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Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds
ANALYTICAL
BIOCHEMISTBY
Separation
110-121 (1974)
59,
and
Determination
Intermediates
of the
and YUKIKO Department
of
Related
Chemistry, University,
Citric
Acid
Cycle
Compounds
TOKUMITSU
Physiological Hokkaido
of 14C-Labelled
AND
Faculty Sapporo,
MICHIO of
UI
Phnrmaceutical Jnpan
Sciences,
Received January 1, 1973; accepted October 29, 1973 A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino. keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. %-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.
In the course of our research on the 2-ketoglutarate-linked substratelevel phosphorylation in the isolated rat liver mit’ochondria (1,2), the need arose to determine the specific radioactivity of the intermediates of the citric acid cycle and related amino and keto acids. The method presented below offers a resolution of a large number of these acids by a simple procedure based on the principle that these are divided broadly into three groups by treatment with an ion exchanger before being analyzed on thin-layer chromatographic plates. 2-Keto acids, which should be handled with a great care owing to lability (e.g., oxalacetic acid) or volatility (e.g., pyruvic acid), were converted enzymatically into the respective amino acids. MATERIALS
AND METHODS
Preparation of IT-Labelled
k’eto Acids
W-Labelled pyruvic, oxalacetic and 2-ketoglutaric acids wcrc prepared from 14C-labelled alanine, aspartic and glutamic acids by enzymic 110 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
SEPARATION
OF
CITRIC
ACID
CYCLE
111
transamination just before use. The reaction mixture consists of 5 pmoles of one of these laC-amino acids, 10 ,.moles of 2-ketoglutarate (or pyrurate in the case of [“Cl2-ketoglutarate preparation), 10 units of glutamic-oxalacetic transsminase (GOT) or glutamic-p\-ruvic transaminase (GPT) and 0.05 M trirthnnolamine buffer (pH 7.6 I in a total volume of 2.0 ml. After being kept at 30°C for 30 min, the reaction was terminated by the addition of 0.2 ml of 3 N HClO, and the deproteinizecl supernatant was neutralized with 20% KOH. The chemical amount of the keto acid formed was &imatcd cnzgmatically while its radioactivity content was analyzed after separation from the “C-amino acid by applying the supernatant to a column of cation-exchanger as described in RESULTS for the separation of amino acid fraction. Prepclrntiorl
of Ion-Esch.unge
Columns
AC 50W-X8(H+-form) and AG 2-X10 (OH--form) (50-100 mesh, BioRad Laboratories) were used. The latter was prepared by a columnwise application of 20 volumes of 1 N SaOH to AG B(Cl--formj followed by a thorough leashing with CO,-free water and was stored without exposure to atmospheric CO, at 0°C until use. The bottom of columns (0.5 X 15 cm) packed with these exchangers was equipped with a small Tygon tubing to which a screw-clamp was attached to control the flow rate. Thin-layer
Chromatography
Fifteen grams of cellulose powder (Cellulose MN300HR, RlacheryNagelI, was homogenized with 100 ml distilled water in a Waring blender for 90 set and the slurry formed was applied to 5 IAates to a thickness of 0.25 mm. Prewashing of the dried plates was performed w&h an appropriate solvent syst’em as described in RESULTS and kept, overnight before use. For the purpose of dctect#in g organic acids on the developed plate, a minute amount (2-3 mg/lOO ml) of dichlorofluorescein was added into the solvent system, which makes the spot visible under ultraviolet light after thorough evaporation of the acidic solvent’. The spots of amino acids were detected by spraying the plate with ninhydrin reagent (0.2% ninhydrin in collidinc (2%) -containing acetone ). The development of the color reaction was facilitated by heating the plate in warm air. The cellulose po\vder on the individual spot was subsequently scraped out of the plate and placed in a vial containing 0.5 ml water. The scintillation fluid consisting of 1:2 mixture of Triton Xl00 and toluene-type scintillator (4 g of PPO and 150 mg of POPOP in 1 liter of toluene) was then added to determine the radioactivit#y in a Beckman Liquid Scintillation Spectrometer, Model LS230.
112
TOKPMITSU
AND
UI
Sources of Reagent 14C-Labelled acids were purchased from New England h’uclear. Enzymes and coenzymes were obtained from Sigma and Boehringer. Other reagents are of analytical grade from commercial sources. RESULTS
Principle
of the Present
Procedure
The principle of the method presented here is that ‘“C-intermediates are first separated into three broad groups, amino acid, keto acid and other organic acid fractions. Each fraction can then be separated into individual acids by a one-dimensional thin-layer chromatography which is far better than two-dimensional ones when a large number of specimens must be handled. The amino acid fraction was first separated from other organic acids by adsorption onto a cation exchanger. Keto acids in the effluent were enzymatically converted into the respective amino acids which were separated from other di- and t,ricarboxylic acids by being again applied to the cation exchange column. These column manipulations proved to be very successful in eliminating the inorganic salts and buffers which, being contained in the incubation medium in a rather large amount, ot.herwise interfere with the subsequent separation on thinlayer chromatograms. The basal conditions studied and worked out for each step of the separation procedure are presented below. Conversion
of Keto Acids to the Colrespondiny
The combination of the GOT and GPT with the reduction of 2-ketoglutaric of t.hree kinds of keto acids
Amino Acids
two transamination reactions catalyzed by glutamic dehydrogenase (Glu-DH) -catalyzed acid results in the quantitative conversion to the corresponding amino acids as follows.
