the nucleotide concentration. We also tested the effects of the divalent cations Ca 2+ and Mg 2+ on the binding of PFK to myofibrils (Fig. 4). For both cations there was a minor increase in th amount of PFK which was bound to the myofibrils as the ion concentration w increased. This difference was too small to be detected electrophoretically.


A

19

B

PFK

*•>

PH

ADP

Fig. 1. Electrophoretic analysis of PFK solubility experiments. 10% polyacrylamide gels run in the presence of SDS and silver-stained. Myofibrillar suspensions were centrifuged for 5min at 13 000 g and the supernatants were diluted 10-fold in SDS sample buffer. Identification of the PFK bands was made by comigration with purified rabbit muscle PFK. PFK purified from Paralabrax nebulifer has been shown to comigrate with purified rabbit PFK (unpublished observations). (A) The left-hand lane is the pH 7 0 supernatant of a white muscle myofibrillar suspension and the right-hand lane is the p H 8 0 eluant from the resuspended pH7-0 pellet. (B) The left-hand lane is the supernatant of the myofibrillar suspension containing 0005 mmol 1~' ADP and the right-hand lane is the supernatant of the myofibrillar suspension containing 0-075 mmol T 1 ADP. Comparison of red and white muscle Homogenates of red muscle contained approximately half the PFK activity per gram fresh mass found in white muscle. In extracts of red muscle prepared identically to white muscle, we discovered that, although the amount of enzyme solubilized in the Triton buffer was similar in both red and white muscle types (40-50%), PFK eluted gradually with each wash of the red myofibrils with

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Fig. 2. Effect of pH on PFK binding to white muscle myofibrils. Suspensions of myofibrils and PFK in myofibril buffer (lOmmolP1 potassium phosphate, 30mmoir J potassium fluoride, 2mmoH~1 EDTA, lmoll" 1 glycerol, 0-5mmoH~J DTT) at varying pH were incubated for 15 min at 20°C. The suspension was centrifuged and the percentage of bound activity determined. There was no loss of PFK activity under these conditions, as demonstrated by the complete recovery of enzyme in the supernatants and pellets of selected samples.

80-

60-

40-

GTP

\ * v - - - ^

20-

^

^

0-

ADP 0-02

0-00 •

i

0-04

0-06

MgATP

J-^^ATP ^ 0-08

010

1

• i • i • Nucleotide concentration (mmoll" )

Fig. 3. Effects of nucleotides on the association of PFK with myofibrils. MYB-PFK, adjusted to pH7-2, with the appropriate concentration of nucleotide was added to myofibrils in the ratio of 0-4 g of myofibrils ml"1 MYB-PFK. The pH of the resulting suspension was 7-2. After 45 min at 20°C, the suspension was centrifuged and the percentage of bound activity determined.


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50 1-0

2-0 1

log concentration (mmoll" x 100)

Fig. 4. Effect of divalent cations on the association of PFK with myofibrils. MYBPFK, adjusted to pH7-2 with the appropriate concentration of Ca or Mg was added to myofibrils in the ratio of 0-4 g of myofibrils ml"1 MYB-PFK. The pH of the resulting suspension was 7-2. After 45min at 20°C, the samples were centrifuged and the percentage of bound activity was determined from the PFK activity in the resuspended pellets.

myofibril buffer at pH7 as well as at pH 8. In white muscle only a small fraction of PFK was released in the pH7 myofibril buffer. Unlike the white muscle PFK (see above), eluted red muscle PFK did not reassociate with the myofibrils at pH7. The contrast between the pH response of the red and white myofibrillar PFK fractions seen in attempts to elute PFK from the myofibrils led us to test whether this effect was due to a difference in the myofibrillar proteins, a difference in the PFKs, or an unknown factor associated with PFK. Comparisons between the binding of PFK in the supernatants of red or white muscle homogenates to either red or white muscle myofibrils revealed that PFK was the source of the variation (Table 1). White muscle PFK binds strongly to both white and red myofibrils whereas only small amounts of red muscle PFK bind to myofibrils of either red or white muscle. Table 1. Comparison of the binding of PFK to myofibrils in red and white muscle % PFK activity bound at pH7-l Red muscle PFK + white muscle myofibrils Red muscle PFK + red muscle myofibrils White muscle PFK + white muscle myofibrils White muscle PFK + red muscle myofibrils

14-7 10-9 91-8 93-2

Percentage of bound activity was calculated from the activity in the 5 min, 13 000 g supernatant divided by the activity in the original suspension. Data are from a single fish.

