Pacific Science (1982), vol. 36, no. 3 © 1983 by the University of Hawaii Press. All rights reserved

Some Catalytic and Regulatory Properties of Pyruvate from the Spadix and Retractor Muscles of pompilius1

JEREMY H. A. FIELDS 2

ABSTRACT: was partially purified from the spadix and retrac­ tor muscles of Nautilus pompilius. In both cases, the was activated by

magnesium and potassium ions with similar affinities (apparent Ka values were 0.63 ± 0.04 mM and 5.8 ± 0.4 mM, respectively, for the enzyme from the spadix; and 0.77 ± 0.06 mM and 6.7 ± 0.8 mM, respectively, for the enzyme from the retractor muscle). The showed normal hyperbolic saturation kinetics for the substrates adenosine Y-diphosphate and phosphoenolpyruvate, and the apparent Km values were identical when measured at saturating concen­ trations of the cosubstrate (apparent Km values were 0.28 ± 0.01 mM and 0.063 ± 0.005 mM, respectively, for the spadix). Adenosine 5'-triphosphate, alanine, and citrate were found to be inhibitors. The enzyme from the spadix was more susceptible to inhibition by alanine than that from the retractor muscle. For the latter enzyme, inhibition by alanine was noncompetitive with respect to phosphoenolpyruvate, but the inhibition was nonlinear; it also decreased the affinity for Mg2+. For the enzyme from the spadix, inhibition by alanine changed the saturation kinetics for phosphoenolpyruvate to sigmoidal form. The affinity for Mg2+ was also decreased by alanine. For both enzymes, fructose-I, 6-bisphosphate at a concentration of 0.05 mM partially reversed the inhibition by alanine, but not that by adenosine Y-triphosphate. The sigmoidal kinetics observed for phosphoenolpyruvate could also be reversed by increasing the concentration of Mg2 +. In general, the properties were found to be similar to those of other pyruvate from the muscle of squid and octopus, except for the observation of inhibition by alanine. These regulatory properties are discussed with respect to potential control of glycolytic flux during muscle activity.

CEPHALOPODS ARE SOME OF the most active 1981; Hochachka et al. 1975). Anaerobic or marine invertebrates but are among the least hypoxic conditions lead to the accumulation studied at a biochemical level. Recently, of octopine from the glycogen reserves several workers have shown that energy meta­ (Greishaber and Gade 1976; Hochachka, bolism in the mantle muscle is based on the Fields, and Hartline 1977) because octopine catabolism of carbohydrates rather than on dehydrogenase (EC 1.5.1.11) is used to main­ the oxidation of fat, since enzymes of the tain redox balance in the cytosol (Ballantyne glycolytic pathways are found in high activity et al. 1981; Fields, Baldwin, and Hochachka whereas those of fatty acid oxidation are 1976; Gade 1980; Robin and van Thoai 1961). low (Ballantyne, Hochachka, and Mommsen Flux through the glycolytic pathway is con­ trolled at the level of (EC 2.7.1.11) and pyruvate kinase (EC 1 This research was conducted as part of the Alpha 2.7.1.40) (Scrutton and Utter 1968; William­ Helix Expedition to the Republic of the son 1965, 1966). A wide range of metabolites Philippines, supported by National Science Foundation grant PCM 77-16269 to J. Arnold. have been shown to affect phosphofructo­ 2 University of Washington, Department of Zoology, kinase (Mansour 1972, Newsholme 1971, Seattle, Washington 98195. Tornheim and Lowenstein 1976), and the 315

