Purification and properties of bovine uterine glutamic dehydrogenase: A comparison with the liver enzyme

Ink J. Biochem., 1975, Vol. 6, pp. 871

to 875.

Pergamon Press. Printed in

Great Britain

PURIFICATION AND PROPERTIES OF BOVINE UTERINE GLUTAMIC DEHYDROGENASE: A COMPARISON WITH THE LIVER ENZYME* NANCY C. Y. Lmt,

EDWARDH. FRIEDENAND ALLEN B. RAW~TCH$

Biochemistry Division, Department of Chemistry, Kent State University, Kent, OH 44242 U.S.A. (Received 22nd July 1975) Abstract-l. L-Glutamate dehydrogenase (GDH) has been isolated from cotyledons of pregnant bovine uterus. 2. Although the specific activity of uterine GDH is relatively low, its enzymatic parameters are similar to those of the liver enzyme. 3. The molecular weight of uterine GDH is approximately 240,000; unlike the liver enzyme, it does not display concentration-dependent aggregation. Alanine dehydrogenase activity is confined to a lower molecular weight (mol. wt = 160,000) form, which appears to be in rapid equilibrium with the larger species. 4. The effects of nucleotides, steroid hormones, and diethylstilbestrol upon the enzymatic activity of uterine GDH have been examined.

INTRODUCTION

source is known to undergo concentration-dependent aggregation to form linear polymers (Olson & Anfinsen, 1952; Eisenberg & Tomkins, 1968). At low protein concentrations (< 1 mg/ml) the monomer is the predominant species. As the concentration is raised, progressively larger aggregates are generated. The reversible association of monomeric (and protomeric) forms is dramatically affected by both substrate and non-substrate ligands; this, in turn, affects the enzyme activity. Little work has been done on the comparative aspects of GDH from different organs of the same animal. Warren et al. (1964) purified glutamate dehydrogenases from human liver and uterus; besides noting that purified uterine enzyme was much less active than crystalline human liver GDH, no other properties of the uterine enzyme were recorded. In the study reported here, GDH has been isolated from pregnant bovine uteri, and the properties of the enzyme isolated from this source have been compared to those of crystalline bovine liver GDH. The rationale for the study was provided by the observation that both the activity and physical state of bovine liver GDH is affected by estrogens as well as by purine nucleotides (Ton&ins & Yielding, 1961). It was thought, therefore, that the isolation and characterization of the enzyme from a steroid-sensitive organ would be of interest.

CRYSTALLINEL-glutamate dehydrogenase (GDH; E.C.1.4.1.24) has been prepared from a wide variety of sources, ranging from bacterial cells to mammalian tissues. Some of the comparative aspects of the enzymes from various sources have been explored (Dewan, 1938; Strecker, 1953; Snoke, 1956; Ton&ins & Yielding, 1961; Warren et al., 1964; Corman et al., 1967; LeJohn, 1967; Stachow & Sanwal, 1967; Wainwright et al., 1967; Bond & Sang, 1968; Janssens & Cohen, 1968; Sedgwick & Frieden, 1968; Arnold & Maier, 1971) and it is clear that the physical molecular-kinetic and regulatory properties of these enzymes vary widely from one species to another. Bovine liver GDH is by far the most extensively studied; even here, however, a clear correlation is lacking between the function and the structural properties of the enzyme. The enzyme from this * From a dissertation submitted by Nancy Lan to the Graduate College of Kent State University in partial firhilment of the requirements for the degree of Doctor of Philosophy. Presented in part at a meeting of the American Society of Biological Chemists. Atlantic Citv. N.J., April 14-18, 1975. Inquiries should be addressed to E. H. Frieden, Department of Chemistry, Kent State University.

t Present address: Department of University of Illinois, Urbana, IL, U.S.A. $ Present address: University of Kansas

KS, U.S.A.