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S. J. ROBERTS, M. S. LOWERY AND G. N. SOMERO B

1

1

c 0-020 H

6

a aa

"o

I act ivity

a ^^

• 0-010-

a

• •

a a •

26 X 0-

a a

a

°

•

•

•

0-0007-35

0-0

0-1 0-2 [Citrate] (mmoll"1)

0-3

Fig. 5. Effects of pH and citrate concentration on the activity of PFKs from white and red muscle. (A) The dependence of PFK activity on pH was determined using the supernatant fractions of white and red muscle homogenized in myofibril buffer. Assays were made at five different pH values in the following reaction mixture: 50mmoll"1 Hepes (adjusted to the appropriate pH with KOH), 15mmoir J MgCl2, lmmolP 1 EDTA, 0-75mmoir' ATP and 4-0mmoll"1 F-6-P. Maximal activity is defined as the activity of the enzyme assayed at the highest pH. Open squares, white muscle PFK; filled squares, red muscle PFK. (B) The inhibition of PFK by citrate was determined using PFK prepared as described above and assayed in the Hepes reaction mixture described in Materials and methods with the addition of 4-Ommoir1 F-6-P. Small samples of a neutralized stock of citrate were added to adjust the concentration of citrate. Open squares, white muscle PFK; filled squares, red muscle PFK.

Characterization of the allosteric properties of PFK from red and white muscle further supported the hypothesis that two different isozymes of PFK occur in these tissues. Red muscle PFK was more readily inhibited than white muscle PFK by both protons and citrate (Fig. 5A,B)- Most strikingly, the strong inhibition of red muscle PFK by protons can be reversed by the addition of micromolar quantities of F-2,6-P2 (Fig. 6A). A comparison of F-6-P saturation curves in the presence and absence of F-2,6-P2 at pH7-2 indicates that F-2,6-P2 increased the affinity of red muscle PFK for F-6-P and changed the kinetics from sigmoidal to hyperbolic. PFK from the white muscle of fish was less inhibited by protons and F-2,6-P2 did not shift its kinetics dramatically even at a lower pH (pH7-l) (Fig. 6B). Discussion

This study establishes a connection between the allosteric properties of PFK and its mode of association with muscle filaments in fish. The same effectors that regulate the enzymatic activity of PFK also influence the binding of PFK to myofibrils. PFK activity was extremely sensitive to small fluctuations in pH. This response has been thoroughly documented in the literature on PFK and is ascribed to a conformational change in the enzyme resulting from protonation at low pH (Trivedi & Danforth, 1966; Bock & Frieden, 1976). Low pH enhances both the allosteric properties of PFK and the dissociation of active PFK tetramers to


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2-0

[Fructose-6-phosphate] (mmol 1 ') Fig. 6. Effect of F-2,6-P2 on the F-6-P saturation curve at low pH. (A) Red muscle PFK was assayed in the Hepes reaction buffer at pH7-2 containing 0-75 mmol I"1 ATP with (filled squares) or without (open squares) 5/.tmoirJ F-2,6-P2. (B) White muscle PFK was assayed in the Hepes reaction buffer at pH 7-2 (triangles) or pH 7-1 (squares) containing 0-75 mmol 1~' ATP with (filled symbols) or without (open symbols) J F-2,6-P2.

