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Characterization of glutamine synthetase from avian liver mitochondria
0020-711X/82/080747-10$03.00/0 Copyright 0 1982 Pergamon Press Ltd
Int. J. Biochem. Vol. 14. pp. 747 to 756, 1982 Printed in Great Britain. All rights reserved
CHARACTERIZATION OF GLUTAMINE SYNTHETASE FROM
AVIAN LIVER
MITOCHONDRIA
JEAN E. VORHABEN, DARWIN D. SMITH and JAMESW. CAMPBELL Department of Biology, William Marsh Rice University, Houston, TX 77001, U.S.A.
(Received 25
Nowmber
1981)
Abstract-l. Glutamine synthetase has been purified to homogeneity from chicken liver mitochondria. 2. The native enzyme is an octamer composed of identical subunits with monomeric mol. wt of 42,000 dalton. 3. Apparent K,s for NH:. ATP and glutamate were 0.5, 0.9 and 6 mM, respectively. D-Glutamate and L-a-hydroxyglutarate were utilized as substrates with activities approx. 40% those obtained with glutamate. Of several nucleotides tested, none were effective replacements for ATP. 4. Heavy metal ions were inhibitory as were Mn*+, Ca2+ and lanthanide ions. 5. Despite its different subcellular localization and physiological function, avian glutamine synthetase is markedly similar to the weakly-bound microsomal rat liver enzyme with respect to a number of physical and chemical properties.
INTRODUCTION Previous studies from our laboratory have shown that the function of glutamine synthetase (EC 6.3.1.2) in livers of uricotelic vertebrates appears to be analogous to that of carbamyl phosphate synthetase 1 (EC 2.7.2.5) in ureotelic animals (Vorhaben & Campbell, 1972, 1976, 1977). Each enzyme catalyzes a reaction which results in the effective removal of mitochondrially-generated ammonia via its conversion to a nitrogenous end-product precursor. This is glutamine in the case of uricoteles and carbamyl phosphate in the. case of ureoteles. The ammonia utilized as substrate by these enzymes is ultimately derived from amino acid nitrogen. During protein catabolism, amino acids undergo transamination with LX-ketoglutarate in the cytosol and in so doing, funnel amino nitrogen into glutamate. Transport of glutamate into the mitochondrion is followed by its oxidative deamination to release ammonia. In keeping with its physiological function, avian liver glutamine synthetase, like carbamyl phosphate synthetase 1, is localized in the mitochondrial matrix, the site of ammonia release via glutamate dehydrogenase (Vorhaben & Campbell, 1977). It shows a marked response to conditions of gluconeogenesis such as starvation or high protein diets (Katunama et al., 1970), a response also analogous to that shown by carbamyl phosphate synthetase 1 in mammalian liver. On the other hand, mammalian liver glutamine synthetase appears to be loosely bound to the endoplasmic reticulum (Wu, 1963) and does not respond to starvation or high dietary protein intake (Katunama et al., 1970), indicating that it is not primarily involved in the disposal of a-amino nitrogen. Although the purification and characterization of glutamine synthetase from a number of higher eukaryotic sources have been reported including rat and hamster liver (Tate et al., 1972; Deuel rt al.. 1978; Tiemier & Milman, 1972) sheep brain (Ronzio et al., 1969) and chick neural retina (Sarkar ct al., 1972), the
properties of the uricotelic liver enzyme have not been investigated in depth (Seyama et al., 1972). Given the differences noted above between the uricotelic and ureotelic enzymes with respect to physiological function and subcellular localization and considering the hypothesis regarding the symbiotic origin of mitochondria, we deemed it important to further characterize the avian liver enzyme. The results of our studies indicate properties of the uricotelic liver enzyme more closely resemble those of glutamine synthetase isolated from other vertebrate tissues rather than from prokaryotic or lower eukaryotic sources. MATERIALSAND
METHODS
Materials
With the exception of NAD and NADH, which were purchased from PL Biochemicals, all biochemical reagents were obtained from Sigma. Potassium phosphate, magnesium chloride, calcium chloride and sucrose were products of Fisher Co. Lanthanum chloride and terbium chloride were obtained from Aldrich Co. Sources of additional materials are given in other sections of Materials and Methods. All other reagents were of the highest purity available commercially. Enzyme
pur@cation
As source material for the purification, White Leghorn hens were obtained from a local commercial supplier. Animals were starved for 4 days before killing by decapitation to induce higher levels of enzyme (Katunama et al., 1970). The procedure utilized for the isolation of avian liver glutamine synthetase was a modification of that used fur the purification of the rat liver enzyme (Tate et al., 1972). All steps were performed at 4-5°C. The initial step in the purification of chicken liver glutamine synthetase was the preparation of mitochondria. These were isolated as follows. Ten percent liver homogenates (from 84 g liver) were prepared in 0.25 M sucrose containing 5 mM HEPES (pH 7.37.4) utilizing a PotterElvehjem homogenizer. The homogenate was centrifuged at 350 9 for 10 min and the loose pellet washed .once with 747
JEAN
748
E.
VORHABEN
100 ml isolating medium. The supernatant fluid and washing were combined and centrifuged at 9000 g for 20 min to sediment mitochondria. Following a single wash, the mitochondrial pellet was resuspended in minimal isolating medium (80ml), lypholized overnight and powdered by means of mortar and pestle. Extraction of the powder (12 g) was accomplished by the addition, with stirring, of 75 ml of 50 mM potassium phosphate buffer (pH 7.4); stirring was continued for 1 hr after which the suspension was centrifuged at 100,OOOg for 60 min. The pellet was extracted a second time with 60ml buffer. The extracts were combined and applied without volume reduction to a hydroxyl apatite column (Bio Rad, 2.6 x 30 cm, previously equilibrated with phosphate buffer) with the aid of a peristaltic pump. Elution was carried out with a linear gradient of 50-500 mM ohosohate buffer CDH7.41.The enzvme was eluted at a phbsphate concentrition 0; 0.21 M which is similar to the elution characteristics of the rat liver enzyme (Tate et al., 1972). After a reduction in volume by pressure filtration (UM 10 filter, Amicon Corp.), the eluate was subjected to gel filtration chromatography on a Bio Gel P-300 column (2.6 x 76cm) equilibrated with a solution of 0.15 M potassium chloride-l mM EDTA brought to a pH of 7.4; elution was with the equilibrating solution. Active fractions were pooled, concentrated by ultrafiltration, dialyzed against 50mM imidazole buffer containing 2mM EDTA (pH 7.4) and applied to a DEAE
et ul.
