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Purification and properties of the pyruvate kinase isozyme M1 from the pig brain
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Pergamon
J. Biochem.
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1357-2725(95)00090-9
Vol. 27, No. II, pp. 114%1151, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. Ail rights reserved 1357-272519s $9.50 + 0.00 Bid.
Pwification and Properties of the Pyruvate Ki Isozyme M, from the Pig Brain GABERT
FARRAR,
WILLIAM
W. FARRAR”
Department
of Biological Sciences, Eastern Kentucky University, Richmond, KY 40475, U.S.A.
There are four pyruvate kinase isozymesin vertebrate tissues,designated as L, M, , M2, ssd R. Although pyravate kiaases have been par&d and characte&ed from pig liver, muade, kidney, and heart, the brain isozymehas not. The ahn of this work was to purify, characteri~ and make an isozymic designation for the pig brain pyruvate kinase. Puriftatlom was by chromatography on phosphocelhdose,Sephadex G200, and bwextraa agarose cobmms. The . moiecalar weight of the native enzyme was determined by sucrose de&ty w The degree of parity, and subunit molecular weight were determined by poiyac@amide gel ele&ophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid W&tric focusing method in sucrosegradients. The pH opthnum, aad kinetics in the preaeaceand abseace of fractose-1,6diphosphate were determined spectrophotometrically. The pmi@Wion &etne used redted in a 382-fold paritlcation of pig brain pyruvate khmse.,aRd a &ml specitlc activity of 191 Units/mg protein. As estimated by scaoniog of the dmn do&cyl s&ate poiyacryhm&le gels, the par&&ion scheme also resulted in a preparatioa that was of at least 98% parity, Pig brain pyruvate kinase has a native molecular weight of approx. 23fI,OWJ,and a s&t&t m&c&r weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimmn was estimated at pH 7.4. Fructose-l&-diphosphate had no et&et on either the K, for phospho(enol)pywvate, or the V,, of the reaction. Pig brain pyruvate kinase is similar in native and subunit molecular weights to the other pyravate kieases from pig &aaes. Like other vertebrate pyruvate kinases, pig brain pyruvate kinase is a tetramer. The pH optimum of 7.4 excludes pig brain pyruvate kinase as a type L isozyme, but cannot disting&h between MI and M, isozymes.An isoelectric point of 8.0, as well as hyperbolic k&tics aud the lack of inactivation by fructose-l&diphosphate, however, are consistent with the pyruvate kinase from pig brain being categorized as a type M, isozyme. Keywords: Pyruvate kinase Brain
Isozymes Pig Purification
Properties
ht. J. Biochem. Cell Biof. (1995) 27, 1145-l 151
The liver isozyme (PyK-L) has been purified from livers of rat (Tanaka et al., 1967; Imamura and Tanaka, 1982), ox (Cardenas and Dyson, 1973), pig (Kutzbach et al., 1973) chicken (Eigenbrodt and Schoner, 1977), and human (Kahn and Marie, 1982). Kinetically, PyK-L isozymes isolated from these sources are activated by fructose-l$&diphosphate (FDP). PyK-M, is the most widely distributed isozyme and has been detected and examined in rat epididymal adipose tissue (Pogson, 1968; Marco et al., 1971; Carbonell et al., 1973) and purified and characterized from hepatoma (Imamura et al., 1972), leucocytes (Van Berkel and Koster, 1973), rat liver (Imamura et ul.,
INTRODUCTION
There are four known isozymic forms of pyruvate kinase (ATP: Pyruvate phosphotransferase; EC 2.7.1.40; PyK) in vertebrates, designated as L, M,, M, (or A), and R. These isozymic forms differ in their chemical, physical, kinetic, electrophoretic, and immunological properties, as well as their tissue distributions. Their parameters have been extensively reviewed (Hall and Cottam, 1978).
*To whom all correspondence should be addressed. Received 6 April 1995; accepted 21 June 1995. 1145
1146
Gabert Farrar and William W. Farrar
1972), and pig kidney (Berglund et al., 1977). Moreover, although PyK-M, is the minor PyK isozyme form in liver, it is nevertheless the major form in kidney. Within the liver, PyK-L is found in parenchymal cells, and PyK-M, in Kupffer cells (Van Berkel et al., 1972). Kinetically, PyK-L and PyK-M, are similar in that both are inhibited by adenosine triphosphate (ATP) and alanine, and activated by FDP. These two isozymes, however, differ in that PyK-L displays sigmoidal kinetics, whereas PyK-M, shows hyperbolic kinetics when phospho-(enol)pyruvate (PEP) is the variable substrate. Moreover, the presence of FDP decreases the K,,, of PEP for PyK-M, much less than it does for the PyK-L isozyme (see Hall and Cottam, 1978). PyK-M, , the skeletal muscle isozyme, has been purified from the muscle of rabbit (Steinmetz and Deal, 1966), rat (Tanaka et al., 1967; Imamura and Tanaka, 1982), ox (Cardenas et al., 1973), chicken (Cardenas et al., 1975), human (Baranowska and Baranowski, 1975; Harkins et al., 1977), dog (Wu et al., 1981; Becker et al., 1986), and pig (Berglund and Humble, 1979) and from the brain of ox (Terlecki, 1989), and partially purified and kinetically studied from guinea-pig (Nicholas and Bachelard, 1974). Kinetically, PyK-M, is not activated by FDP and displays hyperbolic Michaelis-Menten kinetics. Human erythrocyte (Chern et al., 1972; Kahn and Marie, 1982) PyK has also been isolated. As with PyK-L, this isozymic form is activated by FDP and displays sigmoidal kinetics for PEP in the absence of FDP. PyK has been purified from the heart muscle of turtle (Storey and Hochachka, 1974), ox (Parkinson and Easterby, 1977), and pig (Kiffmeyer and Farrar, 1991). PyK isozymes have also been isolated and characterized from several additional pig tissues, including liver (Kutzbach et al., 1973), kidney (Berglund et al., 1977) and muscle (Berglund and Humble, 1977). PyK-M, predominates in skeletal muscle, and brain, and can be found as a minor isozyme component in heart tissue (see Hall and Cottam, 1978). PyK has been isolated and studied from rat brain (Srivastava and Baquer, 1985) where its kinetic properties suggest that it is an isozyme distinct from muscle and liver forms, and from bovine brain (Terlecki, 1989) where its kinetic properties support an M, isozymic designation. In our report we present the purification of PyK from pig brain, and data on its native