inactive dimers. The pH curve for PFK binding to myofibrils (Fig. 2) has the same characteristic steepness described for the effect of pH on the kinetics of PFK (Trivedi & Danforth, 1966; Bock, Gilbert & Frieden, 1975; Hand & Somero, 1983). This contrasts with the modest effect of pH on the association of purified rabbit muscle PFK and actin (Roberts & Somero, 1987). The difference may be due to an intrinsic difference in the proteins from fish and rabbit muscle or it may be a clue that there is another factor present in muscle extracts, missing in the purified PFK and actin preparations, that mediates the pH-dependent interactions between PFK and actin. There is some evidence that pH also regulates the solubility of PFK in crude extracts of mammalian muscle. Poon & Wood (1968) noticed that supernatants of centrifuged rat skeletal muscle homogenates occasionally lacked significant PFK activity. They were able to correlate this finding with the pH of the extraction medium. At pH 8-2, all the PFK activity was located in the supernatant, but at pH7-2 40% of the activity was soluble and at pH6-9 20% of the activity was soluble. Hence, the pH-dependence of PFK binding to myofibrils could be a common mechanism for changing the localization of PFK in muscle fibres. Particularly in a highly glycolytic tissue, such as the white muscle of fish, fluctuations in the cellular pH occur concomitantly with muscle activity. The energy for muscle contraction in this tissue comes almost exclusively from the catabolism of glycogen. In carp, half the glycogen may be depleted in the first 15 s of burst activity, and the concentration of the end-product of anaerobic glycolysis, lactic acid, rises simultaneously (Driedzic & Hochachka, 1976). Milligan & Wood (1987) describe a shift in intracellular pH from 7-56 to 7-27 after exhaustive exercise in the starry flounder, Platichthys stellatus. Such a shift in pH would cause a 55 % increase in the amount of PFK bound to myofibrils according to the in vitro

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data presented in Fig. 2. Measurements of the pH of muscle homogenates of exercised fish described here indicate that the white muscle of P. nebulifer also experiences acidosis as a result of glycolytic metabolism. The consequences of this association are currently undetermined, but association may foster more efficient energy transduction through the co-localization of glycolytic ATP production with the site of ATP hydrolysis. A similar model has been proposed for the localization of creatine phosphokinase on the M line of striated muscle (Turner, Walliman & Eppenberger, 1973; Walliman, Pelloni, Turner & Eppenberger, 1978). Alternatively, the association of PFK with myofibrils may keep some of the PFK in its active tetrameric state when the pH is low enough to cause dissociation of the subunits (Bock & Frieden, 1976). We have previously shown that actin prevents the dissociation of rabbit muscle PFK at low pH (Roberts & Somero, 1987). An interesting comparison with this effect in white muscle is the lack of pHdependent binding of PFK to myofibrils in red muscle. Instead, PFK activity in extracts of red muscle is more sensitive to the pH of the assay medium than a similar extract of white muscle. Unlike white muscle, red muscle depends heavily on the oxidative metabolism of fatty acids. This is reflected in the lower activities of the glycolytic enzymes in red muscle relative to white muscle (Driedzic & Hochachka, 1978). Since red muscle is primarily an aerobic tissue, it is unlikely to have the sudden pH fluctuations associated with the activity of white muscle. We have attempted to uncover the basis for the difference in the binding properties of white and red muscle PFK, and present evidence that pH-dependent myofibril binding is a characteristic of the enzyme, not the myofibrils. In mammals, three isozymes of PFK exist - the forms found in muscle, liver and brain - but there is no difference between PFK from slow twitch and fast glycolytic muscles. The isozymic forms of PFK in fish are unknown, but results from studies on the kinetic and allosteric properties of PFK in extracts from white and red muscle suggest that the enzyme may exist in different forms in the two muscle types. Red muscle PFK catalytic activity is dramatically decreased at low pH compared with PFK in white muscle. However, 5^moll~' F-2,6-P2 is sufficient to restore the activity of red muscle PFK at low pH. Although white muscle PFK is stimulated by F-2,6-P2, the percentage regain of activity is less dramatic than in red muscle PFK. Reversal of pH-dependent loss of PFK activity by F-2,6-P2 has also been described for rabbit muscle PFK (Dobson, Yamamoto & Hochachka, 1986). In this case, the enzyme shows an increased inhibition by ATP at low pH that is counteracted by the addition of F-2,6-P2 to the assay medium. In many aspects the association of PFK with myofibrillar proteins parallels the situation in erythrocytes. PFK binds to erythrocyte membranes in a pH-dependent manner with an apparent pK of 7-1 (Higashi, Richards & Uyeda, 1979). Evidence from the same study suggests that PFK binds to the membrane protein Band 3 which is also the binding site for other glycolytic enzymes in the erythrocyte. Jenkins, Kezdy & Steck (1985) report that the binding of PFK to erythrocyte membranes is inhibited by micromolar concentrations of ATP, ADP and NADH. They suggest that the acidic cytoplasmic domain of Band 3 binds to the polybasic