massie Blue in a 9% acetic acid-4%; methanol solution. Destaining was achieved by diffusion in a 7.5% acetic acid-5% methanol solution. Protein standards and their respective sources were: bovine serum albumin, pepsin, ovalbumin (Sigma); myoglobin and chymotrypsinogen (Mann Research) and catalase (Worthington). Analytical isoelectric focusing was performed in 3% polyacrylamide gels with 2% (w/v) ampholytes (Bio Rad, pH 3-10) according to Maize1 (1971). The pl of glutamine synthetase was also verified by isoelectric focusing using a IlOml LKB preparative isoelectric focusing column and following LKB manual procedures. Analytical
ultracentrijiigation
Determination of the molecular weight of the native enzyme by sedimentation equilibrium analysis was kindly performed by Dr Richard M. Caprioli. Analytical Chemistry Center, University of Texas Medical School at Houston with the technical assistance of Mr Tim Woodward. The analysis was performed with a Beckman Model E analytical ultracentrifuge equipped with U.V.scanning optics. Molecular weights were calculated by the method of meniscus depletion assuming a partial molar volume of 0.74. Amino acid composition
Protein was hydrolyzed in uacuo for 24, 48 and 72 hr with 6 N hydrochloric acid at 110°C. Amino acid analyses were performed at Rice University by Dr Kathleen S. Matthews, Department of Biochemistry, on a Beckman Model 120-C analyzer. Electron microscopy
Negative staining of glutamine synthetase was accomplished essentially as described by Haschemeyer (1970). A 2 ~1 aliquot of the purified enzyme (50 pg/ml) in 50 mM imidazole buffer (pH 7.4) was applied to a 400 mesh copper grid coated with Formvar. After standing for 5 min, residual solution was removed by means of a filter paper wick. Immediately thereafter, 2 ~1 of a 2% uranyl acetate solution was applied and allowed to stand for 5 min. Excess staining solution was removed by again touching to filter paper and the residual material was then allowed to dry. Electron microscopy was performed with a Philips 200 instrument operated at 60 kV. RESULTS
Glutamine synthetase
AND
DISCUSSION
assay
Glutamine synthetase activity was determined colorimetrically by following the formation of y-glutamylhydroxamate in reaction mixtures containing glutamate, hydroxylamine, ATP and Mg *+. Reactant concentrations are given in legends. For the determination of some kinetic parameters of the purified enzyme, a spectrophotometric method was employed which measures NADH oxidation continuously (Shapiro & Stadtman, 1970). The release of ADP was followed in a coupled assay system containing 4 U pyruvate kinase, 1.5 mM phosphoenolpyruvate, 2.5 U lactate dehvdroeenase. 0.1 mM NADH, 100 mM notassium chloride, 16 rnh? maghesium chloride ‘and 50 m-M imidazole buffer (pH 7.4) in a volume of 1 ml. Glutamate, ATP and ammonium chloride concentrations were varied as described in the text. The temperature was 37°C. Doubling the amount of coupling enzymes had no effect on the reaction rate. Electrophoresis
Analytical polyacrylamide gel electrophoresis of the undissociated native enzyme was performed as described by Maize1 (1971) using 5% gels in 50 mM Tris-glycine buffer (pH 8.9). For the dissociated enzyme, SDS-gel electrophoresis was carried out according to the procedure of Weber et al. (1972). Gels were fixed and stained with COO-
Glutamine synthetase from avian liver mitochondria has been purifed over 750-fold by the steps summarized in Table 1. This purification scheme represents a significant improvement with respect to both purity and overall yield compared to that previously reported (Seyama et al., 1972). The binding of the avian enzyme to various affinity chromatography media differs considerably from that reported for glutamine synthetases from microbial sources. For example, the avian enzyme was dissociated from Blu&3epharose by 1 M sodium chloride but not by buffered solutions of 2 mM ADP or ATP whereas the reciprocal conditions hold for the release of glutamine synthetases from blue-green algal and bacterial sources (Stacey et al., 1979; Tuli et al., 1979; and Siede1 & Shelton, 1979). Glutamate-Sepharose has been used in the isolation of yeast glutamine synthetase (Lin & Kapoor, 1978) yet our partially purified enzyme (a post DEAE-cellulose preparation) was not bound to glutamate-Agarose (Sigma) under similar conditions. Binding of the avian enzyme to glutamine-Sepharose (Sigma) was also negative. We might add that attempts to purify the chicken glutamine
Characterization
of glutamine synthetase
749
Table 1. Purification of glutamine synthetase from chicken liver Total protein
(mg) Crude liver homogenate Mitochondrial extract Hydroxylapatite chromatography Bio Gel P-300 chromatography DEAE-cellulose chromatography Blue-Sepharose chromatography
24,412 1345 110 32 15 13
synthetase by the zinc precipitation method of Miller et al. (1974) were unsuccessful. Evidence for the homogeneity of our preparation is given in Fig. 1. Single protein bands appeared when the native enzyme was subjected to polyacrylamide gel electrophoresis in the absence (Gel A) or presence (Gel B) of sodium dodecylsulfate. Our failure to observe multiple bands when the enzyme is electrophoresed under dissociating conditions suggests the avian enzyme is probably composed of identical subunits, a property characteristic of all glutamine synthetases heretofore purified. Isoelectric focusing of our purified enzyme also showed the presence of a single protein as shown by Gel C of Fig. 1. Utilizing preparative isoelectric focusing conditions, a p1 of 6.1 was determined for the purified avian liver enzyme which is significantly higher than the value of 4.2 reported for the sheep brain enzyme (Pamiljans et al., 1962) but within the range of PI’S (5.2-6.1) measured for glutamine synthetases from several bacterial sources (Wedler et al., 1978; Alef et al., 1981; Darrow &
Knotts, 1977). Although evidence for a difference in the isoelectric points of the rat liver and ovine brain enzymes has been presented (Tate & Meister, 1973), no value for the former enzyme was given and hence a comparison of the avian and rat liver enzymes cannot be made. The absorption spectrum of the avian liver enzyme was that of a typical protein without chromophoric groups and the probable absence of a bound adenyl group was suggested by a A28,JA260 ratio of 1.69. Molecular weight of the native enzyme and its subunit
The molecular weight of the monomeric subunit was determined by comparison of the relative migration of avian glutamine synthetase with the mobilities of standard proteins during SDS-gel electrophoresis. From these data, an average mol. wt of 42,000 + 1000 was calculated. A representative plot is shown in Fig. 2. The molecular weight determined for the avian liver monomer is identical to that reported for the chick retinal and liver subunit (Sarkar et al., 1972; Matsuno & Shirasawa, 1978) and is within the range of values determined for other mammalian glutamine synthetase subunits including rat liver, 44,000 (Tate et al., 1972); hamster liver, 42,000 (Tiemier & Milman, 1972) and ovine brain, 49,000 (Tate et al., 1972). From sedimentation equilibrium analyses, the molecular weight of the native enzyme was determined to be 389.000 daltons; on the basis of a monomeric subunit mol. wt of 42,000, a structure composed of nine subunits would be implied. However, as discussed below, electron micrographs of the native enzyme are
Purification
Specific activity
(Ujmg) Recovery 2.15 31.4 371 600 700 1700
100 80.3 77.6 36.5 20.0 41.9
yield (%) 1.0 14.6 173 279 326 789
consistent with a structure composed of eight subunits. Octameric structures would therefore appear to be typical of all eukaryotic glutamine synthetases (Lin & Kapoor, 1978; Tate & Meister, 1971). Moreover equilibrium ultracentrifugation studies with ovine brain and hamster liver glutamine synthetases suggest protein aggregation may occur during analyses leading to artificially high molecular weight determinations (Tiemier & Milman, 1972; Tate & Meister, 1971). Sedimentation equilibrium analysis of the chick neural retinal glutamine synthetase, having a subunit molecular weight identical to that of the avian liver enzyme (42,000), likewise yielded a molecular weight in excess of that predicted by an octameric structure (Tiemier & Milman, 1972). Electron microscopy An electron micrograph of a negatively stained avian liver glutamine synthetase preparation is shown in Fig. 3. The field shows only nine enzyme molecules since higher concentrations of protein lead to aggregated structures. Tetrameric structures having a square projection are evident under low and high magnification with each of the four subunits occupying a corner of the square. Similar forms have been observed in uranyl acetate stained preparations of glutamine synthetase from ovine brain (Haschemeyer, 1968) hamster liver (Tiemier & Milman, 1972) and chick retina (Sarkar et al., 1972). The octameric structure would be generated by one tetrad layered above another. Particles are approx. 100 A on a side which is within the range of dimensions reported for the enzyme isolated from hamster liver, 90 x 90 A (Tiemier & Milman, 1972); chick retina, 90 x 90 x 125 A (Sarkar et al., 1972) and ovine brain, 90 x 90 x 120 A (Haschemeyer, 1968). Measurements of three-dimensional structure cannot be made from our micrographs. Nonetheless, the general appearance and size of the particles are similar to those found for other eukaryotic glutamine synthetases and is in accord with a structure composed of eight rather than nine subunits. Amino acid composition
The amino acid compositions of avian liver and several other eukaryotic glutamine synthetases are compared in Table 2. Hydrolysis was performed in 6N hydrochloric acid rather than methane sulfonic acid to facilitate comparison with other glutamine synthetases hydrolyzed under similar conditions. Marked similarities in amino acid composition are evident, especially with regard to the chicken and mammalian enzymes.
750
JEAN EVORHABEN
rt al.
Table 2. Amino acid composition of glutamine synthetase from eukaryotic sources
Amino acid Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Chicken Chicken Rat* Ovine* Neurospora* crassa liver retina liver brain (mol/mol of subunit?) 27 24 41 9: 39 38 17 17 24 22 10 17 22 20# 175 6/( 15 17
24 24 35 ND 31 35 19 15 20 18 ND 20 20 24 &I 14 19
28 21 48 12 33 42 13 23 25 25 12 21 23 24 20 6 17 20
29 25 41 12 44 41 11 21 22 25 11 21 25 25 20 7 16 18
35 23 40 4 45 30 9 19 35 21 9 15 20 26 22 16 13 27
*Values for the chick retina enzyme taken from (Sarkar et al., 1972), for the rat liver and ovine brain from (Tate et al., 1972) and for Neurospora crassa (Lin & Kapoor, 1978). t Subunit molecular weights are: chicken liver and retina, 42,000 dalton; rat liver, ovine brain and Neurospora crassa, 45,000 dalton. $ Determined by titration with Ellman’s reagent (Habeeb, 1972). $ Extrapolated to zero time of hydrolysis. j/ Determined by the spectrophotometric method of Spande & Witkop (1967). ND = not determined.
Table 3. Substrates of glutamine synthetase Substrate
Relative activity
L-Glutamate, 25 mM L-cr-Hydroxyglutarate, 25 mM D-Glutamate, 25 mM p-Methyl-L-glutamate, 25 mM N-Acetylglutamate, 25 mM N-Carbamyl glutamate, 25 mM L-Aspartate, 25 mM a-Ketoglutarate, 25 mM
100 41 39 16 6 4 6 0
ATP, 1OmM ITP, 10 mM GTP, 10 mM TTP, 10mM
100 10 3 3
Mg”, 10mM MgZi, 2 mM Mn”, 1OmM
100 13 2
Glutamine synthetase activity was determined by measuring y-glutamylhydroxamate in reaction mixtures containing 50 mM imidazole buffer (pH 7.4), 125 mM hydroxylamine, 10 mM nucleotide triphosphate, 20 mM magnesium chloride and 2.2-2.6U glutamine synthetase. For the glutamate analog study, amino acids (and cc-ketoglutarate) were 25 mM; for the nucleotide triphosphate substitution experiments, the L-glutamate concentration was 50mM. The total volume of the reaction mixtures was 0.5 ml and they were incubated for 15 min at 37°C. The reactions were terminated by the addition of 0.75 ml ferric chloride reagent (Pamiljans et al., 1962).