GPT Pyruvate
+ Glutamate
E
2-Ketoglutarate
+
Alanine
GOT
Oxalacetate
+ Glutamate
s
ZKetoglutarate
+ Aspartate
+ 3H+
-
3Glutnmate
Alanine
+ Aspartate
Glu-DH
3(2-Ketoglutarate) Pyruvate
+ 3NADH
+ Oxalacet,ate
+ 2-Ketoglutarate
+ 3NHs +
+ 3NAD+ + Glutamate
In our separation procedure, this enzymic conversion must be conducted in a rather large volume of solution, i.e., with the effluent passing through the column of cation exchanger by which the amino acids originally present in the starting solution have been trapped. (Concentration should be avoided before this step because of lability of keto acids.) It is also advisable, in this cnzymic reaction, to use a buffer which will be readily
SEPARATION
OF
CITRIC
ACID
I I8
CYCLE
2KETOGLUTARATE
-
I
‘1
Bd
ASPARTATE
OXALACETATE
PYRWATE
5
10
20
3
40 MIN
FIG. 1. Enzymatic conversion of 2-ketoglutarnte, oxalacetate and pyruxate to amino acids. 2-Ketoglutaric (panel A), osalacetic (panel B) and pyruvic (panel C) acids, 0.5 bmole each. ~vere enzymatically converted to the corresponding amino acids in the reaction mixture containing 2 pmoles of NADH, 10 pmoles of NH&l. 0.115 N HCl buffered with 0.3 ml of N-ethylmorpholine in a total volume of 8.5 ml (final pH 7.6). Amounts of the keto acids remaining in the reaction mixture after incubation at 25°C (time shown on abscissa in min) are plotted as percent of the initial value (in panel B. the formation of aspartate is also plotted). Units of the enzymes added are shown by symbols specified in panel A.
114
TOKUMITSU
A;“;D
UI
removable before further separation is undertaken on thin-layer plates. Two kinds of volatile buffer, 2,4,6-collidine and N-cthylmorpholine~ were tested. Since collidine was found to be very inhibitory to the glutamic dehydrogenase reaction, we studied the rate of the reaction in x-ethylmorpholine buffer (pH 7.6) as shown in Fig. 1. In this experiment, 7.5 ml of 0.13 N HCl cont,aining pyruvate, oxalacetate and 2-ketoglutarate each in an amount of 0.5 ,lmoles (which mimics the effluent from the cation exchanger in the H+-form) was neutralized to pH 7.6 by the addition of 0.3 ml of N-ethylmorpholine and further added with 10 pmoles of NH,Cl, 2 pmoles of NADH and enzymes to make a final volume of 8.5 ml. At each time of incubation as indicated on abscissa, the reaction was terminated by the addition of 3 K HCIO, and the aliquot, after being neutralized with KOH, was enzymatically analyzed for 2-ketoglutaric acid, oxalacetic and aspartic acids, and pyruvic acid which were plotted in panels A, B and C, respectively, in Fig. 1. In the presence of 1 unit of Glu-DH, the addition of 1 unit of GOT caused a prompt disappearance of 2-ketoglutarate (Fig. IA), while no exact stoichiomctric relationship was maintained between disappearance of oxalacetate and the formation of aspartate under this condition. It is presumably because oxalacetate undergoes a breakdown during incubation before being converted to aspartate. Accordingly, the acceleration of the reaction by the addition of 3 units of GOT was necessary for a quantitative conversion of oxalacetat,e to aspartate (Fig. 1B). On the other hand, much more GPT (up to 10 units) were required for a quantitative conversion of pyruvate to alanine in 30 min (Fig. IC). The stability of these keto acids was studied and is presented in Table 1. Pyruvate and 2-kctoglutarate safely survived the whole incubation period, while a significant reduction of the concentration of oxalacetat’e occurred during 60 min incubation. This result confirms the preceding finding in Fig. 1 that 3 units of GOT was needed for the quantitative conversion of oxalacetate to aspartate; essentially no breakdown of oxalacetate took place up to 10 min incubation during which time almost all of oxalacetate had been converted to aspartate by 3 units of GOT. NO TABLE 1 of 2-Keto
Stability
Acids Oxalacetic
Pyruvic (% -~ O”C, 25 min and 25”C, 25”C, 60 min = Mean
k SEM,
No.
10 min
97.3 100.6
of observations:
f 3.9” + 2.9 6.
2-Ket,oglutaric
of initial) 96.5 X4.6
rf: 3.3 f 3.0
97.3 97.6
* t
1.9 2.6
SEPARATION
OF
CITRIC
ACID
CYCLE
115
degradation of oxalacetate (as well as pyruvate and 2-ketoglutarate) was observed cluring the passage through AG50 column, the procedure to be carried out prior to the enzymic conversion to amino acids, because the medium was meanwhile kept acidic (data not shown). Column
Manipulations
Outline of the column procedure employed in our method is as follows. ilmino acids and cations such as lUg”+, K+ and buffers (Tris or triethanolamine) used for incubation of mitochondria were first separated from anions by their adsorption onto AG-50(H+) column followed by the elution with NH&OH. Amino acids were then separated from cations by applying the eluate directly to the column of iiG-2 in the hydroxide form. (The anion exchanger in the chloride or formate form fails to trap alanine quantitatively.) hmino acids adsorbed were allowed to leave the column by eluting with formic acid. The amino acids prepared from keto acids were also applied to AG-5O(H+) and elutcd therefrom with IXH,OH. Other organic acids which had passed through the columns of XG-50 were separated from nonionized compounds such as sucrose, a major component of mitochondrial preparation, by being adsorbed on AG-2 (HCOO-) followed by ehkion with formic acid. It was found that the adsorption of the ionized compounds onto these three types of ion exchanger columns as well as the passage of nonadsorbable compounds was quant,itative even when the rate of flow was as high as 2 ml/min. (Among the intermediat,es tested, aspartate was the only compound that tended to appear in the effluent when the column of cat,ion exchanger which had adsorbed it was washed wit’h a rather large volume of water. Therefore, the AG-50 column was washed with 5 ml of water following application of amino acid-containing solution.1 In contrast, the elution of the arlsorbcd compounds from rolunms was not quantitative if small amomits of elution fluids were applied. Since it is desirable to get a quantitative elut’ion with as a small volume of the fluid as possible, the efficiency of elution was tested with the flow rate maintained as low as 0.2 ml/min. It was found that 10 ml of l-2 N NH,OH and 40% HCOOH was the minimum volume required for the quantitative elution from the cat)ion and anion exchanger respectively. Based on this result the volume of clution was detcrminccl as sho~~n in the standard procedure.
