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Kinetic properties of pyruvate kinases from turtle liver and heart
Comp. Biochem. Physiol., 1974, Vol. 49B,pp. 15 to 24. Pergamon Press. Printed in Great Britain
K I N E T I C PROPERTIES OF PYRUVATE KINASES FROM T U R T L E LIVER AND HEART E L I Z A B E T H H. K O R N E C K I - G E R R I T Y and DAVID G. PENNEY Department of Biological Sciences, University of Illinois at Chicago Circle, Chicago, Illinois 60680, U.S.A. (Received 14 September 1973)
Abstract--1. Turtle liver pyruvate kinase (PK), which shows aUosteric properties with respect to phosphoenolpyruvate (PEP), is activated by fructose diphosphate (FDP), glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P) and L-serine. Its activity is strongly inhibited (over 85 per cent) by L-alanine [Kiclpp) = 0-09 mM], L-isoleueine [Klcapp~= 0"19 mM], L-valine [Kjapp~= 0-56 mM] and L-cysteine and over 50 per cent by L-methionine, L-tryptophan and L-phenylalanine. 2. Liver PK is inhibited by ATP and this inhibition is strongly influenced by the concentration of Mg*+ (e.g. 4 mM Mgs+ produces 70 per cent inhibition, whereas 50 mM Mg'+ produces 32 per cent inhibition). 3. Citrate, suecinate, a-ketoglutarate, some L-amino acids and other compounds tested produce little or no effect on liver PK activity. 4. Heart PK does not show an allosterie response with varying PEP concentration. It is not activated by FDP. It was found to be inhibited by Lalanine [Kl(app) -- 0"19 mM], but not by L-valine. Very slight inhibition was observed with L-isoleucine. 5. The pH optima for liver PK activity is in the range of pH 6"6-7"0. INTRODUCTION T A N A K A et al. (1965, 1967) demonstrated at least two types of PK in the rat, types L and M, which predominate in liver and muscle, respectively. Type L shows a sigmoidal relationship of reaction velocity to PEP concentration. On the other hand, type M PK shows typical Michaelis-Menten kinetics with PEP. Where FDP causes activation of the L type by increasing its affinity for PEP, the M type shows no activation with FDP. In previous works we (Kornecki-Gerrity & Penney, 1974; Penney & Kornecki, 1973) have reported that turtle liver PK exhibits sigmoidal kinetics with respect to PEP with a K m ~ w i , J above 2 raM. Upon addition of 0.1 mM FDP, a hyperbolic curve is obtained yielding a K m WEVJ value of 0-2 raM. In the presence of 2 mM L-alanine and L-valine, two very potent inhibitors of the turtle liver enzyme, the Km ~'Ev~ values increase to several millimoles. L-Alanine has been shown to inhibit PK from crude extracts of mammalian liver (Llorente et aL, 1970; Stifel & Herman, 1971 ; Carbonell et aL, 1973). In contrast, L-valine did not produce any significant inhibition in such preparations (Stifel & Herman, 1971). Turtle heart PK shows 15
16
ELIZABETH H. KORNECKI-GERRITYAND DAVID G. PENNEY
hyperbolic kinetics both in the presence and absence of F D P with K m(PEP) values of 0.2 mM. L-Alanine was shown to inhibit the heart PK, whereas L-valine produced no effect. This differs strikingly from the mammalian heart PK which is not inhibited by L-alanine (Carbonell et al., 1973). Therefore, we suggested that in turtle liver and heart (a) two (or more) kinetically different forms of PK are present and (b) that these enzymes differ in several respects from the mammalian liver and heart PK isoenzymes. This study was undertaken in order te further characterize turtle heart and liver PK. MATERIALS AND METHODS Both male and female turtles (Pseudemys scripta elegans) of 500-1000 g body weight were used. These turtles were maintained at 20°C and given slices of beef liver once each week. Liver and heart supernate fractions used for P K studies were prepared according to previously described methods (Penney & Kornecki, 1973).
P K assay Enzyme activities were measured at 20°C by the method of Boyer (1962). The assay mixture contained the following components: imidazole C1- buffer (pH 7"0), 1 0 0 m M ; KC1, 1 0 0 m M ; MgC12, 4 m M ; ADP, 4 m M ; PEP, l m M ; N A D H , 0"16mM; lactate dehydrogenase (LDH), 1 unit per ml; and diluted tissue enzyme preparation. The oxidation of N A D H was followed at 340 n m with a Gilford Model 2400 recording Spectrophotometer. Control cuvettes from which either PEP or ADP were omitted compensated for non-specific oxidation of N A D H .
Studies of effects of various intermediates P K assays carried out in the presence of various intermediates had the same component concentrations as given above, with one exception; the concentration of PEP was 0"5 m M instead of 1 raM. PEP plus the intermediate whose effect was being examined were added to the assay mixture simultaneously. After a 5 rain incubation period, the reaction was started by the addition of ADP. Control cuvettes lacked the intermediate under study. Activities were corrected for any N A D H oxidation which occurred during the incubation period, especially when F D P was added. All intermediates were soluble in imidazole C1buffer at a pH of 7"0. All assay mixtures were found to remain within + 0"1 pH unit of the set p H value during the reaction.
Determination of K t (app) Kt(a~p) indicates the concentration of the inhibitor required to slow the reaction to half the rate it shows in the absence of the inhibitor. To determine Kl¢~pp)the reactions were run at various inhibitor concentrations (0'01-2"5 mM) and at a PEP concentration of 0"5 mM.
Determination of p H optima P K assays were performed at a number of pH values ranging from 5'5 to 8"5. The concentration of PEP in the P K assay mixture was 1 mM. Imidazole C1- buffer was used throughout the entire pH range. The pH of each mixture was measured before and after the reaction and was found to be within + 0"1 pH unit for each pH value tested.