Substrate
specijicity
Activity of avian liver glutamine synthetase was dependent upon the presence of L-glutamate, ATP, Mg2+ and NH: or hydroxylamine. Of several glutamate analogs tested, only cr-hydroxyglutarate and D-
glutamate were partially effective as substrates; their activities relative to L-glutamate were each approx. 40%. Data are given in Table 3. Activity with both Land D-isomers of glutamate appears to be characteristic for most if not all glutamine synthetases (Meister, 1974; Monder, 1965). Interaction of the D-isomer with glutamine synthetase has been explained by Meister in terms of a rotation of the molecule which brings the amino and carboxyl groups into the same relative positions observed with L-glutamate (Meister, 1974). Acetylation or carbamylation of the amino nitrogen abolishes activity as does conversion of the amino group to a keto function. L-Aspartate was neither a substrate nor an inhibitor in keeping with Meister’s view that glutamate binds to the active site in a fully extended form with binding sites for the amino group and both carboxyl groups (Meister, 1974). As shown in Table 3, none of the nucleotide triphosphates tested were substitutes for ATP nor was Mn2+ an effective replacement for Mg2+ under the assay conditions employed (biosynthetic formation of y-glutamylhydroxamate at pH 7.4). Manganese ions will however completely support activity of the biosynthetic reaction at pH 4.8 (Monder, 1965) and will also function in the glutamyltransferase assay (Meister, 1974).
A
B
C Fig. 3. Electron micrographs of chicken liver glutamine synthetase. Purified enzyme was negatively stained with uranyl acetate as described under Materials and Methods. Magnifications for the larger and smaller panels were x 290,000 and x 980,000, respectively.
Fig. 1. Electrophoresis of purified glutamine synthetase. Gel A, electrophoresis of 50 ng of the native enzyme on 5% polyacrylamide gels in Tris-glycine (pH 8.9). Gel B, electrophoresis of 50,~~g dissociated enzyme on 7.5% polyacrylamide gels in Tris-glycine, (pH 8.9) and 0.1% SDS. Gel C. 50,~g native enzyme subjected to isoelectric focusing as described under experimental procedures. Migration is from the top (cathode) downward.
751
Characterization of glutamine synthetase
76-
0.2
0.6
0.4 Relative
0.6
1.0
mobility
Fig. 2. Subunit molecular weight of chicken liver glutamine synthetase. SDS-polyacrylamide gel electrophoresis was carried out on 7.5% gels in 0.2 M sodium phosphate buffer (pH 7.2). Standard marker proteins and their molecular weights were: 0 bovine serum albumin, 68,000; n catalase, 58,000: o ovalbumin. 45,000; 0 pepsin, 34.700; A chymotrypsinogin, 25,000; l myoglobin, 17,800; W glutamine synthetase. Logarithms of the molecular weights are plotted against the electrophoretic molibities relative to the Bromophenol Blue tracking dye. Kinetic parameters
Avian glutamine synthetase was found to obey Michaelis-Menten kinetics over a wide range of reactant concentrations. For these experiments a coupled assay system employing pyruvate kinase and lactate dehydrogenase was used so that NADH oxidation could be determined spectrophotometrically. This method of assay allows the reaction to be monitored in a continuous manner and in addition permits the use of substrates at concentrations which are more physiologically relevant. The experimental protocol and evaluation of the kinetic constants were as described by Rudolph & Fromm (1979). A series of initial rate studies were carried out in which one substrate was varied and the remaining two were held at different nonsaturating levels (but maintained at a
753
constant ratio). From double-reciprocal plots of the data (not shown), secondary plots of the intercepts and slopes were made and kinetic constants evaluated. The apparent K,s for NH:, ATP and glutamate determined by this method were 0.50, 0.87 and 6.2 mM, respectively, and fall within the range of values reported for several vertebrate and plant glutamine synthetases (Tate et al., 1972; Deuel et al., 1978; Tiemier & Milman, 1972; Seyama et al., 1972; Pamiljans ef al., 1962; O’Neal & Joy, 1974). Interestingly, from the primary plots, it was found that the Kapp decreased as the concentrations of glutamate and NH: were lowered. With the latter substrates at concentrations of 1.5 mM and 35pM, the apparent K, for ATP decreased to 0.24mM. O’Neal & Joy (1974) have observed a similar decrease in the apparent K, for ATP with lowered concentrations of glutamate in studies with pea leaf glutamine synthetase. In both the coupled assay system used here and the biosynthetic y-glutamylhydroxamate assay used by O’Neal & Joy, the concentration of Mg2+ was held constant. The apparent K, for NH: responded in a manner similar to that of ATP. As the concentrations of ATP and glutamate were lowered to 0.25 and 1.5 mM, the K,,, for NH: decreased to a value of 70pM. The apparent K, for glutamate remained essentially unchanged with decreasing concentrations of ATP and NH:. Whether or not the decreases in K,,, noted for ATP and NH; are physiologically significant cannot be assessed at this time. It should be emphasized however that the K,,, values determined from the secondary plots are derived at infinite concentrations of the remaining substrates. Effect of metabolites
Glutamine synthetase is a key component of nitrogen metabolism in all biological systems and its interaction with various metabolites is well documented. Hence a number of amino acids, nucleotides and other metabolites were examined for their possible effects on avian liver glutamine synthetase activity. Results are given in Table 4. Except for methionine sulfoximine, a well-characterized inhibitor of glutamine synthetase, none of the amino acids tested af-
Table 4. Effect of various compounds synthetase
on avian liver glutamine
Relative activity Compound added None L-Alanine L-Serine Glycine L-Histidine L-Glutamine a-Ketoglutarate NAD Methionine sulfoximine
Concentration (mM) 20 20 20 20 20 20 10 1.0
Mg2+ 100 96 96 94 102 104 104 98 64
Mn” 100 57 69 69 64 147 110 ND ND
Glutamine synthetase activity was determined as described in Table 3 with either 50 mM magnesium chloride or 2 mM manganous chloride. ND = not determined.
754 fected
JEAN E. VORHABEN
activity
when
assay
with
Mg2+
ion.
With
Mn2+ as the divalent cation, inhibitions of 30-35% were found with alanine, serine, glycine and histidine. The partial inhibition observed with these amino acids is typical of many, if not all, glutamine synthetases (Tate et al., 1972; Villafranca & Balakrishnan, 1979; Sawhney & Nicholas, 1978). In general, greater inhibition is observed in the presence of the Mn2+ ion and traces of the latter will reportedly sensitize rat liver preparations to inhibition by amino acids in the presence of excess Mg” (Tate & Meister, 1971). The extent of inhibition varies with the nature of the amino acid and source of enzyme. Although responses of the rat and avian liver enzymes were similar with regard to most of the compounds examined, two differences were noted. Rat liver glutamine synthetase is subject to substantial activation by a-ketoglutarate with either Mgzf or Mn2+ ions (Tate & Meister, 1971) whereas the avian enzyme exhibits no significant stimulation under similar reaction conditions. The absence of a stimulatory effect by cc-ketoglutarate was confirmed by a second assay procedure in which the release of inorganic phosphate was measured. However, by raising the concentration of Mni+ from 2 to lOmM, it was possible to attain an increase in activity of over 2-fold in the presence of the keto acid. Given the high level of Mn2+ required for activation, it is doubtful that the proposed physiological relevance of the cr-ketoglutarate effect (Tate & Meister, 1971) is applicable to the avian enzyme. A second Table 5. Effect of metal ions on avian liver glutamine synthetase Metal
Concentration fmM)
% Inhibition
Cd’+
0.05 0.50
11 13
coz+
5.0 10.0
0 12
CL?+
0.50 5.00
87 90
Fe3+
0.05 0.50
26 69
Hg’+
0.05 0.50
94 100
Ni’+
0.50 5.00
14 59
Pb’+
0.05 0.50
90 90
Zn2+
0.50 5.00
60 84
Glutamine synthetase activity was determined as described in Table 3 with 15 mM magnesium chloride, 25 mM hydroxylamine and metal chlorides at concentrations given in the Table. Controls with p-glutamylhydroxamate showed no inhibitory effects on color development by any of the metal ions tested.
et al.
c Laclg q.CTbCI,l
50
100
m PM 9
150
200
J
4 3 2 tC0Cl~l0,tMnCl~3*,mM I
Fig. 4. Inhibition of glutamine synthetase by Ca*+, Mg”, La3+ and Tb3+ ions. Activity was determined as described in the legend of Table 3 with 10 mM ATP, 15 mM magnesium chloride, 25 mM hydroxylamine and 50mM L-glutamate.