Department of Medical Center,

Physiology, Biochemistry, Kansas City,

EXPEEIMENTAL Coenxymes, nucleotides, steroids and crystalline liver glutamate dehydrogenase were purchased from Nutritional Biochemicals Inc. All coenxyme solutions were made up the same day that they were used. 871

872

NANCY C. Y. LAN,EDWARD H. FRWEN ANDALLEN B. R~wrrcB

Nucleotides and co-factors were obtained from Sigma Chemical Co. Acryhunide, N,N-methylene bisacrylamide and N.N,N.N-tetramethylenediamine were purchased from Eastman Organic Chemicals. Sucrose and ammonium sulfate (special enzyme grade) were obtained from Mann. DEAESephadex was purchased from Pharmacia Corporation. All other chemicals used were reagent grade. Enzyme assay Enzyme activity was measured at 25°C in O-05 M Tris-chloride buffer, pH 7.9, containing 0.1 mM EDTA, 0.05 M NH&l, 0.025 M sodium arsenate, 1.6 x 1O-4 M NADH (or_NADPH) and 4x lo-’ M or-ketoglutarate (for GDH activitv) or uvruvate (for alanine dehydrogenase [ADH] act&y). &al velocities were determined in 1 cm cells at 340 nM against blanks containing all of the reagents except NADH (or NADPH). Protein determinations Routine determinations of protein concentration in solutions of glutamate dehydrogenase were made by measuring the absorbance at 280 nm and assuming an absorbance of 0.97 per mg of enzyme (Olson & Antimen, 1952). However, the biuret or Lowry reaction (Mokrasch & McGilvery, 1956) was used when very accurate protein determinations were required or when a large amount of protein other than glutamate dehydrogenase was known to be present. Bovine serum albumin was used as a standard. Polyacrylamide gel electrophoresis Polyacrylamide gels were prepared by the method described by Davis (1964), with modifications. If electrophoresis was carried out in the presence of diethylstilbestrol (DES) or GTP, electrophoresis buffer, polyacrylamide gels, and staining medium all contained 1O-4 M of DES or 1O-6 M GTP. The gels were stained with Comma&e Blue for protein. To locate the enzyme on gels, the procedure of Dietz & Lubrano (1967) was followed, modified to detect glutamate or alanine dehydrogenase. After removal from electrophoresis tubes, the gels were stained in a medium containina 9.2 ml of 0.05 M Tris-HCl buffer (uH 7.8). 0.21 ml of glutamate or alanine solution (1 M), 65 ml ‘if NAD solution (16 mg/ml), 3 mg of 2,5I-2)-tetrazolium diuhenvl-3-(4.5 dimethvlthiazolvl bromide (Ml’?) and 0.5 & of freshly prepar~ed phenazine methosulfate (1 mg/ml). The tubes were wrapped with alummium foil to avoid light. They were then incubated at 30°C for l-l) hr. GDH (or ADH) active components appeared as characteristic blue bands of varying intensity. Determination of sedimentation coeficients Sedimentation velocity measurements were made in a Spinco model E analytical ultracentrifuge at a speed of 60,000 rev/mm The experiments were performed in 0.1 M phosphate buffer (pH 7-O). The sedimentation coetBcients obtained were corrected to water at 20°C. Amino acid analysis Samples of purified bovine cotyledon and crystalline liver glutamate dehydrogenases were extensively dialyzed against distilled water, hydrolyzed, and analyzed .on a Jeolco model 6AH amino acid analyzer. The ammo

acid content of the cotyledon enzyme was quite similar to the liver enzyme, except that the former somewhat more leucine and less methionine.