molecular weight, subunit molecular weight, quaternary structure, pH optimum (pH,), isoelectric point (PI), and lack of activation by FDP. These parameters are compared with those of other pig tissues PyKs, and, where possible, with rat and bovine brain PyKs in an effort to make an isozymic assignment. MATERIALS
AND
Materials and preparation homogenate (CCH)
METHODS
of the clarified crude
Pig brains were obtained from Slade Packing Co., Cynthiana, Ky and immediately frozen until use. Adenosine diphosphate (ADP), phospho(enol)pyruvate (PEP), fructose- 1,Bdiphosphate (FDP), reduced nicotinamide adenine dinucleotide (NADH), pyruvate, phosphocellulose, Sephadex G200, ampholyte, blue dextranagarose, and the enzymes lactate dehydrogenase (LDH; rabbit muscle), enolase (yeast), aldolase (rabbit muscle), pyruvate kinase (PyK; rabbit muscle), and phosphorylase b (rabbit muscle) were all purchased from Sigma Chemical Co. Ultrapure ammonium sulfate and sucrose were obtained from Schwartz-Mann. All other chemicals were of reagent-grade purity. For preparation of the centrifuged crude homogenate (CCH), appropriate amounts of brain tissue were thawed, weighted and homogenized for 2 min in 2 vol of cold (4°C) 50 mM imidazole, 2 mM MgCl,, pH 6.5, using a glass homogenizer with a motor driven teflon pestle. This crude homogenate was then centrifuged at 40,OOOg at 4°C for 30 min to obtain the CCH. The CCH was used to initiate purification, to obtain the isoelectric point (PI), and to obtain the native molecular weight by sucrose density ultracentrifugation. Protein determination
Protein was estimated by absorbancy at 280/260 nm (Warburg and Christian, 1943), using a Bausch & Lomb Spectronic 2000 spectrophotometer. Assays for PyK and LDH activities
PyK activity was routinely measured using a Bausch & Lomb Spectronic 2000 spectrophotometer by coupling pyruvate formation to the LDH reaction (Bucher and Plleiderer, 1975) and monitoring the disappearance of NADH at 340 nm. The reaction mixture was 50 mM imidazole, 100 mM KCl, 2 mM MgCl,, 1 mM PEP, 1 mM ADP, 0.02 mM NADH, 5 Units of LDH,
Pyruvate
kinase
pH 7.4. Reaction volume was 1.0 ml and the temperature was 22°C. Rabbit muscle LDH activity was measured by monitoring the disappearance of NADH at 340 nm. Final reaction mixture component concentrations were: 50 mM sodium phosphate, 1.0 mM pyruvate, 0.20 mM NADH, pH 6.8. Reaction volume was 1.0 ml and the temperature was 22°C (Farrar and Farrar, 1983). Isoelectric focusing The isoelectric point (PI) for pig brain was determined by the rapid focusing method (Behnke et al., 1975) with slight modifications, in 540% sucrose gradients, using ampholytes in the pH 3-10 range. The reservoir buffers were 3% ethylenediamine and 0.5% H,S04. For focusing, 0.2 ml of the CCH was used. Focusing was performed at 22°C at 5 ma/tube. Usually, focusing of the proteins in the CCH took between 2.5-3.5 hr. Sucrose density gradient centrifugation Linear 5-20% sucrose gradients in 50mM imidazole, 100 mM KCl, 2 mM MgCl,, 1 mM PEP, 1 mM ADP, pH 7.0, were used to determine the SZO,wand the molecular weight of the native enzyme (Martin and Ames, 1961). Rabbit muscle LDH was used as a standard of known S2o.w and molecular weight. Centrifugation was at 40,000 rpm (4°C) for 17 hr in a Beckman L5-75 Preparative Ultracentrifuge using an SW 60 rotor. Sodium dodecyl (lauryl) sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) The subunit molecular weight of pig brain PyK was estimated by SDS-PAGE (Weber and Osborn, 1969) in 8% gels. This method was also used to estimate the degree of purity following the final step of purification. Steady-state kinetics The K, and V,,,,, in the presence and absence of FDP were determined using the reaction mixture described under Assay for PyK activity, but with PEP as the variable substrate at eight concentrations between 0.10 and 2.0 mM. Purified PyK was used. PuriJication of pig brain PyK Purification of pig brain PyK followed the same general procedure as for pig heart PyK (Kiffmeyer and Farrar, 1991) with the exception
from
1147
pig brain
that an additional blue dextran-agarose chromatography step was necessary following gel filtration on Sephadex G200. All steps in the purification, except the preparation of the CCH, and ammonium sulfate dialysis, were performed at room temperature (21-25°C). For phosphocellulose chromatography, a 2.5 cm x 40 cm column was equilibrated with 25 mM imidazole, 1 mM MgCl?, pH 6.5. The CCH was applied to the column and flow-through was with the same buffer. PyK adhered to the phosphocellulose, and after the protein concentration in the eluting fractions became minimal, the KC1 concentration was adjusted to 0.15 M. and then 0.40 M. Additional protein was eluted at each of these KC1 concentrations, but PyK remained on the column. PyK was eluted when the KC1 concentration was made 0.6 M. All fractions containing PyK activity were pooled. The pooled fractions were dialyzed for 12 hr at 4°C against 80% saturated ammonium sulfate in 25 mM imidazole, 100 mM KCl, 1 mM pH 7.0. MgCl,, 2 mM fl-mercaptoethanol, The resulting precipitate was collected by centrifugation. No PyK activity was found in the supernate. The ammonium sulfate precipitate was dissolved in a minimum volume of 25 mM 0.15
.* .I. !’ii .4
z o. I o a” .$ lZ 2 0.05
0
”
.i
IO
Fraction Fig. 1. Sucrose density gradient ultracentrifugation of pig brain PyK (0) with rabbit muscle LDH (0) as a standard. For centrifugation, 0.10 ml each of pig brain CCH and rabbit muscle LDH were layered on top of individual gradients. Following centrifugation, the total volume within each gradient tube was removed in O.lOml fractions (48 fractions from each). Fractions from the pig brain PyK tube were assayed for PyK, and those from the LDH standard tube were assayed for LDH as described in the M a t erials and Methods section. Activity is expressed as AA,,,/min/assay/aliquot of 0.020 ml.