difference noted between the rat and chicken glutamine synthetases is their contrasting response to glutamine; the former is inhibited by this amino acid (Deuel et al., 1978) whereas the latter is activated. A number of nucleotides were screened as possible effecters of avian liver glutamine synthetase. At concentrations of lOmM, the following compounds had little (inhibitions of 5% or less) or no effect on activity with Mg2+ ion: AMP, IMP, UMP, CDP, UDP, CTP, UTP and NAD. Only ADP gave a significant inhibition which appeared to be competitive with respect to ATP (data not shown). A Ki for ADP of 2 mM was determined from these data. Inhibition by metal ions The sensitivity of avian glutamine synthetase to various metal ions was investigated. Results are given in Table 5 and Fig. 4. At concentrations of 50pM, Hg2+ and Pb2+ virtually abolished activity and lo-fold higher concentrations of Cu”, Cd’+, Fe3+ and Zn2+ produced significant inhibition. Ni2+ was a much weaker inhibitor and Co” was essentially noninhibitory. The absence of an inhibitory effect by Co’+ has been observed with at least two other eukaryotic glutamine synthetases (Monder, 196.5; Kanamori & Matsumoto, 1972). Since the avian enzyme contains nine sulfhydryl groups per subunit and is subject to inactivation by thiol reagents such as N-ethylmaleimide and iodoacetamide (inhibitions of 92 and 85x, respectively, at concentrations of 25 mM), the inhibitory effects of some of the heavy metal ions may be accounted for by their interaction with sulfhydryl groups. In addition to the heavy metal ion effects noted above, the avian enzyme was inhibited by relatively low concentrations of manganese and calcium ions as shown in Fig. 4. Inhibitions of 50% were observed at concentrations of approx. 875 and 175pm for Ca2+ and MnZ+, respectively. Mammalian glutamine synthetases are also sensitive to inhibition by Mn2+ ion and Mg 2c does not apparently readily reverse this inhibition (Tate & Meister, 1973; Monder, 1965). In
Characterization
of glutamine synthetase
studies with the rat liver enzyme, substantial inhibition was still evident at Mg’+ to Mn’+ ratios of
1ooO. More potent inhibitors were the trivalent lanthanide ions, La3+ and Tb3’. These ions have ionic radii similar to those of CaZ+ in its different coordination states and have been utilized to probe the calciumbinding sites of a number of proteins including E. co/i glutamine synthetase (Martin & Richardson, 1979; Wedler & D’Aurora, 1974). Extensive physicochemical studies of the latter enzyme have shown a complex picture of metal ion interactions. There are at least two metal binding sites, separated by a distance of approximately 6A. One site interacts with Mg ‘+-ATP; the second metal ion site binds Mn2+ and induces a protein conformational change to an active tightened state. In addition, Mn2 + may participate in the binding of glutamate to the catalytic site. Speculations regarding metal ion interaction with eukaryotic glutamine synthetases are premature at the present time. The situation is obviously complex in that Mn” and Ca2+ ions are inhibitory while Co2+ is not; yet Co2+ and Mn2+ but not Ca2+ will meet the divalent cation requirement and support activity, especially at lower pHs (Monder, 1965). Effects of these metal ions are not readily explainable in terms of competition with Mg2’ ion for ATP since the stability constant of Ca2 ‘-ATP (9300 M- ‘) is lower than those of Co2+ (45,000 M-‘) and Mn2+ (60,200 M-l) and are greater than that of Mg2 ’ (16,600 M - ‘) (Tu & Heller, 1974)*. Further studies are needed to distinguish between metal ion interaction with cysteine residues, with metal-ATP binding site(s) or with yet other protein ligands, such as carboxylate and amino moieties. The objective of the present study was an overall characterization of avian liver glutamine synthetase. Our results indicate that, despite its mitochondrial localization, properties of the chicken enzyme markedly resemble those of the weakly bound microsomal rat liver enzyme and mammalian glutamine synthetases in general. Its octameric structure, amino acid composition, Michaelis constants, subunit molecular weight, substrate specificity and sensitivity to various metal ions and metabolites are similar to those reported for eukaryotic glutamine synthetases. Indeed, the only exception of note is the absence of an activating effect by a-ketoglutarate when assayed with magnesium ion. The physiological significance of the inhibitory effects of alanine, serine and glycine as well as those of Ca2+ and Mn2+ cannot be assessed at the present time. Given its mitochondrial localization and the proposed role of the mitochondrion in regulating intracellular levels of Ca’+ (Borle, 1975), hit is conceivable that Ca’+, rather than Mn2+ ion, functions in the regulation of avian liver glutamine synthetase. In this context, it is interesting to note that carbamyl phosphate synthetase 1, the mammalian complement * Values reported for the stability constants are dependent upon pH variables such as temperature and ionic strength and may differ with the method of determination. However for a given study, the stability constants decrease in the following sequence: Mn2+ > Co2+ > MgZ+ > CaZf (Tu & Heller, 1974). B.T.14/8-E
755
of uricotelic glutamine synthetase, is inhibited by Ca2+ at concentrations which do not interfere with mitochondrial ATP generation and which are pysiologically relevant (Meijer et al., 1981). SUMMARY
Glutamine synthetase has been purified over 750-fold from chicken liver mitochondria. The preparation was judged to be homogeneous on the basis of a single protein band appearing after analytical isoelectrofocusing and after polyacrylamide gel electrophoresis of the enzyme in the presence and absence of SDS. The molecular weight of the native enzyme was 389,000 dalton as determined by sedimentation equilibrium analysis. The molecular weight of its monomeric subunit was 42,000 as determined by SDS-gel electrophoresis. Electron micrographs suggest the enzyme is an octamer, a structure typical of eukaryotic glutamine synthetases. The amino acid composition was also similar to other vertebrate glutamine synthetases. The apparent Michaelis constants determined for ammonia, ATP and glutamate were 0.5, 0.9 and 6 mM, respectively. Of a number of compounds tested for their effect on glutamine synthetase activity, only ADP and methionine sulfoximine gave a significant degree of inhibition when activity was measured by the y-glutamylhydroxamate assay in the presence of Mg2+ ; when Mn2 ’ was substituted for Mg’+, several amino acids (L-alanine, L-serine, L-glycine) were partially inhibitory whereas L-glutamine and ec-ketoglutarate were slightly stimulatory. A number of heavy metal ions were inhibitory as were Ca2’, Mn2+ and lanthanide ions. In general, the physical and chemical properties of the avian liver mitochondrial enzyme reported here resemble those of other eukaryotic glutamine synthetases. Acknowledgements-This work was supported by grants from the National Science Foundation (PCM 75-13161 and BBS 79-25911). REFERENCES
ALEF K., BURKARDTH-J., HORSTMANN .H-J. & ZUMFT W. G. (1981) Molecular characterization of glutamine syn-
thetase from the nitrogen-fixing
phototropic
bacterium
Rhodopseudomonas pahktris. 2. Naturf: 36; 246254. BOHMEH-J., KOPPERSCHLAGER G.. SCHULZJ. & HOFMANN E. (1972) Affinity chromatography of phosphofructokinase using Cibacron blue F3G-A. J. Chromat. 69,
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BRADFORDM. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analvt. Biochek 12, f!48-2j4. . . CAMPBELLJ. W. & VORHABEN J. E. (1976) Avian mitochonrial glutamine metabolism. J. biol. Chem. 251, 781-786. DARROW R. A. & KNOTTY,R. R. (1977) Two forms of glutamine synthetase in free-living root-nodule bacteria. Biochem. biophys. Res. Commun. 78, 554559.
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KATUNAMAN., MATUSUDAY. & KURODAY. (1970) Phylogenie aspects of different regulatory mechanisms of glutamine metabolism. In Advances in Enzyme Regulation (Edited by WEBERG.) Vol. 8, pp. 73-81. Pergamon Press, Oxford. LIN W. S. & KAPOORM. (1978) Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa. Can. J. Biochem. 56, 927-933. LOWRY0. H., ROSEBROIJGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZELJ. V. (1971) Polyacrylamide gel electrophoresis of viral proteins. Meth. Viral. 5, 179-246. MARTINB. R. & RICHARDSON F. S. (1979) Lanthanides as probes for calcium in biological ivstems. 0. Rev. Bio_ _ phys. 12, 181-189. MAT~UNOT. & SHIRASAWA N. (1978) Interferon suvvresses . .. steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina. Biochim. biophys. Acta 538, 188-194.
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SAWHNEYS. K. & NICHOLASD. J. D. (1978) Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica. Biochim. hiophys. Acta 527, 485-489.
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TATE S. S., LEU F-Y., & MEIS~R A. (1972) Rat liver glutamine synthetase. Preparation, properties and mechanism of inhibition by carbamyl phosphate. J. biol. Chem. 247, 5312-5321.
TATE S. S. & MEISTERA. (1971) Regulation of rat liver glutamine synthetase: Activation by cc-ketoglutarate and inhibition by glycine, alanine and carbamyl phosphate. Proc. natn. Acad. Sci. U.S.A. 68, 781-785.
