Purification and properties of hydroxypyruvate: l -Alanine transaminase from rabbit liver

I\RC’HI\,.S

OF

11IOCHI~:MISTltT

Purification

.\NI)

and

HIC)E’H1-SICS

Properties

Transaminase RONALD lkpartment

of Physiological

757-i6Ci

159,

Chemistry,

of Hydroxypyruvate:L-Alanine from

D. FIi;LD University

(1973)

Rabbit

H. J. SALLACH

AND

of Wisconsin

Received

June

Liver’

Medical

School,

~Waclison,

Wisconsin

53706

20, 1973

A procedure is described for the extensive purificatiou of hydroxypyruvate:~alanine transaminase from rabbit liver. On the basis of gel filtration studies, the molecular weight of the enzyme is estimated to be about 41,000 daltons. A similar value was obtained when t,he euzyme was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate indicating that the enzyme consists of a single polypeptide chain. The purified enzyme catalyzes the transamination of glyoxylate as well as hydroxypyruvat,e with L-alanine as the preferred amino donor for both substrates. The two enzymatic activities were not separated during purificat,ion nor by chromat,ographic or electrophoretic procedures. Kinet,ic studies demonstrated t,hat the two a-keto acids are competitive substrates. The above data are consistent with the fact that a single enzyme catalyzes the transamination of both glyoxylate and hydroxypyruvate. The effects of various inhibitors on enzymatic activity were investigated. The enzyme is inhibited by glyceraldehyde-3.phosphate and other

aldehydes. The possible is discrlssed.

role

of hydroxypyruvatr:I,-alanille

transaminase

Scvcral pathways have been demonskated by which the carbon chain of scrinc can be formed from, or converted to, glycolytic intermediates in mammalian systems. In one pathway, 3-P-glycerate is converted via P-hydroxypyruvate to P-scrinc and t’he latter is hydrolyzed by a specific phosphatase to yield serine. Present evidence indicates that t,his route, which is referred to as the phosphorylated pathway, is the primary one for the biosynthesis of serine (cf. l-5). The gluconeogenic role of this pat’hn-ay is precluded by the irreversibility of the last i.e., that catalyzed by phosreaction, phoserinc phosphatase. On the other hand, cbnzymes have been demonstrated in mammalian systems for a pathway in which n-glyccrate is converted t)o scrme via hy1 This investigation was supported by United Slates Public Health Service Research Grant No. NS10287 and by Research Contract No. AT(ll-l)1631 from the U. S. Atomic Energy Commission. 75; Copyright

B 1873 by hcdemic

Press,

Inc.

in gluconeogenesis

droxypyruvate (6). Since n-glycerate can be formed from 2-P-glycerate by the action of a phosphatase (7) and reconverted to this compound by a kinase (S), this route, which is referred to as the nonphosphorylated pathway, could function in either a biosynthetic or gluconeogcnic direction. Enzymes of both pathways have been shown to be under dietary and hormonal control. The hepatic lcvcl of activity of hydroxypyruvatc: L-alanine transaminase (L-alanine: 2- oxoacid aminotransferase EC 2.6.1.12), a key enzyme in the nonphosphorglat,cd pathway, is increased by high protein diets, glucagon, cortisone, and cyclic AhIP while the activity of 3-Pglycerate dchydrogenase, an enzyme of the phosphorylatcd pathway, is decreased under the same conditions (9-11). Conversely, a low protein diet dccrcases the level of hydroxypyruvatc: L-alaninc transaminase and increases the lcvcl of 3-P-glgcerate de-

758

FELD

AND

hydrogenasc. These resulk support the biosynthetic role of t’he phosphorylated pathway and suggest that the nonphosphorylated route may fun&ion in a gluconeogenic direction. In view of the responsiveness of hepatic hydroxypyruvate : L-alanine transaminase to both dietary and hormonal conditions, purification of the enzyme was initiated in order to study its properties and t’o obtain a preparation that would be suitable for the production of an antibody so that synthesis and degradation of the enzyme could be studied. Rabbit liver was chosen as the source because it has a high level of the enzyme which is responsive t,o different dietary conditions. This paper describes a procedure for the extensive purification of the enzyme from this source and certain properties of the enzyme. MATERIALS