Chemicals and enzymes All chemicals and enzymes [acetylcholine, adenosine-5'-diphosphate, adenosine-5'triphosphate, ammonium chloride, arginine, asparagine, choline, citrate, cysteine, F-6-P, FDP, glucose, glucose-l-phosphate (G-l-P), G-6-P, glutamine, glycine, histidine, imidazole
PK
17
F R O M T U R T L E TISSUES
CI-, isoleucine, ~-ketoglutarate, LDH, leucine, methionine, NADH, OH-proline, phenylalanine, PEP, serine, succinate, threonine, tryptophan, urea and valine] were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. All amino acids used were of the L-series (except glycine). RESULTS Table 1 shows the effects of various amino acids on turtle liver PK activity. The greatest PK inhibition (over 90 per cent) is produced by alanine at 2 mM and isoleucine at 2.5 mM. Valine (2 raM), 2.5 mM cysteine and 2 mM methionine TABLE1--THE EFFECT
O F VARIOUS A M I N O ACIDS O N T U R T L E L I V E R P Y R U V A T E K I N A S E
Treatment [PEP] = 0"5 mM Control L-Alanine L-Isoleucine (2"5 mM) L-Valine L-Cysteine (2,5 mM) L-Methionine L-Tryptophan L-Phenylalanine L-Threonine L-Leucine Glycine
Per cent of control PK activity 100 7 (4-11) 8 (6-10) 15 (9-25) 16 (15-16) 22 (20-23) 45 (42-48) 47 (38-60) 56 (49-60) 57 (44-70) 66 (56-72)
53 4* 6 10 3 3 3 6 2 3 7
All amino acids used at 2 mM, unless otherwise designated. *Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. produce liver PK inhibition of over 75 per cent. Both tryptophan and phenylalanine at 2 mM inhibit liver PK activity over 50 per cent. Threonine, leucine and glycine inhibit liver PK activity over 30 per cent. Figure 1 shows the influence of varying concentrations of alanine, isoleucine, valine and serine at 0.5 mM PEP on liver PK activity before and after the addition of 0.1 mM FDP. In the presence of varying concentrations of alanine (Fig. 1A) a hyperbolic curve is obtained and the apparent K i value is 0.09 mM. The addition of 0-1 mM FDP largely overcomes alanine inhibition. Figure 1B shows the effects of isoleucine on liver PK activity. The curve obtained in the absence of FDP is hyperbolic with an apparent Ki of 0-19 mM. The addition of FDP completely reverses the PK inhibition produced by isoleucine. The effect of varying concentrations of valine on liver PK activity is shown in Fig. 1C. In the absence of FDP, a hyperbolic curve is obtained and the apparent K i value for valine is 0.56 mM. Again, with the addition of FDP, inhibition is largely overcome. Figure 1D shows the influence of serine on PK activity. A sigmoidal increase is obtained with increasing serine concentrations. Upon the addition of FDP, the increase in activity
18
ELIZABETH H . KORNECKI-(~EERITY AND DAVID G . PENNEY
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L-Alanine [mM]
2.5
L-isoleucine [mM]
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200 ^ .L
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L-valine [mM]
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FIG. 1. Effect of various concentrations of L-alanine (A), L-isoleucine (B), L-valine (C) and L-serine (D) on liver pyruvate kinase activity in the presence A - - A - - A and absence O - - O - - O of FDP. The concentration of PEP is 0"5 mM.
is seen to be due solely to " F D P - a c t i v a t i o n " (i.e. there is no added stimulation resulting from serine in the presence of FDP). Table 2 shows the effects of alanine, valine and isoleucine on turtle heart P K activity. Alanine at 2 m M produces over 90 per cent inhibition of heart P K activity giving an apparent K i for alanine of 0-19 mM. In the presence of F D P , alanine TABLE 2 - - T H E EFFECT OF L-ALANINE~ L-VALINE AND L-ISOLEUCINE ON TURTLE HEART PYRUVATE KINASE ACTIVITY
Treatment [PEP] = 0'5 mM Control L-Alanine (2 mM) L-Valine (2 mM) L-Isoleucine (2"5 raM)
Per cent of control PK activity n 100 11 (9-15) 93 (89-96) 75 (73-79)
11 4* 4 3
*Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. inhibition of heart P K activity is reversed (Fig. 2). However, 2 m M valine produces no significant inhibition of heart PK. Isoleucine at 2.5 mM, which produces such strong inhibition of liver P K (Table 1), is seen to have only a very slight inhibitory effect on heart P K activity.
PK FROMTURTLETISSLrI~ 100,~
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FIG. 2. Influence of L-alanine at various concentrations on the activity of heart pyruvate kinase in the presence (A--A--A) and absence ( O - - O - - O ) of FDP. The concentration of PEP is 0'5 raM. A T P (3 m M ) in the presence of 4 m M M g ~+ inhibits liver P K activity over 70 per cent (Table 3). Increasing the concentration of M g 2+ to 12.5 m M reduces inhibition to 38 per cent. A still higher Mg 2+ concentration of 50 m M results in a further decrease in P K inhibition to 32 per cent. TABLE 3--TH~ EF~CT OF ATP AND Mg 2+ ION CONCENTRATIONON TURTLELIVER PYRUVATE
Treatment [PEP] = 0"5 mM
KINASE
ACTIVITY
Per cent of control PK activity
Control 100 ATP (3 mM) + Mg 2+ (4 mM) 29 (22-39) ATP (3 m M ) + M g s+ (12.5 mM) 62 (47-77) ATP (3 mM) + Mg ~+ (50 mM) 68 (63-72)
n 11 4* 5 2
*Values given as mean percentages of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. Table 4 shows the effects of three phosphorylated glycolytic intermediates (FDP, G-6-P and F-6-P) and serine on liver P K activity at a P E P concentration of 0.5 mM. T h e addition of F D P (0.1 raM), G-6-P (2 mM) or F-6-P (0.4 raM) exerts a stimulatory effect on the liver P K by increasing its activity over 50 per cent. Serine at 2-5 m M shows a smaller, but nonetheless significant, stimulatory effect (45 per cent higher than control) on liver P K activity. T h e amino acids asparagine, arginine and glutamine at 2 raM, and histidine and OH-proline at 2"5 m M produced no significant effects on liver P K activity.
20
ELIZABETH H. KORNECKI-GERRITY AND DAVID G. PENNEY
The Krebs cycle intermediates, citrate, succinate and ~-ketoglutarate at 2 m M and the glycolytic intermediate G-1-P (2 mM) and glucose (5 raM) also produced little or no effect on liver P K activity. Other compounds tested which showed little or no effect were urea (2 raM), acetylcholine (10 -2 to 10 -~ raM), choline (1 mM) and ammonium chloride (2 raM). TABLE 4 - - T H E EFFECT OF FRUCTOSE-1,6-DIPHOSPHATE, GLUCOSE-6-PHOSPHATE, FRUCTOSE-6-PHOSPHATE AND L-SERINE ON TURTLE LIVER PYRUVATE KINASE ACTIVITY
Treatment [PEP] -~ 0-5 mM
Per cent of control PK activity
n
100 174 (152-202) 169 156 (138-174) 145 (138-158)
18 8* 2 2 5
Control F-1,6-diP (0"1 mM) G-6-P (2 raM) F-6-P (0"4 raM) L-Serine (2"5 raM)
*Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. The effect of pH on liver P K activity was studied (Fig. 3). Maximal activity was observed at pH 6.6-7.0. There was a 20 per cent decrease in activity when the pH was decreased by 0.6 pH unit (pH 6.0). No P K activity could be detected at pH 5.5. At pH 7.5, the P K activity was decreased by 45 per cent.