TATE S. S. & MEISTERA. (1973) Glutamine synthetases of mammalian liver and brain. In The Enzymes of Glutamine Metabolism (Edited by PRUSINERS. & STADTMAN E. R.) pp. 77-127. Academic Press, New York. TIEMIERD. C. & MILMANG. (1972) Chinese hamster liver glutamine synthetase. Purification, physical and biochemical properties. J. biol. Chem. 247, 2272-2277. Tu A. T. & HELLERM. H. (1974) Structure and stability of metal-nucleotide phosphate complexes. In Metal Ions in Biological Sysrems (Edited by SIGELH.) Vol. 1, pp. l-49. Marcel Dekker, New York. TULI R., JAWALI N. & THOMASJ. (1979) A rapid method for the purification of glutamine synthetase from the bluegreen alga Anabaena L-31. Ind. J. exp. Biol. 17, 1239-1241. VILLAFRANCA J. J. & BALAKRISHNAN M. S. (1979) Glutamine synthetase: Biophysical studies of structure-function control relationships. In?. J. Biochem. 10, 565-571. VORHABEN J. E. & CAMPBELLJ. W. (1972) Glutamine synthetase. A mitochondrial enzyme in uricotelic species. J. biol. Chem. 247, 2763-2767.
VORHABENJ. E. & CAMPBELLJ. W. (1977) Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver. J. Cell Biol. 73, 300-310. WEBERK., PRINGLEJ. R. & OSBORNM. (1972) Measurement of molecular weights by electrophoresis on SDSacrylamide gel. Meth. .b%zym.-26, 3-27: WEDLERF. C. & D’AURORAV. (1974) Svectroscovic . . vrobes . of Escherichia cob glutamine synthetase. Rare earth ions by difference absorption. Biochim. biophys. Acta 371, 432-441.
WEDLERF. C., KENNEYR. M.. ASHOURA. E. & CARFI J. (1978) Two regulatory isozymes of glutamine synthetase from Bacillus caldolyticus, an extreme thermophile. Biothem. biophys. Res. Commun. 81, 122-126. Wu C. (1963) Glutamine synthetase. I. A comparative study of its distribution in animals and its inhibition by DL-allohydroxylysine. Comp. Biochem. Physiol. 8, 335-351.
Wu C. (1963) Glutamine synthetase II. The intracellular localization in rat liver. Biochim. biophys. Acta 77, 482-493.
Int. J. Biochem. Vol. 14. pp. 747 to 756, 1982 Printed in Great Britain. All rights reserved
CHARACTERIZATION OF GLUTAMINE SYNTHETASE FROM
AVIAN LIVER
MITOCHONDRIA
JEAN E. VORHABEN, DARWIN D. SMITH and JAMESW. CAMPBELL Department of Biology, William Marsh Rice University, Houston, TX 77001, U.S.A.
(Received 25
Nowmber
1981)
Abstract-l. Glutamine synthetase has been purified to homogeneity from chicken liver mitochondria. 2. The native enzyme is an octamer composed of identical subunits with monomeric mol. wt of 42,000 dalton. 3. Apparent K,s for NH:. ATP and glutamate were 0.5, 0.9 and 6 mM, respectively. D-Glutamate and L-a-hydroxyglutarate were utilized as substrates with activities approx. 40% those obtained with glutamate. Of several nucleotides tested, none were effective replacements for ATP. 4. Heavy metal ions were inhibitory as were Mn*+, Ca2+ and lanthanide ions. 5. Despite its different subcellular localization and physiological function, avian glutamine synthetase is markedly similar to the weakly-bound microsomal rat liver enzyme with respect to a number of physical and chemical properties.
INTRODUCTION Previous studies from our laboratory have shown that the function of glutamine synthetase (EC 6.3.1.2) in livers of uricotelic vertebrates appears to be analogous to that of carbamyl phosphate synthetase 1 (EC 2.7.2.5) in ureotelic animals (Vorhaben & Campbell, 1972, 1976, 1977). Each enzyme catalyzes a reaction which results in the effective removal of mitochondrially-generated ammonia via its conversion to a nitrogenous end-product precursor. This is glutamine in the case of uricoteles and carbamyl phosphate in the. case of ureoteles. The ammonia utilized as substrate by these enzymes is ultimately derived from amino acid nitrogen. During protein catabolism, amino acids undergo transamination with LX-ketoglutarate in the cytosol and in so doing, funnel amino nitrogen into glutamate. Transport of glutamate into the mitochondrion is followed by its oxidative deamination to release ammonia. In keeping with its physiological function, avian liver glutamine synthetase, like carbamyl phosphate synthetase 1, is localized in the mitochondrial matrix, the site of ammonia release via glutamate dehydrogenase (Vorhaben & Campbell, 1977). It shows a marked response to conditions of gluconeogenesis such as starvation or high protein diets (Katunama et al., 1970), a response also analogous to that shown by carbamyl phosphate synthetase 1 in mammalian liver. On the other hand, mammalian liver glutamine synthetase appears to be loosely bound to the endoplasmic reticulum (Wu, 1963) and does not respond to starvation or high dietary protein intake (Katunama et al., 1970), indicating that it is not primarily involved in the disposal of a-amino nitrogen. Although the purification and characterization of glutamine synthetase from a number of higher eukaryotic sources have been reported including rat and hamster liver (Tate et al., 1972; Deuel rt al.. 1978; Tiemier & Milman, 1972) sheep brain (Ronzio et al., 1969) and chick neural retina (Sarkar ct al., 1972), the
properties of the uricotelic liver enzyme have not been investigated in depth (Seyama et al., 1972). Given the differences noted above between the uricotelic and ureotelic enzymes with respect to physiological function and subcellular localization and considering the hypothesis regarding the symbiotic origin of mitochondria, we deemed it important to further characterize the avian liver enzyme. The results of our studies indicate properties of the uricotelic liver enzyme more closely resemble those of glutamine synthetase isolated from other vertebrate tissues rather than from prokaryotic or lower eukaryotic sources. MATERIALSAND
METHODS
Materials
With the exception of NAD and NADH, which were purchased from PL Biochemicals, all biochemical reagents were obtained from Sigma. Potassium phosphate, magnesium chloride, calcium chloride and sucrose were products of Fisher Co. Lanthanum chloride and terbium chloride were obtained from Aldrich Co. Sources of additional materials are given in other sections of Materials and Methods. All other reagents were of the highest purity available commercially. Enzyme
pur@cation
As source material for the purification, White Leghorn hens were obtained from a local commercial supplier. Animals were starved for 4 days before killing by decapitation to induce higher levels of enzyme (Katunama et al., 1970). The procedure utilized for the isolation of avian liver glutamine synthetase was a modification of that used fur the purification of the rat liver enzyme (Tate et al., 1972). All steps were performed at 4-5°C. The initial step in the purification of chicken liver glutamine synthetase was the preparation of mitochondria. These were isolated as follows. Ten percent liver homogenates (from 84 g liver) were prepared in 0.25 M sucrose containing 5 mM HEPES (pH 7.37.4) utilizing a PotterElvehjem homogenizer. The homogenate was centrifuged at 350 9 for 10 min and the loose pellet washed .once with 747
JEAN
748
E.
VORHABEN
100 ml isolating medium. The supernatant fluid and washing were combined and centrifuged at 9000 g for 20 min to sediment mitochondria. Following a single wash, the mitochondrial pellet was resuspended in minimal isolating medium (80ml), lypholized overnight and powdered by means of mortar and pestle. Extraction of the powder (12 g) was accomplished by the addition, with stirring, of 75 ml of 50 mM potassium phosphate buffer (pH 7.4); stirring was continued for 1 hr after which the suspension was centrifuged at 100,OOOg for 60 min. The pellet was extracted a second time with 60ml buffer. The extracts were combined and applied without volume reduction to a hydroxyl apatite column (Bio Rad, 2.6 x 30 cm, previously equilibrated with phosphate buffer) with the aid of a peristaltic pump. Elution was carried out with a linear gradient of 50-500 mM ohosohate buffer CDH7.41.The enzvme was eluted at a phbsphate concentrition 0; 0.21 M which is similar to the elution characteristics of the rat liver enzyme (Tate et al., 1972). After a reduction in volume by pressure filtration (UM 10 filter, Amicon Corp.), the eluate was subjected to gel filtration chromatography on a Bio Gel P-300 column (2.6 x 76cm) equilibrated with a solution of 0.15 M potassium chloride-l mM EDTA brought to a pH of 7.4; elution was with the equilibrating solution. Active fractions were pooled, concentrated by ultrafiltration, dialyzed against 50mM imidazole buffer containing 2mM EDTA (pH 7.4) and applied to a DEAE
et ul.
