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In vivo and in vitro studies on the pathway of modification of mussel pyruvate kinase
Comp. Biochem. Physiol. Vol. 92B, No. 2, pp. 375-380, 1989 Printed in Great Britain
0305-0491/89 $3.00+0.00 © 1989 Pergamon Press pie
IN VIVO AND IN VITRO STUDIES ON THE PATHWAY OF MODIFICATION OF MUSSEL PYRUVATE KINASE D. A. HOLWERDA,M. VELDHUIZEN-TSOERKAN,P. R. VEENHOF and E. EVERS Vakgroep Experimentele Dierkunde, Rijksuniversiteit Utrecht, Padualaan 8, 3584 CH UTRECHT, The Netherlands (Tel: 030 533084) (Received 15 April 1988) Almtraet--l. On aerial exposure, pyruvate kinase is inactivated in various organs of M. edulis; the decrease of activity is slower in muscle than in non-muscular tissue. 2. Anoxic/n vitro incubation of gills results in a rapid inactivation of pyruvate kinase. No change occurs in an aerated medium. 3. Enzyme inactivation is mimicked in part by oxic incubation in an acidified medium containing 5,5-dimethyloxazolidine-2,4-dione, and by the action of calcium ionophore A23187. 4. Incubation of supernatant of gill homogenate results in a slow inactivation of pyruvate kinase that is inhibited by trifluoperazine or EGTA and stimulated by exogenous calmodulin. 5. Addition of ATP plus cAMP stimulates pyruvate kinase inactivation in supernatant of homogenized muscle but not so in a high molecular weight fraction thereof.
INTRODUCTION Pyruvate kinase (EC 2.7.1.40) from adductor muscle and non-muscular tissues of the c o m m o n sea mussel, Mytilus edulis, is an allosteric enzyme. With respect to enzyme modulation, it shares many properties with L-type pyruvate kinase from vertebrates (Holwerda and De Zwaan, 1973), but is, in contrast to the latter, allosterically inhibited at decreased p H (De Zwaan and Hoiwerda, 1972). Also in c o m m o n with the L-type enzyme (Ekman et al., 1976), pyruvate kinase from mussel tissues is regulated by covalent modification (Siebenaller, 1979; Holwerda et al., 1981). The enzyme is believed to play an important regulatory role in the anaerobic metabolism of euryoxic marine molluscs. There is ample evidence that modification of pyruvate kinase from M. edulis and other facultatively anaerobic molluscs is effected by a phosphorylation--dephosphorylation mechanism (Siebenaller, 1979; Holwerda et aL, 1983; Plaxton and Storey, 1984; Plaxton and Storey, 1985). In the regulation of gluconeogenesis, phosphorylation o f L-type pyruvate kinase is elicited by the hormone glucagon (Taunton et al., 1974) that utilizes a cAMP-linked pathway. As yet, little is known about factors triggering the modification of invertebrate pyruvate kinase during anaerobiosis. By the present study we have tried to get more insight into the pathway of enzyme modification, by the use of in vitro incubation o f excised organs and cell-free systems. MATERIALS AND METHODS Sea mussels, Mytilus edulis L., had been collected from the Eastern Scheldt (The Netherlands). Animals were maintained in the laboratory in recirculating natural sea water (2896o) at 13°C without a supply of food. To induce/n vivo anoxia mussels were exposed to air at 13°C. After exposure organs were rapidly dissected out and frozen in liquid nitro-
gen, freeze-dried for 48 hr, and stored dry at - 20°C. In vitro experiments were carried out by incubating excised organs in filtered (Millipore-0.22/zm) natural sea water (SW) or in artificial sea water (ASW), bubbled with air (aerobic condition) or N 2 (anoxic condition) at 15°C. When two conditions were tested within one incubation experiment, each incubation time gills or mantles from four animals were taken and divided in such a way that tissue material from the same animals was compared, using four half gills or mantles per tube (15 ml bath volume). After incubation organ parts were rinsed in ice-cold aqua bidest, frozen in liquid N2, freezedried for 48 hr, and stored at -20°C until use. Standard ASW was composed of: 0.41 M NaCI, 9 mM KCI, 9 mM CaCI2, 23 mM MgCI2, 26mM MgSO4, 10mM HEPES at pH 7.8. Acidified ASW was composed of: 0.39 M NaCI, 9 mM KC1, 9 mM CaCI2, 23 mM MgCI2, 26 mM MgSO4, 20 mM 5,5-dimethyloxazolidine-2,4-dione (DMO), 10 mM imidazole at pH 6.5. Ca-free ASW was composed of: 0.50 M NaCI, 15mM KC1, 2.5raM NaHCO3 at pH 8.0. Dry tissues were homogenized in 30 to 40 volumes (g/ml) of ice-cold buffer (50 mM imidazole-HCl, 25 mM NaF, 5 mM EDTA, 1 mM 1,4-dithioerythritol (DTE), 0.1 mM phenylmethylsulfonyl fluoride at pH 7.0) with an Ultra-Turrax homogenizer. The homogenate was centrifuged at 50,000g for 20min. Two ,volumes of 3.8M (NH4)2SOJI mM DTE at pH 7.0 were added to the supernatant in order to precipitate pyruvate kinase. The suspension was stirred for 1 hr and then centrifuged at 50,000 g for 20 min. The pellet was dissolved in 1 volume of buffer (0.10M imidazole--HC1, 8.3mM MgSO4, 67ram KCI, 5 mM NaF, 0.5 mM DTE at pH 7.0) and chromatographed on a PD-I 0 column (Pharmacia Fine Chemicals), containing Sephadex G-25, in order to remove residual ammonium sulfate. The column was eluted with the same buffer. The first, high molecular weight fraction was taken as the source of enzyme. All fractionation procedures were performed at 0-4°C. Pyruvate kinase (PyK) was assayed using the coupled reaction with NADH and lactate dehydrogenase. The assay buffer was composed of (final concentrations): 0.10M imidazole-HCl (at pH 7.0), 8.3 mM MgSO4, 67 mM KCI, 5 mM NaF, 0.5 mM DTE, 0.2 mM NADH, 2 mM ADP and 5.5 IU per ml of lactate dehydrogenase (dissolved in 50% 375
376
D.A. HOLWERDAet al.
glycerol; ammonium sulfate was found to affect PyK kinetics). The reaction was initiated by addition of the substrate P-enolpyruvate (PEP). PyK activity was determined at 22°C. All assays were done in duplicate. Maximal enzyme activity (Vmax)was measured at 2 mM PEP, in the presence of 0.1 mM fructose-l,6-diphosphate (FDP). Sub-maximal activities (v) were measured at variable PEP concentrations as indicated. As a rule, PyK activity was expressed as the fraction v/V .... as maximal PyK activity is not influenced by modification of the enzyme (Holwerda et al., 1981). Kinetic parameters (S0.5 and nil) were calculated from Hill plots that were determined by linear regression. All biochemicals were purchased from Boehringer, Mannheim. 5,5-Dimethyloxazolidine-2,4-dione was from Sigma Chemical Company. All other chemicals were of reagent grade quality.
vo/Vmax 1.0
0.75
0.50
0.25
0.5
1
1.5
2
IS] (rnM)
Fig. 2. PEP saturation curves of gill Pyk from control ( - - © - - ) and exposed animals (--O--).
