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An automated PCO2 assay for glutamic acid decarboxylase
ANhLYTICAI,
An Automated GARY Radiation
52, 63-68
BIOCHEMISTRY
(1973)
P co2 Assay
for Glutamic
Acid
Decarboxylase
H. ZEMAN, PHILIP Z. SOBOCINSKI, RAYMOND T,. CHAPVT
Biology Deprr,,tmeut, Defense Yuclenr Iitcci\.ctl
Jwe
Armed Agewq, 7, 1972;
Forces Radiobiolog!/ Bethesda. Mlnrl/lnrld accepted
Oclokr
Ah-D
Resenrch .?OOi/,
Institute,
12, 1972
An automated :rss:1y for determination of glutamic acid decarboxylase activity is dexribed. The Pw, of an incubation medium is used as a measure of total COz formed by enzymic decarbosylation of glutamate. Details of manifold construction, reagent composition, and data concerning the reliability of the method are presented.
Glutamic acid dccarbosylase (EC 4.1.1.15) (GAD ) catalyzes the conversion of glutamic acid to y-aminobutyric acid (GABA) and carbon dioxide : HOOCCHNIIzCII?CH2COOII
2 CH~NH?CH,CII&OOH
+ COz.
GAD activity has been determined by either the rate of CO, release or the rate of GABA formation. The liberated CC), is tlsually measured by either volumetric, manometric, or radiometric techniques. GABA formation rates have been measured chromatographically or spettrophotometrically. A recent article (1) describes the spectrophotometric technique and cites numerous references to earlier methods. Although these manual methods provide high sensitivity, they do not facilitate handling large numbers of samples. An automated calorimetric procedure has been reported (2) which permits assay of up to 40 samples per hour. The purpose of this report is to present a new automated procedure for determining GAD activity which is based on measurement’ of the partial pressure of CO, (Pc,,J in a reaction mixture. The CO, formed in a reaction mist’urc ir l’rcsen: according to the following equilibria : CO,
dissolved
+ II20
F? II&O0
F? H+ + IlCO-.
The following equation, derivable from tht Henderson tion (3), relates the Poe, of a liquid to the total CO,. Copyright .411 rights
@ 1973 by Academic Press. of wproduciion in nuy form
63 Inc. rearrved.
Hasselbalch
cqua-
64
ZEMAN,
“o’
(‘~o’r)
SOBOCINSKI,
AND
CHAPCT
16.9 X total COs (~mole/ml) = LYx [antilog (pH - pk”) + I]
where (Y is the solubility of CO, in the liquid apparent, dissociation constant of carbonic acid
[ll
(cm”/ml)
and pK’ is the
issolved] + [H,( ‘O,] pK’ = log [Co2 d,H+,,Hc‘o-, -’ ’ 3
PI
For a given pH and piY’ Eq. 1 represents a linear relation between P c0,, and total CO,. Thus, determination of the rate of Pe+ increase in an incubation medium will provide a measure of the CO, formation rate. In principle, the activity of any decarboxylase can be determined by this method. However, to the best, of our knowledge this principle has not previously been applied to measurements of enzyme activity. METHODS
Reayents. Glutamic Acid: 0.5 M L-glutamic acid, pH 6.5 adjusted wit,h 1.0~W KOH. Acetate Buffer: 0.15 2M sodium acetate, pH 5.0 adjusted with 0.2M acetic acid. (Buffer systems other than acetate were not evaluated.) Substrate: 200 ml acetate buffer added to 150 ml glutamic acid. Pyridoxal Phosphate: 1.25 mg/ml pyridoxal 5’ phosphate (Sigma Chemical Co., St. Louis, MO.). Enzyme: L-glutamic acid decarboxylase, nominal activity 4.5 units/mg. (Sigma Chemical Co., St. Louis, MO.) prepared in acetate buffer. Radioisotope: l-14C-L-glutamic acid, specific activity 52 mCiJmmole (Cal Atomic, California). Radiometric Assay. Radiometric assays were performed by the method of Roberts and Simonsen (4). The incubation mixture contained 0.3 ml labeled substrate, 0.04 ml pyridoxal phosphate, and 0.3 ml enzyme solution. Awtomated Assay. The flow diagram for the automated method is shown in Fig. 1. The analytical system was assembled with the following components: one Sampler II and one Proportioning Pump I (Technicon Corp., Tarrytown, N. Y.), one Acid/Base Analyzer with PcoY module (Radiometer Corp., Copenhagen, Denmark), and one Model MR recorder (E. H. Sargent and Co., Chicago, Illinois). Pump tubing and glass fittings are Technicon designations. The Poe, electrode was calibrated prior to analytical determinations with two gases of known CO, composition, 21.7 Torr and 126.0 Torr. The gases also contained known amounts of oxygen and nitrogen. Buffered enzyme solution is aspirated at the rate of 30 samples per hour (2: 1 sample to wash ratio) with two water samples between each enzyme sample to obtain maximum resolution between sample peaks.