massie Blue in a 9% acetic acid-4%; methanol solution. Destaining was achieved by diffusion in a 7.5% acetic acid-5% methanol solution. Protein standards and their respective sources were: bovine serum albumin, pepsin, ovalbumin (Sigma); myoglobin and chymotrypsinogen (Mann Research) and catalase (Worthington). Analytical isoelectric focusing was performed in 3% polyacrylamide gels with 2% (w/v) ampholytes (Bio Rad, pH 3-10) according to Maize1 (1971). The pl of glutamine synthetase was also verified by isoelectric focusing using a IlOml LKB preparative isoelectric focusing column and following LKB manual procedures. Analytical
ultracentrijiigation
Determination of the molecular weight of the native enzyme by sedimentation equilibrium analysis was kindly performed by Dr Richard M. Caprioli. Analytical Chemistry Center, University of Texas Medical School at Houston with the technical assistance of Mr Tim Woodward. The analysis was performed with a Beckman Model E analytical ultracentrifuge equipped with U.V.scanning optics. Molecular weights were calculated by the method of meniscus depletion assuming a partial molar volume of 0.74. Amino acid composition
Protein was hydrolyzed in uacuo for 24, 48 and 72 hr with 6 N hydrochloric acid at 110°C. Amino acid analyses were performed at Rice University by Dr Kathleen S. Matthews, Department of Biochemistry, on a Beckman Model 120-C analyzer. Electron microscopy
Negative staining of glutamine synthetase was accomplished essentially as described by Haschemeyer (1970). A 2 ~1 aliquot of the purified enzyme (50 pg/ml) in 50 mM imidazole buffer (pH 7.4) was applied to a 400 mesh copper grid coated with Formvar. After standing for 5 min, residual solution was removed by means of a filter paper wick. Immediately thereafter, 2 ~1 of a 2% uranyl acetate solution was applied and allowed to stand for 5 min. Excess staining solution was removed by again touching to filter paper and the residual material was then allowed to dry. Electron microscopy was performed with a Philips 200 instrument operated at 60 kV. RESULTS
Glutamine synthetase
AND
DISCUSSION
assay
Glutamine synthetase activity was determined colorimetrically by following the formation of y-glutamylhydroxamate in reaction mixtures containing glutamate, hydroxylamine, ATP and Mg *+. Reactant concentrations are given in legends. For the determination of some kinetic parameters of the purified enzyme, a spectrophotometric method was employed which measures NADH oxidation continuously (Shapiro & Stadtman, 1970). The release of ADP was followed in a coupled assay system containing 4 U pyruvate kinase, 1.5 mM phosphoenolpyruvate, 2.5 U lactate dehvdroeenase. 0.1 mM NADH, 100 mM notassium chloride, 16 rnh? maghesium chloride ‘and 50 m-M imidazole buffer (pH 7.4) in a volume of 1 ml. Glutamate, ATP and ammonium chloride concentrations were varied as described in the text. The temperature was 37°C. Doubling the amount of coupling enzymes had no effect on the reaction rate. Electrophoresis
Analytical polyacrylamide gel electrophoresis of the undissociated native enzyme was performed as described by Maize1 (1971) using 5% gels in 50 mM Tris-glycine buffer (pH 8.9). For the dissociated enzyme, SDS-gel electrophoresis was carried out according to the procedure of Weber et al. (1972). Gels were fixed and stained with COO-
Glutamine synthetase from avian liver mitochondria has been purifed over 750-fold by the steps summarized in Table 1. This purification scheme represents a significant improvement with respect to both purity and overall yield compared to that previously reported (Seyama et al., 1972). The binding of the avian enzyme to various affinity chromatography media differs considerably from that reported for glutamine synthetases from microbial sources. For example, the avian enzyme was dissociated from Blu&3epharose by 1 M sodium chloride but not by buffered solutions of 2 mM ADP or ATP whereas the reciprocal conditions hold for the release of glutamine synthetases from blue-green algal and bacterial sources (Stacey et al., 1979; Tuli et al., 1979; and Siede1 & Shelton, 1979). Glutamate-Sepharose has been used in the isolation of yeast glutamine synthetase (Lin & Kapoor, 1978) yet our partially purified enzyme (a post DEAE-cellulose preparation) was not bound to glutamate-Agarose (Sigma) under similar conditions. Binding of the avian enzyme to glutamine-Sepharose (Sigma) was also negative. We might add that attempts to purify the chicken glutamine
Characterization
of glutamine synthetase
749
Table 1. Purification of glutamine synthetase from chicken liver Total protein
(mg) Crude liver homogenate Mitochondrial extract Hydroxylapatite chromatography Bio Gel P-300 chromatography DEAE-cellulose chromatography Blue-Sepharose chromatography
24,412 1345 110 32 15 13
synthetase by the zinc precipitation method of Miller et al. (1974) were unsuccessful. Evidence for the homogeneity of our preparation is given in Fig. 1. Single protein bands appeared when the native enzyme was subjected to polyacrylamide gel electrophoresis in the absence (Gel A) or presence (Gel B) of sodium dodecylsulfate. Our failure to observe multiple bands when the enzyme is electrophoresed under dissociating conditions suggests the avian enzyme is probably composed of identical subunits, a property characteristic of all glutamine synthetases heretofore purified. Isoelectric focusing of our purified enzyme also showed the presence of a single protein as shown by Gel C of Fig. 1. Utilizing preparative isoelectric focusing conditions, a p1 of 6.1 was determined for the purified avian liver enzyme which is significantly higher than the value of 4.2 reported for the sheep brain enzyme (Pamiljans et al., 1962) but within the range of PI’S (5.2-6.1) measured for glutamine synthetases from several bacterial sources (Wedler et al., 1978; Alef et al., 1981; Darrow &
Knotts, 1977). Although evidence for a difference in the isoelectric points of the rat liver and ovine brain enzymes has been presented (Tate & Meister, 1973), no value for the former enzyme was given and hence a comparison of the avian and rat liver enzymes cannot be made. The absorption spectrum of the avian liver enzyme was that of a typical protein without chromophoric groups and the probable absence of a bound adenyl group was suggested by a A28,JA260 ratio of 1.69. Molecular weight of the native enzyme and its subunit
The molecular weight of the monomeric subunit was determined by comparison of the relative migration of avian glutamine synthetase with the mobilities of standard proteins during SDS-gel electrophoresis. From these data, an average mol. wt of 42,000 + 1000 was calculated. A representative plot is shown in Fig. 2. The molecular weight determined for the avian liver monomer is identical to that reported for the chick retinal and liver subunit (Sarkar et al., 1972; Matsuno & Shirasawa, 1978) and is within the range of values determined for other mammalian glutamine synthetase subunits including rat liver, 44,000 (Tate et al., 1972); hamster liver, 42,000 (Tiemier & Milman, 1972) and ovine brain, 49,000 (Tate et al., 1972). From sedimentation equilibrium analyses, the molecular weight of the native enzyme was determined to be 389.000 daltons; on the basis of a monomeric subunit mol. wt of 42,000, a structure composed of nine subunits would be implied. However, as discussed below, electron micrographs of the native enzyme are
Purification
Specific activity
(Ujmg) Recovery 2.15 31.4 371 600 700 1700
100 80.3 77.6 36.5 20.0 41.9
yield (%) 1.0 14.6 173 279 326 789
consistent with a structure composed of eight subunits. Octameric structures would therefore appear to be typical of all eukaryotic glutamine synthetases (Lin & Kapoor, 1978; Tate & Meister, 1971). Moreover equilibrium ultracentrifugation studies with ovine brain and hamster liver glutamine synthetases suggest protein aggregation may occur during analyses leading to artificially high molecular weight determinations (Tiemier & Milman, 1972; Tate & Meister, 1971). Sedimentation equilibrium analysis of the chick neural retinal glutamine synthetase, having a subunit molecular weight identical to that of the avian liver enzyme (42,000), likewise yielded a molecular weight in excess of that predicted by an octameric structure (Tiemier & Milman, 1972). Electron microscopy An electron micrograph of a negatively stained avian liver glutamine synthetase preparation is shown in Fig. 3. The field shows only nine enzyme molecules since higher concentrations of protein lead to aggregated structures. Tetrameric structures having a square projection are evident under low and high magnification with each of the four subunits occupying a corner of the square. Similar forms have been observed in uranyl acetate stained preparations of glutamine synthetase from ovine brain (Haschemeyer, 1968) hamster liver (Tiemier & Milman, 1972) and chick retina (Sarkar et al., 1972). The octameric structure would be generated by one tetrad layered above another. Particles are approx. 100 A on a side which is within the range of dimensions reported for the enzyme isolated from hamster liver, 90 x 90 A (Tiemier & Milman, 1972); chick retina, 90 x 90 x 125 A (Sarkar et al., 1972) and ovine brain, 90 x 90 x 120 A (Haschemeyer, 1968). Measurements of three-dimensional structure cannot be made from our micrographs. Nonetheless, the general appearance and size of the particles are similar to those found for other eukaryotic glutamine synthetases and is in accord with a structure composed of eight rather than nine subunits. Amino acid composition
The amino acid compositions of avian liver and several other eukaryotic glutamine synthetases are compared in Table 2. Hydrolysis was performed in 6N hydrochloric acid rather than methane sulfonic acid to facilitate comparison with other glutamine synthetases hydrolyzed under similar conditions. Marked similarities in amino acid composition are evident, especially with regard to the chicken and mammalian enzymes.