When the cellulose illate was dercloped with liquid phenol (500 g phenol added wit’h 150 ml water) in an atmosphere equilibrated with 3% NH&OH (3), most. of amino nc%ls closely related to the citric acid cycle were resolved in small spots with l?i values shown in Table 2. A
116
TOKUMITSU
AXD
TABLE 2 Rf of Amino Acids and Di- and Tricarboxylic
UI
Acids on a Cellulose Platen
R, X 100 after development Water-saturated isopropyl ether: HCOOH (3/l) Citric Malic Aconitic Succinic (Lactic Fumaric
16 29 35 69 76) 87
__.-
with Liquid phenol
Aspartic Glutamic Serine Asparagine Glutamine Alanine Citrulline
-. 22 32 45 52 58 65 72
a The cellulose plate had been prewashed as described in t,ext prior to development.
good separation without tailing of any spot was obtained only when the plate of Cellulose MN300 HR had been prewashed by ascending irrigation with liquid phenol a,nd dried overnight at room temperature. Two dimensional chromatography with a solvent system of n-butanol-acetonediethylamine-H,O (10/10/Z/5) (3) in the first direction followed by the second development with the above phenol syst,em revealed that the spots of glutamic and aspartic acids were not contaminated by any other amino acid. Though threonine and glycine were found to migrate only slightly ahead of asparagine and serine respectively and hence hardly undergo a complete resolution in the one-dimensional system adopted here, the present system proved still valuable because, to our experience, essentially no radioactivity was detected in threonine and glycine when asparagine and glut’amine were significantly labelled in many casesof mitochondrial incubation. Most of the other amino acids ran ahead of citrulline. Rf values of organic acids separated in a new system of one-dimensional thin-layer chromatography are shown in Table 2. The cellulose plate, prewashed by an ascending irrigation with n-butanol-2 K NH,OHethanol (7/2/l) was developed with isopropyl ether-HCOOH (3/l) for about 70 min. This system is superior to other similar acidic solvent mixture such as ether-HCOOH-H,O (4,6) in that the much smaller spreading of the sample spots is obtained after development. In this system, isocitric acid migrates slightly faster than citric acid without sharp resolution in many cases. The Whole Separation
Procedure
The whole procedure for the separation of all the intermediates of the citric acid cycle, worked out on the basis of the foregoing results, is
SEPARATION
Neutral
OF
CITRIC
or acidified
I
to
c~olurnn
I
I
Ad&bed
Eflluent
1 Eluted
wit,h
10 ml of 2 .u NHaOH
I
applied
AG-2(OH-)
column
1 Eluted with 10 ml of 407, HCOOH dried
+ wLhing (5 ml) I submitt,ed to
Enzymic conversion of keto acids to amino acids N-ethylmorpholine (to make the pH around 7) Glu-DH 1 unit, GOT 3 units, GPT 10 units, NH&l 10 pmoles NADH 1 2 pmoles, kept at 25°C for 10 min and then at _ 30°C for 10 min. I
to
1 Effluent + washing (10 ml)
1 Adsorbed
117
CYCLE
sample
applied
AC&5O(H+)
ACID
II Discard
in vacua
I
i
applied
TLC-System Ia (Amino acids)
AG-50(H+)
column
1
1
Adsorbed
Effluent
+ washing
(5 ml)
1 Eluted wit’h 10 ml of 2N NH&H I 4 dried
applied
to
I A<;-2(HCOO-) I
in vacua
column I Ef&nt washing
Adiorbed
1 TLC-System Ia (Amino acids originating from keto acids)
1
Ii
Eluted with 10 ml of 407, HCOOH dried
+ (10 ml)
Discard
1 in vacua
TLC-System IIb (Organic acids) a Liquid prewashed
phenol in 3$‘, NH*OH, as in text. FIG.
b I sopropgl
2. A summary
ether-HCOOH
of the separation
(3/l), method.
with
a llse of plate
118
TOKUMITSU
AND
CI
summarized in Fig. 2. Not only the incubation medium of biological preparation such as isolated mitochondria but also t.he deproteinized extract of mammalian tissues can directly serve as the starting sl)ccimen for this separation method. Cations contained in the starting solution in amounts less than the exchange capacity of the resin used (volume of t,he resin bed: 3 ml) can be removed during these column manipulations, while inorganic anions adhere to the final organic anion fraction. Therefore, nonvolatile inorganic anions used for the deproteinization, such as trichloroacetate or perchlorate, should be eliminated from the sample before application to the first column. For this purpose, the perchloratedeproteinized solution was previously added with KC1 or neut,ralized with KOH. The enzymic conversion of keto acids to amino acids is completed in 30 min at 25°C under this condition (see Fig. 1). To ensure the quantitative conversion, however, the reaction mixture was kept at 25°C for 20 min and then at 30°C for a further 10 min in the standard procedure in Fig. 2. Adsorption to and elut’ion from ion exchange columns is repeated four times in the whole procedure. However, only two columns, each packed with AG-50(H+) and AG-2(OH-), are required for the complete separation of one sample. After first elution of amino acids from AC-50 with NH,OH, the resin in the column in situ is generat,ed by applying 50 ml of 2 N HCI followed by washing with water to be ready for the second adsorption. Likewise, AG-2(OH)-) is converted to the form&e form during elution of amino acids and then directly serves as the adsorbent of organic anions after simple washing with water. It usually takes only 4 hr before the final eluate was obtained, including the time required for the regeneration of resin as \yell as t,he enzymic conversion of keto to amino acids. The final eluat,es are evaporated to dryness under reduced pressure by the concentrator (TAITEC Model TC 8) at 25°C (or lyophilized) and then dissolved in small amounts of water (usually 0.2 ml) before being applied to thin-layer plates. Recoveq
Test
The reaction mixture for the incubation of rat liver mitochondria (mitochondria isolated from 200 mg liver, sucrose 100 pmoles, KC1 700 pmoles, Tris-HCI buffer 100 pmoles and EDTA 100 ,umoles in a total volume of 4.5 ml) was deproteinized by the addition of 0.25 ml of 3 N HCIO,. 14C-Intermediates were added each in an amount of 0.2-0.8 pmoles as indicated in Table 3. To the supernatant of this deproteinized solution was added 0.3 ml of N KOH and the precipitated KCIO, was centrifuged off. The resultant supernatant served as a test solution for