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physiological implications of these have been than that of pyruvate kinase (Fields et al. discussed by Newsholme and Start (1973). 1976, Hochachka et al. 1975). Activities of Pyruvate kinase from the muscle of most citrate synthetase in the spadix and retractor mammals is inhibited by adenosine 5'­ muscle suggest that the retractor muscle has a triphosphate (ATP) (Cardenas and Oyson higher aerobic capacity than does the spadix, 1973, Tanaka et al. 1967), but on the other but the activities of octopine dehydrogenase hand, the enzyme from the heart of the turtle, and kinase are comparable in these the muscle of some fish, and some bivalve tissues (Hochachka et al. 1978). The spadix mollusks is inhibited by alanine and activated muscle can function under hypoxic conditions by fructose-I, 6-bisphosphate (FOP) (Ban­ that lead to the accumulation of octopine nister and Anastasi 1976, Fields et al. 1978, (Hochachka et al. 1977), indicating a high Holwerda and de Zwaan 1973, Mustafa and capacity for anaerobic glycolysis. The high Hochachka 1971, Randall and Anderson activity of pyruvate kinase in these tissues 1975, Somero and Hochachka 1968, Storey suggested that the enzyme from the spadix of and Hochachka 1974). These properties may Nautilus pompilius might have some catalytic be correlated with the necessity to function and regulatory features that differ from those under prolonged hypoxia (Fields et al. 1978, exhibited by pyruvate kinase from the mantle Hochachka and Somero 1973). Few studies muscle of other . This study was have been performed on the control ofglycol­ undertaken to determine whether the catalytic ysis in cephalopods. Storey and Storey (1978) and regulatory properties of pyruvate kinase examined the changes on the concentrations in the retractor and spadix muscles ofNautilus of glycolytic intermediates in the mantle are comparable to those reported for other muscle of Loligo pealeii, and concluded that cephalopod muscles. The data indicated that phosphofructokinase was rate-limiting. The the affinities for the substrates were similar catalytic properties of phosphofructokinase and comparable to other cephalopods. Unlike from the mantle ofthe squid Symplectoteuthis other cephalopods, the enzymes from the oualaniensis and the cuttlefish Sepia officinalis spadix and retractor muscles were inhibited have been determined, and the results indicate by alanine, this inhibition being partially re­ that the regulatory properties are similar versed by FOP. Also, the enzyme from the to those reported for the enzyme isolated spadix was more susceptible to alanine in­ from the muscles of a variety of animals hibition than was that from the retractor (Storey 1981, Storey and Hochachka 1975a). muscle. Pyruvate kinase has been studied in the muscle of three cephalopods, Sym­ MATERIALS AND METHODS plectoteuthis oualaniensis, Octopus cyanea, and Sepia officinalis. The enzymes were Chambered nautilus were obtained from local fishermen in the Tanon Strait, Republic shown to have similar K m (Michaelis con­ stant) values for adenosine 5'-diphosphate of the Philippines, and kept in running sea­ (AOP) and phosphoenolpyruvate (PEP), and water on board the RjV Alpha Helix. The to be inhibited by ATP and citrate (Guderley spadix muscle was dissected from freshly killed animals, frozen, flown to Seattle, and et al. 1976, Storey 1981, Storey and D Hochachka 1975b). The pyruvate kinase from kept frozen at - 20 C until required. All Sepia officinalis was also activated by FOP biochemicals were purchased from Sigma (Storey 1981). Pyruvate kinase occurs in high Chemical Co., St. Louis, Missouri; all other activity in the spadix and retractor muscles of chemicals were of reagent grade. the chambered nautilus, being approximately Enzyme Assay equal to the activity of octopine dehydro­ genase (Hochachka, French, and Meredith Pyruvate kinase catalyzes the reaction 1978). This is surprising since in Symplecto­ teuthis oualaniensis and O. cyanea the activity Phosphoenolpyruvate + AOP -+ of octopine dehydrogenase is much higher Pyruvate + ATP

.".. ,~.",,', .... ' .. ,"'>. ,., ...... 'r.~'''''.,·~".,..,.,,.''',,.,,.-.,,'',,.,.,''"'r>,'''.,.. ,,, .• ~.,,,..,, , .... ",. , .... "". , 1L~ oJ ~" Pyruvate Kinase in Nautilus-FIELDS 317

It was assayed by the method of Bucher and void volume. The other elutes approximately Pfleiderer (1955). Pyruvate formation was fol­ midway through the gradient. The tubes con­ lowed by coupling with lactate dehydrogenase taining peak activity of the enzyme were and measuring the rate of oxidation of re­ pooled, dialyzed against 20 mM potassium duced nicotinamide adenine dinucleotide phosphate, pH 7.0, containing 10 mM 2­

(NADH) by decrease in A34o ' All assays were mercaptoethanol, and this partially purified performed at 25°C with a Varian Superscan preparation was used for all kinetic studies. IBE spectrophotometer equipped with a ther­ No significant difference in the catalytic pro­ mostated cell holder. The temperature was perties of the two peaks of pyruvate kinase maintained by a Haake FK circulating water was found, and all the data presented are from bath. All assays were performed at 25°C. the larger peak. The specific activities ofthese Standard assay conditions were 4 mM adeno­ preparations were 30 units/mg protein for the sine 5'-diphosphate (ADP), 2 mM phospho­ enzyme from the spadix and 12 units/mg pro­

enolpyruvate (PEP), 10 mM MgS04 , 100 mM tein for the enzyme from the retractor muscle. KCI, 0.2 mM NADH, and 5 units of beef 100 mM heart lactate dehydrogenase in Kinetic Analysis imidazole-HCI, pH 7.0.