RESULTS AND DISCUSSION In vivo modification o f pyruvate kinase Figure 1 shows the time-dependent decrease of pyruvate kinase activity (expressed as the ratio of v and Vmax) in four organs of Mytilus edulis during aerial exposure of the mussels. In the gills and midgut gland, activity ratios started decreasing half an hour after the onset of exposure and reached near-minimal values between 4 and 8hr. These amounted to about 20% of the control value. In the the mantle the drop in activity was somewhat retarded and the highest rate of decrease did not occur before the second hour. In the adductor muscle, during the first hour of anoxia, enzyme activity at sub-optimal substrate concentration even increased somewhat. Thereafter it decreased as well, but significantly slower than in the other organs. After
vo/Vmax
0.3
16hr, the ratio in muscle was about 40% of the control value. These results may reflect some organspecific differentiation in rate and mode of anaerobic metabolism in the sea mussel (Holwerda et al., 1984). Substrate saturation curves of gill pyruvate kinase from control and exposed (8 hr) animals are depicted in Fig. 2. It shows that So 5 increased from 0.41 m M to 0 . 9 5 m M phosphoenolpyruvate. The Hill coefficient remained virtually unaltered with values of 2.27 and 2.25, respectively. In vitro modification o f pyruvate kmase To clarify the mechanism mediating pyruvate kinase modification, gill and mantle were incubated in vitro. When gills were held in aerated SW, activity ratio did not change over a period of 4 hr (Fig. 3). In anoxic, NE-bubbled medium a rapid decrease of gill pyruvate kinase activity occurred over the first hour of incubation. After 4 hr the activity ratio had decreased to less than 10% of the control value. The inserted figure shows that under either bathing condition, V~ax remained constant. This result indicates that the anoxic state per se of the isolated organ can trigger enzyme modification. It is therefore unlikely that inactivation of mussel pyruvate kinase will be
Vo/Vma x
0.2
Gill
0.4 °
0.1 '
0.2
/,c.~ Aerial exposure (hr)
6'
'
'
~
, b
8-~--I~.~__ '
"
Fig. 1. Activity ratio of pyruvate kinase (PyK) in four organs of M. edulis as a function of the duration of aerial exposure. - - A - - gill, - - O - - midgut gland, --&--mantle, - - Q - - posterior adductor muscle; v0 at 0.3 mM PEP. Mean ( + SE) of four groups of three mussels each.
I
I
I
0
1
2
I
hr
4
Fig. 3. Activity ratio of PyK from gills incubated in vitro for 0 to 4 hr in aerated SW ( - - O - - ) or N2-SW ( - - 0 - - ) ; v0 at 0.35 mM PEP. Inserted: V~x in either condition, expressed as % of control (0 hr).
°4I vo I V ma x
Modification of mussel pyruvate kinase
Mantle
377
1. Kinetic parameters of pyruvatekinase from gills incubated in vitro for 0 hr (control)and 4 hr in N,-SW. R: activity ratio (v0 at 0.3raM PEP); S0.s:app. KM;n,: Hill-coefficient.Values are the Table
mean ± SE of 4 incubations
R0.3
S0.5
Control
0.41 _+0.02
0.37 -+0.015
2.04_+0.09
nH
Anoxic
0.04 -+ 0.002
1.34 -+ 0.03
2.08 _+ 0.02
0.2
served that the intracellular pH (pHi) fell from 6.96 to 6.70 in adductor muscle of M. edulis and from 6.95 to 6.56 in retractor muscle of Geukensia demissa during 12 hr of anoxic in vitro incubation. Also, in the | I | I isolated ventricle of the whelk Busycon contrarium, 0 1 2 4 hr incubated anoxically, a drop in pHi was observed, Fig. 4. Activity ratio of PyK from mantle incubated in vitro amounting to 0.24 units during 5 hr (Ellington, for 0 to 4 hr in aerated SW (--©--) or N2-SW (--0--); v0 1983b). Interestingly, in the muscle tissue of the worm Sipunculus nudus, pH~ remained constant during the at 0.35 mM PEP. first 12 hr of environmental hypoxia (P6rtner et al., induced by hormonal action, as was found for the 1984a). This was attributed to the intial utilization of the phosphagen phospho-L-arginine, resulting in the vertebrate L-type enzyme (Engstr6m, 1978). Mantle tissue responded much slower in the/n vitro release of a base. In the next 12 hr of anaerobiosis, incubation with respect to the activity ratio (Fig. 4). pHi fell by 0.3 to 0.4 units. Generally, muscle tissues Moreover, aeration of the bathing medium appar- rely on the phosphagen as the main source of ATP ently did not guarantee the oxic state of the tissue, as during the initial stage of anaerobiosis. For adductor in this condition the ratio decreased as well. Beside muscle of M. edulis, Ebberink et al. 0979) found an the difference of in vivo response between mantle and initial rate of degradation of phosphoarginine of gill (Fig. 1), an additional reason for the compara- 1.2/zmol/hr per g wet weight, that decreased extively weak in vitro response in the mantle might be ponentially to zero after 24 hr. The rate of formation found in the geometry of the latter, which is a much of succinate was constant during 24 hr at thicker organ than the gill. Cells inside the tissue 0.15/zmol/hr per g wet weight. Acidosis would, therefore, not occur during the first hours of exposure. In would not be in equilibrium with the medium. In Fig. 5 saturation curves are depicted for gill contrast, gill tissue was not found to contain depyruvate kinase before incubation and after 4 hr of tectable amounts of the phosphagen (Zurburg and anoxic in vitro incubation. Enzyme parameters have Ebberink, 1981) and would rely solely on anaerobic glycolysis for ATP production. Cellular acidosis been collected in Table 1. would then result from the accumulation of organic Effect o f experimental acidification acids as well as from proton-generating hydrolysis of ATP (P6rtner et al., 1984b). In search of the prime stimulus eventually causing Intracellular pH can also be manipulated by incuinactivation of pyruvate kinase, cellular acidosis was considered. For bivalve molluscs and other facul- bation of tissues and cells in the presence of weak tatively anaerobic invertebrates, environmental an- organic acids at lowered pH (Roos and Boron, 1981). oxia results in a drop in extracellular and intracellular In the isolated ventricle of B. contrarium perfused in pH. Wijsman (1975) has reported that, in the extra- artificial sea water, buffered at pH 6.5 and containing pallial fluid ofM. edulis, pH decreased from 7.5 to 7.0 20 mM of the weak acid 5,5-dimethyloxazoduring 8 hr of aerial exposure. Using the nuclear lidine-2,4-dione (DMO), pH~ decreased from 7.15 to magnetic resonance technique, Ellington (1983a) ob- 6.55 within l hr (Ellington, 1985). In the present study, it was investigated whether the inactivation of gill pyruvate kinase during anoxic in vitro incubation could have been mediated by vo/Vmax intracellular acidification. Gill flaps were incubated in 1.o aerated ASW, containing 20 mM DMO and buffered at pH 6.5. Figure 6 (middle curve)shows that the 0.75 activity ratio decreased slowly but steadily, to about 80% of the control after 4 hr. It seems therefore possible that pyruvate kinase inactivation is primarily 0.50 triggered by an intracellular acidosis. The slow in vivo response in adductor muscle, with a slight increase in activity ratio during the first 30 min of exposure (Fig. 0.25 l), could then be due to a retarded drop in pH i or even a slight alkalosis, as a consequence of phosphoarginine degradation. 0.5 1 1.5 ~;"2 * FDP [8] traM)
Fig. 5. PEP saturation curves of PyK from gills incubated
in vitro for 0 hr (--O--) or 4 hr ( - - 0 - - ) in N=-SW.
