Neurochemical Research, Vol. 15, No. 3, 1990, pp. 301-305

A Sensitive Method for the Assay of Glutamine Synthetase N. Seiler ~, J. Reid 1, and B. Knfdgen (Accepted November 22, 1989)

The method for the assay of glutamine synthetase (GlnS) relies on the 7-glutamyl transferase reaction, i.e. the formation of glutamyl-7-hydroxamate from glutamine and hydroxylamine, and the chromatographic separation of the reaction product from the reactants. The method is not only simple and reliable, but also has a sensitivity comparable to those methods applying radioactively labelled substrates. This new procedure has been applied to the assay of GlnS in cultured rat cortical astroglial cells which have been treated with a homologous series of c~,to-bis-(dimethylamino)alkanes. Effects of these drugs on astroglial development are reported. K E Y WORDS: Glutamine synthetase; glutamyl-',/-hydroxamate, HPLC.

INTRODUCTION

ods are of limited sensitivity, unless (at least partially) purified enzyme preparations are used. The method described in the following relies on the 7-glutamyl transfer reaction (11, 19) :

Glutamine synthetase (L-glutamate : ammonia ligase (ADP-forming) ; E.G. 6.3.1.2.) (GlnS) is the major glutamine forming enzyme of vertebrates and, as far as the brain is concerned, generally accepted to be a marker of astroglial cells (1-4) although there have been contradictory reports (5, 6). Maturation of rat cortical astroglial cells is characterized by an increase of GlnS activity (7), and the regulation of this enzyme is the topic of numerous publications (see for example, ref. 8-14). Several methods exist for the determination of GlnS activity; among these are radioisotopic assays, utilizing labeled glutamic acid (9, 15-17), which are chosen for their sensitivity and spectrophotometric methods (11, 12, 18-22) popular because of their convenience. In order to avoid the use of labelled substrates the column chromatographic separation of glutamine (Gln) from the substrates of the enzymatic reaction has been suggested (23, 24). In fact, our previously published separation of the non-essential amino acids (25) could also be used to this purpose as it has the advantages that Gln elutes in front of glutamate without requiring a gradient for elution. Since, however, Gtn is a normal component of tissues and of tissue culture media these chromatographic meth-

glutamine + H2NOH

ADP ; Mn2+ ; AS043glutamyl-7I~ GInS hydroxamate + NH3

and the determination of the reaction product glutamyl",/-hydroxamate (GIuNHOH) by high performance liquid chromatography (HPLC). As an example of the practical applicability of the method, GlnS activity was determined in astroglial cells which had been treated with a homologous series of et,cobis-(dimethylamino)alkanes. These compounds are known to inhibit cellular proliferation and to antagonize the binding of putrescine-bound polystyrene latex spheres to binding sites on the surface of glioblastoma cells (26).

EXPERIMENTAL PROCEDURE Preparation of a, w-bis-( dimetylamino )alkanes. The homologous e~,to-b/s-(dimethylamino)alkanes were obtained from the respective cqtodiamines by permethylation with formaldehyde/sodium borohydride (27). Cultures of Astroblasts. Cultures of astroblasts from hemispheres of newborn rat brain were prepared as was described by Pettman et

301 0364-3190/90/0300-0301506.00/09 1990PlenumPublishingCorporation

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al. (28) with cells from one brain being seeded in 30 Falcon petridishes (35 mm diameter) each containing 2 ml of culture medium. Basal culture medium was Waymouth's MD 705/1 supplemented with sodium pyruvate, antibiotics, and 10% fetal calf serum (Gibco). Drugs were first administered on day 6 dissolved in serum-free medium in doses that gave a final concentration of 0.1, 0.5, and 1 mM. Medium was exchanged twice a week, and at the same time the cells were retreated. Total cell culture time was 38 days. Tissue Sample Preparation. Astrocytes were washed in 0.9% NaCI (37~ and harvested in 0.9% NaC1 at 4~ After centrifugation (0~ C; 4000 rpm ; 5 min) the pellet was either stored at - 80~ C until it was used for analysis, or it was immediately sonicated at + 4~ C in 320 r imidazole-EDTA buffer (68 mg imidazole, and 18.6 mg EDTA per 100 Ixl of water, adjusted to pH 7.0 with HC1). A 60 ~1 aliquot of the cell sonicate was removed for DNA determination, diluted 1:4 with 0.05 M sodium phosphate buffer containing 2 M NaCI (pH 7.4; 4~ C), and left on ice for about 1 h, while the GInS assay was conducted. Two 20 ~1 aliquots were each diluted with 60 r imidazoleEDTA buffer, and used for the estimation of GlnS. In 200 p.1 of the ceiI sonicate RNA and proteins were determined. Homogenates of mouse brain (male CD1 albino mice ; Charles River, St. Aubin-les-Elbeuf, France) were freshly prepared with 10 vol. of the imidazole-EDTA buffer, and subsequently diluted using the same buffer. The reaction mixture had the following composition :