SEPARATION
ltecovery
of W-Intermediates W
i\mino
column
Combined
81.9
CITRIC
TABLE throughout
in the fraction
$CID
119
CYCLE
3 the Present
Separation
of (4; of t,he radioactivity Veto
acids
Following
Throughout
recovered
OF
acids
Method
added) Organic
acids
manipulatious -t 0.65
the entire
Combined
71.5
i
Alanine Aspartat,e Glutamate
72.7 64.8 83.8
+ 3.42 + 2.06 * 4.04
process
(including
95.4
j, 0.61
85.9
+ 0.63
86.4
f
84.9
* 2.17
90.2 X6.6 ’ 85.9
+_ 2.18 k 1.63 + 1.76
77.6 92.7 s3.4
k 2.17 * 0.97 ) l.SS
TLC)
1.59 Pyruvate Oxalacetate P-Ketoglutarate
1.45 Succinate Malate Citrate
the whole procedure of the present separation method. The percent recovery of each intermediate arc listed in Table 3 as the mean value & SEA1 for five observations. DISCUSSION
Several simple methods have been proposed for the chromatographic separation of the intermediates of the citric acid cycle on thin-layer plates (4-g). It is impract,ical, however, to apply these chromatographic methods directly to the biological specimens, because inorganic salts present in the specimen are concentrated on the plate and tend to intcrferc with the separation of organic anions by causing serious tailing. i2s pointed out by Ting and Dugger (4)) preliminary purification of samples, for the purpose of desalting, is an essential step for their sufficient separation by thin-layer chromatography. In the present method, a systematic column procedure with cation- and anion-exchangers was employed for the purpose of not only desaltin g of the biological specimen but also of dividing a large number of intermediates to three subgroups (Fig. 2). This broad fractionation by columns is based on the exhaustive adaorption and elution and, unlike the column chromatographic procedures previously reported (10-12)) does not depend on a slight difference in the ionized state of substances to be separated. Therefore, the recovery of the ‘“C-labelled substances applied was reproducible as shown in Table 3. Each subgroup thus prepared was then successfully separated into individual metabolites by a one-dimensional thin-layer chromatography. Table 3 clearly shows that the present technique is successfully applicable to the reaction mixture of biological preparations such as mito-
120
TOKUMITSU
AND
UI
chondria. This technique also proves useful for an analysis of “Cintermediates in the tissue excised from intact animal or from perfusion apparatus; the deproteinized extract of the tissues contains interfering ions in much less amounts t,han the reaction mixture used for the in vitro experiments which must be fortified with buffers or ions required for enzymic reactions. Since ket,o acids are unstable and hence are liable to undergo brcakdown during separation, a lot of devices has been proposed to convert them into more stable derivatives including 2,4-dinitrophcnylhydrazone (13-15)) phenylhydrazone (15,16), oxime (15)) quinolplhydrazone ( 15)) quinoxaline (17) and semicarhazone (18). Some of these derivatives suffer from disadvantages such as quenching in the liquid scintillation system or separation into multiple spots (due to the presence of stereoisomers or production of artifact) on the thin-layer plate. The preparation of semicarbazones, reported from this laboratory to be useful for the separation of lGpyruvic acid originating from blood lactate (18), was abandoned in the early stage of the development of the present method, because oxalacetic and 2-ketoglutaric acid semicarbazones migrate at comparable rates in all t’he solvent syst,ems so far tested. Instead, the present paper is the first to propose that the enzpmic conversion of keto acids to the corresponding amino acids proves very useful for their separation from other organic acids. A use of N-etl~ylmorl~l~oline, ammonium ion which becomes volatile 171>011 being the quarternary separated from anions, as a buffer in the eneymic conversion made it very easy to separate subsequently the products of the reaction into individual amino acids on thin-layer plates. The principle of the present technique. i.e., a combination of column and t,hin-layer chromatography, has been already applied by one of the of the citric acid present authors to the analysis of “C-intermediates cycle and gluconeogenic pathway in the perfused liver (19,20). REFERENCES 1. TOKT-MITSU.
Y., AND Ur. M. (1973) Biochim. Biophys. Acta 292, 310-324. Y.. AND UI, M. (1973) Biochim. Biophys. Acta 292, 325-337. vex AHOX. E., AND NEHER, R. (1963) J. Chromatogr. 12, 329-341. TIN<;. I. P., .+ND DUWER, W. M. (1965) Anal. Biochem. 12, 561-578. MYERS, W. F.. AND HUANG, K. Y. (1966) Anal. Biochem. 17, 210-213. KNAPPE, E., AND ROHDEW~LD, I. (1965) 2. Aunl. Chem. 210, 183-193. BLEIWEIS, A. S., REEVES. H. C.. .~ND AJL. S. J. (1967) Anal. Biochem. 20, 335-338. HIGGINS, H., AND VON BRAND, T. (1966) And. Biochem. 15, 122-126. VEXEZIALE, C. M.. AND GABRIELLI. F. (1969) Anal. Biochem. 28, 206-215. VARXER. J. E. (1957) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 3, pp. 397-403, Academic Press, Ncm York. GAMBLE. J. L. (1965) J. Biol. Chem. 240, 2668-2672.
2. TOKUMITSU,
3. 4. 5.
6. 7. 8. 9. 10. 11.
SEPARATION
OF
CITRIC
ACID
CYCLE
121
12. LANOGE. Ii., NICKL.~~. IT:. J.. A~UD WILLIAMSON, J. R. (1970) J. Biol. Chew 215, 102-111. 13. EL HAWAKY, M. F. S.. AND THOAIPSON, R. H. S. (1953) Biochem. J. 53, 340-347. 14. ROSAN, R. C., AND NIELAND. M. L. (1963) Anal. &o&em. 6, 125134. 15. BACHELARD. H. S. (1965) Anal. Biochem. 12, 8-17. 16. FREMINET, A., BCRSAUX, E., AND POTART. C.-F. (1972) Biochem. hled. 6, 72-76. 17. MO~BRAY. J., AND OTTAWAY, J. H. (1970) Biochem. J. 120, 171-175. 18. KUSAK.4. M., AND UI, M. (1973) Anal. Biochem. 52, 369-376. 19. UI, M.. CLAUS, T. H., ESTON, J. H.. AIX-D PARK. C. R. (1973) J. Biol. Chem. 248, 5344-5349. 20. UI, M.. EXTON, J. H.. AND PARK, C. R. (1973) J. Biol. Chem. 248, 5350-5359.