Effect o f experimental elevation o f cellular calcium Intracellular pH is supposed to be utilized by cells as a regulator of cellular functions (Moolenaar,
378
D.A. HOLWERDAet al. Vo/Vma x (% of control)
o
100
vo/Vma x (% of control) 100
50 50
I
I
I
0
1
2
!
4 hr Fig. 6. Activity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated ASW, buffered at pH 7.8 (--©--), aerated ASW + 20 mM DMO, buffered at pH 6.5 (--O--), or N2-ASW, buffered at 7.8 (--/k--); v0 at 0.25 mM PEP. In - - O - - and - - O - - , per incubation time, gill flaps were from the same mussels.
1986). One of these functions might be connected with the concentration of cytosolic free Ca 2÷. Elevation of free Ca 2÷, as a result of cellular acidification, could then be suspected as the next step in the mechanism of pyruvate kinase inactivation. It was therefore investigated whether the presence of calcium ionophore A23187 could mimic the decrease of enzyme activity. Figure 7 shows the result of incubations of gill flaps in aerated or in anoxic medium, without or in the presence of A23187. It appears that, in the anoxic medium, the ionophore did not affect the normal drop in activity ratio. In the aerated bath, in the presence of 50/~ M of the ionophore, the ratio slowly decreased to 80% of the control value after 4 hr, whereas no change occurred without A23187. Incubation of gills in aerated Ca-free SW, without or
I
I
hr
4
Fig. 8. Acitivity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated Ca-free ASW, containing 1 mM EGTA, without ( - - O - - ) or with (O--) 50#M A23187 added; v0 at 0.2mM PEP.
with 50/~M A23187, resulted in unaltered activity ratios over the whole period of 4 hr (Fig. 8). The data indicate the possibility that elevation of cytosolic Ca 2+ constitutes a step in the mechanism of pyruvate kinase inactivation during anoxia. It has been shown (Garrison et al., 1984) that treatment of rat hepatocytes with A23187 results in an increase in the phosphorylation state of several cytosolic proteins, including pyruvate kinase. Furthermore, rat liver L-type pyruvate kinase was found to be phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver (Schworer et al., 1985). These authors consider such a protein kinase as a candidate to catalyze in vivo pyruvate kinase inactivation. Also, erythrocyte pyruvate kinase can be phosphorylated via a Ca2+/calmodulindependent mechanism (Nakashima et al., 1982). E n z y m e modification in tissue h o m o g e n a t e s
vo/Vma x (% of control) 100
Q
o
Q
50
I
I
1
2
I
hr
4
Fig. 7. Activity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated SW (--O--), aerated SW + 50 #M A23187 (--O--, these two conditions having, per incubation time, gill flaps from the same mussels), N2-SW (--/k--), or Nz-SW+50/~M A23187 (--A--); v0 at 0.2 mM PEP.
As discussed before, the strong decline of pyruvate kinase activity in isolated gills, incubated anoxically, points to a hormone-independent mechanism of modification. In addition, the effect of A23187 on the activity ratio in isolated gills, incubated aerobically, would indicate to a Ca2+/calmodulin-dependent phosphorylation of the enzyme. Further evidence of the latter mechanism was obtained from incubations of the supernatant fraction of gill homogenate (Fig. 9). Pyruvate kinase activity ratio had decreased to 85% of the control value (not incubated) after 1.5 hr of incubation in the homogenization buffer. The decrease could be prevented in part by the presence of 50 # M of the calmodulin inhibitor trifluoperazine (TFP) and "totally with 100#M TFP. In another experiment, incubation for 45 min resulted in a drop of 12% in activity ratio, which value was increased to 27% when calmodulin at 0.87 p M had been added to the supernatant fraction. In the presence of 10 mM EGTA, virtually no decrease in the activity ratio was observed. However, there are indications of a cAMPdependent mechanism as well, at least for adductor
Modification of mussel pyruvate kinase Gill
Muscle
v o / V m a x (% of control)
~~I
100
379
0.23-- L
O0
._o .~0.19 -;>
a
b
c I c2 c3
b'
d
e
Fig. 9. Activity ratio (as % of control-bar a) of PyK in supernatant of adductor muscle homogenate, incubated for 1½hr (bars b through c3) or 45 min (bars b' through e); v0 at 0.1 mM PEP. (a) not incubated (N = 4, mean 4- SD), (b) no additions (N=4), (ct) plus 25#M TFP ( N = 2 , mean+deviation of the mean), (c:) plus 50#M TFP (N =4), (c3) plus 100#M TFP (N =4), (b') no additions (N = 2), (d) plus 0.87/~M calmodulin (N =2), (e) plus 10 mM EGTA (N = 2).
muscle of M. edulus. It was reported earlier (Holwerda et al., 1981) that cAMP and cGMP concentrations in adductor muscle change during aerial exposure. In addition, incubation of a muscle homogenate with ATP and cAMP resulted in a marked shift of pyruvate kinase units from the first ("active", less phosphorylated) peak to the second ("inactive", more phosphorylated) after ion exchange chromatography (Holwerda et al., 1983). In the present study, gill and muscle homogenates were compared with regard to the decreasing effect of ATP plus cAMP on enzyme activity. Figure 10 shows that the response was considerably stronger (ca 25% decrease) in muscle than in gill (ca 10% decrease). A small decrease also results from the mere incubation without added substances (see, for example, bars b and b' in Fig. 9). This effect can be inhibited by the presence of ATP alone, as illustrated by Fig. 11 (bar c). In this condition, the activity ratio was not different from the non-incubated control (bar a). Interestingly, the spontaneous decrease of the ratio (without substances added), did not occur when the high molecular weight (HMW) fraction--after Sephadex G-25 gel filtration of centrifuged gill homogenate--was incubated. Apparently, the low molecular weight (LMW) fraction contains a factor essential for enzyme modification. Following recombination of the two fractions in the ratio two volumes H M W fraction and one volume L M W fraction, the decreasing effect of ATP plus cAMP was restored in part (Fig. 11, bars e' vs. d). Recombination in the ratio 1 : 2, approaching the initial composition of unseparated supernatant, almost wholly restored the response by ATP plus cAMP (bar e"). Figure I 1 (bar e") shows that the essential factor in the L M W fraction is not the calcium ion. CONCLUSION The central view from our studies is that anaerobiosis brings about such a change in the intracellular milieu as to result eventually in the modification, presumably by phosphory]ation, of pyruvate kinase. We think that the initiating factor will be directly
I 0.15I
a
b
a
b
Fig. 10. Activity ratio of PyK in supematant of adductor muscle and gill homogenates, incubated for 2 hr without (a) or with (b) 2mM ATP+0.1mM cAMP added; v0 at 0.3 mM PEP. Mean + SD (N = 5).