The Km values were estimated by the method ofWilkinson (1961) or by plotting log Protein Assay [v/(V - v)] versus log S (where v is the initial Protein concentration was determined by velocity, Vis the maximal velocity, and S is the measuring the optical density at 260 and 280 concentration of ). The effects of in­ nm (Layne 1957). hibitors were determined by means ofdouble reciprocal plots; inhibition constants were by the method Enzyme Preparation calculated from these and also of Dixon (1953). All kinetic constants were Small portions of the muscle were thawed determined at least twice. and weighed. They were homogenized for 3 min in a Sorval Omnimixer set at maximum speed in 5 vol ice-cold 100 mM Tris-HCl RESULTS AND DISCUSSION buffer, pH 7.5, containing 10 mM 2-mercap­ toethanol. The homogenate was centrifuged Effects ofpH, Cation Requirements, and 20 min, and the pellet at 30,000 x g for Substrate Affinities was discarded. Ammonium sulfate was slowly added to 40% saturation, and the solution was The pH profile for the enzyme showed an stirred for 1 hr at O°C. The suspension was optimum between pH 6.8 and 7.2 when as­ centrifuged as above, and the pellet was dis­ sayed with the standard concentrations of carded. The supernatant was further treated substrates and cofactors. This is similar to the with ammonium sulfate to 60% saturation as pH optimum of pyruvate kinase from other above, then centrifuged, and the supernatant cephalopods (Guderley et al. 1976, Storey was discarded. The pellet was dissolved in 20 and Hochachka 1975b), but lower than that mM potassium phosphate, pH 6.5, containing of bivalves (Livingstone and Bayne 1974, 10 mM 2-mercaptoethanol, dialyzed against I Mustafa and Hochachka 1971, de Zwaan and liter of the same buffer, and then applied to a Holwerda 1972). 5 x 1.5-cm column of phosphocellulose that All previously studied pyruvate kinases had been previously equilibrated with the have an absolute requirement for a divalent same buffer. The enzyme was eluted with a cation that can be met by Mg2+ or Mn2 +, and linear gradient from 0 to 300 mM KCI in 20 they are also activated by K + or NH~ (Kayne mM potassium phosphate, pH 6.5. Itelutes as 1973). Pyruvate kinase from the retractor and two peaks, one of which represented about spadix adhered to this pattern, with satura­ 20% of the total activity and is found in the tion kinetics of K + and Mg2 + following

• ,_ "" " , _.. • ~ ~. J..' ,.~, '''' 318 PACIFIC SCIENCE, Volume 36, July 1982 hyperbolic patterns. The apparent Ka (activa­ tion constant) values for K + were 5.8 ± ~45 D 0.4 mM and 6.7 ± 0.8 mM for the spadix and 2 -0 retractor, respectively; the values for Mg + L 0. o Ol were 0.63 ± 0.04 mM and 0.77 ± 0.06 mM, E respectively. The enzyme was also activated ~ 30 E by Mn2 +, but the initial velocities were about 2 ,,' one-third those obtained with Mg2+, so Mn + (5 E was not studied further. These results are very -3 similar to those obtained from other cephalo­ C\j'::: 15 o pods and other organisms (Guderley et al. 1976; Kayne 1973; Munday, Giles, and Poat 1980; Storey and Hochachka 1975b). The enzymes showed normal Michaelis­ Menten saturation kinetics for ADP and phosphoenolpyruvate. The enzyme from the -20 -10 o 10 20 1/lPEP](mtv1-1) retractor muscle had Km values for phosphoe­ nolpyruvate and ADP of 0.06 0.006 mM ± FIGURE I. Double reciprocal plot showing the pattern and 0.28 ± 0.01 mM, respectively. These did of inhibition by ATP with respect to phosphoenol­ not change when the concentration of the pyruvate (PEP) for pyruvate kinase from the spadix. cosubstrate was altered; a similar situation LEGEND: L>, 0 mM MgATP; .. , 2.5 mM MgATP; 0,5.0 is found in pyruvate kinase from the muscle mM MgATP. OTHER CONDITIONS: 2 mM ADP, 10 mM , MgS04 100 mM KCI, 100 mM imidazole HCI, pH 7.0, of other cephalopods (Guderley et al. 1976, 25°C. Storey 1981, Storey and Hochachka 1975b). In contrast to this, the apparent Km values 300 for phosphoenolpyruvate and ADP ofthe en­ zyme from the spadix did change as the con­ c centration of the cosubstrate was altered; Q) therefore, the K values were calculated from -0 m 2i 22·5 replots of the initial velocity data (Cleland OJ 1971). These were 0.074 mM (range 0.065­ E 0.09, five determinations) for phosphoenol­ c pyruvate and 0.33 mM (range 0.27-0.41, five 'E determinations) for ADP. These results sug­ o 15.0 gest that the reaction mechanism for the E 2- pyruvate kinase from the spadix is different > from the random order binding found in the 1; enzyme from mammalian muscles (Kayne 1973). The data are inadequate to unequivo­ 7-5 cally determine the reaction mechanism for the spadix enzyme. On the other hand, the data are equivocal for the retractor enzyme, which mayor may not have a random binding mechanism. For both enzymes, the Km values -4 -2 2 4 6 8 10 for ADP are similar to those reported for 1I1ADP] (mM-1) Sepia officinalis, Symplectoteuthis oualanien­ sis, and Octopus cyanea, but the apparent Km FIGURE 2. Double reciprocal plot showing the pattern for phosphoenolpyruvate is lower than that of inhibition by ATP with respect to ADP for pyruvate found in Symplectoteuthis oualaniensis (0.15 kinase from the spadix. LEGEND: L>, 0 mM MgATP; .., 2.5 mM MgATP; D, 5.0 mM MgATP. OTHER mM) and O. cyanea (0.25 mM) (Guderley et CONDITIONS: 1.0 mM phosphoenolpyruvate, 10 mM > al. 1976, Storey and Hochachka 1975b), re­ MgS04 100 mM KCI, 100 mM imidazole HCl, pH 7.0, sembling the values reported for pyruvate 25°C. Pyruvate Kinase in Nautilus-FIELDS 319