Gabert Farrar and William W. Farrar
1148 5.0
4.6
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RESULTS AND DISCUSSION
Phb
0
.
Millimeters 4.5 1 0
I 0.1
I 0.2
I
I
0.3
0.4
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Mobility Fig. 2. SDS polyacrylamide (8%) gel electrophoresis of purified pig brain PyK. Standard of known subunit molecular weights are: rabbit muscle phosphorylase b (Phb, MW = 94,000); rabbit muscle PyK (RM PyK, MW = 57,000); yeast enolase (Enolase, MW = 42,000); rabbit muscle LDH (LDH, MW = 36,500); PB PyK is designated as PB PyK (MW = 60,000). Mobilities are relative to the migration distance of bromophenol blue. Inset is a scan of the SDS gel at 500 nm.
imidazole, 100 mM KCl, 1 mM MgCl,, 2 mM p-mercaptoethanol, pH 7.0 and applied to a Sephadex G200 column (1.5 cm x 70 cm) that had been equilibrated with the same buffer. Fractions of 1.O ml were collected. PyK eluted in the void volume and fractions containing substantial PyK activity were pooled. The pooled fractions from gel filtration were dialyzed extensively against 10 mM imidazole, 1 mM MgCl,, pH 7.5, and a blue dextranagarose column was equilibrated with the same buffer. The dialyzed sample was then applied to the column at room temperature and 1.0 ml fractions collected. Under these conditions, PyK adhered, but some proteins eluted. Over 85% of the PyK activity was specifically eluted by the addition of 1.0 mM ADP to the column buffer.
The purification procedure adapted from that used for pig heart PyK (Kiffmeyer and Farrar, 1991) works very well for pig brain PyK. The results of a typical purification from 100 g of pig brain are given in Table 1. As with pig heart PyK (Kiffmeyer and Farrar, 1991) rat brain (Srivastava and Baquer, 1985), and bovine brain (Terlecki, 1989), phosphocellulose column chromatography proved to be the best purification step. The final specific activity of 191 Units/mg protein for pig brain PyK (Table 1) is similar to the 217 Units/mg protein for purified bovine brain PyK (Terlecki, 1989), but somewhat lower than the specific activity of 593 of rat brain PyK (Srivastava and Baquer, 1985). However, this latter difference is most likely due to both the significantly lower assay temperature used for pig brain PyK (22°C) vs that used for rat brain PyK (37°C) and different assay buffers and component concentrations. Figure 1 shows the results of sucrose density ultracentrifugation of pig brain PyK using rabbit muscle LDH as a standard. Using an S20,W of 7.45 for LDH yielded a calculated SzO,W for pig brain PyK of 9.3, which corresponds to a native molecular weight of approx. 230,000, similar to that of PyKs from pig liver (Kutzbach et al., 1973; Berglund et al., 1977) muscle, and kidney (Berglund et al., 1977). SDS-PAGE of the combined PyK-containing fractions from the blue-dextran agarose column showed one major band plus one minor band of lesser molecular weight. The scan of the SDSPAGE gel of purified pig brain PyK revealed that the major band comprised about 98% of the total protein applied, while the minor band contributed -2% (Fig. 2) demonstrating that the purification protocol (Table 1) for pig brain PyK results in essentially homogeneous PyK. The molecular weight of the subunit is estimated to be approx. 60,000 (Fig. 2). These results, along with those from sucrose density ultracentrifugation (Fig. l), demonstrate that native pig
Table 1. Purification scheme for pig brain pyruvate kinase (based on 100 g starting tissue) Total Specific Total % Fold Step units* activity? Yield protein (mg) purification 2758 5256 0.5 100 1 CCH$ 27.3 81 55 P-cellulose 2449 89 1434 15 95.6 52 191 G200 Blue-dextran agarose 1220 6.3 191.1 44 382 *One unit is the production of 1 pmol pyruvate/min. tUnits/mg protein. SClarified crude homogenate.
Pyruvate kinase from pig brain Table 2. Summary of physical chemical, and kinetic parameters Parameter PBV’yK PH-PyK PL-PyK s 2o.w 9.3 1o.4b 8.1’ MW 230,000 237,000b 244,OOOd SU MW 60,000 59,OOOb 6 1,OOOd Subunits 4 4b 4 7.4 7.2b 6.9’ PH, 8.0 8.2b 5.4d, 6.1’ PI FDP act. No Yesb Yes’ Shape of curve0 H Hb S Specific activityh 191 163b 280’ “PB, pig brain; PH. pig heart; PL, pig liver; PM, pig muscle; PK, bKiffmeyer and Farrar (1991). ‘Kutzbach et al. (1973). dBerglund et al. (1977). ‘Berglund and Humble (1979). ‘Kobayashi et al. (1976). rH, hyperbolic; S, sigmoidal. hGiven as Units/mg protein.
brain PyK, like PyKs from other mammalian tissues, exists as a tetramer. The parameters which best distinguish and categorize the mammalian PyK isozymic forms (M, , M,, L, and R) seem to be the pH optimum for activity (pH,), the isoelectric point (PI), and kinetics [shape of the Michaelis-Menten curve (hyperbolic or sigmoidal with PEP as the variable substrate), and activation by FDP] (see Hall and Cottam, 1978, for a review). These parameters for pig brain PyK, and PyK isozymes from other pig tissues are summarized in Table 2. Figure 3 shows that pig brain PyK has a pH, of 7.4. This pH, thus excludes pig brain PyK from being categorized as a type L isozyme, which has a pH, of 6.9 (Kutzbach et al., 1973). At pH 6.9, pig brain PyK has only
1149 for pig pyruvate kinases PM-PyK PK-PyK 9.8 244,oOOd 236,000d 61,000d 59,OOOd 4 4 6.5’. 7.5’ 7.5’ 5.6d, 7.9’ 7.8’ No Yes H H 340 522 pig kidney.