related to anaerobic metabolism. There is some evidence, from literature and this study, that this factor is a cellular acidosis: aerobic in vitro incubation in acidified SW mimics in part the enzymic response to anoxia and the tissue specificity with respect to the magnitude of the in vivo response can be easily understood from tissue-specific differentiation in the degree of acidosis. Evidence has further been presented for both a Ca2÷/calmodulin - and a cAMP-dependent mechanism of enzyme modification. Are both systems operating in vivo, simultaneously or sequentially, or does one of the two lack physiological significance? The situation resembles that for the red blood cell. Erythrocyte pyruvate kinase can be phosphorylated in vitro both by the cAMP- and the Ca2+/ calmodulin-dependent process (Nakashima et al.,
volVmax (%of control) 100, ~ .~ I"11
so 6O
~Yc~
e
es
e"
e "S
Fig. 11. Activity ratio (as % of control-bar a) of PyK in (a fraction of) supernatant of adductor muscle homogenate, incubated for 4 hr; v0 at 0.I mM PEP. (a) not incubated (N = 2, mean + deviation of the mean), (b) no additions (N = 5, mean _ SD), (c) plus 2 mM ATP (N = 5), (d) plus 2 mM ATP+0.1 mM cAMP (N = 5), (e) HMW fraction of supernatant, plus 2 mM ATP + 0.1 mM cAMP (N = 5), (e') HMW- + LMW fractions recombined (2:1), plus 2 mM ATP+0.1 mM cAMP ( N = 5), (e") HMW-+LMW fractions reeombined (I :2), plus 2 mM ATP + 0.1 mM cAMP (N = 5), (e") HMW fraction, plus 2mM ATP+0.1 mM cAMP + 0.5 mM CaCI2 (N = 5).
380
D . A . HOLWERDA et al.
1982), but the red blood cell does not a p p e a r to produce the cyclic nucleotide endogenously. O n the o t h e r h a n d , examples o f second messenger interaction to produce certain physiological responses have been reported ( R a s m u s s e n a n d Barrett, 1984). It remains to be clarified w h e t h e r in vivo phosp h o r y l a t i o n is effected in the various organs of M. edulis by the same process, a n d whether this involves one or more second messengers. REFERENCES
Ebberink R. H. M., Zurburg W. and Zandee D. I. (1979) The energy demand of the posterior adductor muscle of Mytilus edulis in catch during exposure to air. Mar. Biol. Lett. l, 23-31. Ekman P., Dahlquist U., Humble E. and Engstr6m L. (1976) Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3',5'-AMP-stimulated protein kinase. Biochim. biophys. Acta 429, 374-382. Ellington W. R. (1983a) The extent of intracellular acidification during anoxia in the catch muscles of two bivalve molluscs. J. exp. Zool. 227, 313 317. Ellington W. R. (1983b) Phosphorus nuclear magnetic resonance studies of energy metabolism in molluscan tissues. J. comp. Physiol. 153, 159-166. Ellington W. R. (1985) Metabolic impact of experimental reductions of intracellular pH in molluscan cardiac muscle. Molec. Physiol. 7, 155 163. Engstr6m L. (1978) The regulation of liver pyruvate kinase by phosphorylation~lephosphorylation. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. R.), Vol. 13, pp. 29 51. Academic Press, New York. Garrison J. C., Johnsen D. E. and Campanile C. P. (1984) Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+/calmodulin-dependent protein kinases in the response of hepatocytes to angiotensin II and vasopressin. J. biol. Chem. 259, 3283-3292. Holwerda D. A., Kruitwagen, E. C. J. and Bont A. M. Th. de (1981) Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity during anaerobiosis in Mytilus edulis L. Molec. Physiol. 1, 165-171. Holwerda D. A., Veenhof P. R., Heugten H. A. A. van and Zandee D. I. (1983) Modification of mussel pyruvate kinase during anaerobiosis and after temperature acclimation. Molec. Physiol. 3, 225-234. Holwerda D. A., Veenhof P. R and Zwaan A. de (1984) Physiological and biochemical investigations of the ecological relevance of anaerobiosis in bivalves: I. The changes in activity of mussel adductor muscle and mantle pyruvate kinase during aerial exposure and reimmersion. Mar. Biol. Lett. 5, 185-190. Holwerda D. A. and Zwaan A. de (1973) Kinetic and
molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L. Biochim. biophys. Acta 309, 296-306. Moolenaar W. H. (1986) Regulation of cytoplasmic pH by Na+/H + exchange. Trends biochem. Sci. 11, 141-143. Nakashima K., Fujii S., Kaku K. and Kaneko T. (1982) Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase. Biochem. biophys. Res. Comm. 104, 285 289. Plaxton W. C. and Storey K. B. (1984) Phosphorylation in vieo of red-muscle pyruvate kinase from the channelled whelk, Busvcotypus eanalieulatum, in response to anoxic stress. Eur. J. Biochem. 143, 267-272. Plaxton W. C. and Storey K. B. (1985) Tissue specific isozymes of pyruvate kinase in the channelled whelk Busvcotypus canalieulatum: enzyme modification in response to environmental anoxia. J. comp. Physiol. B155, 291 296. P6rtner H. O., Grieshaber M. K. and Heisler N. (1984a) Anaerobiosis and acid--base status in marine invertebrates: effect of environmental hypoxia on extracellular and intracellular pH in Sipunculus nudus L. J. comp. Physiol. B155, 13 20. P6tner H. O., Heisler N. and Grieshaber M. K. (1984b) Anaerobiosis and acid base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism. J. comp. Physiol. 18155, 1-12. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Roos A. and Boron W. F. (1981) lntracellular pH. Physiol. Rev. 61, 296434. Schworer C. M., E1-Maghrabi M. R., Pilkis S. J. and Soderling T. R. (1985) Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase. J. biol. Chem. 260, 13018 13022. Siebenaller J. F. (1979) Regulation of pyruvate kinase in Mytilus edulis by phosphorytation dephosphorylation. Mar. Biol. Lett. 1, 105 110. Taunton O. D., Stifel F. B., Greene H. L. and Herman R. H. (1974) Rapid reciprocal changes in rat hepatic glycolytic enzyme and fructose diphosphatase activities following insulin and glucagon injection. J. biol. Chem. 249, 72287239. Wijsman T. C. M. (1975) pH fluctuations in Mytilus edulis (L.) in relation to shell movements under aerobic conditions. Proc. 9th Eur. Mar. Biol. Syrup. (Edited by Barnes H.), pp. 139 149. Aberdeen University Press, Aberdeen. Zurburg W. and Ebberink R. H. M. (1981)The anaerobic energy demand of Mytilus edulis. Organ specific differences in ATP-supplying processes and metabolic routes. Molec. Physiol. l, 153 164. Zwaan A. de and Holwerda D. A. (1972) The effect of phosphoenolpyruvate, fructose 1,6-diphosphate and pH on allosteric pyruvate kinase in muscle tissue of the bivalve Mytilus edulis L. Biochim. biophys. Acta 276, 430 433.