contains

Purification of the enzyme Uteri from pregnant cattle were wllected at a slaughterhouse, chilled, and kept on ice while being transported to the laboratory. The maternal and fetal parts of the cotyledons were separated from the rest of the uterus and either processed immediately or kept frozen at -60°C until worked up. Ah subsequent steps were carried out at O+C. The procedure used for isolation of the enzyme was a modification of the method described by Arnold & Maier (1971) for rat liver enzyme. After homogenization of 1.5 kg of maternal cotyledon tissue in 45 1 of 0.25 M sucrose, pH 7.2, 0.01 M KHPG,, the mitochondrial fraction (sedimenting between 1600 x g and 13,000 x g) was collected by centrifugation. The mitochondria were resuspended in 150 ml of 0.02 M Tris-HCI, pH 7.8, 0.1 mM EDTA and frozen at -60°C for 15 hr, then thawed and disintegrated by intermittent (5 set) exposure to ultrasound for a total of 60 sec. After centrifugation at 20,000 x g for 20 min, the supematant was adjusted to 50% saturation with ammonium sulfate and allowed to stand for 30 min. The suspension was then centrifuged at 20,000 x g for 20 min. The pellet was suspended in 0.02 M potassium phosphate, pH 7.2, and dialyzed for 12 hr against 10% saturated ammonium sulfate, then centrifuged again. The precipitate was discarded and the ammonium sulfate waS removed from the supematant bv dialvsis aaainst butfer (0.02 M KHPG,. nH 7.2). The diaiysate -&s applied toa DEAE-Sephadex ~01~ (4 x 30 cm) which had been equilibrated with the dialysis buffer. After 150 ml of elBuent had emerged, a linear KCl gradient (W5 M KCL in buffer) was applied. The enzyme-containing fractions were pooled, concentrated, and applied to a 4% agarose column (bed volume = 260 ml). The purifkd enzyme which emerged

from the agarose column had a specitic activity approximately 30 times that of the disrupted mitochondrial supematant. Rechromatographyof the agarose puritkd material on either DEAeSephadex or agarose did not significantly increase the specific activity of the product. In one experiment, GDH was isolated from fetal cotyledon tissue, using the procedure described above. The properties of fetal cotyledon GDH did not differ signitlcantly from those of the enzyme isolated from maternal tissue. RESULTS A summary of the recoveries of protein and enzyme activity obtained at each step of a typical fractionation is shown in Table 1. The overall purification achieved was about 30-fold. As shown in Fig. I, when the material recovered from the DEAESephadex column was chromatographed on 4% agarose, symmetrical curves for the emergence of both protein and GDH activity were obtained; however, GDH activity emerged slightly ahead of the bulk of the protein. The disparity between the elution volumes of GDH activity and protein _ _ persisted

when the fractions

containing

most of the

873

Purification and properties of bovine uterine glutamic dehydrogenase TabIe 1. Swmmary of enzyme purification procedure VOlUme

step

(ml)

Hitochondrlal

supernatant 50%SASP&e. 10% SAS

Filtr.

185

I

units ml (X10

1

Total unita

Protein (lW/lIW

specific Activity

Yield

Purification

FWTWX

(%I

_-

171

31.6

17.6

10.3

100

49.5

341

26.8

44

12.3

84

1.2

43.5

508

24.2

25

20.3

76

2.0

85.2

25

8.3

DEAE-Scphadex Eluate

110

72

7.9

0.85

‘4garose Filtrate-I

35

193

6.7

0.68

284

21.4

27.5

Agarose Filtrate-II

48

69

3.29

0.22

312

10.4

30.3

-~

Effluent, ml. Efllumt

ml

Fig. 1. Chromatography of DEAE-Sephadex-purified cotyledon GHD on 4% agarose (2.2 x 90 cm). The eluting buffer was @02 M phosphate, pH 7.2. -, Protein cont. (kft ordinate); - - -, Relative GDH activity (right ordinate).

Fig. 2. Chromatography of cotyledon GDH on 4% agarose (2.2 x 90 cm). -, Protein cont. (Ieft ordinate); - - -, relative ADH activity (right ordinate); . . ., relative GDH activity (right ordinate).

GDH activity were pooled and rechromatographed on agarose (Fig. 2). ADH activity, on the other hand, emerged from the second cohmm in a peak which coincided exactly with that of protein. When agarose-purified cotyledon (either maternal or fetal) GDH was subjected to electrophoresis in 10% polyacrylamide gels at pH 8.6, only two, closely-spaced bands of equal intensity appeared when the gels were stained with Comassie Blue; under the same conditions of protein and gel concentration, a similar doublet was observed with bovine liver GDH (Fig. 3). When similarly prepared gels were stained for GDH activity a single band appeared in both gels at approx~ately the same position as the doublet; in addition,however, another GDH band, closer to the anode appeared in both preparations. The intensity of this band indicated that it contained a large fraction of the total GDH activity. The segmentation coethcient of purified cotyledon GDH in 0.1 M phosphate buffer, pH 7.0 (C = 10 mg/ml) was 5.06 S (assumed Y= O-75), with no

