Purification and characterization of human meningioma M2-type pyruvate kinase

Clin Biochem, Vol. 26, pp. 383-388, 1993

0009-9120/93 $6.00 + .00 Copyright ©1993 The Canadian Society of Clinical Chemists.

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Purification and Characterization of Human Meningioma M2-Type Pyruvate Kinase ALi A. MELLATI, 1 MERAL YUCEL, 1 NUR ALTINORS, 2 and UFUK GONDOZ~ 1Biology Department, Middle East Technical University, 06531 Ankara and 2Department of Neurosurgery, The Hospital of Sosyal Sigortalar, Ankara, Turkey The M2-type pyruvate kinase was purified from human meningioma by ammonium sulfate precipitation, followed by ion exchange and affinity chromatography. The specific activity of the purified enzyme was 33.4 U/mg with a yield of 6.5%. The enzyme gave a single band with 63,000 -+ 2000 Da upon SDS polyacrylamide gel electrophoresis. On cellulose acetate electrophorasis zymograms, the purified enzyme (M2) showed a single band, while crude extracts gave two broad bands corresponding to pyruvate kinase isozymes. The pl value of purified enzyme was found to be 6.9. With phosphoenol pyruvate as substrate the purified enzyme showed sigmoidal kinetics, while in the presence of 0.6 mM fructose 1,6-diphosphate as modulator it gave a hyperbolic saturation curve with a Km value of 0.53 mM.

KEY WORDS:pyruvate kinase, M2-type; meningioma; brain tumor; isoenzymes;enzymepurification. Introduction kinase (PK; ATP: pyruvate phosp )yruvate photransferase, EC 2.7.1.40) catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. Four isozymes have been described in mammalian tissues: the L form occurs mainly in liver; R in red cells; M1 in muscle, heart, and brain; M2 in kidney and other adult and fetal tissues and tumors (1,2). The normal developmental pattern of PK is changed in tumors. In tumors of various tissues a shift from adult to fetal cellular characteristics has been described (3-5). Ml-type PK, which is the major isozyme in normal brain, is replaced by M2-type in gliomas and meningiomas (6-8). Studies of M1 and M2 type PKs in different species including humans have shown that their properties are not identical, although the enzymes are immunologically closely related (9-11). The M1type PK shows hyperbolic kinetics, is not activated by fructose 1,6-diphosphate (F-1,6-DP) and is not affected by L-alanine (12-14). The M2-type isozyme shows sigmoidal kinetics with regard to PEP, is activated by F-1,6-DP and is inhibited by L-alanine (12,15).

The presence of M2-type PK isozyme in human meningioma could have diagnostic importance. In meningiomas and gliomas a shift from adult M1type PK to fetal M2-type PK was also observed (716). Moreover, the shift of the M1 to M2 type was correlated with the degree of malignancy. The M1 and M2 type PKs are both immunologicaUy related and share a common antigenic reactivity (10); however, they show different kinetic properties. For instance, Ml-type isozyme is not influenced by L-alanine whereas M2-type is strongly inhibited (14). From a clinical point of view, the determination of the percentage of alanine inhibition may be a valuable tool assisting in tumor diagnosis. We report the purification of a pyrnvate kinase from human meningioma and its characterization as M2-type isozyme. Further kinetic characteristics of this isozyme were described in another report (17). Materials and methods

MATERIALS Tumor tissues (fibroblastic meningioma) were obtained from the Department of Neurosurgery, The Hospital of Sosyal Sigortalar in Ankara. Reactive Blue 4-Agarose, carboxymethylcellulose, 6-amino-n-caproic acid, phosphoenolpyruvate ( m o n o p o t a s s i u m salt), MOPS (3-[NMorpholino] propanesulfonic acid) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Adenosine-5'-diphosphate, NADH (disodium salt), lactate dehydrogenase (LDH, rabbit muscle), F-1,6-DP were obtained from Boehringer (Mannheim, Germany). Common chemicals and acrylamide, N:N'-methylene-bis-acrylamide came from Sigma Chemical Company. METHODS

