The measurement of glutamate decarboxylase activity in brain tissues by a simple microradiometric method

ANALYTICAL

BIOCHEMISTRY

65,

449-457 (t 975)

The Measurement Activity

JOSEPH

of Glutamate

Decarboxylase

in Brain Tissues by a Simple Microradiometric Method R. MOSKAL

Department of Chemistry, Uni\lersity of Notre

AND

Biochemistry Dame, Notre

SUBHASH and Biophysics Dame. Indiana

BASU Program, 46556

Received October 12, 1974: accepted December 34, 1974 A simple method was developed for the assay of L-glutamate decarboxylase (GAD) in brain homogenate and subcel~ul~ fractions. In a dual tube assay system, the incubation mixture (0.1 ml) was placed in an inner disposable culture tube (6 x 50 mm) which was then placed in an outer reusable culture tube (10 x 75 mm) and sealed with a serum cap. The procedure utilizes absorption of ‘“CO, (released from [ I-“C]t-glutamate) on a Hyamine hydroxide spotted Whatman 3MM chromatographic paper strip (5 x 40 mm) which is then counted with a MiniVial containing 5 ml of toluene scintillation solution. Only very small tissue samples (0.05-0.4 mg of protein) and minimum manipulations are necessary. The assay method reported here could be used for the measurement of any decarboxyiase-catalyzed reaction, provided that suitable carboxyl labeled substrates are employed.

L-Glutamate decarboxylase (GAD) catalyzes the formation of yaminobutyric acid and 14C0, from [ 1-14C]L-glutamate in the presence of pyridoxal phosphate (1). Potassium hydroxide (2), ethanolamine (3), Hyamine hydroxide (4), and phenylethylamine (5) are commonly used as trapping agents for ‘“COz released during the reaction which is then quantitated by liquid scintillation counting. Early assay methods required the use of relatively large incubation volumes (1.0-2.5 ml) and special incubation flasks (2,6). More recent procedures have avoided those flasks but introduced plastic scintillation vials with caps containing Hyamine hydroxide soaked blotting paper disks for absorption of 14COz (7). Also scintihator plastic adapters (8) to adsorb the radioactive gases and gelatin microtubes fitted with plastic vial caps (with rubber liners) for injection (9) have been used. Paper ~hromatographic (10,l l), thinlayer chromatographic (1 Z), and polyamide layer ( 13) techniques for the separation of dansyl-“C-y-aminobutyric acid and its estimation are also available, but are relatively time consuming assay procedures. This paper reports a simple assay procedure for the determination of GAD activity which is quantitative in the range of 250-500 pmol. 449 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MATERIALS