Solution Imidazole (pH 7.0) Sodium arsenate (pH 7.0) Sodium ADP (pH 7.0) L-Olutamine (pH 7.0) MnC12"4HaO Water

Volume ml 1.3 0.65 1.3 4.9 0.16 5.8

Initial concn, M 1.0 1.0 0.01 0.1 0.1 --

Final concn. mM 80.7 40.3 0.081 30.3 1 --

Hydroxylamine Solution : A 2M stock solution of hydroxylamine hydrochloride is prepared in water. Immediately before use aliquots are diluted 1:1 with 1 M NaOH and then are adjusted to pH 7.0 with conc. NaOH. This solution is diluted 1:1 with water. Incubation Mixture : Cell or tissue homogenate (80 ~1) is mixed with 70 ~1 reaction mixture. Hydroxylamine solution (101xl) is added immediately before the samples are incubated (usually for 20 rain) at 37~ C under gentle shaking. The enzyme reaction is terminated by addition of 801xl 1 M ascorbic acid solution. Samples are kept at 4 ~ C until the chromatographic analyses are performed. Blanks are made as the incubation mixtures, except that instead of the enzyme preparation (i.e. cell or tissue homogenate), 80 ~1 of the imidazole-EDTA buffer added. Determination of Glutamyl-y-Hydroxamate (GluNHOH). Before the samples are applied on the HPLC column (Superspher RP18 (3 Ixm pellicular silica gel with C18-brushes ; 250 mm in length ; 4.6 mm I.D.) Bischoff, Leonberg, Germany, or Merck, Darmstadt, Germany) they are usually diluted with water to a volume of 1.5 ml (final ascorbic acid concn. 0.053 M), in order to improve the base-line of the chromatogram. The injection volume was normally 50 txl. Separation is achieved by isocratic elution (flow rate 1 ml.min -~ at 22~ C) with the following eluent : 650 ml 0.2 M phosphoric acid ; 270 ml 0.2 M sodium acetate (pH 4.50) ; 80 ml methanol ; 2.59 g sodium dodecylsulphate. For the preparation of all solutions water distilled over phosphoric acid was used. (Commercial sources for water suitable for HPLC are

available). Samples can be applied (automatically) every 30 min without column regeneration. The column eluate is mixed in a T-piece at a 1:1 ratio with ophthaldehyde-2-mercaptoethanol reagent (see below) and then passed through a reaction coil (2 min length, 0.5 mm I.D.) which is heated at 45" C. Fluorescence intensity is continuously monitored at 455 nm (fluorescence excitation at 345 nm). o-Phthalaldehyde-2-Mercaptoethanol Reagent : 50 g boric acid, 31.5 g sodium hydroxide, 3 ml of Brij-35 solution (30 %) (Merck, Darmstadt, Germany), 3 ml 2-mercaptoethanol and 0.4 g o-phthalaldehyde (in 10 ml of mehtanol) per 1000 ml of water. Equipment : A Varian Vista 5500 liquid chromatograph wa s used in conjunction with a Varian 9090 Auto Sampler, a Milton-Roy Fluoro Monitor III and a LDC/Milton Roy C-10 integrator (LDC, Paris, France). The o-phathaldehyde-2-mercaptoethanol reagent was pumped using an Instrument Minipump (Milton Roy). Standard Solutions of Glutamyl-'/-Hydroxamate : GIuNHOH was obtained from Sigma Chemical Co. (St. Louis, U.S.A.). Stock solutions (10 IxM) were prepared twice per week in 0.05 M ascorbic acid and stored at 4~ C. Dilutions to standard solutions (usually containing 1 nmol GIuNHOH per ml 0.05 M ascorbic acid) were prepared immediately before chromatographic separations were started. Determination of DNA, RNA, and Proteins. The DNA content was evaluated using the fluorimetric method of Labarca and Paigen (29). Bisbenzimide trihydrochloride (Hoechst 33258) was obtained from Sigma. RNA and protein were determined after separation from DNA using a modified Schmidt-Thannhauser procedure (30). RNA was determined spectrophotometrically, protein by a modified Lowry procedure (31). Calf thymus DNA, ribonucleic acid from bakers yeast, and bovine serum albumine (Sigma) were used as standards.