0305-0491/89 $3.00+0.00 © 1989 Pergamon Press pie
IN VIVO AND IN VITRO STUDIES ON THE PATHWAY OF MODIFICATION OF MUSSEL PYRUVATE KINASE D. A. HOLWERDA,M. VELDHUIZEN-TSOERKAN,P. R. VEENHOF and E. EVERS Vakgroep Experimentele Dierkunde, Rijksuniversiteit Utrecht, Padualaan 8, 3584 CH UTRECHT, The Netherlands (Tel: 030 533084) (Received 15 April 1988) Almtraet--l. On aerial exposure, pyruvate kinase is inactivated in various organs of M. edulis; the decrease of activity is slower in muscle than in non-muscular tissue. 2. Anoxic/n vitro incubation of gills results in a rapid inactivation of pyruvate kinase. No change occurs in an aerated medium. 3. Enzyme inactivation is mimicked in part by oxic incubation in an acidified medium containing 5,5-dimethyloxazolidine-2,4-dione, and by the action of calcium ionophore A23187. 4. Incubation of supernatant of gill homogenate results in a slow inactivation of pyruvate kinase that is inhibited by trifluoperazine or EGTA and stimulated by exogenous calmodulin. 5. Addition of ATP plus cAMP stimulates pyruvate kinase inactivation in supernatant of homogenized muscle but not so in a high molecular weight fraction thereof.
INTRODUCTION Pyruvate kinase (EC 2.7.1.40) from adductor muscle and non-muscular tissues of the c o m m o n sea mussel, Mytilus edulis, is an allosteric enzyme. With respect to enzyme modulation, it shares many properties with L-type pyruvate kinase from vertebrates (Holwerda and De Zwaan, 1973), but is, in contrast to the latter, allosterically inhibited at decreased p H (De Zwaan and Hoiwerda, 1972). Also in c o m m o n with the L-type enzyme (Ekman et al., 1976), pyruvate kinase from mussel tissues is regulated by covalent modification (Siebenaller, 1979; Holwerda et al., 1981). The enzyme is believed to play an important regulatory role in the anaerobic metabolism of euryoxic marine molluscs. There is ample evidence that modification of pyruvate kinase from M. edulis and other facultatively anaerobic molluscs is effected by a phosphorylation--dephosphorylation mechanism (Siebenaller, 1979; Holwerda et aL, 1983; Plaxton and Storey, 1984; Plaxton and Storey, 1985). In the regulation of gluconeogenesis, phosphorylation o f L-type pyruvate kinase is elicited by the hormone glucagon (Taunton et al., 1974) that utilizes a cAMP-linked pathway. As yet, little is known about factors triggering the modification of invertebrate pyruvate kinase during anaerobiosis. By the present study we have tried to get more insight into the pathway of enzyme modification, by the use of in vitro incubation o f excised organs and cell-free systems. MATERIALS AND METHODS Sea mussels, Mytilus edulis L., had been collected from the Eastern Scheldt (The Netherlands). Animals were maintained in the laboratory in recirculating natural sea water (2896o) at 13°C without a supply of food. To induce/n vivo anoxia mussels were exposed to air at 13°C. After exposure organs were rapidly dissected out and frozen in liquid nitro-
gen, freeze-dried for 48 hr, and stored dry at - 20°C. In vitro experiments were carried out by incubating excised organs in filtered (Millipore-0.22/zm) natural sea water (SW) or in artificial sea water (ASW), bubbled with air (aerobic condition) or N 2 (anoxic condition) at 15°C. When two conditions were tested within one incubation experiment, each incubation time gills or mantles from four animals were taken and divided in such a way that tissue material from the same animals was compared, using four half gills or mantles per tube (15 ml bath volume). After incubation organ parts were rinsed in ice-cold aqua bidest, frozen in liquid N2, freezedried for 48 hr, and stored at -20°C until use. Standard ASW was composed of: 0.41 M NaCI, 9 mM KCI, 9 mM CaCI2, 23 mM MgCI2, 26mM MgSO4, 10mM HEPES at pH 7.8. Acidified ASW was composed of: 0.39 M NaCI, 9 mM KC1, 9 mM CaCI2, 23 mM MgCI2, 26 mM MgSO4, 20 mM 5,5-dimethyloxazolidine-2,4-dione (DMO), 10 mM imidazole at pH 6.5. Ca-free ASW was composed of: 0.50 M NaCI, 15mM KC1, 2.5raM NaHCO3 at pH 8.0. Dry tissues were homogenized in 30 to 40 volumes (g/ml) of ice-cold buffer (50 mM imidazole-HCl, 25 mM NaF, 5 mM EDTA, 1 mM 1,4-dithioerythritol (DTE), 0.1 mM phenylmethylsulfonyl fluoride at pH 7.0) with an Ultra-Turrax homogenizer. The homogenate was centrifuged at 50,000g for 20min. Two ,volumes of 3.8M (NH4)2SOJI mM DTE at pH 7.0 were added to the supernatant in order to precipitate pyruvate kinase. The suspension was stirred for 1 hr and then centrifuged at 50,000 g for 20 min. The pellet was dissolved in 1 volume of buffer (0.10M imidazole--HC1, 8.3mM MgSO4, 67ram KCI, 5 mM NaF, 0.5 mM DTE at pH 7.0) and chromatographed on a PD-I 0 column (Pharmacia Fine Chemicals), containing Sephadex G-25, in order to remove residual ammonium sulfate. The column was eluted with the same buffer. The first, high molecular weight fraction was taken as the source of enzyme. All fractionation procedures were performed at 0-4°C. Pyruvate kinase (PyK) was assayed using the coupled reaction with NADH and lactate dehydrogenase. The assay buffer was composed of (final concentrations): 0.10M imidazole-HCl (at pH 7.0), 8.3 mM MgSO4, 67 mM KCI, 5 mM NaF, 0.5 mM DTE, 0.2 mM NADH, 2 mM ADP and 5.5 IU per ml of lactate dehydrogenase (dissolved in 50% 375
376
D.A. HOLWERDAet al.
glycerol; ammonium sulfate was found to affect PyK kinetics). The reaction was initiated by addition of the substrate P-enolpyruvate (PEP). PyK activity was determined at 22°C. All assays were done in duplicate. Maximal enzyme activity (Vmax)was measured at 2 mM PEP, in the presence of 0.1 mM fructose-l,6-diphosphate (FDP). Sub-maximal activities (v) were measured at variable PEP concentrations as indicated. As a rule, PyK activity was expressed as the fraction v/V .... as maximal PyK activity is not influenced by modification of the enzyme (Holwerda et al., 1981). Kinetic parameters (S0.5 and nil) were calculated from Hill plots that were determined by linear regression. All biochemicals were purchased from Boehringer, Mannheim. 5,5-Dimethyloxazolidine-2,4-dione was from Sigma Chemical Company. All other chemicals were of reagent grade quality.
vo/Vmax 1.0
0.75
0.50
0.25
0.5
1
1.5
2
IS] (rnM)
Fig. 2. PEP saturation curves of gill Pyk from control ( - - © - - ) and exposed animals (--O--).