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BASU

METHODS

The following materials were obtained from commercial sources: [l14C]DL-glutamic acid (8.45 mCi/mmol) from New England Nuclear (Boston, MA); L-glutamic acid, pyridoxal-5’-monophosphate and HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) from Sigma Chemical Co. (St. Louis, MO); Triton X-100 from Rohm and Haas; Hyamine hydroxide (benzyldimethyl{2-[2-(p1,1,3.3-tetramethylbutylcresoxy) ethoxylethyl} ammonium hydroxide) from Amersham/Searle and Co. (Arlington Heights, IL); PP0(2,5-diphenyloxazole) and Me,POPOP (1,4-bis [2-(4-methyl-5-phenyloxalolyl)] benzene) from Packard Instrument Co. (Downers Grove, ILL); reagent grade 2ethyoxyl-ethanol, ethyleneglycol, 1,4-dioxane, and toluene from J. T. Baker Chemical Co. (Phillipsburg, NJ); napthalene from Coleman and Bell (Cincinnati, OH); rubber serum caps, disposable culture tubes (6 x 50 mm), reusable Pyrex culture tubes without lips (10 X 75 mm), Whatman 3 MM chromatographic paper and microsyringes from Scientific Products (McGraw Park, IL). Miniature scintillation vials (5 ml capacity) were purchased from Research Products International (Elk Grove Village, IL) and fertilized chicken eggs were obtained from Liechty Hatchery (South Bend, IN). Tissue homogenization. Brains from 7- to 2 1-day-old chick embryos were dissected and homogenized in 0.32 M sucrose containing 14 mM 2mercaptoethanol and 1.0 mM EDTA at pH 7.0 with ten strokes of a Teflon glass homogenizer. Further fractionation for the isolation of synaptosomal fraction was performed according to the previously published method (14). A Polytron IO-ST homogenizer was used in an attempt to isolate Golgi type membranes from embryonic chicken brain (15). Homogenization was performed within 30 min after dissection and all steps were carried out between 0 and 5”. Assay procedure. The enzyme was assayed within 2 hr after homogenization with a diffusion tube system as shown in Fig. 1. Complete incubation mixtures contained the following components in final volumes of 0.1 ml; Triton X-100, 75 ,ug; HEPES buffer, 0.1 M, pH 7.2; pyridoxal 5’monophosphate, 0.5; mrvr enzyme fraction, 0.1-0.4 mg of protein. As shown in Fig. 1 the incubation mixture was taken up in an inner disposable minitube (6 X 50 mm), which was then placed in an outer reusable Pyrex culture tube (10 X 75 mm). A strip of Whatman 3MM chromatographic paper (5 X 60 mm) containing 25 ~1 of 1 M Hyamine hydroxide (in methanol) was introduced into the outer test tube, and after flusing with N, the tube system was closed with a tightly fitting serum cap. The enzyme reaction was started by injecting 30 ~1 (0.15 pmol) of [ I-‘“C]L-glutamic acid (0.1 X IO” to 0.4 X lo6 dpm/pmol) with

GAD ASSAY

BY A MICRORADIOMETRIC

METHOD

451

------Syringe(0.25ml)

------

l~4C]Glutamate(25-50~ll

e-------Serum

cap

------Culture

tube( 10x75

-------Hyamme

hydroxide

-------Whatmon

mm1 IOM(25pl)

No 3MM pap45

---~---Disposable

culture

Incubation

mwturs

x60

tube(6x50 (50-100

mm) mm)

pl 1

FIG. I. Diagrams of r4COz diffusion system and MiniVial counting.

arrangement for scintillation

a semimicrosyringe through the serum cap into the miniculture tube. The dual-tube system was incubated at 37” for 15-30 min, and the reaction was stopped by placing the whole system in a boiling water bath for 5 min. A postincubation period of 3 hr at 37” after the reaction was terminated was used to allow maximum absorption of 14C0, on the Hyamine hydroxide-spotted filter paper strip. The strip was shortened to 4 cm and placed in a MiniVial containing 5 ml of toluene scintillator solution, type IV (Table 1). The MiniVial was then placed in a 20 ml empty TABLE SCINTILLATOR

Name Type 1

Type II Type III Type IV Type V

SYSTEMS

Solvent used to make up volume to 1 liter

Chemicals PPO MqPOPOP Naphthalene 2-Ethoxyethanol Ethylene glycol PPO Me,POPOP Naphthalene PPO MsPOPOP Triton X- IO0 PPO Me,POPOP PPO Me,POPOP

1

SOLVENT

8.0 g 0.6 g 15o.og 100 ml 20 ml 7.0 g 0.6 g 15o.og 5.5 g 0.1 g 333 ml 4.0 g 0.15 g 1.3 g 0.05 g

1.4 Dioxane

Toluene

Toluene Toluene Toluene

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glass vial, and the amount of radioactivity was determined using a Packard scintillation counter, model 3375. A special adapter for the MiniVial can be used but is not necessary. Efect of age on GAD activity in embryonic chicken brains. Chick embryos of the indicated ages were dissected and the brains were frozen at - 18”. On the day of the experiment, the tissues of various ages were thawed and then homogenized with 3 vol of 0.32 M sucrose. Aliquots of each preparation were assayed for GAD activity under the above mentioned assay conditions. The rate of reaction remained constant with time of incubation up to 90 min and was proportional between 0.05 and 0.4 mg of protein/O.1 ml incubation volume. Optimal activity was observed in 2 I -day-old embryonic chicken brain (Fig. 5) in the presence or absence of pyridoxal-5’-phosphate. RESULTS