750
JEAN EVORHABEN
rt al.
Table 2. Amino acid composition of glutamine synthetase from eukaryotic sources
Amino acid Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Chicken Chicken Rat* Ovine* Neurospora* crassa liver retina liver brain (mol/mol of subunit?) 27 24 41 9: 39 38 17 17 24 22 10 17 22 20# 175 6/( 15 17
24 24 35 ND 31 35 19 15 20 18 ND 20 20 24 &I 14 19
28 21 48 12 33 42 13 23 25 25 12 21 23 24 20 6 17 20
29 25 41 12 44 41 11 21 22 25 11 21 25 25 20 7 16 18
35 23 40 4 45 30 9 19 35 21 9 15 20 26 22 16 13 27
*Values for the chick retina enzyme taken from (Sarkar et al., 1972), for the rat liver and ovine brain from (Tate et al., 1972) and for Neurospora crassa (Lin & Kapoor, 1978). t Subunit molecular weights are: chicken liver and retina, 42,000 dalton; rat liver, ovine brain and Neurospora crassa, 45,000 dalton. $ Determined by titration with Ellman’s reagent (Habeeb, 1972). $ Extrapolated to zero time of hydrolysis. j/ Determined by the spectrophotometric method of Spande & Witkop (1967). ND = not determined.
Table 3. Substrates of glutamine synthetase Substrate
Relative activity
L-Glutamate, 25 mM L-cr-Hydroxyglutarate, 25 mM D-Glutamate, 25 mM p-Methyl-L-glutamate, 25 mM N-Acetylglutamate, 25 mM N-Carbamyl glutamate, 25 mM L-Aspartate, 25 mM a-Ketoglutarate, 25 mM
100 41 39 16 6 4 6 0
ATP, 1OmM ITP, 10 mM GTP, 10 mM TTP, 10mM
100 10 3 3
Mg”, 10mM MgZi, 2 mM Mn”, 1OmM
100 13 2
Glutamine synthetase activity was determined by measuring y-glutamylhydroxamate in reaction mixtures containing 50 mM imidazole buffer (pH 7.4), 125 mM hydroxylamine, 10 mM nucleotide triphosphate, 20 mM magnesium chloride and 2.2-2.6U glutamine synthetase. For the glutamate analog study, amino acids (and cc-ketoglutarate) were 25 mM; for the nucleotide triphosphate substitution experiments, the L-glutamate concentration was 50mM. The total volume of the reaction mixtures was 0.5 ml and they were incubated for 15 min at 37°C. The reactions were terminated by the addition of 0.75 ml ferric chloride reagent (Pamiljans et al., 1962).
Substrate
specijicity
Activity of avian liver glutamine synthetase was dependent upon the presence of L-glutamate, ATP, Mg2+ and NH: or hydroxylamine. Of several glutamate analogs tested, only cr-hydroxyglutarate and D-
glutamate were partially effective as substrates; their activities relative to L-glutamate were each approx. 40%. Data are given in Table 3. Activity with both Land D-isomers of glutamate appears to be characteristic for most if not all glutamine synthetases (Meister, 1974; Monder, 1965). Interaction of the D-isomer with glutamine synthetase has been explained by Meister in terms of a rotation of the molecule which brings the amino and carboxyl groups into the same relative positions observed with L-glutamate (Meister, 1974). Acetylation or carbamylation of the amino nitrogen abolishes activity as does conversion of the amino group to a keto function. L-Aspartate was neither a substrate nor an inhibitor in keeping with Meister’s view that glutamate binds to the active site in a fully extended form with binding sites for the amino group and both carboxyl groups (Meister, 1974). As shown in Table 3, none of the nucleotide triphosphates tested were substitutes for ATP nor was Mn2+ an effective replacement for Mg2+ under the assay conditions employed (biosynthetic formation of y-glutamylhydroxamate at pH 7.4). Manganese ions will however completely support activity of the biosynthetic reaction at pH 4.8 (Monder, 1965) and will also function in the glutamyltransferase assay (Meister, 1974).
A
B
C Fig. 3. Electron micrographs of chicken liver glutamine synthetase. Purified enzyme was negatively stained with uranyl acetate as described under Materials and Methods. Magnifications for the larger and smaller panels were x 290,000 and x 980,000, respectively.
Fig. 1. Electrophoresis of purified glutamine synthetase. Gel A, electrophoresis of 50 ng of the native enzyme on 5% polyacrylamide gels in Tris-glycine (pH 8.9). Gel B, electrophoresis of 50,~~g dissociated enzyme on 7.5% polyacrylamide gels in Tris-glycine, (pH 8.9) and 0.1% SDS. Gel C. 50,~g native enzyme subjected to isoelectric focusing as described under experimental procedures. Migration is from the top (cathode) downward.
751
Characterization of glutamine synthetase
76-
0.2
0.6
0.4 Relative
0.6
1.0
mobility
Fig. 2. Subunit molecular weight of chicken liver glutamine synthetase. SDS-polyacrylamide gel electrophoresis was carried out on 7.5% gels in 0.2 M sodium phosphate buffer (pH 7.2). Standard marker proteins and their molecular weights were: 0 bovine serum albumin, 68,000; n catalase, 58,000: o ovalbumin. 45,000; 0 pepsin, 34.700; A chymotrypsinogin, 25,000; l myoglobin, 17,800; W glutamine synthetase. Logarithms of the molecular weights are plotted against the electrophoretic molibities relative to the Bromophenol Blue tracking dye. Kinetic parameters
Avian glutamine synthetase was found to obey Michaelis-Menten kinetics over a wide range of reactant concentrations. For these experiments a coupled assay system employing pyruvate kinase and lactate dehydrogenase was used so that NADH oxidation could be determined spectrophotometrically. This method of assay allows the reaction to be monitored in a continuous manner and in addition permits the use of substrates at concentrations which are more physiologically relevant. The experimental protocol and evaluation of the kinetic constants were as described by Rudolph & Fromm (1979). A series of initial rate studies were carried out in which one substrate was varied and the remaining two were held at different nonsaturating levels (but maintained at a
753
constant ratio). From double-reciprocal plots of the data (not shown), secondary plots of the intercepts and slopes were made and kinetic constants evaluated. The apparent K,s for NH:, ATP and glutamate determined by this method were 0.50, 0.87 and 6.2 mM, respectively, and fall within the range of values reported for several vertebrate and plant glutamine synthetases (Tate et al., 1972; Deuel et al., 1978; Tiemier & Milman, 1972; Seyama et al., 1972; Pamiljans ef al., 1962; O’Neal & Joy, 1974). Interestingly, from the primary plots, it was found that the Kapp decreased as the concentrations of glutamate and NH: were lowered. With the latter substrates at concentrations of 1.5 mM and 35pM, the apparent K, for ATP decreased to 0.24mM. O’Neal & Joy (1974) have observed a similar decrease in the apparent K, for ATP with lowered concentrations of glutamate in studies with pea leaf glutamine synthetase. In both the coupled assay system used here and the biosynthetic y-glutamylhydroxamate assay used by O’Neal & Joy, the concentration of Mg2+ was held constant. The apparent K, for NH: responded in a manner similar to that of ATP. As the concentrations of ATP and glutamate were lowered to 0.25 and 1.5 mM, the K,,, for NH: decreased to a value of 70pM. The apparent K, for glutamate remained essentially unchanged with decreasing concentrations of ATP and NH:. Whether or not the decreases in K,,, noted for ATP and NH; are physiologically significant cannot be assessed at this time. It should be emphasized however that the K,,, values determined from the secondary plots are derived at infinite concentrations of the remaining substrates. Effect of metabolites
Glutamine synthetase is a key component of nitrogen metabolism in all biological systems and its interaction with various metabolites is well documented. Hence a number of amino acids, nucleotides and other metabolites were examined for their possible effects on avian liver glutamine synthetase activity. Results are given in Table 4. Except for methionine sulfoximine, a well-characterized inhibitor of glutamine synthetase, none of the amino acids tested af-
Table 4. Effect of various compounds synthetase
on avian liver glutamine
Relative activity Compound added None L-Alanine L-Serine Glycine L-Histidine L-Glutamine a-Ketoglutarate NAD Methionine sulfoximine
Concentration (mM) 20 20 20 20 20 20 10 1.0
Mg2+ 100 96 96 94 102 104 104 98 64
Mn” 100 57 69 69 64 147 110 ND ND
Glutamine synthetase activity was determined as described in Table 3 with either 50 mM magnesium chloride or 2 mM manganous chloride. ND = not determined.
754 fected
JEAN E. VORHABEN
activity
when
assay
with
Mg2+
ion.