AND

METHODS

Materials The lithium salt of hydroxypyruvate was prepared according to the method of Dickens and Williamson (12). [2J%]Hydroxypyruvate was synthesized as described previously (13). The sodium salt of glyoxylic acid was purchased from Sigma and the 2-‘%-labeled sodium salt of glyoxylic acid from Calbiochem. Glyoxylate reductase (EC 1.1.1.27) (n-glyceric dehydrogenase) from spinach leaves and lactate dehydrogenase (EC 1.1.1.27) from rabbit muscle were obtained from Sigma. Acrylamide and S,Ai’methylenebisacrylamide were products of Eastman Kodak Co. DEAE-Sephadex, CM-Sephadex, C&l00 and G-150 Sephadex were purchased from Pharmacia and were prepared as per the inst,ructions provided. Hypatite C (hydroxylapatite) was puchased from Clarkson Chemical Co. Ampholines were obtained from LKB-Products AB. Calcium phosphate gel was prepared according to the method of Keilen and Hartree (14) and t,hen equilibrated with the appropriate buffer. All other chemicals used were of the highest grade available commercially.

Spectrophotometric

Enzyme

Assays

The standard conditions described in detail previously were used for the assay of hydroxypyruvate:L-alanine transaminase (2). The assay is based on the quantitative reduction of unreacted hydroxypyruvate in the presence of excess NADH by spinach glyoxylate reductase prior to the determination of pyruvate with lactate de-

SALLACII hydrogenase. The only modification of the earlier procedure was that the reaction was terminated at, the end of 6 min. Glyoxylate:L-alanine transaminase was assayed by the same procedure except t,hat hydroxypyruvate was replaced by equimolnr amounts of glyoxylate and 0.3 M Tris-HCl buffer (pH 8.3) was substituted for the 1.0 M potassium phosphate buffer (pH 7.4); the Tris buffer was used since it was found, as reported (151, that with a Tris:glyoxylateratiogreater than 100: 1, glyoxylat,e is complexed with the Tris and therefore is not available as a substrate for lactate dehydrogenase. Hence, in this assay, the pyruvate formed in the transamination reaction could be determined directly. Units of enzymatic activity are internat,ional units (IU), i.e., that amount of enzyme that catalyzes the transformation of 1 rmole of substrate/min. Specific activity is defined as number of units/mg protein.

Euxyme Assays Ilt~dizing Radioactive Substrates The incubation system used in experiments in which X-labeled hydroxypyruvate or glyoxylate served as substrates was the same as that, given above. The reaction was terminated by the addition of 0.1 ml of 4.5 N perchloric acid. Precipitated protein was removed by centrifugation and the supernatant solutions were neutralized with 1 N KOH. After chilling, insoluble potassium perchlorate was removed by centrifugation and 1 ml aliquots of each reaction mixture were placed on columns (0.5 X G cm) of Dowex-1 (acetate form) poured in disposable Pasteur pipettes. The columns were washed with four 0.5 ml aliquots of distilled water. A 0.5 ml aliquot, of the total column eluate was placed in 10 ml of Bray’s solut,ion (16) and counted in a Packard model 3950 Liquid Scintillation Spectrometer. Control experiments demonstrated that radioactive cu-keto acids were removed quantitatively by this procedure. fi:speriments employing complete reaction mixtures, but, with heated enzyme, demonstrated that the rate of nonenzymatic transamination was negligible under these experimental conditions.