A comparison of counting efficiency as measured with mini and regular scintillation vials was made (Table 2); the best result was obtained using a MiniVial placed inside a regular 20 ml empty vial after the dualtube system was flushed with N,. In the presence of air there was only a 2-3% loss of counting efficiency as compared to a NZ flushing. For routine assay procedures we omitted Nz flushing. Table 3 compares the effects of injection of 2 N H2S04, 12.5% TCA, and immersion into a boiling water bath for 2 and 5 min on the evolution of 14C0, during the termination of the reaction. Of the three methods used, sulfuric acid inTABLE COMPARISON

OF COUNTING

EFFICIENCY

2

AS MEASURED

Paper strip placed in

WITH

DIFFERENT

VIALS~

14COz measured nmolesimg proteimhr

A. 20 ml counting vial containing 15 ml of type IV scintillator solution

33.9

B. MiniVial containing 5 ml of type IV scintillator solution was placed inside a 20 ml vial. Dual-tube system was flushed with Nz

40.2

C. Same as B, except Nz was omitted

39.1

u Complete incubation mixtures contained the following components (micromoles) in final volumes of 0.1 ml: Triton X-100,75 pg; HEPES buffer, pH 7.2, 10; pyridoxal-Y-PO, (PLP), 0.05; [ lJ4C]L-glutamic acid (430,000 dpm/Fmole), 0.15: 21-day-old embryonic chicken brain homogenate (0.32 mg of protein/incubation mixture). The mixtures were incubated for 30 min at 37” and assayed according to the method oescribed in the text except that the paper strips were counted under the above conditions.

GAD ASSAY

BY A MICRORADIOMETRIC TABLE

COMPARISON

OF DIFFERENT

METHODS

METHOD

453

3

FOR TERMINATION

OF ENZYMATIC

REACTIONS

WO, released Termination

method

Minus PLP

Plus PLP

nmolesimg proteinihr 13.8 32.2 15.8 43.6 12.0 30.0 15.0 44.6

2 min at 100” 5 min at 100” 12.5% TCA (0.1 ml) 2 N H2S04 (0.1 ml)

n Incubation mixtures contained the following components (micromoles) in final volumes of 0.085 ml: HEPES buffer, pH 7.2, 10; Triton X-100. 75 pg; pyridoxal-S-PO, (PLP), 0.05; [I-W]r-glutamate (144.000 dpmipmol), 0.15: synaptosomal fraction from 19-day-old embryonic chicken brain (0.24 mg of protein/incubation mixture). The mixtures were incubated at 37” for 30 min and then assayed according to the method described in the text except that the reactions were stopped in the above ways.

jection led to the maximum 14C0, evolution but was not significantly better than immersion in boiling water for 5 min. The latter method was used for our routine assay work because of its minimum time requirement. Various aliquots of 1 N Hyamine hydroxide (lo-50 ~1) were spotted onto the filter paper strips to test the maximum absorption of released r4C0, (Fig. 2) by GAD in the presence and absence of pyridoxal phosphate. 25 ~1 (25 pmol) was found to be the saturating amount for absorption of 14C0, up to 50 nmol. Furthermore, at higher concentrations of Hyamine hydroxide, quenching of radioactivity was not ob-

HYAMINE

HYDROXIDE

(~1)

FIG. 2. Effect of Hyamine hydroxide concentration on the absorption of released ‘CO,. incubation conditions were the same as described in Table 3, except that varied concentrations of Hyamine hydroxide were applied onto the paper strips.

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6

IO

. 36-

0

2

4

6

I

24

T I ME (hrs.)