25

adenylate activation site on PFK and, therefore, binding to erythrocyte membranes is inhibited by these anionic modulators. The same concentration range of nucleotides also inhibits the association of PFK with myofibrils (Fig. 3). There is some specificity in the ability of various nucleotides to prevent binding. GTP is a significantly less effective inhibitor than ATP or ADP. Such specificity is expected from the binding of nucleotides to the adenine regulatory site of PFK, and suggests that the adenine regulatory site is also the site where actin binds to PFK. The divalent cations Ca 2+ and Mg 2+ have little effect on the association of PFK and myofibrils, other than a slight increase in the fraction of enzyme bound at higher Ca 2+ or Mg 2+ concentrations (Fig. 4). Since PFK binding is inhibited by tropomyosin and troponin (Roberts, 1986), the actin-binding proteins responsible for the Ca2+-sensitivity of the actin-myosin interaction, PFK probably binds to a different site on thin filaments. Thus, binding may be relatively unaffected by Ca 2+ -mediated changes in thin filaments. For comparison, aldolase, a glycolytic enzyme known to bind to all three thin filament proteins, is strongly inhibited from binding to filaments in the presence of calcium (Walsh et al. 1980; Clarke, Stephan, Morton & Weidemann, 1983). The biochemical consequences of PFK localization to myofibrils can only be inferred from studies on the purified proteins in rabbit muscle. In this system, actin stabilizes the PFK tetramer and activates PFK, rendering it less sensitive to inhibition by ATP and increasing its affinity for F-6-P (Hand & Somero, 1983; Kuo et al. 1986; Liou & Anderson, 1980; Luther & Lee, 1986; Roberts & Somero, 1987). Activation of PFK during times of energy stress, i.e. during muscle contraction, through the increased binding of PFK to myofibrils as pH decreases, may be a unique adaptation of this glycolytic enzyme that allows the more efficient production of ATP. We thank Mr Ronald R. McConnaughey and Mr John O'Sullivan for their assistance in obtaining fish. This research was supported by National Science Foundation grant PCM83-00983. SJR and MSL were supported during part of this study by National Science Foundation graduate fellowships. References P. E. & FRIEDEN, C. (1974). pH-induced cold lability of rabbit muscle phosphofructokinase. Biochemistry, N.Y. 13, 4191-4196. BOCK, P. E. & FRIEDEN, C. (1976). Phosphofructokinase. I. Mechanism of the pH-dependent inactivation and reactivation of the rabbit muscle enzyme. J. biol. Chem. 251, 5630-5636. BOCK, P. E., GILBERT, H. R. & FRIEDEN, C. (1975). Analysis of the cold lability behavior of rabbit muscle phosphofructokinase. Biochem. biophys. Res. Commun. 66, 564-569. CLARKE, F. M., SHAW, D. & MORTON, D. J. (1980). Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Biochem. J. 186, 105-109. CLARKE, F., STEPHAN, P., MORTON, D. & WEIDEMANN, J. (1983). The role of actin and associated structural proteins in the organization of glycolytic enzymes. In Actin: Structure and Function in Muscle and Non-Muscle Cells (ed. C. G. dos Remedios & J. A. Barden), pp. 249-257. Sydney: Academic Press. DOBSON, G. P., YAMAMATO, E. & HOCHACHKA, P. W. (1986). Phosphofructokinase control in muscle: nature and reversal of pH-dependent ATP inhibition. Am. J. Physiol. 250, R71-R76. BOCK,