BIOCHEMISTBY
Separation
110-121 (1974)
59,
and
Determination
Intermediates
of the
and YUKIKO Department
of
Related
Chemistry, University,
Citric
Acid
Cycle
Compounds
TOKUMITSU
Physiological Hokkaido
of 14C-Labelled
AND
Faculty Sapporo,
MICHIO of
UI
Phnrmaceutical Jnpan
Sciences,
Received January 1, 1973; accepted October 29, 1973 A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino. keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. %-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.
In the course of our research on the 2-ketoglutarate-linked substratelevel phosphorylation in the isolated rat liver mit’ochondria (1,2), the need arose to determine the specific radioactivity of the intermediates of the citric acid cycle and related amino and keto acids. The method presented below offers a resolution of a large number of these acids by a simple procedure based on the principle that these are divided broadly into three groups by treatment with an ion exchanger before being analyzed on thin-layer chromatographic plates. 2-Keto acids, which should be handled with a great care owing to lability (e.g., oxalacetic acid) or volatility (e.g., pyruvic acid), were converted enzymatically into the respective amino acids. MATERIALS
AND METHODS
Preparation of IT-Labelled
k’eto Acids
W-Labelled pyruvic, oxalacetic and 2-ketoglutaric acids wcrc prepared from 14C-labelled alanine, aspartic and glutamic acids by enzymic 110 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
SEPARATION
OF
CITRIC
ACID
CYCLE
111
transamination just before use. The reaction mixture consists of 5 pmoles of one of these laC-amino acids, 10 ,.moles of 2-ketoglutarate (or pyrurate in the case of [“Cl2-ketoglutarate preparation), 10 units of glutamic-oxalacetic transsminase (GOT) or glutamic-p\-ruvic transaminase (GPT) and 0.05 M trirthnnolamine buffer (pH 7.6 I in a total volume of 2.0 ml. After being kept at 30°C for 30 min, the reaction was terminated by the addition of 0.2 ml of 3 N HClO, and the deproteinizecl supernatant was neutralized with 20% KOH. The chemical amount of the keto acid formed was &imatcd cnzgmatically while its radioactivity content was analyzed after separation from the “C-amino acid by applying the supernatant to a column of cation-exchanger as described in RESULTS for the separation of amino acid fraction. Prepclrntiorl
of Ion-Esch.unge
Columns
AC 50W-X8(H+-form) and AG 2-X10 (OH--form) (50-100 mesh, BioRad Laboratories) were used. The latter was prepared by a columnwise application of 20 volumes of 1 N SaOH to AG B(Cl--formj followed by a thorough leashing with CO,-free water and was stored without exposure to atmospheric CO, at 0°C until use. The bottom of columns (0.5 X 15 cm) packed with these exchangers was equipped with a small Tygon tubing to which a screw-clamp was attached to control the flow rate. Thin-layer
Chromatography
Fifteen grams of cellulose powder (Cellulose MN300HR, RlacheryNagelI, was homogenized with 100 ml distilled water in a Waring blender for 90 set and the slurry formed was applied to 5 IAates to a thickness of 0.25 mm. Prewashing of the dried plates was performed w&h an appropriate solvent syst’em as described in RESULTS and kept, overnight before use. For the purpose of dctect#in g organic acids on the developed plate, a minute amount (2-3 mg/lOO ml) of dichlorofluorescein was added into the solvent system, which makes the spot visible under ultraviolet light after thorough evaporation of the acidic solvent’. The spots of amino acids were detected by spraying the plate with ninhydrin reagent (0.2% ninhydrin in collidinc (2%) -containing acetone ). The development of the color reaction was facilitated by heating the plate in warm air. The cellulose po\vder on the individual spot was subsequently scraped out of the plate and placed in a vial containing 0.5 ml water. The scintillation fluid consisting of 1:2 mixture of Triton Xl00 and toluene-type scintillator (4 g of PPO and 150 mg of POPOP in 1 liter of toluene) was then added to determine the radioactivit#y in a Beckman Liquid Scintillation Spectrometer, Model LS230.
112
TOKPMITSU
AND
UI
Sources of Reagent 14C-Labelled acids were purchased from New England h’uclear. Enzymes and coenzymes were obtained from Sigma and Boehringer. Other reagents are of analytical grade from commercial sources. RESULTS
Principle
of the Present
Procedure
The principle of the method presented here is that ‘“C-intermediates are first separated into three broad groups, amino acid, keto acid and other organic acid fractions. Each fraction can then be separated into individual acids by a one-dimensional thin-layer chromatography which is far better than two-dimensional ones when a large number of specimens must be handled. The amino acid fraction was first separated from other organic acids by adsorption onto a cation exchanger. Keto acids in the effluent were enzymatically converted into the respective amino acids which were separated from other di- and t,ricarboxylic acids by being again applied to the cation exchange column. These column manipulations proved to be very successful in eliminating the inorganic salts and buffers which, being contained in the incubation medium in a rather large amount, ot.herwise interfere with the subsequent separation on thinlayer chromatograms. The basal conditions studied and worked out for each step of the separation procedure are presented below. Conversion
of Keto Acids to the Colrespondiny
The combination of the GOT and GPT with the reduction of 2-ketoglutaric of t.hree kinds of keto acids
Amino Acids
two transamination reactions catalyzed by glutamic dehydrogenase (Glu-DH) -catalyzed acid results in the quantitative conversion to the corresponding amino acids as follows.