100 -
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I
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I
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I
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7.5
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F I a . 3. T h e effect of p H o n the activities of turtle liver p y r u v a t e kinase. E a c h
point is an actual observation. The concentration of PEP is 1 mM.
PK
FROM TURTLE TISSUES
21
DISCUSSION Alanine is a very potent inhibitor of turtle liver PK activity as shown by its apparent K i value of 0.09 raM. Similar results have been found for the unpurified rat liver PK (Llorente et al., 1970; Stifel & Herman, 1971 ; Carbonell et al., 1973), the mammalian liver L type PK (Taylor et al., 1969; Llorente et al., 1970; Costa et al., 1972; Imamura et aL, 1972; Irving & Williams, 1973; Carbonell et al., 1973) and the mammalian liver M S type (or class A) PK (Jim6nez de Asfia et al., 1971 ; Carbonell et al., 1973). With the addition of FDP, alanine inhibition of the turtle liver PK is relieved. This is also observed with the mammalian L type PK. Isoleucine and valine also strongly inhibit turtle liver PK activity as shown by their Kitapp~ values of 0.19 and 0.56 mM, respectively. FDP completely overcomes this inhibition. In contrast, such strong inhibition has not been observed for the unpurified mammalian liver PK or the purified liver L and M S isoenzymes which are only slightly inhibited by relatively high concentrations of L-isoleucine and L-valine (Seubert et al., 1968; Stifel & Herman, 1971; Imamura et al., 1972; Carbonell et al., 1973). Apparently the strong inhibition of turtle liver PK activity produced by these two amino acids at low concentrations is unique. Turtle heart PK is inhibited by alanine, as is the liver PK, with an apparent K i of 0.19 mM. Also, as with the liver enzyme this inhibition by alanine is completely reversible by FDP. However, the activity achieved in the presence of both alanine and FDP is no higher than in the presence or absence of FDP alone (KorneckiGerrity & Penney, 1974), indicating that FDP is not an activator of turtle heart PK. In contrast, Flanders et al. (1971) report that FDP is an activator of frog heart PK. Interestingly, mammalian heart PK extracts are not inhibited by alanine (Carbonell et aL, 1973), while frog heart PK, like turtle heart PK, is also inhibited by L-alanine at a low concentration. Turtle heart PK inhibition by alanine is therefore similar to that of mammalian liver L and M S type PKs (Jim6nez de Asda et al., 1971; Imamura et al., 1972; Balinsky et al., 1973; Carbonell et al., 1973; Irving & Williams, 1973) and also to that of frog heart PK. However, turtle heart PK activity is not significantly inhibited by 2 m M valine or 2.5 m M isoleucine. Likewise, the effect of these amino acids at similar concentrations on the mammalian liver L and M S type PK isoenzymes is insignificant (Carbonell et al., 1973). From the data obtained it appears that turtle heart PK differs considerably from mammalian heart PK, but has some properties in common with the mammalian liver L and M S isoenzymes. In addition to inhibition of turtle liver PK by L-alanine, isoleucine and valine, other amino acids also cause substantial inhibition. For example, cysteine, methionine, tryptophan and phenylalanine inhibit turtle liver PK activity by over 50 per cent, whereas a 30 per cent inhibition occurs with threonine, leucine and glycine. Somewhat different responses from those found here were observed with crude rat liver extracts and purified L-type PK where L-methionine, L-tryptophan, L-phenylalanine, L-threonine, L-leucine and glycine were found not to inhibit significantly at similar concentrations (Seubert et al., 1968; Stifel & Herman, 1971 ; Carbonell et al., 1973). However, the rat liver M 2 PK isoenzyme has been reported
22
ELIZABETH H. KORNECKI-GERRITY AND DAVID G. PENNEY
to be inhibited by phenylalanine, methionine, threonine and tryptophan in addition to alanine (Jim6nez de Asfia et aL, 1971 ; Carbonell et al., 1973). Cysteine, at 1 mM, has also been reported to produce 95 per cent inhibition of rat liver L type PK and 60 per cent inhibition of rat liver M 2 type PK by Carbonell et al. (1973), although this was not observed with the latter isoenzyme by Jim6nez de Asfia et al. (1971). Thus it appears that in addition to differences between turtle liver and mammalian liver PK's based on sensitivity to alanine, valine and isoleucine, differences may also exist with respect to sensitivity to a number of other amino acids. Turtle liver PK is also inhibited by 3 mM ATP and the inhibition is greatest (71 per cent) when the magnesium concentration is low (4 mM). At a high Mg 2+ concentration of 50 mM, the ATP inhibition is reduced, although still detectable (32 per cent). Similar results were obtained by Wood (1968) who demonstrated that the inhibition of rabbit muscle pyruvate kinase by 5 mM ATP was dependent on the magnesium concentration. This inhibition at low Mg 2+ has been explained as a magnesium chelating effect (Wood, 1968). It was similarly noted by Irving & Williams (1973) that the inhibition by ATP of rabbit liver L type PK could be attributed to chelation of Mg 2+ in the assay medium. The M2 type PK, on the other hand, remained sensitive to the ATP inhibition even in the presence of excess Mg 2+ (Jim6nez de Asfia et al., 1971 ; Imamura et al., 1972; Irving & Williams, 1973). As previously shown, 0.1 mM FDP greatly increases turtle liver PK activity (Penney & Kornecki, 1973; Kornecki-Gerrity & Penney, 1974). It now appears that G-6-P, F-6-P and serine have the same effect. However, FDP of the four compounds tested is the strongest activator, since a concentration one-fourth that of F-6-P (0.4 mM) and about one-twentieth that of G-6-P (2.0 mM) and serine (2.5 mM) also resuks in over a 1-1.5 fold increase in activity. In this regard, activation of the crude mammalian liver PK has been shown to occur at FDP concentrations as low as 0.5/zM (Llorente et al., 1970). Similar results were obtained by Koster & Hulsmann (1970) who observed that 0.5 mM G-6-P and F-6-P produced about a twofold increase in rat liver L PK activity. However, G-6-P at 5 mM had no significant affect on the activity of rat liver M, PK (Jim6nez de Asfla et al., 1971). L-Serine at 1 mM was also reported by Stifel & Herman (1971) to increase, somewhat, crude rat liver PK activity. With purified liver PK preparations only the M 2 type showed slight activation by L-serine (Carbonell et al., 1973). The addition of various other amino acids (asparagine, arginine, glutamine, histidine and OH-proline) and certain metabolic intermediates (citrate, succinate, a-ketoglutarate, glucose, G-l-P, urea, acetylcholine and NH4C1) produced no significant effect on turtle liver PK activity. L-Histidine (0"1-1 mM) ,however, has been observed to stimulate activity of crude rat liver PK (Stifel & Herman, 1971), whereas citrate and succinate did not (Seubert et al., 1968; Sehoner et al., 1970). Turtle liver PK was found to have a pH optima in the range of 6.6-7-0, somewhat similar to the pH optima of 6.5 for the mammalian liver L form (Imamura et al., 1972; Irving & Williams, 1973), although lower than the pH optima of 7.0-7.5 for the liver M S type PK (Imamura et al., 1972).