RESULTS AND DISCUSSION In vivo modification o f pyruvate kinase Figure 1 shows the time-dependent decrease of pyruvate kinase activity (expressed as the ratio of v and Vmax) in four organs of Mytilus edulis during aerial exposure of the mussels. In the gills and midgut gland, activity ratios started decreasing half an hour after the onset of exposure and reached near-minimal values between 4 and 8hr. These amounted to about 20% of the control value. In the the mantle the drop in activity was somewhat retarded and the highest rate of decrease did not occur before the second hour. In the adductor muscle, during the first hour of anoxia, enzyme activity at sub-optimal substrate concentration even increased somewhat. Thereafter it decreased as well, but significantly slower than in the other organs. After
vo/Vmax
0.3
16hr, the ratio in muscle was about 40% of the control value. These results may reflect some organspecific differentiation in rate and mode of anaerobic metabolism in the sea mussel (Holwerda et al., 1984). Substrate saturation curves of gill pyruvate kinase from control and exposed (8 hr) animals are depicted in Fig. 2. It shows that So 5 increased from 0.41 m M to 0 . 9 5 m M phosphoenolpyruvate. The Hill coefficient remained virtually unaltered with values of 2.27 and 2.25, respectively. In vitro modification o f pyruvate kmase To clarify the mechanism mediating pyruvate kinase modification, gill and mantle were incubated in vitro. When gills were held in aerated SW, activity ratio did not change over a period of 4 hr (Fig. 3). In anoxic, NE-bubbled medium a rapid decrease of gill pyruvate kinase activity occurred over the first hour of incubation. After 4 hr the activity ratio had decreased to less than 10% of the control value. The inserted figure shows that under either bathing condition, V~ax remained constant. This result indicates that the anoxic state per se of the isolated organ can trigger enzyme modification. It is therefore unlikely that inactivation of mussel pyruvate kinase will be
Vo/Vma x
0.2
Gill
0.4 °
0.1 '
0.2
/,c.~ Aerial exposure (hr)
6'
'
'
~
, b
8-~--I~.~__ '
"
Fig. 1. Activity ratio of pyruvate kinase (PyK) in four organs of M. edulis as a function of the duration of aerial exposure. - - A - - gill, - - O - - midgut gland, --&--mantle, - - Q - - posterior adductor muscle; v0 at 0.3 mM PEP. Mean ( + SE) of four groups of three mussels each.
I
I
I
0
1
2
I
hr
4
Fig. 3. Activity ratio of PyK from gills incubated in vitro for 0 to 4 hr in aerated SW ( - - O - - ) or N2-SW ( - - 0 - - ) ; v0 at 0.35 mM PEP. Inserted: V~x in either condition, expressed as % of control (0 hr).
°4I vo I V ma x
Modification of mussel pyruvate kinase
Mantle
377
1. Kinetic parameters of pyruvatekinase from gills incubated in vitro for 0 hr (control)and 4 hr in N,-SW. R: activity ratio (v0 at 0.3raM PEP); S0.s:app. KM;n,: Hill-coefficient.Values are the Table
mean ± SE of 4 incubations
R0.3
S0.5
Control
0.41 _+0.02
0.37 -+0.015
2.04_+0.09
nH
Anoxic
0.04 -+ 0.002
1.34 -+ 0.03
2.08 _+ 0.02
0.2
served that the intracellular pH (pHi) fell from 6.96 to 6.70 in adductor muscle of M. edulis and from 6.95 to 6.56 in retractor muscle of Geukensia demissa during 12 hr of anoxic in vitro incubation. Also, in the | I | I isolated ventricle of the whelk Busycon contrarium, 0 1 2 4 hr incubated anoxically, a drop in pHi was observed, Fig. 4. Activity ratio of PyK from mantle incubated in vitro amounting to 0.24 units during 5 hr (Ellington, for 0 to 4 hr in aerated SW (--©--) or N2-SW (--0--); v0 1983b). Interestingly, in the muscle tissue of the worm Sipunculus nudus, pH~ remained constant during the at 0.35 mM PEP. first 12 hr of environmental hypoxia (P6rtner et al., induced by hormonal action, as was found for the 1984a). This was attributed to the intial utilization of the phosphagen phospho-L-arginine, resulting in the vertebrate L-type enzyme (Engstr6m, 1978). Mantle tissue responded much slower in the/n vitro release of a base. In the next 12 hr of anaerobiosis, incubation with respect to the activity ratio (Fig. 4). pHi fell by 0.3 to 0.4 units. Generally, muscle tissues Moreover, aeration of the bathing medium appar- rely on the phosphagen as the main source of ATP ently did not guarantee the oxic state of the tissue, as during the initial stage of anaerobiosis. For adductor in this condition the ratio decreased as well. Beside muscle of M. edulis, Ebberink et al. 0979) found an the difference of in vivo response between mantle and initial rate of degradation of phosphoarginine of gill (Fig. 1), an additional reason for the compara- 1.2/zmol/hr per g wet weight, that decreased extively weak in vitro response in the mantle might be ponentially to zero after 24 hr. The rate of formation found in the geometry of the latter, which is a much of succinate was constant during 24 hr at thicker organ than the gill. Cells inside the tissue 0.15/zmol/hr per g wet weight. Acidosis would, therefore, not occur during the first hours of exposure. In would not be in equilibrium with the medium. In Fig. 5 saturation curves are depicted for gill contrast, gill tissue was not found to contain depyruvate kinase before incubation and after 4 hr of tectable amounts of the phosphagen (Zurburg and anoxic in vitro incubation. Enzyme parameters have Ebberink, 1981) and would rely solely on anaerobic glycolysis for ATP production. Cellular acidosis been collected in Table 1. would then result from the accumulation of organic Effect o f experimental acidification acids as well as from proton-generating hydrolysis of ATP (P6rtner et al., 1984b). In search of the prime stimulus eventually causing Intracellular pH can also be manipulated by incuinactivation of pyruvate kinase, cellular acidosis was considered. For bivalve molluscs and other facul- bation of tissues and cells in the presence of weak tatively anaerobic invertebrates, environmental an- organic acids at lowered pH (Roos and Boron, 1981). oxia results in a drop in extracellular and intracellular In the isolated ventricle of B. contrarium perfused in pH. Wijsman (1975) has reported that, in the extra- artificial sea water, buffered at pH 6.5 and containing pallial fluid ofM. edulis, pH decreased from 7.5 to 7.0 20 mM of the weak acid 5,5-dimethyloxazoduring 8 hr of aerial exposure. Using the nuclear lidine-2,4-dione (DMO), pH~ decreased from 7.15 to magnetic resonance technique, Ellington (1983a) ob- 6.55 within l hr (Ellington, 1985). In the present study, it was investigated whether the inactivation of gill pyruvate kinase during anoxic in vitro incubation could have been mediated by vo/Vmax intracellular acidification. Gill flaps were incubated in 1.o aerated ASW, containing 20 mM DMO and buffered at pH 6.5. Figure 6 (middle curve)shows that the 0.75 activity ratio decreased slowly but steadily, to about 80% of the control after 4 hr. It seems therefore possible that pyruvate kinase inactivation is primarily 0.50 triggered by an intracellular acidosis. The slow in vivo response in adductor muscle, with a slight increase in activity ratio during the first 30 min of exposure (Fig. 0.25 l), could then be due to a retarded drop in pH i or even a slight alkalosis, as a consequence of phosphoarginine degradation. 0.5 1 1.5 ~;"2 * FDP [8] traM)
Fig. 5. PEP saturation curves of PyK from gills incubated
in vitro for 0 hr (--O--) or 4 hr ( - - 0 - - ) in N=-SW.