kinase from the mantle muscle of Sepia 100 officinalis (0.07 mM) (Storey 1981) and from the adductor muscle of bivalves (Holwerda and de Zwaan 1973, Mustafa and Hochachka 75

1971, de Zwaan and Holwerda 1972). >, +-' > 50 +-' Regulation by Metabolites u eu Like other pyruvate kinases, the enzyme ~ ° 25 was inhibited by MgATP and citrate. Phos­ phoarginine at a concentration of20 mM had no effect on the enzymes, unlike pyruvate kinase from the mantle ofOctopus cyanea and 20 40 60 Sepia officinalis (Guderley et al. 1976, Storey [alanine] (mM) 1981). In the case of the spadix enzyme, in­ hibition by ATP was noncompetitive with re­ FIGURE 4. Effect ofalanine on the activity ofpyruvate spect to ADP and competitive with respect to kinase from the retractor muscle at varying concentration of MgS0 . LEGEND: ", 5 mM MgS0 ; .. , 10 mM phosphoenolpyruvate (Figures 1, 2), the ap­ 4 4 MgS04 ; 0, 15 mM MgS04 . OTHER CONDITIONS: parent K j (inhibition constant) value being 0.4 mM phosphoenolpyruvate, 2.0 mM ADP, 100 mM 1.5 mM (conditions: 100 mM KCl, 10 mM KCI, 100 mM imidazole HCI, pH 7.0, 25°C. MgS04 , 2 mM ADP). In the case ofthe retrac­ tor enzyme, the pattern of inhibition by ATP .rc 312 was mixed noncompetitive with respect to .~ both phosphoenolpyruvate and ADP, the ap­ 0 '- 26.0 0. parent K j value being 3 mM (conditions: 100 OJ mM KCl, 10 mM MgS04 , 2 mM ADP). The E 20·8 inhibition by citrate was noncompetitive with "c respect to both substrates, but the apparent K E 156 j (5 value was found to be between 20 and 25 mM E .3 1004 > 100 5.2

75 0·5 10 15 2·0 25 3·0 35 40 [ADP] (mt'v1J >, ...... > FIGURE 5. Effect of alanine on the saturation kinetics ...... u 50 of ADP for pyruvate kinase from the spadix muscle. ru LEGEND: 0, 0 mM alanine; .. , 10 mM alanine; ", 20 mM alanine. OTHER CONDITIONS: 1.0 mM phosphoenol­

pyruvate, 10 mM MgS04 , 100 mM KCI, 100 mM imi­ 25 dazole HCI, pH 7.0, 25°C.

for both enzymes; therefore, it is unlikely that this has any physiological relevance, and it 5 10 15 20 was not studied further. Both enzymes were [alanine] (mM) inhibited by alanine (Figures 3, 4), with the spadix enzyme being inhibited to a greater FIGURE 3. Effect ofalanine on the activity ofpyruvate extent. It was found that the degree of in­ kinase from the spadix at varying concentrations of hibition by alanine was changed by altering MgS04 · LEGEND: ", 5 mM MgS04 ; .. , 10 mM MgS04 ; 2 0,15 mM MgS04 . OTHER CONDITIONS: 0.4 mM phos­ the concentration of Mg +; inhibition was phoenolpyruvate, 2.0 mM ADP, 100 mM KCI, 100 mM more pronounced at lower concentrations imidazole HCI, pH 7.0, 25°C. and more reduced at higher concentrations

" "'... ,",r ~,~, ,,,--..,,,, '" ,., ff"' 'r-r , u-·.. . "" . "r' ,.,.. -, , ". ,_~".. ... """__ ~ " 320 PACIFIC SCIENCE, Volume 36, July 1982

31-5 ______0 o ,....,270 A---~------~ 1: ! '(j) -022·5 .~:/~!-----­ L / Q. 0/ / Ol ~., ~ E 18·0 Ii .,. I. I'/ .~ 13·5 ~ /;:/ o ~ A / ~ jt! > /1 At:::!