75% of the activity at pH 7.4. However, the pH, is within the pH, range for types M, (7.5) and M, (7.5) (Berglund and Humble, 1979; see also Table 2). The pH, value alone is therefore unable to completely distinguish the isozyme category for pig brain PyK. The p1 of approx. 8.0 (Fig. 4) is similar to those of 7.8 and 7.9 reported for pig muscle PyK (type M, ) and pig kidney PyK (type M2), respectively (see Table 1 in Kobayashi et al., 1976). However, Berglund and Humble (1977a) reported a p1 of 5.6 for pig kidney PyK. The pI of Mz is therefore uncertain and cannot be used to distinguish between M, and Mz PyK isozymes in the pig. It may be of interest that the p1 pattern (Fig. 4) also shows two minor PyK peaks, one with a pI of about 7.4 and another
r
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.
./ ../
0.I b.6 _-
I
I
I
I
I
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6.8
7.0
7.2
7.4
1.6
7.8
PH Fig. 3. pH optimum (pHo) for pig brain PyK. Assay mixtures are as described under the Material and Methods section, except that the pH was varied between 6.7 and 7.7. Reactions were initiated by the addition of 0.25 pg of purified pig brain PyK. Activity is expressed as AA,,/min.
Fraction Fig. 4. Isoelectric focusing and isoelectric point (~1) of pig brain PyK. For focusing, each isoelectric focusing tube was prepared with ampholyte (pH range 3-10) and 0.2Oml of CCH in a 640% sucrose gradient. Sucrose solutions were prepared in deionized water. Focusing was at 22’C for 3.0-3.5 hr at 5 mA per tube. Following focusing, 0.10 ml fractions were collected and assayed for PyK activity (a), and pH (0). PyK was assayed as described in the Materials and Methods section. Final reaction volume was 1.Oml, and activity is given as AA,,/min.
Gabert Farrar and William W. Farrar
II.50 30
-
/
20 :
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./I
5
l/+ 10
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0
/’ 2
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4
6
8
10
12
l/PEP, mM Fig. 5. Kinetics of pig brain PyK with PEP as the variable substrate. Purified pig brain PyK (0.45 pg) was assayed in the standard reaction mixture given in the Materials and Methods section except that PEP was varied between 0.10 and 2.0 mM. Duplicate assays were performed at each PEP concentration. Activity is given as AA,,/min.
of about 6.5. Since isoelectric focusing was performed using the CCH, these pl data may be evidence that pig brain also has small amounts of other PyK isozymes, perhaps M, and L. The M, PyK has also been detected in ox brain (Standholm et al., 1976). Kinetically, PyK isozymes M,, L, and R are activated by FDP, and M, and M2 isozymes display hyperbolic steady-state kinetics with PEP as the variable substrate (see Hall and Cottam, 1978). Figure 5 shows that the Lineweaver-Burk plot of pig brain PyK with PEP as the variable substrate between 0.1-2.0 mM are linear, which indicates hyperbolic Michaelis-Menten kinetics. Moreover, the presence of 2 mM FDP resulted in no activation within the same PEP concentration range. These two kinetic results taken together lend good evidence that the PyK isozyme purified and examined in this study is M, . The various physical and kinetic properties for five isolated and studied PyKs from pig tissues are summarized in Table 2. In comparing the obtained data on pig brain PyK with that of other pig PyKs, especially the hyperbolic kinetics, lack of FDP activation, and p1, it appears that the best isozymic designation for pig brain PyK, based on these data, is as an M, isozyme. Acknowledgemenrs-This work was supported by an Eastern Kentucky University Faculty Research Grant. REFERENCES Baranowska B. and Baranowski T. (1975) Kinetic properties of human muscle pyruvate kinase. Molec. cell. Biochem. 6, 197-201.
Becker K. J., Geyer H., Eigenbrodt E. and Schoner W. (1986) Purification of pyruvate kinase isoenzymes type M, and M, from dog (Canis ,@riluris) and comparison of their properties with those from chicken and rat. Camp. Biochem. Physiol. 83B, 823-829. Behnke J. N., Massey T. H., Dagher S. M. and Deal W. C. Jr (1975) Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equipment: a two-peak transition in the approach-to-equilibrium. Anal. Biochem. 69, l-9. Berglund L. and Humble E. (1979) Kinetic properties of pyruvate kinase type A from kidney and type M from muscle. Arch. Biochem. Biophys. 195, 347-361. Berglund L., Ljungstrom 0. and Engstrom L. (I 977) Purification and characterization of pig kidney pyruvate kinase (type A). J. Biol. Chem. 252, 610861 I I. Bucher T. and Pflieder G. (1955) Pyruvate kinase from muscle. In Methods in Enzymology (Edited by Colowick S. W. and Kaplan N. 0.) Vol. I, pp. 435440, Academic Press, New York. Carbonell J., Feliu J. E., Marco R. and Sols A. (1973) Pyruvate kinase: classesof regulatory isoenzymes in mammalian tissues. Eur. J. Biochem. 37, 148-156. Cardenas J. M. and Dyson R. D. (1973) Bovine pyruvate kinases. II. Purification of the liver isozyme and its hybridization with skeletal muscle pyruvate kinase. J. Biol.