AU’OMATED
Pco,
ASSAY
FOR
G5
GAD
PROPORTIONING
ACID/BASE ANALYZER
TUBE -81)
SIZE
‘21 3)
“J
WATER
BATH
(4, 5)
16) Ioi lb) IC) md) el if 1
FIG.
glass
1. Flow diagram fittings are Technicon
of
the PC”? designation.
automated
assay
for
REAGENT
,056 ,081 ,015 ,040
FROM ELECTRODE DISTILLED WATER PYRIDOXAL PHOSPHATE SUBSTRATE
,020 ,040
PC02 STANDARD SAMPLE
GAS
3 WAY STOPCOCK 4” POLYETHYLENE TUBING PC02 ELECTRODE ELECTRODE CHAMBER GLASS “T” PCO? STANDARD GAS
GAD.
Pump
tubing
and
(37°C) The enzyme solution enters a ga Y segmented and preincubatcd stream containing pyridoxal phosphate and substrate. The relative volumes of the substrate, enzyme, and pyridoxal phosphate solutions in the reaction mixture are 1: 1:0.14, and the final substrate concentration is 0.1 M L-glutamic acid. The P ,,* standard gas (21.7 Torr) introduced into the system at atmospheric pressure maintains sample separation and establishes in the incubation medium a baseline PccIl within the maximum sensitivity range of the sensor assembly. After a delay of approximately 2.5 min (at’ 37”C,1 the segmented stream passesto a “debubbler” device for phase separation. The liquid phase passesthrough the electrode assembly where CO, diffuses through the semipermeable membrane of the electrode. The electrode output is simultaneously recorded on the strip chart recorder operated at a speed of 10 in./hr. The circulating water bath used to supply a constanttemperature jacket for the Pco, electrode also supplied the constant temperature required for the enzyme-substrate reaction mixture. RESULTS
AND
DISCCSSION
Figure 2 shows a typical strip chart recording for various concentrations of GAD. An enzyme solution containing 500 pg/ml was diluted to 400, 250, 125, and 62.5 ,Lg/ml. Samples were assayed first. in increasing order of concentration, followed by five repetitions of the 125 jLg/ml solu-
66
ZEMAN,
SOBOCINSKI,
AND
CHAPrT
_I TIME
-
FIG. 2. Logarithmic tracing of PW levels for GAD. Enzyme solution concentrations used were The sample rate was 30 per hour (2: 1 sample to ples between each enzyme sample. Solutions were of concentration, followed by five repetitions of samples in decreasing order of concentration. The for 20 min to obtain the steady state.
various concentrations of purified 62.5, 125. 250, 400, and 500 fig/ml. wash ratio) with two water samassa.. ed first in increasing or,dcr the 125 pg/ml solution, then by 125 &ml soltrtion was aspirated
then by samples in decreasing order of concentration. Finally, t,he 125 pg/ml solution was aspirated for 20 min to obtain a steady state. shown in Fig. 3, is The PCO, for the various enzyme concentrations, linear over the entire range of concentrations used. The steady state
tion,
30
25 -
0 0
100
200 GAD
FIG.
Fig.
2.