Polyacrylamide

Gel Electrophoresis

Gels were prepared by the method of Ornstein and Davis (17) except that t,he sample gel was eliminated. A 50 ~1 sample of enzyme (25 pg of protein) was applied in 10% sucrose and the samples were electrophoresed at 3 ma/tube for 2 hr at, 4°C. To locate enzymatic activity, the gels were cut longitudinally and t,he flat surface of the gel was covered with moistened, blotted cellulose acetate strips (Sephraphore 111; Gelman Instrllmrnt Co.) whirh had been soaked in a solut,icm

RABBIT

LIVER

I-IY~~~OSYPYRUVATF:~AT~:L-ALANINE

containing 0.1 31pyruvate, 0.1 M L-serint, 5 mg/rnl

TRANSAMINASl~:

7;is

Livers wrc diwd and homogenized in a Waring Blcndor for six 30.see intervals in 2 vol (K/V) of homogenizing buffer consisting of 0.154 Jr KC1 containing 0.01 11 potassium phosphate buffer (pH 7.4). The homogenate (Fraction A) was centrifuged at 14,600~/for 30 min. The resulting supcrnatant solution was then centrifuged at 105,000~ in a Bwkman L2-6.5 B ultm wntrifuge for 45 min and the residue diseardcd. A 0.5$& CTAB” sol&on in :I ratio of 1: 1 (v,‘v) was added slowly to t’hc supernatant solution. The suspcwion was allowed to equilibrate \vith stirring for 30 min prior to rclmoval of the prwipitatc by centrifugation at 14,600
portions of 0.05 11 potassium phosphate buffer (pH 7.4); the first 100 ml of eluate was discarded and the remaining six combined (Fraction E). To t’his solution, solid ammonium sulfate (50 g/100 ml) was added. The resulting suspension was equilibrated for 30 min prior to the recovery of the precipitate by centrifugation at 14,600g for 20 min. The O-50% ammonium sulfate precipitate was dissolved in a minimal volume of 0.01 11 potassium phosphate buffer (pH 7.4) and dialvzed against the same buffer until the dial&ate gave a negative test for NH4+ as determined with Kessler’s reagent. The dialyzed solution was applied to a CX Sephadex column (3 X 54 cm) that had been previously equilibrated w&h 0.05 31 potassium phosphate buffer (pH 6.6). Thr column was then washed with t’he same buffer until the eluatc was free of prot,ein as determined by absorbance at 280 nm. The column was then eluted with 0.01 JI sodium pyrophosphat’e buffer (pH 7.4). Fractions of 3 ml were collected and those containing enzymatic activity were pooled and concentrated by ultrafiltrat’ion (Diaflo ultrafiltration cell, Amicon Corp.) using a UM-2 filter (Fraction F). The concentrated enzyme solution was dialyzed against’ 50 vol of 0.01 Y potassium phosphate buffer (pH 7.4) with hourly changes for 3 hr and then was applied to a DEAE-Scphadex column (2.5 x 25 cm) t’hat had been previously equilibrated with 0.01 nr potassium phosphatc buffer (pH 7.4) conbaining lo-* 11 pyridoxal phosphate. It was found that, without pyridoxal phosphate in t’hc equilibrating buffer that the enzyme was r(‘solved into apoenzyme and coenzyme. The column was washed with the equilibrating buffer and fractions of 2 ml were collected. The fractions comprising the main enzyme peak were pooled and concentrated bJ ultrafiltrat’ion (Fraction G). The concentrated enzyme solutjion from the previous step was applied to a hydroxylapatite column (1.2 x 13 cm) that had becln previously equilibrated with 0.01 11 potassium phosphate buffer (pH 6.6). The column was washed with t’he same buffer until t,here was no absorbance at 280 nm in the effluent solution. The column was then eluted \\-ith 0.01 .II sodium pyrophosphate buff(,r (pH 7.4)

and the fractions cont,aining c~nz~rnatic: activity were pooled (Fraction H). This fraction ret,ains full enzyme activity 1’01 longer than a month when stowd at -I”(‘ and was the fraction used in all experiments unless otherwise st)ated. A t)!pical purification scheme is show\-n in TabIt> I. Properties

of the Puri$ed

Enzyme

The molccula,r n-eight of hydroxypyruvate : L-alaninc transaminasc was Mimated using Scphadex (;-lo0 ad approTABLE SUMM.IR~

OF

~~----~

Purification

ENZYMF: step

~~ IIomogenate CTAB supernatant 18.5-355; Ammonium

I PURIFIC.\TION EllZYlX fraction

Spe$ic ap . . . . t.,

A B c

0 .3 0.5 1.0

100 99 81

1.5 2.1 14.3 19.0

63 29 23 l!)