evidence for the resolution of a more rapidly sedimenting component. At the same protein wn~n~ation, smolwfor liver GDH was 25.05. When cotyledon GDH was subjected to electrophoresis in a series of gels containing different concentrations of polyacrylamide, and the gels were then stained for GDH, the mobility of the enzyme increased with decreasing gel concentration (Fig. 4); it was accompanied by a series of smaller, GDH-active components. At this same protein concentration (2 mg/ml) liver GDH remained at the origin at all gel concentrations employed. From its behavior in disc gel electrophoresis, the molecular weight of the major cotyledon GDH component was estimated to be 240,000; the minor, more rapidly migrating components appeared to be charge isomers with molecular weights approximating 160,000. In gels containing O-1% SDS (Shapiro et al. (1967)), all forms of cotyledon GDH, like liver GDH, dissociate into protomers of about 52,000 daltons. Purified cotyledon GDH can use either NADH or NADPH as a coenzyme; however, the rate of

874

NANCY C. Y. Lm, Eowm

H. FRIEDENAMDALLW B. RAwrrcn

reduction of or-ketoglutaric acid with NADPH is only about # that with NADH. Using a series of overlapping buffers which covered the pH range 4.2-8.9, the optimum pH for a-ketoglutarate reduction was found to be 7-8-7.9 at 25°C. The apparent Michaelis constants for NH+, and (Yketoglutarate at the same temperature are 3 x lo-*M and 5 x lo-*M respectively. The & for NADH, extrapolated from data obtained at low concentrations, was 3.0 x 1O-4 M. Concentrations of NADH in excess of 160 I*_M were inhibitory. These parameters are quite similar to those reported for bovine liver GDH (Frieden, 1963) which is also inhibited by excess NADH. One of the characteristic properties of bovine liver GDH is its sensitivity to purine nucleotides and some steroid hormones (Frieden, 1963). With few exceptions, mammalian GDH’s are inhibited by GTP and estrogens, and are stimulated by ADP. To some extent, these properties are shared by cotyledon GDH. In the presence of 1.2 x 1O-6 M GTP, enzyme activity is inhibited by SO%, while diethyl stilbestrol is essentially completely inhibitory at 3 x 1O-5 M (Fig. 5). In our experience, however, the inhibition produced by estradiol (40% at 5 x 10esM) was substantially less than the inhibition of liver GDH produced by the same estradiol concentration, while progesterone had a negligible (< 10%) effect. Maximum stimulation by ADP was observed to be only about 20% over the range 1-5 x 10e4 M. Gel electrophoresis experiments indicated that the inhibitory effects of GTP and DES are accompanied by dissociation into smaller molecules of increased electrophoretic mobility. Struck & Sizer (1960) discovered some years ago that crystalline liver GDH can use other amino acids, including alanine, as substrate. Tomkins et al. (1961) reported that the dissociation of high molecular

/o_o/o-oo-

gl.OiQ,O,O

/o-o-o-

3 /

Effactor

I

Cone. x 10’

Fig. 5. Effects of DES (0, b and GTP (a, 0) on cotyledon GDH activity. The substrates were a-ketoglutarate (0, & and pyruvate (0, 0).

weight aggregates of liver GDH by DES resulted in a 2-fold increase in ADH activity, even as GDH activity decreased. Purified cotyledon GDH, can also use pyruvate as a substrate, although the rate of NADH oxidation with pyruvate as substrate is only about one-fifth that with a-ketoglutarate. In part, this may be due to the choice of pH (7.9) for these experiments, since the optimum pH for monocarboxylic acid oxidation has been reported to be between 9.5 and 10. On agarose columns, cotyledon ADH activity always emerges later than does GDH (Fig. 2) and, as noted previously, coincides exactly with the protein peak. In gel electrophoresis experiments (Fig. 6) only the faster moving of the several GDH bands showed ADH activity. DISCUSSION The