RESULTS For the assay of GlnS in tissues and cell h o m o g e hates basically the incubation conditions suggested by Miller et al. (12) have been f o l l o w e d in our procedure. H o w e v e r , G I u N H O H is formed f r o m Gln and hydroxy l a m i n e not only by the e n z y m e - c a t a l y z e d reaction, but also in e n z y m e - f r e e solutions. In order to decrease the " b l a n k " w e decreased the concentrations of Gtn and h y d r o x y l a m i n e in the assay mixture by a factor of 0.5. U n d e r our conditions the formation of G l u N H O H was linear with time for m o r e than 60 min (Figure 1A). Norm a l l y an incubation time of 20 min was chosen. The proportionality between the amount of tissue in the incubation mixture and the formation of G I u N H O H was established for m o u s e brain h o m o g e n a t e s . In the range b e t w e e n 0.4 Ixg and 8 ~zg per assay of w h o l e brain tissue there w a s a linear relationship b e t w e e n the two m e n tioned parameters (Figure 1B). H i g h e r amounts of tissue w e r e not used. For the separation from other c o m p o n e n t s of the reaction mixture and the sensitive determination o f G l u N H O H , in principle a p r e v i o u s l y published m e t h o d (25) was used. The separation on a reversed-phase colu m n of the ion pairs formed by amino acids with do-

Glutamine Synthetase Assay

303

6 >i--

>
g of mouse brain tissue (incubation time : 20 min). B. Blank (incubation of a standard assay mixture without tissue).

0

I 0

I 2 TISSUE

I 4 PER ASSAY (,ug)

I 8

Fig. 1. A. Relationship between glutamyl-q,-hydroxamate (GluNHOH) formation and time of incubation in a standard assay containing 4 >g of mouse brain tissue. B. Relationship between glutamyl-y-hydroxamate (GluNHOH) formation and the amount of mouse brain tissue in standard assays (incubation time 20 rain).

decylsulphate and their fluorimetric determination after reaction with o-phthalaldehyde - 2-mercaptoethanol is utilized in this method. However, in order to separate small amounts of GluNHOH from large amounts of Gln, it was necessary to use a column with a rather high performance and to develop an eluent which allowed GluNHOH to be eluted in front of Gin. As is shown in Figure 2 this has been achieved by isocratic elution, using a coIumn fiIled with Superspher RP18 (3 b~m bead diameter; C18-brushes) (Merck, Darmstadt, Germany) and in comparison with the original method, only a slightly modified eluent. Samples can be run every 30 min for more than 24 h without column regeneration. It is only necessary to wash the column occasionally with water, followed by methanol. This is indicated when separations start to become deteriorated. Two major problems had to be solved which are, in principle, common to all methods utilizing the 3,-glu-

tamyl transfer reaction : the afore-mentioned non-enzymatic formation of GIuNHOH and its instability. In order to minimize the non-enzymatic formation of GluNHOH, hydroxylamine was not added to the mixture until immediately before incubation was commenced, and storage of incubated samples (at 4 ~ C) was kept as short as possible (less than 24 h). The instability of GIuNHOH is most probably due to autoxidation. By using 1 M ascorbic acid for the termination of the enzyme reaction, the problem of autoxidation was largely solved. But even in the presence of ascorbic acid a slow degradation of GIuNHOH was observed. Standards containing 1 nmoI GIuNHOH per ml 0.053 M ascorbic acid showed at room temperature a loss of about 0.9% per h. In order to match for the instability of GluNHOH and for its non-enzymatic formation a standard was run after each group consisting of a blank and four tissue-containing samples. The mean value of the standards run either side each group of samples was used for the calculation of the enzymatically formed GluNHOH. In spite of the somewhat unfavorable characteristics of GluNHOH, the method is quite reproducible. Standards containing 1 nmol.m1-1 GIuNHOH run on different days varied by only + 2.6% of the mean value. The mean standard deviation of duplicate assays of 35 different astroglia homogenates was _+ 3.4%. The method is rather sensitive. Taking a signal-tonoise ratio of 3 as detection limit (i.e. 3 times more

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Seiler, R e i d , and K n 6 d g e n