Purification of PK from human meningioma Correspondence:Prof. Ufuk GOndtiz. Manuscript received June 8, 1992; revised November 11, 1992, February 2, 1993, March 25, 1993; accepted March 31, 1993. CLINICALBIOCHEMISTRY,VOLUME26, OCTOBER1993

The purification was carried out according to previously reported methods with some modifications (10,16). A piece (45 g) of human fibroblastic meningioma 383

MELLATI, YUCEL, ALTINORS, AND GUNDUZ

was washed free of blood in 100 mM Tris-HC1 buffer (pH 7.0) at room temperature. All subsequent steps were performed at 4 °C. The tissue was homogenized for 1.5 min in a Waring blender in five volumes of 100 mM Tris-HC1 buffer (pH 7.0) containing 1 mM EDTA and 20 mM MgC12. The homogenate was centrifuged at 10,000 x g for 15 min. The supernatant was removed and centrifuged at 108,000 x g for 1 h. The supernatant was brought to 40% saturation by solid (NH4)2SO4 and the precipitate was suspended in 20 mL of H20 and stirred for 1 h. The solution was desalted on a Sephadex G-25 column (35 × 2.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 5.5) containing 1 mM 6-aminocaproic acid, 1 mM 2-mercaptoethanol, and 5 mM MgC12. Active fractions were collected and applied to a CMcellulose column (22 x 1.8 cm) equilibrated with the above buffer. PK was eluted with a linear KC1 gradient (0 to 0.4 M) in 200 mL of the same buffer. PK containing fractions were combined and dialyzed overnight against 1 L of 20 mM Tris-HC1 buffer (pH 7.0) containing 1 mM 2-mercaptoethanol, 1 mM 6-aminocaproic acid, and 5 mM MgC12. The dialyzed PK was applied onto a Blue 4-Agarose column (1.5 x 8 cm) previously equilibrated with dialysis buffer. The column was washed with dialysis buffer until the eluate showed no absorption at 280 nm. PK was then eluted by a gradient of KC1 from 0 to 500 mM, in 100 mL of the above buffer containing 0.4 mM F-1,6-DP. Three fractions that had the highest PK activity were collected and stored in 2 M ammonium sulfate or 25% ethyleneglycol at 4 °C.

Enzymatic assay PK activity was measured in the coupled LDH assay as described by Bficher and Pfleiderer (18). The assay mixture contained, in a final volume of 0.6 mL: 25 mM Tris-HC1 buffer (pH 7.2); 0.21 mM NADH; 1.5 units of LDH; 10 mM 2-mercaptoethanol; 5 mM MgC12; 50 mM MOPS (pH 7.0); 0.6 mM F-1,6-DP; 2 mM ADP; different concentrations of PEP (0.05-5.0 mM); and 10 ~L enzyme. The mixture was incubated for 3 min at 30 °C. Reactions were initiated by the addition of enzyme. The activity was measured by following the decrease of absorbance of NADH at 340 nm. One unit of PK activity was defined as the amount of enzyme converting 1 ~mol of PEP per min at 30 °C, and specific activity was given as enzyme units/mg of protein. The protein concentration was determined according to Lowry et al. (19) with crystalline bovine serum albumin as standard.

Cellulose acetate electrophoresis Electrophoresis was carried out according to Cardenas and Dyson (20). Separation of PK isozymes on cellulose acetate strips (Sartorius GmbH, 3400 Gottingen, Germany, 7 x 7 cm) was carried out for 4 384

h at 85 V (12 V/cm) at room temperature in an LKB electrophoresis apparatus (LKB Produkter, Bromma, Sweden). After electrophoresis the cellulose acetate paper was removed and the bands of PK activity were visualized by pressing the cellulose acetate strips against agar reagent assay plate, containing components of the LDH coupled assay, and allowed to stay at 30 °C for 30 min. The reaction was followed visually by observing changes in intensity of fluorescence of the pyridine nucleotide (NADH) upon illumination with short-range UV lamp. After bands appeared, the patterns were photographed by Bioblock Scientific (B.P.III-67403 Illkirch, Cedex, France) UV Trait using a 667 Polaroid film.