FIG. 3. The effect of postincubation time on the absorption of ‘%O,. Incubation conditions were the same as described in Table 3. After 30 min at 37”. the incubation mixtures were kept at 100” for 5 min, then kept at 37” for indicated times and counted in type IV scintillator solution.

1 0

2

4

6

8 TIME

IO

I’

I 24

(hrs.1

4. Counting efficiency of different scintillation mixtures. Incubation conditions were the same as described in Table 2 except that 19-day-old embryonic chicken brain homogenate (0.3 mg) was used as enzyme source. After 3 hours postincubation period the filter paper strips were counted in five different types of scintillation mixture as described in the text. (A-A) type I; (a--n) type II; (H-m) type III; (O-O) type IV; (0-O) type V. FIG.

GAD

ASSAY

BY A MICRORADIOME’IRIC TABLE

COMPARISON

Protein mgi0.2 ml 0.2 0.4 0.8

OF HYAMINE SOLUTION

METHOD

455

4

HYDROXIDE AS TO2

PAPER AND ABSORBERS

Hyamine hydroxide*

ETHANOLAMINE

Ethanolamine-?-methoxyethanolb

nmoles lVOz absorbed/30 min 0.38 0.11 4.34 8.82 15.78

1.23 2.40 4.48

a Complete incubation mixtures contained the following components (m~cromoles) in final volumes of 0.2 mi; Triton X-100, 150 pg; HEPES buffer, pH 7.2, 20; pyridoxal-S-PO, (PLP), 0.1; [I-‘*C]L-glutamic acid (4.3 x lo5 dpm/pmole), 0.3; 21-day-oldembryonic chicken brain homogenates were used as indicated. The mixtures were incubated for 30 min at 37” and then assayed according to the method described in the text. b Incubation conditions were the same as described in experiment A except that ‘“CO, released was absorbed in a liquid mixture of ethanolamine-2-methoxyethanol (2 : 1) placed in an inner chamber ofthe reaction vessel (according to the method of Russell and Snyder (3)).

served. Because a methanolic solution of Hyamine hydroxide was spotted on Whatman 3MM chromatographic paper strips instead of using Hyamine hydroxide solution directly in the tube, it was necessary to determine the postincubation period yielding maximum absorption of YQ. As shown in Fig. 3 there was only 510% increase in the absorption of ‘*CO2 during the first hour of the postincubation step. However, a 3 hr postincubation period was introduced for our routine assay procedure. The rate of diffusion and stabilization of Hyamine hydroxide-14Cbicarbonate in the scintillator liquid was studied over 24 hr (Fig. 4), using five different scintillator solutions (Table 1). The type IV mixture (toluene solution containing 4.0 g PPO and 0.15 g Me, POPOP/liter) was found to be the most satisfactory, and disintegrations per minute (dpm) did not change over the 24-hr counting period. A linear increase in 14C02 absorption by our method (Table 4A) and by the method of Russell and Snyder (Table 4 B) was observed when tested at different protein concentrations (0.2-0.8 mg of protein~O.2 ml incubation volume). It is apparent that our Hyamine hydroxide spotted paper strips absorbed ‘*CO, 3.6 times more efficiently than a liquid mixture placed inside the reaction vessel according to Russell and Snyder (3). DISCUSSION

For determining decarboxylase activities, various methods have been published on the quantitative measurement of released ‘*C02. The assay system reported here is economical and simpler to manipulate than