Effect o f experimental elevation o f cellular calcium Intracellular pH is supposed to be utilized by cells as a regulator of cellular functions (Moolenaar,
378
D.A. HOLWERDAet al. Vo/Vma x (% of control)
o
100
vo/Vma x (% of control) 100
50 50
I
I
I
0
1
2
!
4 hr Fig. 6. Activity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated ASW, buffered at pH 7.8 (--©--), aerated ASW + 20 mM DMO, buffered at pH 6.5 (--O--), or N2-ASW, buffered at 7.8 (--/k--); v0 at 0.25 mM PEP. In - - O - - and - - O - - , per incubation time, gill flaps were from the same mussels.
1986). One of these functions might be connected with the concentration of cytosolic free Ca 2÷. Elevation of free Ca 2÷, as a result of cellular acidification, could then be suspected as the next step in the mechanism of pyruvate kinase inactivation. It was therefore investigated whether the presence of calcium ionophore A23187 could mimic the decrease of enzyme activity. Figure 7 shows the result of incubations of gill flaps in aerated or in anoxic medium, without or in the presence of A23187. It appears that, in the anoxic medium, the ionophore did not affect the normal drop in activity ratio. In the aerated bath, in the presence of 50/~ M of the ionophore, the ratio slowly decreased to 80% of the control value after 4 hr, whereas no change occurred without A23187. Incubation of gills in aerated Ca-free SW, without or
I
I
hr
4
Fig. 8. Acitivity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated Ca-free ASW, containing 1 mM EGTA, without ( - - O - - ) or with (O--) 50#M A23187 added; v0 at 0.2mM PEP.
with 50/~M A23187, resulted in unaltered activity ratios over the whole period of 4 hr (Fig. 8). The data indicate the possibility that elevation of cytosolic Ca 2+ constitutes a step in the mechanism of pyruvate kinase inactivation during anoxia. It has been shown (Garrison et al., 1984) that treatment of rat hepatocytes with A23187 results in an increase in the phosphorylation state of several cytosolic proteins, including pyruvate kinase. Furthermore, rat liver L-type pyruvate kinase was found to be phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver (Schworer et al., 1985). These authors consider such a protein kinase as a candidate to catalyze in vivo pyruvate kinase inactivation. Also, erythrocyte pyruvate kinase can be phosphorylated via a Ca2+/calmodulindependent mechanism (Nakashima et al., 1982). E n z y m e modification in tissue h o m o g e n a t e s
vo/Vma x (% of control) 100
Q
o
Q
50
I
I
1
2
I
hr
4
Fig. 7. Activity ratio (as % of control) of PyK from gills incubated in vitro for 0 to 4 hr in aerated SW (--O--), aerated SW + 50 #M A23187 (--O--, these two conditions having, per incubation time, gill flaps from the same mussels), N2-SW (--/k--), or Nz-SW+50/~M A23187 (--A--); v0 at 0.2 mM PEP.
As discussed before, the strong decline of pyruvate kinase activity in isolated gills, incubated anoxically, points to a hormone-independent mechanism of modification. In addition, the effect of A23187 on the activity ratio in isolated gills, incubated aerobically, would indicate to a Ca2+/calmodulin-dependent phosphorylation of the enzyme. Further evidence of the latter mechanism was obtained from incubations of the supernatant fraction of gill homogenate (Fig. 9). Pyruvate kinase activity ratio had decreased to 85% of the control value (not incubated) after 1.5 hr of incubation in the homogenization buffer. The decrease could be prevented in part by the presence of 50 # M of the calmodulin inhibitor trifluoperazine (TFP) and "totally with 100#M TFP. In another experiment, incubation for 45 min resulted in a drop of 12% in activity ratio, which value was increased to 27% when calmodulin at 0.87 p M had been added to the supernatant fraction. In the presence of 10 mM EGTA, virtually no decrease in the activity ratio was observed. However, there are indications of a cAMPdependent mechanism as well, at least for adductor
Modification of mussel pyruvate kinase Gill
Muscle
v o / V m a x (% of control)
~~I
100
379
0.23-- L
O0
._o .~0.19 -;>
a
b
c I c2 c3
b'
d
e
Fig. 9. Activity ratio (as % of control-bar a) of PyK in supernatant of adductor muscle homogenate, incubated for 1½hr (bars b through c3) or 45 min (bars b' through e); v0 at 0.1 mM PEP. (a) not incubated (N = 4, mean 4- SD), (b) no additions (N=4), (ct) plus 25#M TFP ( N = 2 , mean+deviation of the mean), (c:) plus 50#M TFP (N =4), (c3) plus 100#M TFP (N =4), (b') no additions (N = 2), (d) plus 0.87/~M calmodulin (N =2), (e) plus 10 mM EGTA (N = 2).
muscle of M. edulus. It was reported earlier (Holwerda et al., 1981) that cAMP and cGMP concentrations in adductor muscle change during aerial exposure. In addition, incubation of a muscle homogenate with ATP and cAMP resulted in a marked shift of pyruvate kinase units from the first ("active", less phosphorylated) peak to the second ("inactive", more phosphorylated) after ion exchange chromatography (Holwerda et al., 1983). In the present study, gill and muscle homogenates were compared with regard to the decreasing effect of ATP plus cAMP on enzyme activity. Figure 10 shows that the response was considerably stronger (ca 25% decrease) in muscle than in gill (ca 10% decrease). A small decrease also results from the mere incubation without added substances (see, for example, bars b and b' in Fig. 9). This effect can be inhibited by the presence of ATP alone, as illustrated by Fig. 11 (bar c). In this condition, the activity ratio was not different from the non-incubated control (bar a). Interestingly, the spontaneous decrease of the ratio (without substances added), did not occur when the high molecular weight (HMW) fraction--after Sephadex G-25 gel filtration of centrifuged gill homogenate--was incubated. Apparently, the low molecular weight (LMW) fraction contains a factor essential for enzyme modification. Following recombination of the two fractions in the ratio two volumes H M W fraction and one volume L M W fraction, the decreasing effect of ATP plus cAMP was restored in part (Fig. 11, bars e' vs. d). Recombination in the ratio 1 : 2, approaching the initial composition of unseparated supernatant, almost wholly restored the response by ATP plus cAMP (bar e"). Figure I 1 (bar e") shows that the essential factor in the L M W fraction is not the calcium ion. CONCLUSION The central view from our studies is that anaerobiosis brings about such a change in the intracellular milieu as to result eventually in the modification, presumably by phosphory]ation, of pyruvate kinase. We think that the initiating factor will be directly
I 0.15I
a
b
a
b
Fig. 10. Activity ratio of PyK in supematant of adductor muscle and gill homogenates, incubated for 2 hr without (a) or with (b) 2mM ATP+0.1mM cAMP added; v0 at 0.3 mM PEP. Mean + SD (N = 5).