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Cardenas J. M., Dyson R. D. and Strandholm J. J. (1973) Bovine pyruvate kinases. I. Purification and characterization of the skeletal muscle isozyme. J. Biol. Chem. 248, 693 16937. Cardenas J. M., Blachly E. G., Ceccotti P. L. and Dyson R. D. (1975) Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes. Biochemistry 14, 2247-2252. Chern C. J., Rittenberg M. B. and Black J. A. (1972) Purification of human erythrocyte pyruvate kinase. J. Biol. Chem. 247, 7173-7180. Eigenbrodt E. and Schoner W. (1977) Purification and properties of the pyruvate kinase isoenzymes type L and M2 from chicken liver. Hoppe-Seyler’s Z. Physiol. Chem. 358, 103331046. Farrar W. W. and Farrar Y. J. K. (1983) Energy enzymes from the flight muscle of the house sparrow, Passer domesticus-I. Activity levels of the glycolytic enzymes from the soluble fraction. Camp. Biochem. Biophys. 74B, 549955 I. Hall E. R. and Cottam G. L. (1978) Isoenzymes of pyruvate kinase in vertebrates: their physical, chemical, kinetic and immunological properties. Int. J. Biochem. 9, 785-793. Harkins R. N., Black J. A. and Rittenberg M. B. (1977) Purification and characterization of human muscle pyruvate kinase. Can. J. Biochem. 55, 301-307. Imamura K. and Tanaka T. (1982) Pyruvate kinase isozymes from rat. In Methods in Enzymology (Edited by Wood W. A.), Vol. 90, pp. 150-165. Academic Press, New York. Kahn A. and Marie J. (1982) Pyruvate kinases from human erythrocytes and liver. In Methodr in Enzymology (Edited by Wood W. A.), Vol. 90, pp. 131-139. Academic Press, New York. Kiffmeyer W. R. and Farrar W. W. (1991) Purification and properties of pig heart pyruvate kinase. J. Prof. Chem. 10, 585-59 I.
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Vol. 27, No. II, pp. 114%1151, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. Ail rights reserved 1357-272519s $9.50 + 0.00 Bid.
Pwification and Properties of the Pyruvate Ki Isozyme M, from the Pig Brain GABERT
FARRAR,
WILLIAM
W. FARRAR”
Department
of Biological Sciences, Eastern Kentucky University, Richmond, KY 40475, U.S.A.
There are four pyruvate kinase isozymesin vertebrate tissues,designated as L, M, , M2, ssd R. Although pyravate kiaases have been par&d and characte&ed from pig liver, muade, kidney, and heart, the brain isozymehas not. The ahn of this work was to purify, characteri~ and make an isozymic designation for the pig brain pyruvate kinase. Puriftatlom was by chromatography on phosphocelhdose,Sephadex G200, and bwextraa agarose cobmms. The . moiecalar weight of the native enzyme was determined by sucrose de&ty w The degree of parity, and subunit molecular weight were determined by poiyac@amide gel ele&ophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid W&tric focusing method in sucrosegradients. The pH opthnum, aad kinetics in the preaeaceand abseace of fractose-1,6diphosphate were determined spectrophotometrically. The pmi@Wion &etne used redted in a 382-fold paritlcation of pig brain pyruvate khmse.,aRd a &ml specitlc activity of 191 Units/mg protein. As estimated by scaoniog of the dmn do&cyl s&ate poiyacryhm&le gels, the par&&ion scheme also resulted in a preparatioa that was of at least 98% parity, Pig brain pyruvate kinase has a native molecular weight of approx. 23fI,OWJ,and a s&t&t m&c&r weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimmn was estimated at pH 7.4. Fructose-l&-diphosphate had no et&et on either the K, for phospho(enol)pywvate, or the V,, of the reaction. Pig brain pyruvate kinase is similar in native and subunit molecular weights to the other pyravate kieases from pig &aaes. Like other vertebrate pyruvate kinases, pig brain pyruvate kinase is a tetramer. The pH optimum of 7.4 excludes pig brain pyruvate kinase as a type L isozyme, but cannot disting&h between MI and M, isozymes.An isoelectric point of 8.0, as well as hyperbolic k&tics aud the lack of inactivation by fructose-l&diphosphate, however, are consistent with the pyruvate kinase from pig brain being categorized as a type M, isozyme. Keywords: Pyruvate kinase Brain
Isozymes Pig Purification
Properties
ht. J. Biochem. Cell Biof. (1995) 27, 1145-l 151
The liver isozyme (PyK-L) has been purified from livers of rat (Tanaka et al., 1967; Imamura and Tanaka, 1982), ox (Cardenas and Dyson, 1973), pig (Kutzbach et al., 1973) chicken (Eigenbrodt and Schoner, 1977), and human (Kahn and Marie, 1982). Kinetically, PyK-L isozymes isolated from these sources are activated by fructose-l$&diphosphate (FDP). PyK-M, is the most widely distributed isozyme and has been detected and examined in rat epididymal adipose tissue (Pogson, 1968; Marco et al., 1971; Carbonell et al., 1973) and purified and characterized from hepatoma (Imamura et al., 1972), leucocytes (Van Berkel and Koster, 1973), rat liver (Imamura et ul.,
INTRODUCTION
There are four known isozymic forms of pyruvate kinase (ATP: Pyruvate phosphotransferase; EC 2.7.1.40; PyK) in vertebrates, designated as L, M,, M, (or A), and R. These isozymic forms differ in their chemical, physical, kinetic, electrophoretic, and immunological properties, as well as their tissue distributions. Their parameters have been extensively reviewed (Hall and Cottam, 1978).