3. PCoZ levels
as a function
of GAD
300
400
500
(pg,‘ml)
concentration
for
the
assays
shown
in
AUTOMATED
Pco2
ASSAY
FOR
67
GAD
Pool exhibited some drift, reaching a value of 12.8 Torr after 15 min. The mean and standard deviation of the seven assays of the 125 pg/ml solution were 11.8 + 0.4 Torr. This indicates that at. the sample rate of 30 per hour, peak PCo, is approximately 93% of the maximum obtainable Poe, for any particular GAD concentration. The reason for t,he spurious peak at the termination of the steady state is unknown. However, it does not appear to interfere with the lincaritp or sensitivity of the system. Radiometric assay of various GAD solutions also yielded a linear relation bet’ween evolved CO, and enzyme solution concentration. The enzyme activity was found to be 2.5 ;mole CXIL/mg/min. Figure 4 shows the results of P(:,,? assays of various GAD solutions plotted against the reaults of radiometric assays of the same solutions. Concentrations used were 1.3, 6.7, 13.3, 33.3, 62.5, 125, and 250 pg/‘ml. There is a linear relationship between the radiometrically determined activity and the Poe, changes observed. The correlation coefficient between the two methods is r = 0.9959.
0
2
4
6
8
10
12
ACTIVITY
FIG. various cubation
4. Relation between PcoZ level GAD solutions. Enzyme activity mixture described in Methods.
and radiometrically per reaction refrra
det.ermined activity to the radiometric
of in-
68
ZEMAN,
SOBOCINSICI,
AND
CHAPCT
ACKNOWLEDGMENT We wish to thank Thomas K. Dalton for his skillful
technical assistance.
REFERENCES 1. Cozz.4s1,
I. (1970) Ad. Biochena. 33, 125-131. 2. GAMIER. M.. AND GONNARD. P. (1968) Bull. Sot. Chim. Biol. 50(4), 933-937. 3. HENRY. R. J. (1964) “Cliniral Chemistry: Principles and Techniques,” p. 435, Harper rind Row Puhlishr~rs, New York. 4. ROBERTS. E., AND SI~ZONRET. D. G. (1963) Biochem. Phnrmncol. 12, 113-134.
An Automated GARY Radiation
52, 63-68
BIOCHEMISTRY
(1973)
P co2 Assay
for Glutamic
Acid
Decarboxylase
H. ZEMAN, PHILIP Z. SOBOCINSKI, RAYMOND T,. CHAPVT
Biology Deprr,,tmeut, Defense Yuclenr Iitcci\.ctl
Jwe
Armed Agewq, 7, 1972;
Forces Radiobiolog!/ Bethesda. Mlnrl/lnrld accepted
Oclokr
Ah-D
Resenrch .?OOi/,
Institute,
12, 1972
An automated :rss:1y for determination of glutamic acid decarboxylase activity is dexribed. The Pw, of an incubation medium is used as a measure of total COz formed by enzymic decarbosylation of glutamate. Details of manifold construction, reagent composition, and data concerning the reliability of the method are presented.
Glutamic acid dccarbosylase (EC 4.1.1.15) (GAD ) catalyzes the conversion of glutamic acid to y-aminobutyric acid (GABA) and carbon dioxide : HOOCCHNIIzCII?CH2COOII
2 CH~NH?CH,CII&OOH
+ COz.
GAD activity has been determined by either the rate of CO, release or the rate of GABA formation. The liberated CC), is tlsually measured by either volumetric, manometric, or radiometric techniques. GABA formation rates have been measured chromatographically or spettrophotometrically. A recent article (1) describes the spectrophotometric technique and cites numerous references to earlier methods. Although these manual methods provide high sensitivity, they do not facilitate handling large numbers of samples. An automated calorimetric procedure has been reported (2) which permits assay of up to 40 samples per hour. The purpose of this report is to present a new automated procedure for determining GAD activity which is based on measurement’ of the partial pressure of CO, (Pc,,J in a reaction mixture. The CO, formed in a reaction mist’urc ir l’rcsen: according to the following equilibria : CO,
dissolved
+ II20
F? II&O0
F? H+ + IlCO-.