related to anaerobic metabolism. There is some evidence, from literature and this study, that this factor is a cellular acidosis: aerobic in vitro incubation in acidified SW mimics in part the enzymic response to anoxia and the tissue specificity with respect to the magnitude of the in vivo response can be easily understood from tissue-specific differentiation in the degree of acidosis. Evidence has further been presented for both a Ca2÷/calmodulin - and a cAMP-dependent mechanism of enzyme modification. Are both systems operating in vivo, simultaneously or sequentially, or does one of the two lack physiological significance? The situation resembles that for the red blood cell. Erythrocyte pyruvate kinase can be phosphorylated in vitro both by the cAMP- and the Ca2+/ calmodulin-dependent process (Nakashima et al.,
volVmax (%of control) 100, ~ .~ I"11
so 6O
~Yc~
e
es
e"
e "S
Fig. 11. Activity ratio (as % of control-bar a) of PyK in (a fraction of) supernatant of adductor muscle homogenate, incubated for 4 hr; v0 at 0.I mM PEP. (a) not incubated (N = 2, mean + deviation of the mean), (b) no additions (N = 5, mean _ SD), (c) plus 2 mM ATP (N = 5), (d) plus 2 mM ATP+0.1 mM cAMP (N = 5), (e) HMW fraction of supernatant, plus 2 mM ATP + 0.1 mM cAMP (N = 5), (e') HMW- + LMW fractions recombined (2:1), plus 2 mM ATP+0.1 mM cAMP ( N = 5), (e") HMW-+LMW fractions reeombined (I :2), plus 2 mM ATP + 0.1 mM cAMP (N = 5), (e") HMW fraction, plus 2mM ATP+0.1 mM cAMP + 0.5 mM CaCI2 (N = 5).
380
D . A . HOLWERDA et al.
1982), but the red blood cell does not a p p e a r to produce the cyclic nucleotide endogenously. O n the o t h e r h a n d , examples o f second messenger interaction to produce certain physiological responses have been reported ( R a s m u s s e n a n d Barrett, 1984). It remains to be clarified w h e t h e r in vivo phosp h o r y l a t i o n is effected in the various organs of M. edulis by the same process, a n d whether this involves one or more second messengers. REFERENCES
Ebberink R. H. M., Zurburg W. and Zandee D. I. (1979) The energy demand of the posterior adductor muscle of Mytilus edulis in catch during exposure to air. Mar. Biol. Lett. l, 23-31. Ekman P., Dahlquist U., Humble E. and Engstr6m L. (1976) Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3',5'-AMP-stimulated protein kinase. Biochim. biophys. Acta 429, 374-382. Ellington W. R. (1983a) The extent of intracellular acidification during anoxia in the catch muscles of two bivalve molluscs. J. exp. Zool. 227, 313 317. Ellington W. R. (1983b) Phosphorus nuclear magnetic resonance studies of energy metabolism in molluscan tissues. J. comp. Physiol. 153, 159-166. Ellington W. R. (1985) Metabolic impact of experimental reductions of intracellular pH in molluscan cardiac muscle. Molec. Physiol. 7, 155 163. Engstr6m L. (1978) The regulation of liver pyruvate kinase by phosphorylation~lephosphorylation. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. R.), Vol. 13, pp. 29 51. Academic Press, New York. Garrison J. C., Johnsen D. E. and Campanile C. P. (1984) Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+/calmodulin-dependent protein kinases in the response of hepatocytes to angiotensin II and vasopressin. J. biol. Chem. 259, 3283-3292. Holwerda D. A., Kruitwagen, E. C. J. and Bont A. M. Th. de (1981) Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity during anaerobiosis in Mytilus edulis L. Molec. Physiol. 1, 165-171. Holwerda D. A., Veenhof P. R., Heugten H. A. A. van and Zandee D. I. (1983) Modification of mussel pyruvate kinase during anaerobiosis and after temperature acclimation. Molec. Physiol. 3, 225-234. Holwerda D. A., Veenhof P. R and Zwaan A. de (1984) Physiological and biochemical investigations of the ecological relevance of anaerobiosis in bivalves: I. The changes in activity of mussel adductor muscle and mantle pyruvate kinase during aerial exposure and reimmersion. Mar. Biol. Lett. 5, 185-190. Holwerda D. A. and Zwaan A. de (1973) Kinetic and
molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L. Biochim. biophys. Acta 309, 296-306. Moolenaar W. H. (1986) Regulation of cytoplasmic pH by Na+/H + exchange. Trends biochem. Sci. 11, 141-143. Nakashima K., Fujii S., Kaku K. and Kaneko T. (1982) Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase. Biochem. biophys. Res. Comm. 104, 285 289. Plaxton W. C. and Storey K. B. (1984) Phosphorylation in vieo of red-muscle pyruvate kinase from the channelled whelk, Busvcotypus eanalieulatum, in response to anoxic stress. Eur. J. Biochem. 143, 267-272. Plaxton W. C. and Storey K. B. (1985) Tissue specific isozymes of pyruvate kinase in the channelled whelk Busvcotypus canalieulatum: enzyme modification in response to environmental anoxia. J. comp. Physiol. B155, 291 296. P6rtner H. O., Grieshaber M. K. and Heisler N. (1984a) Anaerobiosis and acid--base status in marine invertebrates: effect of environmental hypoxia on extracellular and intracellular pH in Sipunculus nudus L. J. comp. Physiol. B155, 13 20. P6tner H. O., Heisler N. and Grieshaber M. K. (1984b) Anaerobiosis and acid base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism. J. comp. Physiol. 18155, 1-12. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Roos A. and Boron W. F. (1981) lntracellular pH. Physiol. Rev. 61, 296434. Schworer C. M., E1-Maghrabi M. R., Pilkis S. J. and Soderling T. R. (1985) Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase. J. biol. Chem. 260, 13018 13022. Siebenaller J. F. (1979) Regulation of pyruvate kinase in Mytilus edulis by phosphorytation dephosphorylation. Mar. Biol. Lett. 1, 105 110. Taunton O. D., Stifel F. B., Greene H. L. and Herman R. H. (1974) Rapid reciprocal changes in rat hepatic glycolytic enzyme and fructose diphosphatase activities following insulin and glucagon injection. J. biol. Chem. 249, 72287239. Wijsman T. C. M. (1975) pH fluctuations in Mytilus edulis (L.) in relation to shell movements under aerobic conditions. Proc. 9th Eur. Mar. Biol. Syrup. (Edited by Barnes H.), pp. 139 149. Aberdeen University Press, Aberdeen. Zurburg W. and Ebberink R. H. M. (1981)The anaerobic energy demand of Mytilus edulis. Organ specific differences in ATP-supplying processes and metabolic routes. Molec. Physiol. l, 153 164. Zwaan A. de and Holwerda D. A. (1972) The effect of phosphoenolpyruvate, fructose 1,6-diphosphate and pH on allosteric pyruvate kinase in muscle tissue of the bivalve Mytilus edulis L. Biochim. biophys. Acta 276, 430 433.