118.0

5

sulfate precipitate Sephadcx (:-lo0 fraction ([>a), (PC)& Gel eluate CM-Sephadex fraction DEAli:-Sephadex fraction Hydroxylapatite eluate

1.0

12

14

I).IT.\

1) 1,: v (; H

16

18

Percent

20

FIG. 1. Estimation of the molecular weight of purified hydroxypyruvate (HPA) : L-alanine transaminase by gel filtration. A Sephadex G-100 column (2.5 X 40 cm) was used and V0 was determined with Blue Dextran 2000. Proteins used as standards are given above. Other experimentzal conditions are described in Materials and hrth

RABBIT

LIVER

HYI~ROSYPYRUVATE:L-ALANINE

TRSNSAMINASE

761

priat,e standards of known molecular weight Wig. 1). Assuming that the proteins used for the calibration of the column are of the same shape and that the partial specific volumc is t,he same for each, a value of 41,500 Z!Z 2000 was calculated for t.he molecular weight’ of the transaminasc. Disc-gel elcctrophoresis of the purified enzyme on 7Cj, acrylamidc gel at pH S.9 as described by Ornstein and Davis (17) gave a single, but rather broad, protein band. However, enzymatic activity coincided with t,hr: protein band (Fig. 2). Several different buffer syst’ems in the pH range, S-9.5, were investigated in an att’empt, to obtain a sharper protein band, but the results were not improved. Elcctrophorosis using the method of Williams and Rcisfeld (32) proved unsatisfactory because the protein precipitated at pH 4.5. To gain addit,ional insight into the purity of the enzTmc>, as n-cl1 as information with respect, to Its possible subunit nature, it was

FIG. 3. Sodilutl dodccyl sulfate polyacrylamide gel electrophoresis of purified transaminase and known standards. (;el T, purified t,ransaminase (C) and carbonic anhydrase (11); Gel II, purified transaminase (C) with bovine serum albumin (A), ovaibumin (B), carbonic anhydrase (1))) and cyfochrome c (IS). Experimental conditions are described in Materials and Methods.

subjected to SDS gel A~ctrophoresis (Fig. 3). One major prot,ein band was observed lvith a small amount of a second protein cow taminant’. Bovine serum albumin (SS,OOO), ovalbumin (43,000), carbonic anhydrase (29,000), and cytochrome c (12,400) were used as standards to estimate the molecular weight of the major protein band (Fig. 4jA value of 40,SOOwas obtained for this protein band ahich is very close to that estimated for the holoc~nzymc~ (41,.500) by Sephadex (i-100 chromatography. This r+ sult suggests that the t ransaminasc is a single polypcptidc chain. Substrate FOG. 2. Polyacrylamide disc gel eleetrophoresis of purified enzyme. The gels were stoincd for protein (A) and enzymatic activity (B). Electrophoresis and activity stain were done us described in nlaterials and bZethods.

SpeciJicity

In the early stages of our work on the purification of hydroxypyruvatc:L-alanine transaminaw, it \vas obscrvcd that :I very active glyoxylatcl: I,-alaninc tranxaminase and a loss wtivc glyoxylatc:: I,-glutamate

762

FELL,

ANI

1 SALLhCTI

2 E a E

-305

. /l i

004I E 003-

kI

5.. % ”

-20=

-10

FIG. 4. Estimation of hydroxypyruvate (HPA) by SDS gel electrophoresis. different protein standards molecular weight, using reference (RF = 1.0). are described in Materials

the molecular weight of : L-alanine transaminase The values of the are plotted against log carbonic anhydrase as a Experimental conditions and Methods.