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W. R. & HOCHACHKA, P. W. (1976). Control of energy metabolism in fish white muscle. Am. J. Physiol. 230, 579-582. DRIEDZIC, W. R. & HOCHACHKA, P. W. (1978). Metabolism in fish during exercise. In Fish Physiology, vol. VII (ed. W. S. Hoar & D. J. Randall), pp. 503-543. New York: Academic Press. HAND, S. C. & SOMERO, G. N. (1983). Phosphofructokinase of the hibernator Citellus beecheyi: temperature and pH regulation of activities via influences on the tetramer-dimer equilibrium. Physiol. Zool. 56, 380-388. HIGASHI, T., RICHARDS, C. S. & UYEDA, K. (1979). The interaction of phosphofructokinase with erythrocyte membranes. J. biol. Chem. 254, 9542-9550. HULTIN, H. O. (1975). Effect of environment on kinetic characteristics of chicken lactate dehydrogenase. In Isozymes. II. Physiological Function (ed. C. L. Markert), pp. 69-85. New York: Academic Press. JENKINS, J. D., KEZDY, F. J. & STECK, T. L. (1985). Mode of interaction of phosphofructokinase with the erythrocyte membrane. J. biol. Chem. 260, 10426-10433. JOHNSTON, I. A., DAVISON, W. A. & GOLDSPINK, G. (1977). Energy metabolism of carp swimming muscles. J. comp. Physiol. 114, 203-216. JOHNSTON, I. A. & MOON, T. M. (1980). Exercise training in skeletal muscle of brook trout (Salvelinus fontinalis). J. exp. Biol. 87, 177-194. JOHNSTON, I. A. & SALAMONSKI, J. (1984). Power output and force-velocity relationship of red and white muscle fibres from the Pacific blue marlin {Makaira nigricans). J. exp. Biol. Ill, 171-177. Kuo, H.-J., MALENCIK, D. A., Liou, R.-S. & ANDERSON, S. R. (1986). Factors affecting the activation of rabbit muscle phosphofructokinase by actin. Biochemistry, N.Y. 25, 1278-1286. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Liou, R.-S. & ANDERSON, S. (1980). Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments. Biochemistry, N.Y. 19, 2684-2688. LOCKWOOD, A. H., TRIVETTE, D. D. & PENDERGAST, M. (1981). Molecular events in cAMPmediated reverse transformation. In The Organization of the Cytoplasm, Cold Spring Harb. symp. quant. Biol. 46, 909-919. LOVE, R. M. (ed.) (1970). The Chemical Biology of Fishes, vol. 1, pp. 222-257. London: Academic Press. LOWERY, M. S., ROBERTS, S. J. & SOMERO, G. N. (1987). Effects of starvation on the activities and localization of glycolytic enzymes in the white muscle of the barred sand bass, Paralabrax nebulifer. Physiol. Zool. 60, 538-549. LUTHER, M. A. & LEE, J. C. (1986). The role of phosphorylation in the interaction of rabbit muscle phosphofructokinase with F-actin. J. biol. Chem. 261, 1753-1759. MILLIGAN, C. L. & WOOD, C. M. (1987). Muscle and liver intracellular acid-base and metabolite status after strenuous activity in the inactive, benthic starry flounder Platichthys stellatus. Physiol. Zool. 60, 54-68. MORRISSEY, J. H. (1981). Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem. 117, 307-310. NADAL-GINARD, B. & MARKERT, C. L. (1975). Use of affinity chromatography for purification of lactate dehydrogenase and for assessing the homology and function of the A and B subuhits. In Isozymes. II. Physiological Function (ed. C. L. Markert), pp. 45-67. New York: Academic Press. POON, W. M. & WOOD, T. (1968). Soluble and insoluble phosphofructokinase in rat muscle. Biochem. J. 110, 792-794. ROBERTS, S. J. (1986). The association of the glycolytic enzyme phosphofructokinase with filamentous actin. Doctoral dissertation, University of California, San Diego, pp. 35-68. ROBERTS, S. J. & SOMERO, G. N. (1987). Binding of phosphofructokinase to filamentous actin. Biochemistry, N.Y. 26, 3437-3442. TRIVEDI, B. & DANFORTH, W. H. (1966). Effect of pH on the kinetics of frog muscle phosphofructokinase. 7. biol. Chem. 241,4110-4114. DRIEDZIC,


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D. C.,WALLIMAN,T. & EPPENBERGER, H. M. (1973). A protein that binds specifically to the M-line of skeletal muscle is identified as the muscle form of creatine kinase. Proc. natn. Acad. Sci. U.S.A. 70, 702-705. WALSH, T. P., WINZOR, D. J., CLARKE, F. M., MASTERS, C. J. & MORTON, D. J. (1980). Binding of aldolase to actin-containingfilaments- evidence of interaction with the regulatory proteins of skeletal muscle. Biochem. J. 186, 89-98. WALLIMAN, T., PELLONI, G., TURNER, D. C. & EPPENBERGER, H. M. (1978). Monovalent antibodies against MM-creatine kinase remove the M line from myofibrils. Proc. natn. Acad. Sci. U.S.A. 75, 4296-4300.

TURNER,