AL:.'/

"""-- --'- --'-- L-___~ 1-0 2·0 3·0 40 [PEP] (mM)

FIGURE 6. Effect ofalanine on the saturation kinetics of phosphoenolpyruvate (PEP) for pyruvate kinase from the spadix muscle. LEGEND: 0, 0 mM alanine; .. , 2.5 mM alanine; ", 5.0 mM alanine; ., 10 mM alanine + 0.05 mM FDP.

OTHER CONDITIONS: 2.0 mM ADP, 5 mM MgS04 , 100 mM KCI, 100 mM imidazole HCl, pH 7.0, 25°C.

(Figures 3, 4). In the presence of alanine, the affinity for Mg2+ for both enzymes. The retractor muscle pyruvate kinase displayed enzyme from the retractor muscle retained normal saturation kinetics for phosphoenol­ normal hyperbolic saturation kinetics in the pyruvate, but the saturation kinetics for ADP presence of alanine, but the enzyme from the were anomalous with apparent substrate in­ spadix had sigmoidal saturation kinetics in hibition at high concentrations of ADP. The the presence of alanine (Figure 7). When the spadix enzyme showed similar saturation data for inhibition by alanine were plotted kinetics for ADP in the presence of alanine, according to Dixon (1953), it was found that but the saturation kinetics for phosphoenol­ the inhibition was nonlinear; therefore, no in­ pyruvate were changed to a sigmoidal pattern hibition constants for alanine were obtained (Figures 5, 6). Alanine decreased the apparent for the enzymes. Fructose-I, 6-bisphosphate affinity for phosphoenolpyruvate of both en­ partially reverses the inhibition by alanine zymes; 50 mM alanine increases the apparent (Tables I, 2), and in the case of the spadix Km about fourfold in the case of the retractor enzyme, it changes the saturation curves for enzyme, and 10 mM alanine increases the ap­ Mg2+ and phosphoenolpyruvate to hyper­ parent SO.5 about tenfold in the case of the bolic form (Figures 6, 7). In the presence of spadix enzyme (Table I). Adenosine 5'­ 40 mM alanine the apparent Ka for FDP was diphosphate has been shown to chelate Mg2+ 0.002-0.003 mM for the spadix enzyme (con­ (Cumme, Horn, and Achilles 1973); therefore, ditions: 2 mM ADP, 0.4 mM phosphoenol­ it is possible that the apparent inhibition by pyruvate, 100 mM KCI, 10 mM MgS04 ), and high concentrations of ADP was due to a the effect was maximal at a concentration of combination of chelation of free Mg2+ by 0.05 mM. In the absence ofalanine, FDP had ADP and a decrease in the affinity of the no effect on the enzymes, unlike the pyruvate enzyme for Mg2+. As shown in Table 2, kinase from Sepia officinalis (Storey 1981). alanine profoundly decreases the apparent The regulation of pyruvate kinase by Pyruvate Kinase in Nautilus-FIELDS 321

TABLE I

EFFECTS OF ALANINE AND FDP ON THE AFFINITY FOR PHOSPHOENOLPYRUVATE OF PYRUVATE KINASE FROM THE SPADIX AND RETRACTOR MUSCLES

RETRACTOR SPADIX (Km , mM ± SE) (SO.5' mM) h* Control 0.060 ± 0.006 0.063 1.0 0.05 mM FDP 0.057 ± 0.009 0.061 1.0 50 mM alanine 0.28 ± 0.02 50 mM alanine + 0.05 mM FDP 0.046 ± 0.005 5 mM alanine 0.40 1.6 10 mM alanine 0.49 1.6 10 mM alanine 0.05 mM FDP + 0.066 1.0

CONDITIONS: 2.0 mM ADP, 10 mM MgSO., 100 mM KCI, 100 mM imidazole HCI, pH 7.0, 250C. *Hill constant.

TABLE 2

EFFECTS OF ALANINE AND FDP ON THE AFFINITY FOR Mg2+ OF PYRUVATE KINASE FROM THE SPADIX AND RETRACTOR MUSCLES

RETRACTOR SPADIX (Km , ml\1 ± SE) (SO.5, mM) h* Control 0.77 ± 0.06 0.63 1.0 0.05 mM FDP 0.80 ± 0.08 0.62 1.0 50 mM alanine 3.72 ± 0.17 50 mM alanine + 0.05 mM FDP 0.69 ± 0.08 5 mM alanine 5.5 1.7 10 mM alanine 7.8 I.7 10 mM alanine + 0.05 mM FDP 0.78 1.0

CONDITIONS: 2.0 mM ADP, 1.0 mM phosphoenolpyruvate, 100 mM KCI, 100 mM imidazole HCI, pH 7.0, 250C. *Hill conslant.