specific activity of purified bovine placental cotyledon glutamate dehydrogenase is about 0.3 unit per mg of protein; the amount recovered corresponded to 24 mu/g of tissue. When an additional 1 min homogenization was carried out after the sonication of the thawed mitochondrial fraction, there was no significant increase of the total glutamate dehydrogenase activity. This suggests that the low glutamate dehydrogenase activity found in cotyledon tissue is not due to incomplete disruption of the mitochondria, but rather to intrinsically low levels of enzyme activity. This finding is consistent with the report by Warren et al. (1964) that the specific activity of human uterine GDH is significantly lower than that of human liver GDH. Despite the considerable disparity between the specific activities of cotyiedon and liver GDHs, there appears to be little difference in their kinetic properties in the absence of effecters. The observed optimum pH for a-ketoglutarate reduction by cotyledon GDH (78-7.9) lies between the extremes of 7.6 and 8.4 reported for liver GDH (Olson & Anfinsen, 1952; Strecker, 1953). However, a major difference. between liver and cotyledon GDHs appears when their relative sensitivities to ovarian hormones are compared. At comparable concentrations, inhibition of liver GDH by estradiol and progesterone was found to be 72 and 58% respectively (Ton&ins et al., 1961), whereas inhibition of cotyledon GDH by estradiol never exceeded 40% and that by progesterone was insigmficant. Preincubation of steroid and enzyme for 60 min did not increase the effect. The possibility that GDH isolated from uterine tissue contained bound estrogen (which might have accounted for its low specific activity) seems unlikely, since repeated extraction of an enzyme solution with ethyl ether did not result in increased activity. Another sign&ant difference between bovine liver and cotyledon enzymes appear to reside in the inability of the latter to undergo concentration-

Origin

Origin

10% 9% -c’ ** 3c

8%

7%

7%

Cathode

6% W

6%

5 % * Cathode

Origin -

Cathode

5%

Origin a

Fig. 3. Polyacrylamide gel electrophoresis of bovine C$H’s at pH 8.6. The gels were stained for protein. From left to right: Bovine liver GDH, maternal cotyledon GDH and fetal cotyledon GDH. The protein concentration was 0.4 mg/ml. Fig. liver 8.6.

4. Polyacrylamide gel electrophoresis of bovine kmoer) \.* , and bovine cotvledon (lower) GDH’s at uH The gel concentration was varied from 10 % to +/,. The protein concentration was 2 mg/ml.

Fig. 6. Polyacrylamide gel electrophoresis of cotyledon GDH at pH 8.6. The gel concentration was 8%. Gel A was stained for GDH activity and gel B was’kined for ADH activity.

[to face p. 8741

Purification and properties of bovin ie uterine glutamic dehydrogenase or ligand-dependent association into very large ohgomers. In this respect, cotyledon GDH is similar to the enzymes from dogIish liver and rat liver, which aho fail to form large oligomers. It is noteworthy that while ADP has only a slight (20-30x) stimulatory effect upon bovine cotyldeon GDH, rat liver GDH is nearly ten times as active in the presence of ADP than in its absence. Although oligomer formation does not appear to be a characteristic property of cotyledon GDH, the appearance of several GDH-active species in gels, together with the unusual behavior of the enzyme on agarose filtration displayed in Fig. 2, suggests that the enzyme consists of at least two forms of different sizes which appear to be in rapid equilibrium as judged from its sedimentation behavior. ADH activity appears to be the exclusive property of the smaller (mol. wtw 160,000) species, which exists as a group of isoenzymes of different net charges. Acknowledgement-This work was supported, in part, by a National Institutes of Health Grant (No. AM14553). ABR is the recipient of Research Career Development Award AM70473. REXERENCES ARNOLD H. & MAIER K. P. (1971) Crystallization

and some properties of glutamate dehydrogenase from rat liver. Biochim. biopbvs. Acta 251. 133-410. BOND P. A. & SANG J. H. (1968)*Glutamate dehydrogenase of Drosophila larvae. J. Insect. Physiol. 14, 341-359. CORMAN L., Pmscorr L. M. t KAPLAN N. 0. (1967) Purification and kinetic characteristics of dogfish liver glutamic dehydrogenase. J. BioZ. Chem. 242, 1383-1390. DAVISB. J. (1964) Disk Electrophoresis. II. Method and application to human serum proteins. Ann. N, Y. Acad. Sci. 121,404X7. DEWAN J. G. (1938) CLXXKIII. The l(f) glutamic dehydrogenase of animal tissues. B&&em. J. 32,