P K FROM TURTLE TISSUES
23
T h e P K present in turtle liver appears to differ considerably from that in turtle heart. T h e liver form shows sigmoidal kinetics with respect to P E P and activation by FDP. In contrast, the heart form displays hyperbolic kinetics and is not activated by FDP. While both enzymes are inhibited by alanine, only the liver form is inhibited by valine and isoleucine as well as a n u m b e r of other amino acids. This behavior with regard to strong inhibition by valine and isoleucine is unlike any mammalian P K isoenzyme which has been described. In addition, the strong inhibition of the turtle heart enzyme by alanine is at variance with data on the mammalian heart PK. Therefore, the turtle enzymes from liver and heart appear to differ in several important respects from the P K ' s of mammalian liver and heart.
REFERENCES BALINSKY D., CAYANm E. & BE~SOHN H. (1973) Comparative kinetic study of human pyruvate kinases isolated from adult and fetal livers and from hepatoma. Biochemistry 12, 863-870. BoYea P. D. (1962) Pyruvate kinase. In TheEnzymes (Edited by Bo'~R P. D., LARDYH. & MYRBACKK.), Vol. 6, pp. 95-113. Academic Press, New York. CARBONELLJ., FELIUJ. E., M ~ c o R. & SOLSA. (1973) Pyruvate kinase. Classes of regulatory isoenzymes in mammalian tissues. Eur.y. Biochem. 37, 148-156. COSTAL., JIM~SZ DE AS0A L., ROZENQtmTE., BADeE. & CASML~ATTIH. (1972) Allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex. Biochim. biopkys. Acta 289, 128-136. FLANDEaS L. E., BAMBURGJ. R. & SALLACHH. J. (1971) Pyruvate kinase isoenzymes in adult tissue and eggs of Rana pipiens--II. Physical and kinetic studies of purified skeletal and heart muscle pyruvate kinases. Biochim. biophys. Acta 242, 566-579. IMAMURAK., TANIUCHIK. & TANAKAT. (1972) Multi_molecular forms of pyruvate kinase--II. Purification of Mi-type pyruvate kinase from Yoshida Ascites Hepatoma 130 cells and comparative studies on the enzymological and immunological properties of the three types of pyruvate kinases, L, M and M2. ft. Biochem. (Tokyo) 72, 1001-1015. IRVING M. G. & WILLIAMS J. F. (1973) Kinetic studies on the regulation of rabbit liver pyruvate kinase. Biochem. ft. 131, 287-301. JIM~NEZ DE ASt~A L., ROZENOURT E., DEVALLEJ. J. & CARMINATTI H. (1971) Some kinetic differences between the M isoenzymes of pyruvate kinase from liver and muscle. Biochim. biophys. Acta 235, 326-334. KORNECKI-GEImITYE. & Pm~-Ey D. (1974) The effect of phosphoenolpyruvate, fructose-1,6diphosphate, alanine and valine on pyruvate kinase from turtle liver and heart. Comp. Biochem. Physiol. (In press.) KOSTEa J. F. & HULSMANNW. C. (1970) The influence of inorganic phosphate and phosphorylated hexoses on the activity of pyruvate Idnase. Archs Biochem. Biophys. 141, 98-101. LLOrmNTEP., Mnaco R. & SOLSA. (1970) Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads. Eur. ft. Biochem. 13, 45-54. PSNI~Y D. G. & KOaNECKI E. H. (1973) Activities, intra-cellular localization and kinetic properties of phosphoenolpyruvate carboxykinase, pyruvate kinase and malate dehydrogenase in turtle (Pseudemys scripta elegans) liver, heart and skeletal muscle. Comp. Biochem. Physiol. 46B, 405--415. SCHONER W., HAAG U. & SEUBERT W. (1970) Regulation of carbohydrate metabolism by cortisol independent of the de novo synthesis of enzymes in rat liver. Hoppe-Seyler's Z. physiol. Chem. 351, 1071-1088.
24
ELIZABETH H. KORNECKI-GEBRITYAND DAVID G. PENNEY
SEUBERT W., HENNING H. V., SCHONER W. • L'AGE M. (1968) Effects of cortisol on the levels of metabolites and enzymes controlling glucose production from pyruvate. Adv. Enzyme. Reg. 6, 153-180. STIFLE F. & HERMAN R. (1971) Effect of L-histidine on human and rat jejunal pyruvate kinase activity. Can. J. Biochem. 49, 1105-1116. TANAKA T., HARANO Y., MORIMURA H. & MOBI R. (1965) Evidence for the presence of two types of pyruvate kinase in rat liver. Biochem. biophys. Res. Commun. 21, 55-60. TANAKAT., HARANOY., SUE F. & MORIMURAH. (1967) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem. (Tokyo) 62, 71-87. TAYLOR C. B., MORRIS H. P. & WEBER G. (1969) A comparison of the properties of pyruvate kinase from hepatoma 3924-A, normal liver and muscle. Life Sci. 8, 635-644. WOOD T. (1968) The inhibition of pyruvate kinase by ATP. Biochem. biophys. Res. Commun. 31, 779-785.
Key Word Index--Pyruvate kinase; turtle (Pseudemys scripta elegans); heart; liver; kinetic properties.