*To whom all correspondence should be addressed. Received 6 April 1995; accepted 21 June 1995. 1145
1146
Gabert Farrar and William W. Farrar
1972), and pig kidney (Berglund et al., 1977). Moreover, although PyK-M, is the minor PyK isozyme form in liver, it is nevertheless the major form in kidney. Within the liver, PyK-L is found in parenchymal cells, and PyK-M, in Kupffer cells (Van Berkel et al., 1972). Kinetically, PyK-L and PyK-M, are similar in that both are inhibited by adenosine triphosphate (ATP) and alanine, and activated by FDP. These two isozymes, however, differ in that PyK-L displays sigmoidal kinetics, whereas PyK-M, shows hyperbolic kinetics when phospho-(enol)pyruvate (PEP) is the variable substrate. Moreover, the presence of FDP decreases the K,,, of PEP for PyK-M, much less than it does for the PyK-L isozyme (see Hall and Cottam, 1978). PyK-M, , the skeletal muscle isozyme, has been purified from the muscle of rabbit (Steinmetz and Deal, 1966), rat (Tanaka et al., 1967; Imamura and Tanaka, 1982), ox (Cardenas et al., 1973), chicken (Cardenas et al., 1975), human (Baranowska and Baranowski, 1975; Harkins et al., 1977), dog (Wu et al., 1981; Becker et al., 1986), and pig (Berglund and Humble, 1979) and from the brain of ox (Terlecki, 1989), and partially purified and kinetically studied from guinea-pig (Nicholas and Bachelard, 1974). Kinetically, PyK-M, is not activated by FDP and displays hyperbolic Michaelis-Menten kinetics. Human erythrocyte (Chern et al., 1972; Kahn and Marie, 1982) PyK has also been isolated. As with PyK-L, this isozymic form is activated by FDP and displays sigmoidal kinetics for PEP in the absence of FDP. PyK has been purified from the heart muscle of turtle (Storey and Hochachka, 1974), ox (Parkinson and Easterby, 1977), and pig (Kiffmeyer and Farrar, 1991). PyK isozymes have also been isolated and characterized from several additional pig tissues, including liver (Kutzbach et al., 1973), kidney (Berglund et al., 1977) and muscle (Berglund and Humble, 1977). PyK-M, predominates in skeletal muscle, and brain, and can be found as a minor isozyme component in heart tissue (see Hall and Cottam, 1978). PyK has been isolated and studied from rat brain (Srivastava and Baquer, 1985) where its kinetic properties suggest that it is an isozyme distinct from muscle and liver forms, and from bovine brain (Terlecki, 1989) where its kinetic properties support an M, isozymic designation. In our report we present the purification of PyK from pig brain, and data on its native
molecular weight, subunit molecular weight, quaternary structure, pH optimum (pH,), isoelectric point (PI), and lack of activation by FDP. These parameters are compared with those of other pig tissues PyKs, and, where possible, with rat and bovine brain PyKs in an effort to make an isozymic assignment. MATERIALS
AND
Materials and preparation homogenate (CCH)
METHODS
of the clarified crude
Pig brains were obtained from Slade Packing Co., Cynthiana, Ky and immediately frozen until use. Adenosine diphosphate (ADP), phospho(enol)pyruvate (PEP), fructose- 1,Bdiphosphate (FDP), reduced nicotinamide adenine dinucleotide (NADH), pyruvate, phosphocellulose, Sephadex G200, ampholyte, blue dextranagarose, and the enzymes lactate dehydrogenase (LDH; rabbit muscle), enolase (yeast), aldolase (rabbit muscle), pyruvate kinase (PyK; rabbit muscle), and phosphorylase b (rabbit muscle) were all purchased from Sigma Chemical Co. Ultrapure ammonium sulfate and sucrose were obtained from Schwartz-Mann. All other chemicals were of reagent-grade purity. For preparation of the centrifuged crude homogenate (CCH), appropriate amounts of brain tissue were thawed, weighted and homogenized for 2 min in 2 vol of cold (4°C) 50 mM imidazole, 2 mM MgCl,, pH 6.5, using a glass homogenizer with a motor driven teflon pestle. This crude homogenate was then centrifuged at 40,OOOg at 4°C for 30 min to obtain the CCH. The CCH was used to initiate purification, to obtain the isoelectric point (PI), and to obtain the native molecular weight by sucrose density ultracentrifugation. Protein determination
Protein was estimated by absorbancy at 280/260 nm (Warburg and Christian, 1943), using a Bausch & Lomb Spectronic 2000 spectrophotometer. Assays for PyK and LDH activities
PyK activity was routinely measured using a Bausch & Lomb Spectronic 2000 spectrophotometer by coupling pyruvate formation to the LDH reaction (Bucher and Plleiderer, 1975) and monitoring the disappearance of NADH at 340 nm. The reaction mixture was 50 mM imidazole, 100 mM KCl, 2 mM MgCl,, 1 mM PEP, 1 mM ADP, 0.02 mM NADH, 5 Units of LDH,
Pyruvate
kinase
pH 7.4. Reaction volume was 1.0 ml and the temperature was 22°C. Rabbit muscle LDH activity was measured by monitoring the disappearance of NADH at 340 nm. Final reaction mixture component concentrations were: 50 mM sodium phosphate, 1.0 mM pyruvate, 0.20 mM NADH, pH 6.8. Reaction volume was 1.0 ml and the temperature was 22°C (Farrar and Farrar, 1983). Isoelectric focusing The isoelectric point (PI) for pig brain was determined by the rapid focusing method (Behnke et al., 1975) with slight modifications, in 540% sucrose gradients, using ampholytes in the pH 3-10 range. The reservoir buffers were 3% ethylenediamine and 0.5% H,S04. For focusing, 0.2 ml of the CCH was used. Focusing was performed at 22°C at 5 ma/tube. Usually, focusing of the proteins in the CCH took between 2.5-3.5 hr. Sucrose density gradient centrifugation Linear 5-20% sucrose gradients in 50mM imidazole, 100 mM KCl, 2 mM MgCl,, 1 mM PEP, 1 mM ADP, pH 7.0, were used to determine the SZO,wand the molecular weight of the native enzyme (Martin and Ames, 1961). Rabbit muscle LDH was used as a standard of known S2o.w and molecular weight. Centrifugation was at 40,000 rpm (4°C) for 17 hr in a Beckman L5-75 Preparative Ultracentrifuge using an SW 60 rotor. Sodium dodecyl (lauryl) sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) The subunit molecular weight of pig brain PyK was estimated by SDS-PAGE (Weber and Osborn, 1969) in 8% gels. This method was also used to estimate the degree of purity following the final step of purification. Steady-state kinetics The K, and V,,,,, in the presence and absence of FDP were determined using the reaction mixture described under Assay for PyK activity, but with PEP as the variable substrate at eight concentrations between 0.10 and 2.0 mM. Purified PyK was used. PuriJication of pig brain PyK Purification of pig brain PyK followed the same general procedure as for pig heart PyK (Kiffmeyer and Farrar, 1991) with the exception
from
1147
pig brain
that an additional blue dextran-agarose chromatography step was necessary following gel filtration on Sephadex G200. All steps in the purification, except the preparation of the CCH, and ammonium sulfate dialysis, were performed at room temperature (21-25°C). For phosphocellulose chromatography, a 2.5 cm x 40 cm column was equilibrated with 25 mM imidazole, 1 mM MgCl?, pH 6.5. The CCH was applied to the column and flow-through was with the same buffer. PyK adhered to the phosphocellulose, and after the protein concentration in the eluting fractions became minimal, the KC1 concentration was adjusted to 0.15 M. and then 0.40 M. Additional protein was eluted at each of these KC1 concentrations, but PyK remained on the column. PyK was eluted when the KC1 concentration was made 0.6 M. All fractions containing PyK activity were pooled. The pooled fractions were dialyzed for 12 hr at 4°C against 80% saturated ammonium sulfate in 25 mM imidazole, 100 mM KCl, 1 mM pH 7.0. MgCl,, 2 mM fl-mercaptoethanol, The resulting precipitate was collected by centrifugation. No PyK activity was found in the supernate. The ammonium sulfate precipitate was dissolved in a minimum volume of 25 mM 0.15
.* .I. !’ii .4
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Fraction Fig. 1. Sucrose density gradient ultracentrifugation of pig brain PyK (0) with rabbit muscle LDH (0) as a standard. For centrifugation, 0.10 ml each of pig brain CCH and rabbit muscle LDH were layered on top of individual gradients. Following centrifugation, the total volume within each gradient tube was removed in O.lOml fractions (48 fractions from each). Fractions from the pig brain PyK tube were assayed for PyK, and those from the LDH standard tube were assayed for LDH as described in the M a t erials and Methods section. Activity is expressed as AA,,,/min/assay/aliquot of 0.020 ml.