GPT Pyruvate
+ Glutamate
E
2-Ketoglutarate
+
Alanine
GOT
Oxalacetate
+ Glutamate
s
ZKetoglutarate
+ Aspartate
+ 3H+
-
3Glutnmate
Alanine
+ Aspartate
Glu-DH
3(2-Ketoglutarate) Pyruvate
+ 3NADH
+ Oxalacet,ate
+ 2-Ketoglutarate
+ 3NHs +
+ 3NAD+ + Glutamate
In our separation procedure, this enzymic conversion must be conducted in a rather large volume of solution, i.e., with the effluent passing through the column of cation exchanger by which the amino acids originally present in the starting solution have been trapped. (Concentration should be avoided before this step because of lability of keto acids.) It is also advisable, in this cnzymic reaction, to use a buffer which will be readily
SEPARATION
OF
CITRIC
ACID
I I8
CYCLE
2KETOGLUTARATE
-
I
‘1
Bd
ASPARTATE
OXALACETATE
PYRWATE
5
10
20
3
40 MIN
FIG. 1. Enzymatic conversion of 2-ketoglutarnte, oxalacetate and pyruxate to amino acids. 2-Ketoglutaric (panel A), osalacetic (panel B) and pyruvic (panel C) acids, 0.5 bmole each. ~vere enzymatically converted to the corresponding amino acids in the reaction mixture containing 2 pmoles of NADH, 10 pmoles of NH&l. 0.115 N HCl buffered with 0.3 ml of N-ethylmorpholine in a total volume of 8.5 ml (final pH 7.6). Amounts of the keto acids remaining in the reaction mixture after incubation at 25°C (time shown on abscissa in min) are plotted as percent of the initial value (in panel B. the formation of aspartate is also plotted). Units of the enzymes added are shown by symbols specified in panel A.
114
TOKUMITSU
A;“;D
UI
removable before further separation is undertaken on thin-layer plates. Two kinds of volatile buffer, 2,4,6-collidine and N-cthylmorpholine~ were tested. Since collidine was found to be very inhibitory to the glutamic dehydrogenase reaction, we studied the rate of the reaction in x-ethylmorpholine buffer (pH 7.6) as shown in Fig. 1. In this experiment, 7.5 ml of 0.13 N HCl cont,aining pyruvate, oxalacetate and 2-ketoglutarate each in an amount of 0.5 ,lmoles (which mimics the effluent from the cation exchanger in the H+-form) was neutralized to pH 7.6 by the addition of 0.3 ml of N-ethylmorpholine and further added with 10 pmoles of NH,Cl, 2 pmoles of NADH and enzymes to make a final volume of 8.5 ml. At each time of incubation as indicated on abscissa, the reaction was terminated by the addition of 3 K HCIO, and the aliquot, after being neutralized with KOH, was enzymatically analyzed for 2-ketoglutaric acid, oxalacetic and aspartic acids, and pyruvic acid which were plotted in panels A, B and C, respectively, in Fig. 1. In the presence of 1 unit of Glu-DH, the addition of 1 unit of GOT caused a prompt disappearance of 2-ketoglutarate (Fig. IA), while no exact stoichiomctric relationship was maintained between disappearance of oxalacetate and the formation of aspartate under this condition. It is presumably because oxalacetate undergoes a breakdown during incubation before being converted to aspartate. Accordingly, the acceleration of the reaction by the addition of 3 units of GOT was necessary for a quantitative conversion of oxalacetat,e to aspartate (Fig. 1B). On the other hand, much more GPT (up to 10 units) were required for a quantitative conversion of pyruvate to alanine in 30 min (Fig. IC). The stability of these keto acids was studied and is presented in Table 1. Pyruvate and 2-kctoglutarate safely survived the whole incubation period, while a significant reduction of the concentration of oxalacetat’e occurred during 60 min incubation. This result confirms the preceding finding in Fig. 1 that 3 units of GOT was needed for the quantitative conversion of oxalacetate to aspartate; essentially no breakdown of oxalacetate took place up to 10 min incubation during which time almost all of oxalacetate had been converted to aspartate by 3 units of GOT. NO TABLE 1 of 2-Keto
Stability
Acids Oxalacetic
Pyruvic (% -~ O”C, 25 min and 25”C, 25”C, 60 min = Mean
k SEM,
No.
10 min
97.3 100.6
of observations:
f 3.9” + 2.9 6.
2-Ket,oglutaric
of initial) 96.5 X4.6
rf: 3.3 f 3.0
97.3 97.6
* t
1.9 2.6
SEPARATION
OF
CITRIC
ACID
CYCLE
115
degradation of oxalacetate (as well as pyruvate and 2-ketoglutarate) was observed cluring the passage through AG50 column, the procedure to be carried out prior to the enzymic conversion to amino acids, because the medium was meanwhile kept acidic (data not shown). Column
Manipulations
Outline of the column procedure employed in our method is as follows. ilmino acids and cations such as lUg”+, K+ and buffers (Tris or triethanolamine) used for incubation of mitochondria were first separated from anions by their adsorption onto AG-50(H+) column followed by the elution with NH&OH. Amino acids were then separated from cations by applying the eluate directly to the column of iiG-2 in the hydroxide form. (The anion exchanger in the chloride or formate form fails to trap alanine quantitatively.) hmino acids adsorbed were allowed to leave the column by eluting with formic acid. The amino acids prepared from keto acids were also applied to AG-5O(H+) and elutcd therefrom with IXH,OH. Other organic acids which had passed through the columns of XG-50 were separated from nonionized compounds such as sucrose, a major component of mitochondrial preparation, by being adsorbed on AG-2 (HCOO-) followed by ehkion with formic acid. It was found that the adsorption of the ionized compounds onto these three types of ion exchanger columns as well as the passage of nonadsorbable compounds was quant,itative even when the rate of flow was as high as 2 ml/min. (Among the intermediat,es tested, aspartate was the only compound that tended to appear in the effluent when the column of cat,ion exchanger which had adsorbed it was washed wit’h a rather large volume of water. Therefore, the AG-50 column was washed with 5 ml of water following application of amino acid-containing solution.1 In contrast, the elution of the arlsorbcd compounds from rolunms was not quantitative if small amomits of elution fluids were applied. Since it is desirable to get a quantitative elut’ion with as a small volume of the fluid as possible, the efficiency of elution was tested with the flow rate maintained as low as 0.2 ml/min. It was found that 10 ml of l-2 N NH,OH and 40% HCOOH was the minimum volume required for the quantitative elution from the cat)ion and anion exchanger respectively. Based on this result the volume of clution was detcrminccl as sho~~n in the standard procedure.