The following equation, derivable from tht Henderson tion (3), relates the Poe, of a liquid to the total CO,. Copyright .411 rights
@ 1973 by Academic Press. of wproduciion in nuy form
63 Inc. rearrved.
Hasselbalch
cqua-
64
ZEMAN,
“o’
(‘~o’r)
SOBOCINSKI,
AND
CHAPCT
16.9 X total COs (~mole/ml) = LYx [antilog (pH - pk”) + I]
where (Y is the solubility of CO, in the liquid apparent, dissociation constant of carbonic acid
[ll
(cm”/ml)
and pK’ is the
issolved] + [H,( ‘O,] pK’ = log [Co2 d,H+,,Hc‘o-, -’ ’ 3
PI
For a given pH and piY’ Eq. 1 represents a linear relation between P c0,, and total CO,. Thus, determination of the rate of Pe+ increase in an incubation medium will provide a measure of the CO, formation rate. In principle, the activity of any decarboxylase can be determined by this method. However, to the best, of our knowledge this principle has not previously been applied to measurements of enzyme activity. METHODS
Reayents. Glutamic Acid: 0.5 M L-glutamic acid, pH 6.5 adjusted wit,h 1.0~W KOH. Acetate Buffer: 0.15 2M sodium acetate, pH 5.0 adjusted with 0.2M acetic acid. (Buffer systems other than acetate were not evaluated.) Substrate: 200 ml acetate buffer added to 150 ml glutamic acid. Pyridoxal Phosphate: 1.25 mg/ml pyridoxal 5’ phosphate (Sigma Chemical Co., St. Louis, MO.). Enzyme: L-glutamic acid decarboxylase, nominal activity 4.5 units/mg. (Sigma Chemical Co., St. Louis, MO.) prepared in acetate buffer. Radioisotope: l-14C-L-glutamic acid, specific activity 52 mCiJmmole (Cal Atomic, California). Radiometric Assay. Radiometric assays were performed by the method of Roberts and Simonsen (4). The incubation mixture contained 0.3 ml labeled substrate, 0.04 ml pyridoxal phosphate, and 0.3 ml enzyme solution. Awtomated Assay. The flow diagram for the automated method is shown in Fig. 1. The analytical system was assembled with the following components: one Sampler II and one Proportioning Pump I (Technicon Corp., Tarrytown, N. Y.), one Acid/Base Analyzer with PcoY module (Radiometer Corp., Copenhagen, Denmark), and one Model MR recorder (E. H. Sargent and Co., Chicago, Illinois). Pump tubing and glass fittings are Technicon designations. The Poe, electrode was calibrated prior to analytical determinations with two gases of known CO, composition, 21.7 Torr and 126.0 Torr. The gases also contained known amounts of oxygen and nitrogen. Buffered enzyme solution is aspirated at the rate of 30 samples per hour (2: 1 sample to wash ratio) with two water samples between each enzyme sample to obtain maximum resolution between sample peaks.
AU’OMATED
Pco,
ASSAY
FOR
G5
GAD
PROPORTIONING
ACID/BASE ANALYZER
TUBE -81)
SIZE
‘21 3)
“J
WATER
BATH
(4, 5)
16) Ioi lb) IC) md) el if 1
FIG.
glass
1. Flow diagram fittings are Technicon
of
the PC”? designation.
automated
assay
for
REAGENT
,056 ,081 ,015 ,040
FROM ELECTRODE DISTILLED WATER PYRIDOXAL PHOSPHATE SUBSTRATE
,020 ,040
PC02 STANDARD SAMPLE
GAS
3 WAY STOPCOCK 4” POLYETHYLENE TUBING PC02 ELECTRODE ELECTRODE CHAMBER GLASS “T” PCO? STANDARD GAS
GAD.