GluNHOH formed by the enzyme-catalyzed reaction than is formed chemically) the following values are obtained: Minimum measurable amount of G l u N H O H formed in a standard assay per 20 min of incubation : 0.45 nmol. This amount corresponds to 15 pmol GluNHOH per 50 >1, i.e. the volume applied on the HPLC column after dilution of the assay mixture to 1.5 ml. (For details, see Experimental Procedures). Less extensive dilution of the sample allows an increase in the sensitivity of the method by a factor of 2-3. The formation of 0.45 nmol GIuN H O H in a standard assay requires the presence of about 1.6 ~xg mouse brain tissue, or of 0.5 rxg astroglial proteins (of 5-day-old cultures). Exposure of the astroglial cells to c~,(o-bis-(dimethylamino)alkanes caused at a concentration of 0.1 m M dramatic morphological changes, whereas cells died at concentrations I> 0.5 mM, except in the case of 1.2bis(dimethylamino)ethane. (An account of the morphological changes will be published later). In addition to phenotypic changes the cells showed distinct biochemical changes when treated w i t h l . 2 bis(dimethylamino)ethane at 0.5 m M and above (Table I). RNA and protein content of the cells, as well as the activity of GlnS were significantly elevated by the treatment.

manding. A single high-pressure pump with a suitable pulse dampener can be employed since a gradient is unnecessary for elution. Automated sample application, although an advantage as it allows 24 h operation, is not a requirement. Finally, a chart-recorder may be substituted for an integrator and peak heights rather than areas under the curve determined instead. In practical applications the method proved valuable : Only about i ~xg of astroglial protein was required for duplicate enzyme assays. This allowed us to use the remaining (90 %) cells of each dish for DNA, RNA, and protein determinations. For comparison : spectrophotometric methods require about 200 >g of astroglial protein for a single GlnS assay. Thus, it is evident that our method is well suited to substitute for methods which utilize [14C]glutamate as substrate. Our observations concerning the effects of 1,2-bis(dimethylamino) ethane and its higher homologues are yet too preliminary to draw conclusions. Several interpretations are possible. Among these the induction of differentiative processes and the selection of a cell type with a higher RNA, protein, and GInS content due to higher resistance against the drug, are the most likely ones.

ACKNOWLEDGMENTS DISCUSSION

The method presented in this paper is simple, sensitive, and reliable, and although relatively sophisticated chromatographic equipment has been used in our experiments, the actual requirements are considerably less de-

The astroglial cells were cultured in the laboratory of Dr. Monique Sensenbrenner at the Centre de Neurochimie du CNRS, Strasbourg. Our grateful thanks to her and to Dr. Brigitte Pettman for demonstrating the technique of tissue culture and to their continued support and guidance during the course of this work. Thanks also to Mrs. Marie-France Knoetgen for her expert technical advice.

Table I. RNA. Protein, and Glutamine Synthetase Activity in Rat Cortical Astrocytes. Effect of Treatment with a,to-bis-Dimethylaminoalkanes

Compound None 1,2-Dimethylaminoethane 1,3-Dimethylaminopropane 1,4-Dimethylaminobutane 1,5-Dimethylaminopentane 1,6-Dimethylaminobexane 1,7-Dimethylaminoheptane

Concn. mM

RNA Ixg per Ixg DNA

Protein Ixg per tzg DNA

--

1.2 • 0.2

(5)

0.1 0.5 1.0 0.1 0.1 0.1 0.1 0.1

1.2 1.3 1.7 1.4 1.2 1.2 1.4 1.8

(2) (3) (3) (2) (3) (3) (2) (1)

15 15 19 22 19 16 17 16.5 21

- 0.1 - 0.2* -+ 0.2* -+ 0.1" • 0.1 • 0.1 • 0.2

_+ 1 -+ 1 -+ 1" -+ 1" • 3* • i +_ 1 -+ 0.4

(5)

(2) (3) (3) (2) (3) (3) (2) (1)

Glutamine synthetase activity nmol'(0.DNA)-l"h -~ 30_+ 11 26 -4- 15 50 -+ 12 53 -+ 4" 35 _-2-21 26 _-2 4 28 -+ 3 27 • 8 29

(5)

(2) (3) (3) (2) (3) (3) (2) (1)

Mean values - S.D. (number of samples in parentheses). Rat cortical astrocytes were grown in 2 ml dishes for 5 days in the presence of 10% fetal calf serum. On day 6 the drugs were added (dissolved in 20 :~1 of serum-free culture medium) and the cells were grown for further 32 days. Culture medium was changed against fetal calf serum and drug containing fresh medium twice a week. The asterisk indicates a statistically significant (P -< 0.01) difference between untreated and drug-treated cells.

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