When the cellulose illate was dercloped with liquid phenol (500 g phenol added wit’h 150 ml water) in an atmosphere equilibrated with 3% NH&OH (3), most. of amino nc%ls closely related to the citric acid cycle were resolved in small spots with l?i values shown in Table 2. A
116
TOKUMITSU
AXD
TABLE 2 Rf of Amino Acids and Di- and Tricarboxylic
UI
Acids on a Cellulose Platen
R, X 100 after development Water-saturated isopropyl ether: HCOOH (3/l) Citric Malic Aconitic Succinic (Lactic Fumaric
16 29 35 69 76) 87
__.-
with Liquid phenol
Aspartic Glutamic Serine Asparagine Glutamine Alanine Citrulline
-. 22 32 45 52 58 65 72
a The cellulose plate had been prewashed as described in t,ext prior to development.
good separation without tailing of any spot was obtained only when the plate of Cellulose MN300 HR had been prewashed by ascending irrigation with liquid phenol a,nd dried overnight at room temperature. Two dimensional chromatography with a solvent system of n-butanol-acetonediethylamine-H,O (10/10/Z/5) (3) in the first direction followed by the second development with the above phenol syst,em revealed that the spots of glutamic and aspartic acids were not contaminated by any other amino acid. Though threonine and glycine were found to migrate only slightly ahead of asparagine and serine respectively and hence hardly undergo a complete resolution in the one-dimensional system adopted here, the present system proved still valuable because, to our experience, essentially no radioactivity was detected in threonine and glycine when asparagine and glut’amine were significantly labelled in many casesof mitochondrial incubation. Most of the other amino acids ran ahead of citrulline. Rf values of organic acids separated in a new system of one-dimensional thin-layer chromatography are shown in Table 2. The cellulose plate, prewashed by an ascending irrigation with n-butanol-2 K NH,OHethanol (7/2/l) was developed with isopropyl ether-HCOOH (3/l) for about 70 min. This system is superior to other similar acidic solvent mixture such as ether-HCOOH-H,O (4,6) in that the much smaller spreading of the sample spots is obtained after development. In this system, isocitric acid migrates slightly faster than citric acid without sharp resolution in many cases. The Whole Separation
Procedure
The whole procedure for the separation of all the intermediates of the citric acid cycle, worked out on the basis of the foregoing results, is
SEPARATION
Neutral
OF
CITRIC
or acidified
I
to
c~olurnn
I
I
Ad&bed
Eflluent
1 Eluted
wit,h
10 ml of 2 .u NHaOH
I
applied
AG-2(OH-)
column
1 Eluted with 10 ml of 407, HCOOH dried
+ wLhing (5 ml) I submitt,ed to
Enzymic conversion of keto acids to amino acids N-ethylmorpholine (to make the pH around 7) Glu-DH 1 unit, GOT 3 units, GPT 10 units, NH&l 10 pmoles NADH 1 2 pmoles, kept at 25°C for 10 min and then at _ 30°C for 10 min. I
to
1 Effluent + washing (10 ml)
1 Adsorbed
117
CYCLE
sample
applied
AC&5O(H+)
ACID
II Discard
in vacua
I
i
applied
TLC-System Ia (Amino acids)
AG-50(H+)
column
1
1
Adsorbed
Effluent
+ washing
(5 ml)
1 Eluted wit’h 10 ml of 2N NH&H I 4 dried
applied
to
I A<;-2(HCOO-) I
in vacua
column I Ef&nt washing
Adiorbed
1 TLC-System Ia (Amino acids originating from keto acids)
1
Ii
Eluted with 10 ml of 407, HCOOH dried
+ (10 ml)
Discard
1 in vacua
TLC-System IIb (Organic acids) a Liquid prewashed
phenol in 3$‘, NH*OH, as in text. FIG.
b I sopropgl
2. A summary
ether-HCOOH
of the separation
(3/l), method.
with
a llse of plate
118
TOKUMITSU
AND
CI
summarized in Fig. 2. Not only the incubation medium of biological preparation such as isolated mitochondria but also t.he deproteinized extract of mammalian tissues can directly serve as the starting sl)ccimen for this separation method. Cations contained in the starting solution in amounts less than the exchange capacity of the resin used (volume of t,he resin bed: 3 ml) can be removed during these column manipulations, while inorganic anions adhere to the final organic anion fraction. Therefore, nonvolatile inorganic anions used for the deproteinization, such as trichloroacetate or perchlorate, should be eliminated from the sample before application to the first column. For this purpose, the perchloratedeproteinized solution was previously added with KC1 or neut,ralized with KOH. The enzymic conversion of keto acids to amino acids is completed in 30 min at 25°C under this condition (see Fig. 1). To ensure the quantitative conversion, however, the reaction mixture was kept at 25°C for 20 min and then at 30°C for a further 10 min in the standard procedure in Fig. 2. Adsorption to and elut’ion from ion exchange columns is repeated four times in the whole procedure. However, only two columns, each packed with AG-50(H+) and AG-2(OH-), are required for the complete separation of one sample. After first elution of amino acids from AC-50 with NH,OH, the resin in the column in situ is generat,ed by applying 50 ml of 2 N HCI followed by washing with water to be ready for the second adsorption. Likewise, AG-2(OH)-) is converted to the form&e form during elution of amino acids and then directly serves as the adsorbent of organic anions after simple washing with water. It usually takes only 4 hr before the final eluate was obtained, including the time required for the regeneration of resin as \yell as t,he enzymic conversion of keto to amino acids. The final eluat,es are evaporated to dryness under reduced pressure by the concentrator (TAITEC Model TC 8) at 25°C (or lyophilized) and then dissolved in small amounts of water (usually 0.2 ml) before being applied to thin-layer plates. Recoveq
Test
The reaction mixture for the incubation of rat liver mitochondria (mitochondria isolated from 200 mg liver, sucrose 100 pmoles, KC1 700 pmoles, Tris-HCI buffer 100 pmoles and EDTA 100 ,umoles in a total volume of 4.5 ml) was deproteinized by the addition of 0.25 ml of 3 N HCIO,. 14C-Intermediates were added each in an amount of 0.2-0.8 pmoles as indicated in Table 3. To the supernatant of this deproteinized solution was added 0.3 ml of N KOH and the precipitated KCIO, was centrifuged off. The resultant supernatant served as a test solution for