Gabert Farrar and William W. Farrar
1148 5.0
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RESULTS AND DISCUSSION
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Mobility Fig. 2. SDS polyacrylamide (8%) gel electrophoresis of purified pig brain PyK. Standard of known subunit molecular weights are: rabbit muscle phosphorylase b (Phb, MW = 94,000); rabbit muscle PyK (RM PyK, MW = 57,000); yeast enolase (Enolase, MW = 42,000); rabbit muscle LDH (LDH, MW = 36,500); PB PyK is designated as PB PyK (MW = 60,000). Mobilities are relative to the migration distance of bromophenol blue. Inset is a scan of the SDS gel at 500 nm.
imidazole, 100 mM KCl, 1 mM MgCl,, 2 mM p-mercaptoethanol, pH 7.0 and applied to a Sephadex G200 column (1.5 cm x 70 cm) that had been equilibrated with the same buffer. Fractions of 1.O ml were collected. PyK eluted in the void volume and fractions containing substantial PyK activity were pooled. The pooled fractions from gel filtration were dialyzed extensively against 10 mM imidazole, 1 mM MgCl,, pH 7.5, and a blue dextranagarose column was equilibrated with the same buffer. The dialyzed sample was then applied to the column at room temperature and 1.0 ml fractions collected. Under these conditions, PyK adhered, but some proteins eluted. Over 85% of the PyK activity was specifically eluted by the addition of 1.0 mM ADP to the column buffer.
The purification procedure adapted from that used for pig heart PyK (Kiffmeyer and Farrar, 1991) works very well for pig brain PyK. The results of a typical purification from 100 g of pig brain are given in Table 1. As with pig heart PyK (Kiffmeyer and Farrar, 1991) rat brain (Srivastava and Baquer, 1985), and bovine brain (Terlecki, 1989), phosphocellulose column chromatography proved to be the best purification step. The final specific activity of 191 Units/mg protein for pig brain PyK (Table 1) is similar to the 217 Units/mg protein for purified bovine brain PyK (Terlecki, 1989), but somewhat lower than the specific activity of 593 of rat brain PyK (Srivastava and Baquer, 1985). However, this latter difference is most likely due to both the significantly lower assay temperature used for pig brain PyK (22°C) vs that used for rat brain PyK (37°C) and different assay buffers and component concentrations. Figure 1 shows the results of sucrose density ultracentrifugation of pig brain PyK using rabbit muscle LDH as a standard. Using an S20,W of 7.45 for LDH yielded a calculated SzO,W for pig brain PyK of 9.3, which corresponds to a native molecular weight of approx. 230,000, similar to that of PyKs from pig liver (Kutzbach et al., 1973; Berglund et al., 1977) muscle, and kidney (Berglund et al., 1977). SDS-PAGE of the combined PyK-containing fractions from the blue-dextran agarose column showed one major band plus one minor band of lesser molecular weight. The scan of the SDSPAGE gel of purified pig brain PyK revealed that the major band comprised about 98% of the total protein applied, while the minor band contributed -2% (Fig. 2) demonstrating that the purification protocol (Table 1) for pig brain PyK results in essentially homogeneous PyK. The molecular weight of the subunit is estimated to be approx. 60,000 (Fig. 2). These results, along with those from sucrose density ultracentrifugation (Fig. l), demonstrate that native pig
Table 1. Purification scheme for pig brain pyruvate kinase (based on 100 g starting tissue) Total Specific Total % Fold Step units* activity? Yield protein (mg) purification 2758 5256 0.5 100 1 CCH$ 27.3 81 55 P-cellulose 2449 89 1434 15 95.6 52 191 G200 Blue-dextran agarose 1220 6.3 191.1 44 382 *One unit is the production of 1 pmol pyruvate/min. tUnits/mg protein. SClarified crude homogenate.
Pyruvate kinase from pig brain Table 2. Summary of physical chemical, and kinetic parameters Parameter PBV’yK PH-PyK PL-PyK s 2o.w 9.3 1o.4b 8.1’ MW 230,000 237,000b 244,OOOd SU MW 60,000 59,OOOb 6 1,OOOd Subunits 4 4b 4 7.4 7.2b 6.9’ PH, 8.0 8.2b 5.4d, 6.1’ PI FDP act. No Yesb Yes’ Shape of curve0 H Hb S Specific activityh 191 163b 280’ “PB, pig brain; PH. pig heart; PL, pig liver; PM, pig muscle; PK, bKiffmeyer and Farrar (1991). ‘Kutzbach et al. (1973). dBerglund et al. (1977). ‘Berglund and Humble (1979). ‘Kobayashi et al. (1976). rH, hyperbolic; S, sigmoidal. hGiven as Units/mg protein.
brain PyK, like PyKs from other mammalian tissues, exists as a tetramer. The parameters which best distinguish and categorize the mammalian PyK isozymic forms (M, , M,, L, and R) seem to be the pH optimum for activity (pH,), the isoelectric point (PI), and kinetics [shape of the Michaelis-Menten curve (hyperbolic or sigmoidal with PEP as the variable substrate), and activation by FDP] (see Hall and Cottam, 1978, for a review). These parameters for pig brain PyK, and PyK isozymes from other pig tissues are summarized in Table 2. Figure 3 shows that pig brain PyK has a pH, of 7.4. This pH, thus excludes pig brain PyK from being categorized as a type L isozyme, which has a pH, of 6.9 (Kutzbach et al., 1973). At pH 6.9, pig brain PyK has only
1149 for pig pyruvate kinases PM-PyK PK-PyK 9.8 244,oOOd 236,000d 61,000d 59,OOOd 4 4 6.5’. 7.5’ 7.5’ 5.6d, 7.9’ 7.8’ No Yes H H 340 522 pig kidney.