Pump
tubing
and
(37°C) The enzyme solution enters a ga Y segmented and preincubatcd stream containing pyridoxal phosphate and substrate. The relative volumes of the substrate, enzyme, and pyridoxal phosphate solutions in the reaction mixture are 1: 1:0.14, and the final substrate concentration is 0.1 M L-glutamic acid. The P ,,* standard gas (21.7 Torr) introduced into the system at atmospheric pressure maintains sample separation and establishes in the incubation medium a baseline PccIl within the maximum sensitivity range of the sensor assembly. After a delay of approximately 2.5 min (at’ 37”C,1 the segmented stream passesto a “debubbler” device for phase separation. The liquid phase passesthrough the electrode assembly where CO, diffuses through the semipermeable membrane of the electrode. The electrode output is simultaneously recorded on the strip chart recorder operated at a speed of 10 in./hr. The circulating water bath used to supply a constanttemperature jacket for the Pco, electrode also supplied the constant temperature required for the enzyme-substrate reaction mixture. RESULTS
AND
DISCCSSION
Figure 2 shows a typical strip chart recording for various concentrations of GAD. An enzyme solution containing 500 pg/ml was diluted to 400, 250, 125, and 62.5 ,Lg/ml. Samples were assayed first. in increasing order of concentration, followed by five repetitions of the 125 jLg/ml solu-
66
ZEMAN,
SOBOCINSKI,
AND
CHAPrT
_I TIME
-
FIG. 2. Logarithmic tracing of PW levels for GAD. Enzyme solution concentrations used were The sample rate was 30 per hour (2: 1 sample to ples between each enzyme sample. Solutions were of concentration, followed by five repetitions of samples in decreasing order of concentration. The for 20 min to obtain the steady state.
various concentrations of purified 62.5, 125. 250, 400, and 500 fig/ml. wash ratio) with two water samassa.. ed first in increasing or,dcr the 125 pg/ml solution, then by 125 &ml soltrtion was aspirated
then by samples in decreasing order of concentration. Finally, t,he 125 pg/ml solution was aspirated for 20 min to obtain a steady state. shown in Fig. 3, is The PCO, for the various enzyme concentrations, linear over the entire range of concentrations used. The steady state
tion,
30
25 -
0 0
100
200 GAD
FIG.
Fig.
2.
3. PCoZ levels
as a function
of GAD
300
400
500
(pg,‘ml)
concentration
for
the
assays
shown
in
AUTOMATED
Pco2
ASSAY
FOR
67
GAD
Pool exhibited some drift, reaching a value of 12.8 Torr after 15 min. The mean and standard deviation of the seven assays of the 125 pg/ml solution were 11.8 + 0.4 Torr. This indicates that at. the sample rate of 30 per hour, peak PCo, is approximately 93% of the maximum obtainable Poe, for any particular GAD concentration. The reason for t,he spurious peak at the termination of the steady state is unknown. However, it does not appear to interfere with the lincaritp or sensitivity of the system. Radiometric assay of various GAD solutions also yielded a linear relation bet’ween evolved CO, and enzyme solution concentration. The enzyme activity was found to be 2.5 ;mole CXIL/mg/min. Figure 4 shows the results of P(:,,? assays of various GAD solutions plotted against the reaults of radiometric assays of the same solutions. Concentrations used were 1.3, 6.7, 13.3, 33.3, 62.5, 125, and 250 pg/‘ml. There is a linear relationship between the radiometrically determined activity and the Poe, changes observed. The correlation coefficient between the two methods is r = 0.9959.
0
2
4
6
8
10
12
ACTIVITY
FIG. various cubation
4. Relation between PcoZ level GAD solutions. Enzyme activity mixture described in Methods.
and radiometrically per reaction refrra
det.ermined activity to the radiometric
of in-
68
ZEMAN,
SOBOCINSICI,
AND
CHAPCT
ACKNOWLEDGMENT We wish to thank Thomas K. Dalton for his skillful
technical assistance.
REFERENCES 1. Cozz.4s1,
I. (1970) Ad. Biochena. 33, 125-131. 2. GAMIER. M.. AND GONNARD. P. (1968) Bull. Sot. Chim. Biol. 50(4), 933-937. 3. HENRY. R. J. (1964) “Cliniral Chemistry: Principles and Techniques,” p. 435, Harper rind Row Puhlishr~rs, New York. 4. ROBERTS. E., AND SI~ZONRET. D. G. (1963) Biochem. Phnrmncol. 12, 113-134.