SDS-Polyacrylamide gel electrophoresis SDS-Gel electrophoresis was carried out using 4.7% stacking and 12,5% separatory polyacrylamide slab gels according to Laemli (21). The subunit molecular weight was determined according to Weber and Osborn (22). The gels were run for 10 h at room temperature at 20 mA and stained with Coomassie Brillant Blue.

I soelectrofoc us ing The pI value of M2-type pyruvate kinase was determined by isoelectrofocusing according to O'Farrell (23) in 5% ampholyte (pH 3-10, 5-8) for 10 h at 400 V, and 8 h at 800 V at room temperature. After electrophoresis the gels were removed, sliced into 0.5-cm pieces added into tubes containing 2 mL H20. The tubes were closed, left for 1 h, and after vortexing the pH of the solution was measured. Apparent pH versus distance from the cathode (cm) was drawn. The protein band was detected by both staining with Coomassie Blue and scanning with a CAMAG Model 750241 densitometer (Scientific Instrument Inc., Basel, Switzerland). The position of the protein band was compared with the control gels electrophoresed under the same conditions and the corresponding pI value was determined.

Results PURIFICATION OF M2-TYPE PYRUVATE KINASE

Table i shows the results of the purification of the M2-type PK from human meningioma. The purification was carried out on samples from five different patients and reproducible results were obtained. The enzyme was purified 49.3-fold with an overall yield of 6.5% and total specific activity of 33.4 U/mg of protein. The specific activity of the peak fractions was 112.5 U/mg protein, representing a 166.2-fold purification. By CM-cellulose column chromatography the resolution of M1 and M2 isozymes was achieved (Figure 1). Ml-type isozyme eluted at 0 to 0.1 M KC1 gradient and its activity was independent of F-1,6-DP. Affinity c h r o m a t o g r a p h y on Blue CLINICAL BIOCHEMISTRY, VOLUME 26, OCTOBER 1993

MENINGIOMA M2-TYPE PYRUVATEKINASE TABLE 1

Purification of M2-Type Pyruvate Kinase From Human Meningioma

Purification Steps 10,000 x g supernatant 108,000 × g supernatant CM-Cellulose column chromatography Affinity chromatography on Blue 4-Agarose Peak fraction

Total Enzyme Activity (U)

Total Protein (mg)

1,003 994

1,482 726

305.9

142.3

65.1 24.75

4-Agarose proved to be an efficient method in the purification of tumor enzyme (Figure 2). ELECTROPHORETIC CHARACTERIZATION OF PURIFIED PYRUVATE KINASE

The electrophoretic behavior of purified P K from h u m a n meningioma was tested in three different systems: SDS-polyacrylamide gel electrophoresis (Figure 3), cellulose acetate electrophoresis, and isoelectrofocusing in polyacrylamide gel (not shown). The single band obtained with the purified enzyme on SDS-polyacrylamide gel suggested that the subunits were very similar or identical in molecular weight (MW). The subunit MW was calculated as 63,000 -+ 2000 by interpolation of the best-fit line through the points plotted for the mobilities of the standard proteins run under the same conditions. On cellulose acetate zymograms crude extracts gave two broad bands (data not given) while a single band was observed with the purified enzyme. The purified M2-type P K from h u m a n meningioma was

Specific Activity (U/rag)