alanine and FDP is typical of the enzyme vate kinase has been proposed in the case of found in vertebrate liver (Cardenas and bivalve mollusks. These organisms produce Dyson 1973, Kayne 1973, Seubert and alanine, succinate, and volatile fatty acids Schoner 1971, Tanaka et aL 1967), the hepa­ during anoxia, and it has been suggested that topancreas of octopus or crustacea (Giles, alanine inhibits pyruvate kinase after the early Poat, and Munday 1976, 1977; Guderley et aL stage of anoxia, channelling phosphoenol­ 1976), and the mantle tissue of bivalves pyruvate toward succinate (Hochachka and (Livingstone and Bayne 1974, Mustafa and Mustafa, 1972; Hochachka, Fields, and Hochachka 1971). In these tissues, inhibition Mustafa 1973; de Zwaan, Kluytmans, and by alanine is considered to playa role in pre­ Zandee 1976). The available evidence indi­ venting futile cycling at this level during cates that octopine is the major end gluconeogenesis, There is no evidence as yet of glycolysis in the spadix (Hochachka et al. that the muscles of bivalves or cephalopods 1977) and retractor muscle (Hochachka, un­ have any capability for gluconeogenesis as published observations). This does not re­ has been recently shown for the skeletal quire any special regulatory properties of muscles of humans (Hermansen and Vaage pyruvate kinase, least of all inhibition of 1977), although this possibility cannot be dis­ the enzyme, so another explanation must be counted as an explanation for the regulatory sought. A third possibility is that this method properties ofpyruvate kinase from the spadix. of regulating pyruvate kinase makes it very Another role for alanine inhibition of pyru- responsive to changes in glycolytic flux 322 PACIFIC SCIENCE, Volume 36, July 1982

329 the turtle (Storey and Hochachka 1974), and .. various fishes (Bannister and Anastasi 1976, .~ 28-2 QJ Fields et al. 1978, Randall and Anderson ..... o 1975). The turtle and the porpoise are capable 2i. 235 of withstanding prolonged anoxic periods in OJ E a dive (Hochachka and Storey 1975), but it is ., 188 c uncertain whether all the fish studied could E be exposed to prolonged hypoxic stresses or a 14·1 short bursts of high activity dependent on E anaerobic metabolism. In all these cases, the .3 904 > thesis of Munday et al. (1980) is a potential explanation of the phenomenon of alanine 47 inhibition of muscle pyruvate kinase. The effect of alanine on the affinity of the 4 8 12 16 20 24 28 enzyme for Mg2+ is intriguing, but little can [MgS0 ] (mM) be said about the physiological relevance of 4 this phenomenon. The concentration of free FIGURE 7. Etfect ofalanine and FDPon the saturation Mg2 + in the spadix is unknown; in frog sar­ kinetics of MgS04 for pyruvate kinase from the spadix torius and gastrocnemius muscle the free muscle. LEGEND: 0, 0 mM alanine, or 0 mM alanine + 0.05 mM FDP; "', 5 mM alanine; ., 10 mM Mg2+ has been estimated to be about 0.6 mM alanine; ., 10 mM alanine + 0.05 mM FDP. OTHER (Gupta and Moore 1980), and there is no CONDITIONS: 2.0 mM ADP, 1.0 mM phosphoenolpyru­ evidence as yet that the concentration of free vate, 100 mM KCl, 100 mM imidazole HCl, pH 7.0, 25°C. Mg2 + varies as physiological status is changed (Gupta and Moore 1980). Hence, it is uncer­ mediated by changes in the concentration of tain whether the pyruvate kinase from the FDP (Munday et al. 1980). In this context, the spadix or retractor muscle is regulated directly amount of alanine would change little and by changes in the concentration ofMg2+. For provide a constant background oflowenzyme complete comprehension of the variations of activity. Any increase in the concentration of glycolytic control in cephalopods, further FDP that might occur when glycolytic flux studies on the energy metabolism of the increases (Williamson 1966) would activate muscle will be necessary. pyruvate kinase, bringing about a close couple between phosphofructokinase and pyruvate kinase (Hochachka and Somero 1973). In this ACKNOWLEDGMENTS context, it is interesting to note that the activity of citrate synthetase is higher in the The help of the crew of the Alpha Helix is retractor muscle than in the spadix, and the deeply appreciated. retractor also has a higher mitochondrial con­ tent than the spadix (Hochachka et al. 1978). This suggests that the retractor muscle may LITERATURE CITED have a greater capacity for aerobic metabo­ lism, and that the spadix may rely extensively BALLANTYNE, J. S., P. W. HOCHACHKA, and on anaerobic metabolism during activity. T. P. MOMMSEN. 1981. Studies on the Thus, the spadix may experience a greater metabolism of migratory squid, Loligo change in glycolytic flux than the retractor opalescens: Enzymes of tissues and heart muscle, and this may be correlated with the mitochondria. Mar. BioI. Lett. 2: 75-85. increased sensitivity to alanine. This may be a BANNISTER, J. Y., and A. ANASTASI. 1976. The partial explanation of the occurrence of the effect of fructose-l,6-diphosphate on phenomenon ofalanine inhibition ofpyruvate pyruvate kinase from white muscle of three kinase in the muscle of some vertebrates such species of Mediterranean fish. Compo as the porpoise (Hochachka and Storey 1975), Biochem. Physiol. 55B: 449-451.