SEPARATION
ltecovery
of W-Intermediates W
i\mino
column
Combined
81.9
CITRIC
TABLE throughout
in the fraction
$CID
119
CYCLE
3 the Present
Separation
of (4; of t,he radioactivity Veto
acids
Following
Throughout
recovered
OF
acids
Method
added) Organic
acids
manipulatious -t 0.65
the entire
Combined
71.5
i
Alanine Aspartat,e Glutamate
72.7 64.8 83.8
+ 3.42 + 2.06 * 4.04
process
(including
95.4
j, 0.61
85.9
+ 0.63
86.4
f
84.9
* 2.17
90.2 X6.6 ’ 85.9
+_ 2.18 k 1.63 + 1.76
77.6 92.7 s3.4
k 2.17 * 0.97 ) l.SS
TLC)
1.59 Pyruvate Oxalacetate P-Ketoglutarate
1.45 Succinate Malate Citrate
the whole procedure of the present separation method. The percent recovery of each intermediate arc listed in Table 3 as the mean value & SEA1 for five observations. DISCUSSION
Several simple methods have been proposed for the chromatographic separation of the intermediates of the citric acid cycle on thin-layer plates (4-g). It is impract,ical, however, to apply these chromatographic methods directly to the biological specimens, because inorganic salts present in the specimen are concentrated on the plate and tend to intcrferc with the separation of organic anions by causing serious tailing. i2s pointed out by Ting and Dugger (4)) preliminary purification of samples, for the purpose of desalting, is an essential step for their sufficient separation by thin-layer chromatography. In the present method, a systematic column procedure with cation- and anion-exchangers was employed for the purpose of not only desaltin g of the biological specimen but also of dividing a large number of intermediates to three subgroups (Fig. 2). This broad fractionation by columns is based on the exhaustive adaorption and elution and, unlike the column chromatographic procedures previously reported (10-12)) does not depend on a slight difference in the ionized state of substances to be separated. Therefore, the recovery of the ‘“C-labelled substances applied was reproducible as shown in Table 3. Each subgroup thus prepared was then successfully separated into individual metabolites by a one-dimensional thin-layer chromatography. Table 3 clearly shows that the present technique is successfully applicable to the reaction mixture of biological preparations such as mito-
120
TOKUMITSU
AND
UI
chondria. This technique also proves useful for an analysis of “Cintermediates in the tissue excised from intact animal or from perfusion apparatus; the deproteinized extract of the tissues contains interfering ions in much less amounts t,han the reaction mixture used for the in vitro experiments which must be fortified with buffers or ions required for enzymic reactions. Since ket,o acids are unstable and hence are liable to undergo brcakdown during separation, a lot of devices has been proposed to convert them into more stable derivatives including 2,4-dinitrophcnylhydrazone (13-15)) phenylhydrazone (15,16), oxime (15)) quinolplhydrazone ( 15)) quinoxaline (17) and semicarhazone (18). Some of these derivatives suffer from disadvantages such as quenching in the liquid scintillation system or separation into multiple spots (due to the presence of stereoisomers or production of artifact) on the thin-layer plate. The preparation of semicarbazones, reported from this laboratory to be useful for the separation of lGpyruvic acid originating from blood lactate (18), was abandoned in the early stage of the development of the present method, because oxalacetic and 2-ketoglutaric acid semicarbazones migrate at comparable rates in all t’he solvent syst,ems so far tested. Instead, the present paper is the first to propose that the enzpmic conversion of keto acids to the corresponding amino acids proves very useful for their separation from other organic acids. A use of N-etl~ylmorl~l~oline, ammonium ion which becomes volatile 171>011 being the quarternary separated from anions, as a buffer in the eneymic conversion made it very easy to separate subsequently the products of the reaction into individual amino acids on thin-layer plates. The principle of the present technique. i.e., a combination of column and t,hin-layer chromatography, has been already applied by one of the of the citric acid present authors to the analysis of “C-intermediates cycle and gluconeogenic pathway in the perfused liver (19,20). REFERENCES 1. TOKT-MITSU.
Y., AND Ur. M. (1973) Biochim. Biophys. Acta 292, 310-324. Y.. AND UI, M. (1973) Biochim. Biophys. Acta 292, 325-337. vex AHOX. E., AND NEHER, R. (1963) J. Chromatogr. 12, 329-341. TIN<;. I. P., .+ND DUWER, W. M. (1965) Anal. Biochem. 12, 561-578. MYERS, W. F.. AND HUANG, K. Y. (1966) Anal. Biochem. 17, 210-213. KNAPPE, E., AND ROHDEW~LD, I. (1965) 2. Aunl. Chem. 210, 183-193. BLEIWEIS, A. S., REEVES. H. C.. .~ND AJL. S. J. (1967) Anal. Biochem. 20, 335-338. HIGGINS, H., AND VON BRAND, T. (1966) And. Biochem. 15, 122-126. VEXEZIALE, C. M.. AND GABRIELLI. F. (1969) Anal. Biochem. 28, 206-215. VARXER. J. E. (1957) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 3, pp. 397-403, Academic Press, Ncm York. GAMBLE. J. L. (1965) J. Biol. Chem. 240, 2668-2672.
2. TOKUMITSU,
3. 4. 5.
6. 7. 8. 9. 10. 11.
SEPARATION
OF
CITRIC
ACID
CYCLE
121
12. LANOGE. Ii., NICKL.~~. IT:. J.. A~UD WILLIAMSON, J. R. (1970) J. Biol. Chew 215, 102-111. 13. EL HAWAKY, M. F. S.. AND THOAIPSON, R. H. S. (1953) Biochem. J. 53, 340-347. 14. ROSAN, R. C., AND NIELAND. M. L. (1963) Anal. &o&em. 6, 125134. 15. BACHELARD. H. S. (1965) Anal. Biochem. 12, 8-17. 16. FREMINET, A., BCRSAUX, E., AND POTART. C.-F. (1972) Biochem. hled. 6, 72-76. 17. MO~BRAY. J., AND OTTAWAY, J. H. (1970) Biochem. J. 120, 171-175. 18. KUSAK.4. M., AND UI, M. (1973) Anal. Biochem. 52, 369-376. 19. UI, M.. CLAUS, T. H., ESTON, J. H.. AIX-D PARK. C. R. (1973) J. Biol. Chem. 248, 5344-5349. 20. UI, M.. EXTON, J. H.. AND PARK, C. R. (1973) J. Biol. Chem. 248, 5350-5359.