75% of the activity at pH 7.4. However, the pH, is within the pH, range for types M, (7.5) and M, (7.5) (Berglund and Humble, 1979; see also Table 2). The pH, value alone is therefore unable to completely distinguish the isozyme category for pig brain PyK. The p1 of approx. 8.0 (Fig. 4) is similar to those of 7.8 and 7.9 reported for pig muscle PyK (type M, ) and pig kidney PyK (type M2), respectively (see Table 1 in Kobayashi et al., 1976). However, Berglund and Humble (1977a) reported a p1 of 5.6 for pig kidney PyK. The pI of Mz is therefore uncertain and cannot be used to distinguish between M, and Mz PyK isozymes in the pig. It may be of interest that the p1 pattern (Fig. 4) also shows two minor PyK peaks, one with a pI of about 7.4 and another
r
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./ ../
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I
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6.8
7.0
7.2
7.4
1.6
7.8
PH Fig. 3. pH optimum (pHo) for pig brain PyK. Assay mixtures are as described under the Material and Methods section, except that the pH was varied between 6.7 and 7.7. Reactions were initiated by the addition of 0.25 pg of purified pig brain PyK. Activity is expressed as AA,,/min.
Fraction Fig. 4. Isoelectric focusing and isoelectric point (~1) of pig brain PyK. For focusing, each isoelectric focusing tube was prepared with ampholyte (pH range 3-10) and 0.2Oml of CCH in a 640% sucrose gradient. Sucrose solutions were prepared in deionized water. Focusing was at 22’C for 3.0-3.5 hr at 5 mA per tube. Following focusing, 0.10 ml fractions were collected and assayed for PyK activity (a), and pH (0). PyK was assayed as described in the Materials and Methods section. Final reaction volume was 1.Oml, and activity is given as AA,,/min.
Gabert Farrar and William W. Farrar
II.50 30
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l/+ 10
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6
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l/PEP, mM Fig. 5. Kinetics of pig brain PyK with PEP as the variable substrate. Purified pig brain PyK (0.45 pg) was assayed in the standard reaction mixture given in the Materials and Methods section except that PEP was varied between 0.10 and 2.0 mM. Duplicate assays were performed at each PEP concentration. Activity is given as AA,,/min.
of about 6.5. Since isoelectric focusing was performed using the CCH, these pl data may be evidence that pig brain also has small amounts of other PyK isozymes, perhaps M, and L. The M, PyK has also been detected in ox brain (Standholm et al., 1976). Kinetically, PyK isozymes M,, L, and R are activated by FDP, and M, and M2 isozymes display hyperbolic steady-state kinetics with PEP as the variable substrate (see Hall and Cottam, 1978). Figure 5 shows that the Lineweaver-Burk plot of pig brain PyK with PEP as the variable substrate between 0.1-2.0 mM are linear, which indicates hyperbolic Michaelis-Menten kinetics. Moreover, the presence of 2 mM FDP resulted in no activation within the same PEP concentration range. These two kinetic results taken together lend good evidence that the PyK isozyme purified and examined in this study is M, . The various physical and kinetic properties for five isolated and studied PyKs from pig tissues are summarized in Table 2. In comparing the obtained data on pig brain PyK with that of other pig PyKs, especially the hyperbolic kinetics, lack of FDP activation, and p1, it appears that the best isozymic designation for pig brain PyK, based on these data, is as an M, isozyme. Acknowledgemenrs-This work was supported by an Eastern Kentucky University Faculty Research Grant. REFERENCES Baranowska B. and Baranowski T. (1975) Kinetic properties of human muscle pyruvate kinase. Molec. cell. Biochem. 6, 197-201.
Becker K. J., Geyer H., Eigenbrodt E. and Schoner W. (1986) Purification of pyruvate kinase isoenzymes type M, and M, from dog (Canis ,@riluris) and comparison of their properties with those from chicken and rat. Camp. Biochem. Physiol. 83B, 823-829. Behnke J. N., Massey T. H., Dagher S. M. and Deal W. C. Jr (1975) Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equipment: a two-peak transition in the approach-to-equilibrium. Anal. Biochem. 69, l-9. Berglund L. and Humble E. (1979) Kinetic properties of pyruvate kinase type A from kidney and type M from muscle. Arch. Biochem. Biophys. 195, 347-361. Berglund L., Ljungstrom 0. and Engstrom L. (I 977) Purification and characterization of pig kidney pyruvate kinase (type A). J. Biol. Chem. 252, 610861 I I. Bucher T. and Pflieder G. (1955) Pyruvate kinase from muscle. In Methods in Enzymology (Edited by Colowick S. W. and Kaplan N. 0.) Vol. I, pp. 435440, Academic Press, New York. Carbonell J., Feliu J. E., Marco R. and Sols A. (1973) Pyruvate kinase: classesof regulatory isoenzymes in mammalian tissues. Eur. J. Biochem. 37, 148-156. Cardenas J. M. and Dyson R. D. (1973) Bovine pyruvate kinases. II. Purification of the liver isozyme and its hybridization with skeletal muscle pyruvate kinase. J. Biol.
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Weber K. and Osborn M. (1969) The reliability of molecular weight determination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Gem. 244, 44064412. Wu S. W. N., Wong S. C. and Yeung D. (1981) Purification and properties of dog muscle pyruvate kinase. J. Biochem. 13, 147-152.