1.95

0.22

Purification Factor

0.677 1.37

1 2.02

2.15

3.18

33.4 112.5

49.3 166.2

electrofocused. A single band with a pI value of 6.9 was obtained. DETERMINATION OF K~ AND Vm~ VALUES OF M2TYPE PK FOR IT8 SUBSTRATES Purified h u m a n meningioma P K showed sigmoidal kinetics in the presence of 0 - 0 . 3 m M F-1,6-DP, to varying concentrations of PEP, and at a fixed concentration of ADP (2 mM). In the presence of 0.6 m M F-1,6-DP however, the kinetic response to P E P concentration was normalized to a hyperbolic form in the Michaelis-Menten plot (Figure 4). The cooperativity as indicated by a Hill coeffÉcient of n = 2.3, decreased (n = 1.03) in the presence of 0.6 m M F-1,6-DP (Figure 5). In Table 2 the Km and Vm~ values of the enzyme are given. In the presence of 0.6 mM F-1,6-DP, the Km value for P E P was 0.53 mM (Figure 6). By keeping the P E P concentration fixed at 2 mM, the K ~ for ADP was calculated as 0.58 m M (data not shown). STABILITY OF ENZYME The stability of the purified enzyme upon storage was investigated. The enzyme solution was sus-

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Figure 1 - - CM-Cellulose column chromatography of isozymes of pyruvate kinase from human meningioma. Column dimensions: 22 x 1.8 cm; flow rate: 40 mlJh; fraction volume: 2.5 mL; wash buffer: 50 mM potassium phosphate buffer (pH 5.5), 1 mM 6-Rminocaproic acid, 1 mM 2-mercaptoethanol, 5 mM MgC12. ({~--Q) absorbance at 280 nm; ( A - - A ) pyruvate kinase activity (U/mL). CLINICAL BIOCHEMISTRY,VOLUME26, OCTOBER 1993

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Figure 2 - - Blue 4-Agarose column chromatography of M2-type pyruvate kinase from human meningioma. Column dimensions: 1.5 x 8 cm; flow rate: 20 mlJh; fraction volume: 1.5 mL; wash buffer: Tris-HC1 (pH 7.0), 1 mM 2-mercaptoethanol, 1 mM 6-aminocaproic acid, 5 mM MgC12; gradient: 0.0-500 mM KCI in above buffer plus 0.4 mM F-1,6-DP. ({~--O): absorbance at 280 nm; (A_-A): pyruvate kinase activity (U/mL). 385

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0.2 mM F-1,6-DP under the same conditions the enzyme was more stable and lost only 15% of its activity over the same period. In the presence of 25% ethylene glycol the enzyme lost only 10% of its original activity over this period. "-'r

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Discussion

Figure 3 - - SDS-polyacrylamide slab gel electrophoresis of purified and crude pyruvate kinases from human meningioma during procedures described in Table 1: 1) standard proteins (bovine serum albumin, MW 67,000; egg albumin, MW 45,000; trypsin, MW 23,300; ~-lactoglobulin, MW 18,400; egg lysozyme, MW 14,300); 2) PK eluted from affinity column; 3) PK eluted from CM-cellulose column; 4) enzyme after ammonium sulfate fractionation; 5) supernatant of ultracentrifugation; 6) homogenate. pended in: (a) 2 M ammonium sulfate; (b) 2 M ammonium sulfate containing 0.2 m M F-1,6-DP; and (c) 25% (v/v) ethylene glycol. The solutions were kept at 4 °C and the activity measurements were carried out every week for 3 months. The enzyme, which was suspended in 2 M ammonium sulfate without F-1,6DP, was unstable and lost up to 75% of its original activity after 87 days. However, in the presence of