1378-1385. DIETZ A. A. & LUBRANO T. (1967) Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Anal. Biochem. 20, 246257. EISENBIXGH. & TOMKINSG. M. (1968) Molecular weight of the subunits, oIigomeric, and associated forms of bovine Iiver glutamate dehydrogenase. J. Mol. Biol. 31, 37-49. FRIEDENC. (1963) GGlutamate dehydrogenase. In i?& Enzymes (Edited by BOYER P. D., LARDY H. & MYRBACK IQ 2nd Edn., Vol. 7, p. 1. Academic Press,

New York.

815

HEDR~cK J. L. & SMITH A. J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel efectrophoresis. Arch. Biochem. biophys. 126, 155464. JAN~~ENS P. A. & COHENP. P. (1968) Nitrogen metabolism in the African lung&h. Comp. Biochem. Physiol. 24, 879-886. LEJOHNH. B. (1967) AMP-Activation of an allosteric N~~e~ndent glutamate dehydrogenase. B&hem. biophys. Res. Commun. 28,96-102. MOKRQCH L. C. & MCGILVERY R. W. (1956) Purification and properties of fructose-l ,6-diphosphate. J. Biol. Chem. 221,909-917. OLSON J. A. & ANFINSENC. B. (1952) The crystallization and characterization of L-glutamic acid dehydrogenase. J. Biol. Chem. 197,67-79. SIDGW~CKK. A. & ~+I~DEN C. (1968) The moIecular weight and some kinetic properties of crystalline rat liver glutamate dehydrogenase. Biochem. biophys. Res. Commun. 32,392-397. SHAPIRO A. L., VINUELA E. & MAIZEL J. V. (1967) Molecular weight estimation of pofypeptide chains by electrophoresis in SDS-polvacrvlamide gels. B&hem. biophys. Res. Commun. i8,8151820. SNOKEJ. E. (1956) Chicken liver glutamic dehydrogenase. J. BioZ. Chem. 223,271-276. STACHOWC. S. & SANWAL B. D. (1967) Regulation, puritication, and some properties of the NAD-spe&c glutamate dehydrogenase of Neurospora. Biochim. biophys. Acta 139, 294-307. STRECKERH. J. (1953) Glutamic dehydrogenase. Archs Biochem. biophys. 46, 128-140. STRUCK J., Jr. & SZER I. W. (1960) The substrate specificity of dutamic acid dehydrogenase. Archs Biochem. Biophys. 86,260-266. TOMKINSG. M. & YIELDINGK. L. (1961) Regulation of the euzymic activity of glutamic dehydrogenase mediated by changes in its structure. Cold Spring Harb. Symp. quant. Bioi. XXVI, 331-341. TOMKINSG. M., YXELDINGK. L. & CURRAN J. (1961) Steroid hormone activation of r.-alanine oxidation catalyzed by a subunit of crystalline glutamic dehydrogenase. Proc. natn. Acad. Sci., U.S. 47, 270-278. WAINWRIGHT S. D., BRIGHT-ASARL P. & C-BELL J. C. (1967) Exploratory studies of the liver glutamic dehydrogen~ of the hagfish Maxine ~lutinosa. Lack of regtdation of &tivity -by AbP and diethylstiibestrol in a uhvsioloaical saline. Can. J. Biochem. 45,614-618. - _ WARRENJ. C., CARR D. 0. & GRISOLIAS. (1964) Effect of cofactors, oeetrogens, and magnesium ions on the activity and stability of human glutamate dehydrogenase. Biochem. J. 93,409-419. Key Word Index--Adenosine diphosphate; alanine dehydrogensse; dietbyl stilbestrol; estradiol; glutamic dehydrogenase; guanosine ~pho~ha~; a-ketoglutarate; pyruvate; progesterone; uterus.