~ • ..., r '-~ ,~ ~. ,,,.. ,.,...., • • , "'" • Pyruvate Kinase in Nautilus-FIELDS 323

BUCHER, T, and G. PFLEIDERER. 1955. GUPTA, R K., and R D. MOORE. 1980. P Pyruvate kinase from muscle. Meth. NMR studies of intracellular free Mg in Enzymol. 1:435-441. intact frog skeletal muscle. J. BioI. Chern. CARDENAS, J. M., and R. D. DYSON. 1973. 255: 3987-3993. Bovine pyruvate kinases. II. Purification of HERMANSEN, L., and O. VAAGE. 1977. Lactate the liver isozyme and its hybridization with disappearance and glycogen synthesis in skeletal muscle pyruvate kinase. J. BioI. human muscle after maximal exercise. Chern. 248: 6398-6944. Amer. J. Physiol. 233: E422-E429. CLELAND, W. W. 1971. Steady state kinetics. HOCHACHKA, P. W., and T MUSTAFA. 1972. Pages 1-65 in P. D. Boyer, ed. The en­ Invertebrate facultative anaerobiosis. Sci­ zymes. 3rd Ed. Vol. II. Academic Press, ence 178: 1056-1060. New York. HOCHACHKA, P. W., and G. N. SOMERO. 1973. CUM ME, G. A., A HORN, and W. ACHILLES. Strategies ofbiochemical adaptation. W. B. 1973. Metal complex formation and its im­ Saunders, Philadelphia. portance for enzyme regulation. Acta BioI. HOCHACHKA, P. W., and K. B. STOREY. 1975. Med. Germ. 31 :349-364. Metabolic consequences of diving in ani­ DIXON, M. 1953. The determination of mals and man. Science 187:613-621. constants. Biochem. J. HOCHACHKA, P. W., J. H. A. FIELDS, and P. H. 55:170-171. HARTLINE. 1977. Octopine as an end prod­ FIELDS, J. H. A, J. BALDWIN, and P. W. uct of anaerobic glycolysis in the cham­ HOCHACHKA. 1976. On the role of octopine bered nautilus. Science 195: 72-74. dehydrogenase in cephalopod mantle HOCHACHKA, P. W., J. FIELDS, and T Mus­ muscle metabolism. Can. J. Zool. 54: 871­ TAFA. 1973. Animal life without oxygen: 878. Basic biochemical mechanisms. Amer. FIELDS, J. H. A, W. R. DRIEDZIC, C. J. Zool. 13: 543-555. FRENCH, and P. W. HOCHACHKA. 1978. HOCHACHKA, P. W., C. J. FRENCH, and J. Some kinetic properties of skeletal muscle MEREDITH. 1978. Metabolic and ultrastruc­ pyruvate kinase from air-breathing and tural organization in Nautilus muscles. J. water-breathingfish ofthe Amazon. Can. J. Exp. Zool. 205: 51-62. Zool. 56:751-758. HOCHACHKA, P. W., T W. MOON, T. Mus­ GADE, G. 1980. Biological role of octopine TAFA, and K. B. STOREY. 1975. Metabolic formation in marine molluscs. Mar. BioI. sources ofpower for mantle muscle ofa fast Lett. 1: 121-135. swimming squid. Compo Biochem. Physiol. GILES, I. G., P. C. POAT, and K. A MUNDAY. 52B: 151-158. 1976. The regulation of pyruvate kinase in HOLWERDA, D. A., and A. DE ZWAAN. 1973. the of Carcinus maenas. Kinetic characteristics of allosteric pyru­ Compo Biochem. Physiol. 55B: 423-427. vate kinase from muscle tissue of the sea ---. 1977. An investigation ofthe interac­ mussel Mytilus edulis L. Biochem. Biophys. tions of the allostric modifiers of pyruvate Acta 309: 296-306. kinase with the enzyme from Carcinus KAYNE, F. J. 1973. Pyruvate kinase. Pages maenas hepatopancreas. Biochem. J. 165: 353-382 in P. D. Boyer, ed. The enzymes. 97-105. 3rd Ed. Vol. VIII. Academic Press, New GREISHABER, M., and G. GADE. 1976. The York. biological role of octopine in the squid LAYNE, E. 1957. Spectrophotometric and tur­ Loligo vulgaris (Lamarck). J. Compo Phy­ bidimetric methods for measuring proteins. siol. 108: 225-232. Meth. Enzymol. 3: 447-454. GUDERLEY, H. E., K. B. STOREY, J. H. A LIVINGSTONE, D. R, and B. L. BAYNE. 1974. FIELDS, and P. W. HOCHACHKA. 1976. Cata­ Pyruvate kinase from the mantle tissue of lytic and regulatory properties of pyruvate Mytilus edulis L. Compo Biochem. Physiol. kinase isozymes from octopus mantle 48B: 481-497. muscle and liver. Can. J. Zool. 54: 863-870. MANSOUR, T 1972. Phosphofructokinase. 324 PACIFIC SCIENCE, Volume 36, July 1982