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Ml-type P K isozyme, which is dominant in normal adult brain, is replaced by M2-type P K in brain tumor tissues (7,8,16,24). M2-type P K from h u m a n meningioma is described here. The subunit M W was 63,000 -+ 2000 Da. This value is close to that reported for the enzyme from rat lung (25) and h u m a n liver (26). The purified enzyme had a pl of 6.9, which is lower than that reported (16,27) for dog lung tumor enzyme (pI 7.3) and the M2-type enzyme from chicken liver (pl 8.3),but higher than the pI values reported (25) for M2-type enzymes from rat lung (pI 5.6) and pig kidney (pl 5.8).A pI value of 6.7 was determined for mouse spleen P K by Ibsen et al. (11). The differences in pl values suggest primary structural differences between isozymes from different sources. The crude enzyme gave two broad bands upon cellulose acetate electrophoresis that indicated the existence of M 1 and M 2 isozymes and possibly the hybrid forms in meningioma. Purified enzyme gave a single band on cellulose acetate that corresponded to M2-type P K because it moves faster on cellulose acetate than the Ml-type (7,28). The separation of the TABLE 2

Effect of Different Concentrations of F-I,6-DP on K m and Vmax Values of M2-Type Pyruvate Kinase From Human Meningioma

0

I I

I 3 PEP, mM

I 5

Figure 4 - - Michaelis-Menten plots for purified enzyme in the absence (A~A) and presence of 0.3 ~ (O--O) and 0.6 mM ( 0 - - 0 ) F-1,6-DP. [ADP]: 2 mM. 386

Concentrations of F-1,6-DP

K~ (raM)

V~, (mM/min)

Without 0.3 mM 0.6 mM

1.8 0.65 ± 0.02 0.53 ± 0.02

4.8 6.0 7.3

CLINICALBIOCHEMISTRY,VOLUME 26, OCTOBER 1993

MENINGIOMA M2-TYPE PYRUVATE KINASE that the break in the curve was due to the presence of different hybrids of P K with different affinitiesfor the substrate PEP. The absence of such breaks m a y indicate that the purified M2-type enzyme was not significantly contaminated with other isozymes. O n the basis of electrophoretic and kinetic data, it can be suggested that purified enzyme is M2-type PK. This study also supports the idea that h u m a n meningiomas contain both M 1 and M2-type PK; however the M2-type is the predominant form. The proportion of M2-type P K to Ml-type P K ai~r C M cellulose column chromatography was found to be 1.88. Additional studies are necessary for further characterization of the h u m a n meningioma PK.

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Figure 6 -- Lineweaver-Burk plot for PEP as substrate, in the presence of 0.3 m M (C)--C))and 0.6 m M (Q---O) F-1,6-DP. [ADP]: 2 raM. two isozymes was achieved by CM-cellulose column chromatography (Figure i). The kinetic properties of the purified P K from hum a n meningioma are largely consistent with results reported for M2-type P K from other tissues (1,13,28,29). The purified enzyme showed sigmoidal kinetics with respect to PEP in the absence of F-1,6DP (Figure 4). The same kind of kinetic behavior was observed for M2-type enzyme from Yoshida ascites hepatoma (29) and for the enzyme from kidney cortex (13). In the presence of 0.6 mM F-1,6-DP, the sigmoidal plot was converted to a hyperbolic form. The positive cooperativity indicated by the Hill coefficient, which was n = 2.3 in the absence of F-1,6DP, decreased by the addition of F-1,6-DP to approximately n = 1. The Km value of 0.53 for PEP was higher than the values found by various groups for M2-type PK from different sources (11,25,28,29). The affinity of tumor enzyme toward substrate (PEP) may be low. However, Feliu (30) reported that the Km (PEP) values for PK may change depending on the experimental conditions. Thus the Km values may be considered as comparable only when they are determined under the same conditions. A linear Lineweaver-Burk Plot was obtained for PEP hydrolysis by M2-type PK (Figure 6); however biphasic lines were obtained for PK from human meningioma by other workers (7). It was previously suggested

CLINICALBIOCHEMISTRY,VOLUME26, OCTOBER1993

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CLINICAL BIOCHEMISTRY,VOLUME 26, OCTOBER 1993