With
Mn2+ as the divalent cation, inhibitions of 30-35% were found with alanine, serine, glycine and histidine. The partial inhibition observed with these amino acids is typical of many, if not all, glutamine synthetases (Tate et al., 1972; Villafranca & Balakrishnan, 1979; Sawhney & Nicholas, 1978). In general, greater inhibition is observed in the presence of the Mn2+ ion and traces of the latter will reportedly sensitize rat liver preparations to inhibition by amino acids in the presence of excess Mg” (Tate & Meister, 1971). The extent of inhibition varies with the nature of the amino acid and source of enzyme. Although responses of the rat and avian liver enzymes were similar with regard to most of the compounds examined, two differences were noted. Rat liver glutamine synthetase is subject to substantial activation by a-ketoglutarate with either Mgzf or Mn2+ ions (Tate & Meister, 1971) whereas the avian enzyme exhibits no significant stimulation under similar reaction conditions. The absence of a stimulatory effect by cc-ketoglutarate was confirmed by a second assay procedure in which the release of inorganic phosphate was measured. However, by raising the concentration of Mni+ from 2 to lOmM, it was possible to attain an increase in activity of over 2-fold in the presence of the keto acid. Given the high level of Mn2+ required for activation, it is doubtful that the proposed physiological relevance of the cr-ketoglutarate effect (Tate & Meister, 1971) is applicable to the avian enzyme. A second Table 5. Effect of metal ions on avian liver glutamine synthetase Metal
Concentration fmM)
% Inhibition
Cd’+
0.05 0.50
11 13
coz+
5.0 10.0
0 12
CL?+
0.50 5.00
87 90
Fe3+
0.05 0.50
26 69
Hg’+
0.05 0.50
94 100
Ni’+
0.50 5.00
14 59
Pb’+
0.05 0.50
90 90
Zn2+
0.50 5.00
60 84
Glutamine synthetase activity was determined as described in Table 3 with 15 mM magnesium chloride, 25 mM hydroxylamine and metal chlorides at concentrations given in the Table. Controls with p-glutamylhydroxamate showed no inhibitory effects on color development by any of the metal ions tested.
et al.
c Laclg q.CTbCI,l
50
100
m PM 9
150
200
J
4 3 2 tC0Cl~l0,tMnCl~3*,mM I
Fig. 4. Inhibition of glutamine synthetase by Ca*+, Mg”, La3+ and Tb3+ ions. Activity was determined as described in the legend of Table 3 with 10 mM ATP, 15 mM magnesium chloride, 25 mM hydroxylamine and 50mM L-glutamate.
difference noted between the rat and chicken glutamine synthetases is their contrasting response to glutamine; the former is inhibited by this amino acid (Deuel et al., 1978) whereas the latter is activated. A number of nucleotides were screened as possible effecters of avian liver glutamine synthetase. At concentrations of lOmM, the following compounds had little (inhibitions of 5% or less) or no effect on activity with Mg2+ ion: AMP, IMP, UMP, CDP, UDP, CTP, UTP and NAD. Only ADP gave a significant inhibition which appeared to be competitive with respect to ATP (data not shown). A Ki for ADP of 2 mM was determined from these data. Inhibition by metal ions The sensitivity of avian glutamine synthetase to various metal ions was investigated. Results are given in Table 5 and Fig. 4. At concentrations of 50pM, Hg2+ and Pb2+ virtually abolished activity and lo-fold higher concentrations of Cu”, Cd’+, Fe3+ and Zn2+ produced significant inhibition. Ni2+ was a much weaker inhibitor and Co” was essentially noninhibitory. The absence of an inhibitory effect by Co’+ has been observed with at least two other eukaryotic glutamine synthetases (Monder, 196.5; Kanamori & Matsumoto, 1972). Since the avian enzyme contains nine sulfhydryl groups per subunit and is subject to inactivation by thiol reagents such as N-ethylmaleimide and iodoacetamide (inhibitions of 92 and 85x, respectively, at concentrations of 25 mM), the inhibitory effects of some of the heavy metal ions may be accounted for by their interaction with sulfhydryl groups. In addition to the heavy metal ion effects noted above, the avian enzyme was inhibited by relatively low concentrations of manganese and calcium ions as shown in Fig. 4. Inhibitions of 50% were observed at concentrations of approx. 875 and 175pm for Ca2+ and MnZ+, respectively. Mammalian glutamine synthetases are also sensitive to inhibition by Mn2+ ion and Mg 2c does not apparently readily reverse this inhibition (Tate & Meister, 1973; Monder, 1965). In
Characterization
of glutamine synthetase
studies with the rat liver enzyme, substantial inhibition was still evident at Mg’+ to Mn’+ ratios of
1ooO. More potent inhibitors were the trivalent lanthanide ions, La3+ and Tb3’. These ions have ionic radii similar to those of CaZ+ in its different coordination states and have been utilized to probe the calciumbinding sites of a number of proteins including E. co/i glutamine synthetase (Martin & Richardson, 1979; Wedler & D’Aurora, 1974). Extensive physicochemical studies of the latter enzyme have shown a complex picture of metal ion interactions. There are at least two metal binding sites, separated by a distance of approximately 6A. One site interacts with Mg ‘+-ATP; the second metal ion site binds Mn2+ and induces a protein conformational change to an active tightened state. In addition, Mn2 + may participate in the binding of glutamate to the catalytic site. Speculations regarding metal ion interaction with eukaryotic glutamine synthetases are premature at the present time. The situation is obviously complex in that Mn” and Ca2+ ions are inhibitory while Co2+ is not; yet Co2+ and Mn2+ but not Ca2+ will meet the divalent cation requirement and support activity, especially at lower pHs (Monder, 1965). Effects of these metal ions are not readily explainable in terms of competition with Mg2’ ion for ATP since the stability constant of Ca2 ‘-ATP (9300 M- ‘) is lower than those of Co2+ (45,000 M-‘) and Mn2+ (60,200 M-l) and are greater than that of Mg2 ’ (16,600 M - ‘) (Tu & Heller, 1974)*. Further studies are needed to distinguish between metal ion interaction with cysteine residues, with metal-ATP binding site(s) or with yet other protein ligands, such as carboxylate and amino moieties. The objective of the present study was an overall characterization of avian liver glutamine synthetase. Our results indicate that, despite its mitochondrial localization, properties of the chicken enzyme markedly resemble those of the weakly bound microsomal rat liver enzyme and mammalian glutamine synthetases in general. Its octameric structure, amino acid composition, Michaelis constants, subunit molecular weight, substrate specificity and sensitivity to various metal ions and metabolites are similar to those reported for eukaryotic glutamine synthetases. Indeed, the only exception of note is the absence of an activating effect by a-ketoglutarate when assayed with magnesium ion. The physiological significance of the inhibitory effects of alanine, serine and glycine as well as those of Ca2+ and Mn2+ cannot be assessed at the present time. Given its mitochondrial localization and the proposed role of the mitochondrion in regulating intracellular levels of Ca’+ (Borle, 1975), hit is conceivable that Ca’+, rather than Mn2+ ion, functions in the regulation of avian liver glutamine synthetase. In this context, it is interesting to note that carbamyl phosphate synthetase 1, the mammalian complement * Values reported for the stability constants are dependent upon pH variables such as temperature and ionic strength and may differ with the method of determination. However for a given study, the stability constants decrease in the following sequence: Mn2+ > Co2+ > MgZ+ > CaZf (Tu & Heller, 1974). B.T.14/8-E
755
of uricotelic glutamine synthetase, is inhibited by Ca2+ at concentrations which do not interfere with mitochondrial ATP generation and which are pysiologically relevant (Meijer et al., 1981). SUMMARY
Glutamine synthetase has been purified over 750-fold from chicken liver mitochondria. The preparation was judged to be homogeneous on the basis of a single protein band appearing after analytical isoelectrofocusing and after polyacrylamide gel electrophoresis of the enzyme in the presence and absence of SDS. The molecular weight of the native enzyme was 389,000 dalton as determined by sedimentation equilibrium analysis. The molecular weight of its monomeric subunit was 42,000 as determined by SDS-gel electrophoresis. Electron micrographs suggest the enzyme is an octamer, a structure typical of eukaryotic glutamine synthetases. The amino acid composition was also similar to other vertebrate glutamine synthetases. The apparent Michaelis constants determined for ammonia, ATP and glutamate were 0.5, 0.9 and 6 mM, respectively. Of a number of compounds tested for their effect on glutamine synthetase activity, only ADP and methionine sulfoximine gave a significant degree of inhibition when activity was measured by the y-glutamylhydroxamate assay in the presence of Mg2+ ; when Mn2 ’ was substituted for Mg’+, several amino acids (L-alanine, L-serine, L-glycine) were partially inhibitory whereas L-glutamine and ec-ketoglutarate were slightly stimulatory. A number of heavy metal ions were inhibitory as were Ca2’, Mn2+ and lanthanide ions. In general, the physical and chemical properties of the avian liver mitochondrial enzyme reported here resemble those of other eukaryotic glutamine synthetases. Acknowledgements-This work was supported by grants from the National Science Foundation (PCM 75-13161 and BBS 79-25911). REFERENCES
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