K I N E T I C PROPERTIES OF PYRUVATE KINASES FROM T U R T L E LIVER AND HEART E L I Z A B E T H H. K O R N E C K I - G E R R I T Y and DAVID G. PENNEY Department of Biological Sciences, University of Illinois at Chicago Circle, Chicago, Illinois 60680, U.S.A. (Received 14 September 1973)
Abstract--1. Turtle liver pyruvate kinase (PK), which shows aUosteric properties with respect to phosphoenolpyruvate (PEP), is activated by fructose diphosphate (FDP), glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P) and L-serine. Its activity is strongly inhibited (over 85 per cent) by L-alanine [Kiclpp) = 0-09 mM], L-isoleueine [Klcapp~= 0"19 mM], L-valine [Kjapp~= 0-56 mM] and L-cysteine and over 50 per cent by L-methionine, L-tryptophan and L-phenylalanine. 2. Liver PK is inhibited by ATP and this inhibition is strongly influenced by the concentration of Mg*+ (e.g. 4 mM Mgs+ produces 70 per cent inhibition, whereas 50 mM Mg'+ produces 32 per cent inhibition). 3. Citrate, suecinate, a-ketoglutarate, some L-amino acids and other compounds tested produce little or no effect on liver PK activity. 4. Heart PK does not show an allosterie response with varying PEP concentration. It is not activated by FDP. It was found to be inhibited by Lalanine [Kl(app) -- 0"19 mM], but not by L-valine. Very slight inhibition was observed with L-isoleucine. 5. The pH optima for liver PK activity is in the range of pH 6"6-7"0. INTRODUCTION T A N A K A et al. (1965, 1967) demonstrated at least two types of PK in the rat, types L and M, which predominate in liver and muscle, respectively. Type L shows a sigmoidal relationship of reaction velocity to PEP concentration. On the other hand, type M PK shows typical Michaelis-Menten kinetics with PEP. Where FDP causes activation of the L type by increasing its affinity for PEP, the M type shows no activation with FDP. In previous works we (Kornecki-Gerrity & Penney, 1974; Penney & Kornecki, 1973) have reported that turtle liver PK exhibits sigmoidal kinetics with respect to PEP with a K m ~ w i , J above 2 raM. Upon addition of 0.1 mM FDP, a hyperbolic curve is obtained yielding a K m WEVJ value of 0-2 raM. In the presence of 2 mM L-alanine and L-valine, two very potent inhibitors of the turtle liver enzyme, the Km ~'Ev~ values increase to several millimoles. L-Alanine has been shown to inhibit PK from crude extracts of mammalian liver (Llorente et aL, 1970; Stifel & Herman, 1971 ; Carbonell et aL, 1973). In contrast, L-valine did not produce any significant inhibition in such preparations (Stifel & Herman, 1971). Turtle heart PK shows 15
16
ELIZABETH H. KORNECKI-GERRITYAND DAVID G. PENNEY
hyperbolic kinetics both in the presence and absence of F D P with K m(PEP) values of 0.2 mM. L-Alanine was shown to inhibit the heart PK, whereas L-valine produced no effect. This differs strikingly from the mammalian heart PK which is not inhibited by L-alanine (Carbonell et al., 1973). Therefore, we suggested that in turtle liver and heart (a) two (or more) kinetically different forms of PK are present and (b) that these enzymes differ in several respects from the mammalian liver and heart PK isoenzymes. This study was undertaken in order te further characterize turtle heart and liver PK. MATERIALS AND METHODS Both male and female turtles (Pseudemys scripta elegans) of 500-1000 g body weight were used. These turtles were maintained at 20°C and given slices of beef liver once each week. Liver and heart supernate fractions used for P K studies were prepared according to previously described methods (Penney & Kornecki, 1973).
P K assay Enzyme activities were measured at 20°C by the method of Boyer (1962). The assay mixture contained the following components: imidazole C1- buffer (pH 7"0), 1 0 0 m M ; KC1, 1 0 0 m M ; MgC12, 4 m M ; ADP, 4 m M ; PEP, l m M ; N A D H , 0"16mM; lactate dehydrogenase (LDH), 1 unit per ml; and diluted tissue enzyme preparation. The oxidation of N A D H was followed at 340 n m with a Gilford Model 2400 recording Spectrophotometer. Control cuvettes from which either PEP or ADP were omitted compensated for non-specific oxidation of N A D H .
Studies of effects of various intermediates P K assays carried out in the presence of various intermediates had the same component concentrations as given above, with one exception; the concentration of PEP was 0"5 m M instead of 1 raM. PEP plus the intermediate whose effect was being examined were added to the assay mixture simultaneously. After a 5 rain incubation period, the reaction was started by the addition of ADP. Control cuvettes lacked the intermediate under study. Activities were corrected for any N A D H oxidation which occurred during the incubation period, especially when F D P was added. All intermediates were soluble in imidazole C1buffer at a pH of 7"0. All assay mixtures were found to remain within + 0"1 pH unit of the set p H value during the reaction.
Determination of K t (app) Kt(a~p) indicates the concentration of the inhibitor required to slow the reaction to half the rate it shows in the absence of the inhibitor. To determine Kl¢~pp)the reactions were run at various inhibitor concentrations (0'01-2"5 mM) and at a PEP concentration of 0"5 mM.
Determination of p H optima P K assays were performed at a number of pH values ranging from 5'5 to 8"5. The concentration of PEP in the P K assay mixture was 1 mM. Imidazole C1- buffer was used throughout the entire pH range. The pH of each mixture was measured before and after the reaction and was found to be within + 0"1 pH unit for each pH value tested.