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many reported previously. It requires no special equipment, the results are reproducible, and the method is sensitive enough to detect as little as 250 pmoles of 14C02 released. This microassay should be advantageous to use when a very small amount of tissue is available, such as brain biopsy samples and cultured neuronal cells (16). It appears that Hyamine hydroxide spotted paper strips give a larger area for absorption than a liquid mixture placed inside the reaction vessel according to Russell and Snyder (3). It was proposed by Roberts and Kuriyama (17) that approximately 50-70% of the total GAD activity in embryonic chicken brain homogenates is present in the presynaptic endings. These findings strongly suggested the presence of GAD activity in other neuronal sites. For careful studies of subcellular localization of GAD activity in neuronal cells it was first necessary to develop a simple, accurate microsassay method. Kuriyama et al. (18) previously reported the effect of age on GAD activity in embryonic chicken brain cerebellum. Using our microtechnique, we have estimated GAD activity in brain homogenates and synaptosomal fractions (in the presence and absence of PLP) prepared from various ages of chick embryos. The pattern in total brain homogenate (Fig. 5) was very similar to the results in cerebellum obtained by Kuriyama et al. (18). The specific activity of GAD increased with the increase in age of embryos and the sharp increase at 17 days of age coincided with the increase in activity of galactosylceramide biosynthesis (19). It is interesting to note that galactocerebroside is one of the major constituents of myelin.

AGE (daya

)

FIG. 5. Effect of chicken embryonic age on L-glutamate decarboxylase activity. Incubation conditions were the same as described in Table 2. except that homogenates from embryonic chicken brain of various ages were used as enzyme source (0.15-0.27 mg of protein).

GAD

ASSAY

BY

A

MICRORADIOMETRIC

457

METHOD

ACKNOWLEDGMENT This work was supported by NIH Research Grant NS-09541-04 and a Grant-in-Aid from Miles Laboratories, Inc., Elkhart, IN (to S.B.) and forms part of the requirement for a Ph.D. (J.R.M.) from the University of Notre Dame.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Roberts, E., and Frankel. S. (I 95 1) J. Biol. Cltctn. 190, 505-S 1‘.I. Roberts, R., and Simonsen. D. B. ( 1963) Biochem. Pi7arnrrrcol. 12, 1 l3- 134. Russell, D., and Snyder. S. H. (1968) Proc. Nnt. Acad. SL.~. USA 60, 1420-1427. Passmann. J. M., Radin. N. S., and Cooper. J. A. D. (1956) And. Chem. 28. 484-486. Woeller, F. H. (1961) Anal. Biochem. 2, 508-5 I I. Wu. J-Y.. Matsuda, T.. and Roberts, E. (1973) J. Biol. Chem. 248, 3029-3034. Jones, R. D.. Hampton, J. K.. Jr.. and Preslock, J. P. (1972) Anal. Biochetn. 49, 147-154. 8. Wilson, S. H., and Kronick, M. N. (1971) Anal. ~i~~i~r~n. 43, 460-467. 9. Chalmers, A., McGeer, E. G., Wickson, V., and McGeer. P. L.. (1970) Cornp. Gear. Pharmacol.

4, 385-390.

IO. Casola. L., and DiMatteo. G. (1972) Anal. Biochem. 49, 41 h-429. I 1. Kravitz, E. A. (1962) J. Neurochem. 9, 363-370. 12. Wood. A. W.. McCrea, M. E.. and Seegmiller, J. E. (1973) At7ul. Biochem. 48, 581-587. 13. Osborne, N. N. (1971) Comp. Grn. P/narmacul. 2, 433-438. 14. Basu, S., Kaufman, B., and Roseman, S. (1973) J. Biol. Chem. 248, 1388-1394. 15. Kennan, T. W., Morre, D. J., and Basu, S. (1974) J. Biof. Cfzem. 249, 3 10-3 15. 16. Moskal, J. R., Gardner, D. A.. and Basu. S. ( 1975) Biochem Biophys. Res. Commun., 61, 75 l-758. 17. Roberts, E., and Kuriyama, K. (1968) Brairr Rps. 8, l-35. 18. Kuriyama, K., Sisken. B., Simonsen, D. G.. Haber, B., and Roberts. E. (1968) Brain Res.

11, 412-430.

19. Basu, S., Shultz, A. M.. Basu. M., and Roseman, S. (1971) J. Bid. 4371-4779.

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246,