Curro Top. Cel!. Regu!. 5: 1-45. ---. 1975a. Redox regulation of muscle MUNDAY, K. A, I. G. GILES, and P. C. POAT. phosphofructokinase in a fast swimming 1980. Review of the comparative biochem­ squid. Compo Biochem. Physio!. 52B: 159­ istry of pyruvate kinase. Compo Biochem. 163. Physio!. 67B: 403-431. ---. 1975b. Squid muscle pyruvate kinase: MUSTAFA, T., and P. W. HOCHACHKA. 1971. Control properties in a tissue with an active Catalytic and regulatory properties of py­ a-GP cycle. Compo Biochem. Physio!. 52B: ruvate kinase in tissues ofa marine bivalve. 187-191. J. Bio!. Chern. 246: 3196-3203. STOREY, K. B., and J. M. STOREY. 1978. NEWSHOLME, E. A. 1971. The regulation of Energy metabolism in the mantle muscle of phosphofructokinase in muscle. Cardio!. the squid, Loligo pealeii. J. Compo Physio!. 56:22-34. 123:169-175. NEWSHOLME, E. A., and C. START. 1973. TANAKA, T., Y HARANO, F. SUE, and H. Regulation in metabolism. Wiley, New MORIMURA. 1967. Crystallization, charac­ York and London. terization and metabolic regulation of two RANDALL, R. F., and P. J. ANDERSON. 1975. types of pyruvate kinase isolated from rat Purification of properties of pyruvate tissues. J. Biochem. (Tokyo) 62: 71-91. kinase of sturgeon muscle. Biochem. J. TORNHEIM, K., and J. M. LOWENSTEIN. 1976. 145: 569-573. Control of phosphofructokinase from rat ROBIN, Y, and N. VAN THOA!. 1961. Metabo­ skeletal muscle: Effects of fructose diphos­ lisme des derives guanidyles. X. Metabo­ phate, AMP, ATP, and citrate. J. Bio!. lisme de l'octopine: Son role biologique. Chern. 251: 7322-7328. Biochem. Biophys. Acta 52: 233-240. WILKINSON, G. N. 1961. Statistical esti­ SCRUTTON, M. c., and M. F. UTTER. 1968. mations in . Biochem. J. The regulation of glycolysis and gluco­ 80: 324-332. neogenesis in animal tissues. Ann. Rev. WILLIAMSON, J. R. 1965. Glycolytic control Biochem. 37: 249-302. mechanisms: Inhibition ofglycolysis by ac­ SEUBERT, W., and W. SCHONER. 1971. The etate and pyruvate in isolated perfused rat regulation of pyruvate kinase. Curr. Top. heart. J. Bio!. Chern. 240: 2308-2321. Cell. Regu!. 3: 237-267. ---. 1966. Glycolytic control mech­ SOMERO, G. N., and P. W. HOCHACHKA. 1968. anisms. II. Kinetics ofintermediate changes The effect of temperature on catalytic and during the aerobic-anoxic transition in regulatory functions of pyruvate kinases of perfused rat heart. J. Bio!. Chern. 241: the rainbow trout and the Antarctic fish, 5026-5036. Trematomus bernachii. Biochem. J. 110: DE ZWAAN, A, and D. A HOLWERDA. 1972. 395-400. The effect of phosphoenolpyruvate, fruc­ STOREY, K. B. 1981. Effects of arginine phos­ tose-I,6-diphosphate and pH on allosteric phate and octopine on glycolytic enzymes pyruvate kinase in muscle tissue of the activities from Sepia officinalis mantle bivalve Mytilus edulis L. Biochem. Biophys. muscle. J. Compo Physio!. 142:501-507. Acta 276: 430-433. STOREY, K. B., and P. W. HOCHACHKA. 1974. DE ZWAAN, A., J. H. F. M. KLUYTMANS, and Enzymes of energy metabolism in a ver­ D. I. ZANDEE. 1976. Facultative anaer­ tebrate facultative anaerobe, Pseudemys obiosis in molluscs. Biochem. Soc. Symp. scripta: Turtle heart pyruvate kinase. J. 41: 133-168. Bio!. Chern. 249: 1423-1427.

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