Chemicals and enzymes All chemicals and enzymes [acetylcholine, adenosine-5'-diphosphate, adenosine-5'triphosphate, ammonium chloride, arginine, asparagine, choline, citrate, cysteine, F-6-P, FDP, glucose, glucose-l-phosphate (G-l-P), G-6-P, glutamine, glycine, histidine, imidazole
PK
17
F R O M T U R T L E TISSUES
CI-, isoleucine, ~-ketoglutarate, LDH, leucine, methionine, NADH, OH-proline, phenylalanine, PEP, serine, succinate, threonine, tryptophan, urea and valine] were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. All amino acids used were of the L-series (except glycine). RESULTS Table 1 shows the effects of various amino acids on turtle liver PK activity. The greatest PK inhibition (over 90 per cent) is produced by alanine at 2 mM and isoleucine at 2.5 mM. Valine (2 raM), 2.5 mM cysteine and 2 mM methionine TABLE1--THE EFFECT
O F VARIOUS A M I N O ACIDS O N T U R T L E L I V E R P Y R U V A T E K I N A S E
Treatment [PEP] = 0"5 mM Control L-Alanine L-Isoleucine (2"5 mM) L-Valine L-Cysteine (2,5 mM) L-Methionine L-Tryptophan L-Phenylalanine L-Threonine L-Leucine Glycine
Per cent of control PK activity 100 7 (4-11) 8 (6-10) 15 (9-25) 16 (15-16) 22 (20-23) 45 (42-48) 47 (38-60) 56 (49-60) 57 (44-70) 66 (56-72)
53 4* 6 10 3 3 3 6 2 3 7
All amino acids used at 2 mM, unless otherwise designated. *Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. produce liver PK inhibition of over 75 per cent. Both tryptophan and phenylalanine at 2 mM inhibit liver PK activity over 50 per cent. Threonine, leucine and glycine inhibit liver PK activity over 30 per cent. Figure 1 shows the influence of varying concentrations of alanine, isoleucine, valine and serine at 0.5 mM PEP on liver PK activity before and after the addition of 0.1 mM FDP. In the presence of varying concentrations of alanine (Fig. 1A) a hyperbolic curve is obtained and the apparent K i value is 0.09 mM. The addition of 0-1 mM FDP largely overcomes alanine inhibition. Figure 1B shows the effects of isoleucine on liver PK activity. The curve obtained in the absence of FDP is hyperbolic with an apparent Ki of 0-19 mM. The addition of FDP completely reverses the PK inhibition produced by isoleucine. The effect of varying concentrations of valine on liver PK activity is shown in Fig. 1C. In the absence of FDP, a hyperbolic curve is obtained and the apparent K i value for valine is 0.56 mM. Again, with the addition of FDP, inhibition is largely overcome. Figure 1D shows the influence of serine on PK activity. A sigmoidal increase is obtained with increasing serine concentrations. Upon the addition of FDP, the increase in activity
18
ELIZABETH H . KORNECKI-(~EERITY AND DAVID G . PENNEY
(A)
L . .
(B)
200~-
200:
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~
~- J' ^
I,-,-
.<
.~. 100~
Ki(app)=O.OgmM
100~
Ki(app)=O.19mM
f
1
2
L-Alanine [mM]
2.5
L-isoleucine [mM]
(c)
200 ^ .L
2
1
(D)
&
,>-
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< 10C
~ sol
20C
~ •
•
•
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;
,
"
I
I
I
L-valine [mM]
I
2
I
I
I
I I
I
|
f
I
f /3 ~L 2 2.5
L-serine [mM]
FIG. 1. Effect of various concentrations of L-alanine (A), L-isoleucine (B), L-valine (C) and L-serine (D) on liver pyruvate kinase activity in the presence A - - A - - A and absence O - - O - - O of FDP. The concentration of PEP is 0"5 mM.
is seen to be due solely to " F D P - a c t i v a t i o n " (i.e. there is no added stimulation resulting from serine in the presence of FDP). Table 2 shows the effects of alanine, valine and isoleucine on turtle heart P K activity. Alanine at 2 m M produces over 90 per cent inhibition of heart P K activity giving an apparent K i for alanine of 0-19 mM. In the presence of F D P , alanine TABLE 2 - - T H E EFFECT OF L-ALANINE~ L-VALINE AND L-ISOLEUCINE ON TURTLE HEART PYRUVATE KINASE ACTIVITY
Treatment [PEP] = 0'5 mM Control L-Alanine (2 mM) L-Valine (2 mM) L-Isoleucine (2"5 raM)
Per cent of control PK activity n 100 11 (9-15) 93 (89-96) 75 (73-79)
11 4* 4 3
*Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. inhibition of heart P K activity is reversed (Fig. 2). However, 2 m M valine produces no significant inhibition of heart PK. Isoleucine at 2.5 mM, which produces such strong inhibition of liver P K (Table 1), is seen to have only a very slight inhibitory effect on heart P K activity.
PK FROMTURTLETISSLrI~ 100,~
^
A
A
I 0.5
I
A I
19 A Ll
__
>-
_> ~50. <
I.-t.u
0 I
1.0 1.5 L-alonlne [mM]
I
2.0
FIG. 2. Influence of L-alanine at various concentrations on the activity of heart pyruvate kinase in the presence (A--A--A) and absence ( O - - O - - O ) of FDP. The concentration of PEP is 0'5 raM. A T P (3 m M ) in the presence of 4 m M M g ~+ inhibits liver P K activity over 70 per cent (Table 3). Increasing the concentration of M g 2+ to 12.5 m M reduces inhibition to 38 per cent. A still higher Mg 2+ concentration of 50 m M results in a further decrease in P K inhibition to 32 per cent. TABLE 3--TH~ EF~CT OF ATP AND Mg 2+ ION CONCENTRATIONON TURTLELIVER PYRUVATE
Treatment [PEP] = 0"5 mM
KINASE
ACTIVITY
Per cent of control PK activity
Control 100 ATP (3 mM) + Mg 2+ (4 mM) 29 (22-39) ATP (3 m M ) + M g s+ (12.5 mM) 62 (47-77) ATP (3 mM) + Mg ~+ (50 mM) 68 (63-72)
n 11 4* 5 2
*Values given as mean percentages of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. Table 4 shows the effects of three phosphorylated glycolytic intermediates (FDP, G-6-P and F-6-P) and serine on liver P K activity at a P E P concentration of 0.5 mM. T h e addition of F D P (0.1 raM), G-6-P (2 mM) or F-6-P (0.4 raM) exerts a stimulatory effect on the liver P K by increasing its activity over 50 per cent. Serine at 2-5 m M shows a smaller, but nonetheless significant, stimulatory effect (45 per cent higher than control) on liver P K activity. T h e amino acids asparagine, arginine and glutamine at 2 raM, and histidine and OH-proline at 2"5 m M produced no significant effects on liver P K activity.
20
ELIZABETH H. KORNECKI-GERRITY AND DAVID G. PENNEY
The Krebs cycle intermediates, citrate, succinate and ~-ketoglutarate at 2 m M and the glycolytic intermediate G-1-P (2 mM) and glucose (5 raM) also produced little or no effect on liver P K activity. Other compounds tested which showed little or no effect were urea (2 raM), acetylcholine (10 -2 to 10 -~ raM), choline (1 mM) and ammonium chloride (2 raM). TABLE 4 - - T H E EFFECT OF FRUCTOSE-1,6-DIPHOSPHATE, GLUCOSE-6-PHOSPHATE, FRUCTOSE-6-PHOSPHATE AND L-SERINE ON TURTLE LIVER PYRUVATE KINASE ACTIVITY
Treatment [PEP] -~ 0-5 mM
Per cent of control PK activity
n
100 174 (152-202) 169 156 (138-174) 145 (138-158)
18 8* 2 2 5
Control F-1,6-diP (0"1 mM) G-6-P (2 raM) F-6-P (0"4 raM) L-Serine (2"5 raM)
*Values given as mean percentage of the control. The range is indicated by the numbers in parentheses. Number of observations given by n. The effect of pH on liver P K activity was studied (Fig. 3). Maximal activity was observed at pH 6.6-7.0. There was a 20 per cent decrease in activity when the pH was decreased by 0.6 pH unit (pH 6.0). No P K activity could be detected at pH 5.5. At pH 7.5, the P K activity was decreased by 45 per cent.
100 -
75U .<
50 u.J
25'
5~5
I
6.0
I
6.5
I
I
7.0
7.5
I
8.0
I
8.5
I
9.0
pH
F I a . 3. T h e effect of p H o n the activities of turtle liver p y r u v a t e kinase. E a c h
point is an actual observation. The concentration of PEP is 1 mM.
PK
FROM TURTLE TISSUES
21
DISCUSSION Alanine is a very potent inhibitor of turtle liver PK activity as shown by its apparent K i value of 0.09 raM. Similar results have been found for the unpurified rat liver PK (Llorente et al., 1970; Stifel & Herman, 1971 ; Carbonell et al., 1973), the mammalian liver L type PK (Taylor et al., 1969; Llorente et al., 1970; Costa et al., 1972; Imamura et aL, 1972; Irving & Williams, 1973; Carbonell et al., 1973) and the mammalian liver M S type (or class A) PK (Jim6nez de Asfia et al., 1971 ; Carbonell et al., 1973). With the addition of FDP, alanine inhibition of the turtle liver PK is relieved. This is also observed with the mammalian L type PK. Isoleucine and valine also strongly inhibit turtle liver PK activity as shown by their Kitapp~ values of 0.19 and 0.56 mM, respectively. FDP completely overcomes this inhibition. In contrast, such strong inhibition has not been observed for the unpurified mammalian liver PK or the purified liver L and M S isoenzymes which are only slightly inhibited by relatively high concentrations of L-isoleucine and L-valine (Seubert et al., 1968; Stifel & Herman, 1971; Imamura et al., 1972; Carbonell et al., 1973). Apparently the strong inhibition of turtle liver PK activity produced by these two amino acids at low concentrations is unique. Turtle heart PK is inhibited by alanine, as is the liver PK, with an apparent K i of 0.19 mM. Also, as with the liver enzyme this inhibition by alanine is completely reversible by FDP. However, the activity achieved in the presence of both alanine and FDP is no higher than in the presence or absence of FDP alone (KorneckiGerrity & Penney, 1974), indicating that FDP is not an activator of turtle heart PK. In contrast, Flanders et al. (1971) report that FDP is an activator of frog heart PK. Interestingly, mammalian heart PK extracts are not inhibited by alanine (Carbonell et aL, 1973), while frog heart PK, like turtle heart PK, is also inhibited by L-alanine at a low concentration. Turtle heart PK inhibition by alanine is therefore similar to that of mammalian liver L and M S type PKs (Jim6nez de Asda et al., 1971; Imamura et al., 1972; Balinsky et al., 1973; Carbonell et al., 1973; Irving & Williams, 1973) and also to that of frog heart PK. However, turtle heart PK activity is not significantly inhibited by 2 m M valine or 2.5 m M isoleucine. Likewise, the effect of these amino acids at similar concentrations on the mammalian liver L and M S type PK isoenzymes is insignificant (Carbonell et al., 1973). From the data obtained it appears that turtle heart PK differs considerably from mammalian heart PK, but has some properties in common with the mammalian liver L and M S isoenzymes. In addition to inhibition of turtle liver PK by L-alanine, isoleucine and valine, other amino acids also cause substantial inhibition. For example, cysteine, methionine, tryptophan and phenylalanine inhibit turtle liver PK activity by over 50 per cent, whereas a 30 per cent inhibition occurs with threonine, leucine and glycine. Somewhat different responses from those found here were observed with crude rat liver extracts and purified L-type PK where L-methionine, L-tryptophan, L-phenylalanine, L-threonine, L-leucine and glycine were found not to inhibit significantly at similar concentrations (Seubert et al., 1968; Stifel & Herman, 1971 ; Carbonell et al., 1973). However, the rat liver M 2 PK isoenzyme has been reported
22
ELIZABETH H. KORNECKI-GERRITY AND DAVID G. PENNEY
to be inhibited by phenylalanine, methionine, threonine and tryptophan in addition to alanine (Jim6nez de Asfia et aL, 1971 ; Carbonell et al., 1973). Cysteine, at 1 mM, has also been reported to produce 95 per cent inhibition of rat liver L type PK and 60 per cent inhibition of rat liver M 2 type PK by Carbonell et al. (1973), although this was not observed with the latter isoenzyme by Jim6nez de Asfia et al. (1971). Thus it appears that in addition to differences between turtle liver and mammalian liver PK's based on sensitivity to alanine, valine and isoleucine, differences may also exist with respect to sensitivity to a number of other amino acids. Turtle liver PK is also inhibited by 3 mM ATP and the inhibition is greatest (71 per cent) when the magnesium concentration is low (4 mM). At a high Mg 2+ concentration of 50 mM, the ATP inhibition is reduced, although still detectable (32 per cent). Similar results were obtained by Wood (1968) who demonstrated that the inhibition of rabbit muscle pyruvate kinase by 5 mM ATP was dependent on the magnesium concentration. This inhibition at low Mg 2+ has been explained as a magnesium chelating effect (Wood, 1968). It was similarly noted by Irving & Williams (1973) that the inhibition by ATP of rabbit liver L type PK could be attributed to chelation of Mg 2+ in the assay medium. The M2 type PK, on the other hand, remained sensitive to the ATP inhibition even in the presence of excess Mg 2+ (Jim6nez de Asfia et al., 1971 ; Imamura et al., 1972; Irving & Williams, 1973). As previously shown, 0.1 mM FDP greatly increases turtle liver PK activity (Penney & Kornecki, 1973; Kornecki-Gerrity & Penney, 1974). It now appears that G-6-P, F-6-P and serine have the same effect. However, FDP of the four compounds tested is the strongest activator, since a concentration one-fourth that of F-6-P (0.4 mM) and about one-twentieth that of G-6-P (2.0 mM) and serine (2.5 mM) also resuks in over a 1-1.5 fold increase in activity. In this regard, activation of the crude mammalian liver PK has been shown to occur at FDP concentrations as low as 0.5/zM (Llorente et al., 1970). Similar results were obtained by Koster & Hulsmann (1970) who observed that 0.5 mM G-6-P and F-6-P produced about a twofold increase in rat liver L PK activity. However, G-6-P at 5 mM had no significant affect on the activity of rat liver M, PK (Jim6nez de Asfla et al., 1971). L-Serine at 1 mM was also reported by Stifel & Herman (1971) to increase, somewhat, crude rat liver PK activity. With purified liver PK preparations only the M 2 type showed slight activation by L-serine (Carbonell et al., 1973). The addition of various other amino acids (asparagine, arginine, glutamine, histidine and OH-proline) and certain metabolic intermediates (citrate, succinate, a-ketoglutarate, glucose, G-l-P, urea, acetylcholine and NH4C1) produced no significant effect on turtle liver PK activity. L-Histidine (0"1-1 mM) ,however, has been observed to stimulate activity of crude rat liver PK (Stifel & Herman, 1971), whereas citrate and succinate did not (Seubert et al., 1968; Sehoner et al., 1970). Turtle liver PK was found to have a pH optima in the range of 6.6-7-0, somewhat similar to the pH optima of 6.5 for the mammalian liver L form (Imamura et al., 1972; Irving & Williams, 1973), although lower than the pH optima of 7.0-7.5 for the liver M S type PK (Imamura et al., 1972).
P K FROM TURTLE TISSUES
23
T h e P K present in turtle liver appears to differ considerably from that in turtle heart. T h e liver form shows sigmoidal kinetics with respect to P E P and activation by FDP. In contrast, the heart form displays hyperbolic kinetics and is not activated by FDP. While both enzymes are inhibited by alanine, only the liver form is inhibited by valine and isoleucine as well as a n u m b e r of other amino acids. This behavior with regard to strong inhibition by valine and isoleucine is unlike any mammalian P K isoenzyme which has been described. In addition, the strong inhibition of the turtle heart enzyme by alanine is at variance with data on the mammalian heart PK. Therefore, the turtle enzymes from liver and heart appear to differ in several important respects from the P K ' s of mammalian liver and heart.
REFERENCES BALINSKY D., CAYANm E. & BE~SOHN H. (1973) Comparative kinetic study of human pyruvate kinases isolated from adult and fetal livers and from hepatoma. Biochemistry 12, 863-870. BoYea P. D. (1962) Pyruvate kinase. In TheEnzymes (Edited by Bo'~R P. D., LARDYH. & MYRBACKK.), Vol. 6, pp. 95-113. Academic Press, New York. CARBONELLJ., FELIUJ. E., M ~ c o R. & SOLSA. (1973) Pyruvate kinase. Classes of regulatory isoenzymes in mammalian tissues. Eur.y. Biochem. 37, 148-156. COSTAL., JIM~SZ DE AS0A L., ROZENQtmTE., BADeE. & CASML~ATTIH. (1972) Allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex. Biochim. biopkys. Acta 289, 128-136. FLANDEaS L. E., BAMBURGJ. R. & SALLACHH. J. (1971) Pyruvate kinase isoenzymes in adult tissue and eggs of Rana pipiens--II. Physical and kinetic studies of purified skeletal and heart muscle pyruvate kinases. Biochim. biophys. Acta 242, 566-579. IMAMURAK., TANIUCHIK. & TANAKAT. (1972) Multi_molecular forms of pyruvate kinase--II. Purification of Mi-type pyruvate kinase from Yoshida Ascites Hepatoma 130 cells and comparative studies on the enzymological and immunological properties of the three types of pyruvate kinases, L, M and M2. ft. Biochem. (Tokyo) 72, 1001-1015. IRVING M. G. & WILLIAMS J. F. (1973) Kinetic studies on the regulation of rabbit liver pyruvate kinase. Biochem. ft. 131, 287-301. JIM~NEZ DE ASt~A L., ROZENOURT E., DEVALLEJ. J. & CARMINATTI H. (1971) Some kinetic differences between the M isoenzymes of pyruvate kinase from liver and muscle. Biochim. biophys. Acta 235, 326-334. KORNECKI-GEImITYE. & Pm~-Ey D. (1974) The effect of phosphoenolpyruvate, fructose-1,6diphosphate, alanine and valine on pyruvate kinase from turtle liver and heart. Comp. Biochem. Physiol. (In press.) KOSTEa J. F. & HULSMANNW. C. (1970) The influence of inorganic phosphate and phosphorylated hexoses on the activity of pyruvate Idnase. Archs Biochem. Biophys. 141, 98-101. LLOrmNTEP., Mnaco R. & SOLSA. (1970) Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads. Eur. ft. Biochem. 13, 45-54. PSNI~Y D. G. & KOaNECKI E. H. (1973) Activities, intra-cellular localization and kinetic properties of phosphoenolpyruvate carboxykinase, pyruvate kinase and malate dehydrogenase in turtle (Pseudemys scripta elegans) liver, heart and skeletal muscle. Comp. Biochem. Physiol. 46B, 405--415. SCHONER W., HAAG U. & SEUBERT W. (1970) Regulation of carbohydrate metabolism by cortisol independent of the de novo synthesis of enzymes in rat liver. Hoppe-Seyler's Z. physiol. Chem. 351, 1071-1088.
24
ELIZABETH H. KORNECKI-GEBRITYAND DAVID G. PENNEY
SEUBERT W., HENNING H. V., SCHONER W. • L'AGE M. (1968) Effects of cortisol on the levels of metabolites and enzymes controlling glucose production from pyruvate. Adv. Enzyme. Reg. 6, 153-180. STIFLE F. & HERMAN R. (1971) Effect of L-histidine on human and rat jejunal pyruvate kinase activity. Can. J. Biochem. 49, 1105-1116. TANAKA T., HARANO Y., MORIMURA H. & MOBI R. (1965) Evidence for the presence of two types of pyruvate kinase in rat liver. Biochem. biophys. Res. Commun. 21, 55-60. TANAKAT., HARANOY., SUE F. & MORIMURAH. (1967) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem. (Tokyo) 62, 71-87. TAYLOR C. B., MORRIS H. P. & WEBER G. (1969) A comparison of the properties of pyruvate kinase from hepatoma 3924-A, normal liver and muscle. Life Sci. 8, 635-644. WOOD T. (1968) The inhibition of pyruvate kinase by ATP. Biochem. biophys. Res. Commun. 31, 779-785.
Key Word Index--Pyruvate kinase; turtle (Pseudemys scripta elegans); heart; liver; kinetic properties.