RF

transaminasc were present in the enzyme preparations. Although the latter act.ivity was removed upon further purification, the glyox?;late: L-alanine transaminase could not be separated from tIhr hydroxypyruvate: I,alaninc transaminase. Although the ratio of the two activities did not remain constant in the initial steps of the purification, it was essentially constant during the final stages of purification. For example, ratios of 2.05, 2.07, and 2.09 were found for Fraction F, G, and H, respectively. The change in ratio during the initial steps of the purification was probably due to the presence of the glyoxylate: L-glutamate transaminase since it has becln shown that this transaminase in human (23) and rat (24) liver usesalaninc as an amino donor as well as glutamate. In view of the fact’ that the glyoxylate:~alanine transaminasc was not separated from the hydroxypyruvate: L-alanine transaminase during purification, the following additional experiments were carried out with the purified enzyme to determine if t,he t,wo activities reside in the same enzyme molecule. protein

on

Chromatography Sephadex

of G-150

the gave

purified a single

symmetrical protein peak (Fig. 5). Furthermore, when the individual fractions of the peak were assayed for enzymatic activity, a

Y zi 4 4

-40

yQ

-20

F? aji 2 5 (I)

’ %I

3oGiz-&-130140 FRACTION NUMBER

g

%

fz

a

poo2a 001+%A 0

-60 t 2

FIG. 5. Chromatography of hydroxypyruvate (HPA) : I,-alanine and glyoxylate: L-alaninc transaminase activities on Sephadex G-150. A 1.0 mg sample of enzyme was placed on a Sephadex G-150 column (2 X 40 cm) equilibrated with 0.01 M potassium phosphate buffer (pH 7.4) and the column was eluted with the same buffer. Fractions of 1.0 ml were collected and assayed for both enzyme activities as described in Materials and Methods.

constant specific act,ivity for both transaminaseswas observed. Two milligrams of the purified enzyme wcrc subjected to isoclcctric focusing using a pH gradient of 7-10. After obt’aining constant wattage, 1 ml fractions were collected and analyzed for enzymatic activity. There was no separation by this technique in that both glyoxylatc::L-alaninc and hydroxypyruvate: L-alanine transaminase activities were obtSained in the same fractions. The pH of the individual fract’ions was dct’ermined and these results indicatn that the prot,ein has an apparent isoelectric point of 7.9. The purified enzyme preparation was subjected to disc gel clectrophorcsis and t,hc completed gel was t’hen sliced 1lorizontall.v into 1 mm slices. One-half of each slice was assaycld individually for enzymat.ic activit!. in the following manner. The half-slipes wcrc incubated at 37°C for 10 min in the standard assay mixture minus the: a-k&o acid substrate. After this trcatmcnt, the assay for enzymatic activity was initiatcbd by the addition of the appropriate a-keto acid and allowed to proceed for 30 min. Half of each 1 mm slice was assayed for glyoxylate: L-alanine transaminase while the other

half

vats:

L-alaninc

was

assayed trnnsaminaso.

for

hydroxypyruTdcntical

pat

RABBIT

LIVER

HYDROXYPYRUVATE:L-ALANINE 0.50

^

0.40 a 030

020

0.10

k 4 = 0 3 " 6

6 a GEL SLICE NUMBER FIG. 6. Electrophoresis of hydroxypyruvate:~alanine and glyoxylate: L-alanine transaminase activities in polyacrylamide gel. Conditions for electrophoresis are described in Materials and Methods. Enzymatic activities were assayed as described in the text.

terns of activity were obtained for the two different substrat’es (Fig. 6). Since no physical separation of the two activities was achieved by the above mcthods, kinetic properties were investigated. Competition experiments, with either hydroxypyruvate or glyoxylatc as the inhibitor, were carried out by using one 14Clabeled oc-keto acid as the variable substrate with the other, nonlabcled wketo acid as the inhibitor; enzyme activity was determined by measuring the amount of radioact’ivity mcorporated into either serine or glycine (see hlaterials and Methods). The results of these studies are shown in Fig. 7 and are indicat,ive of competitive inhibition. Since the above data are consistent with the fact that a single enzyme catalyzes the transamination of both hydroxypyruvate and glyoxylate acids, the rclativc affinities of t,he two cy-keto acids were determined. Assuming Michaelis-Mentrn kinetics, plots of l/X vs l/V gave a K, of 1.6 ml1 for hydroxypyruvate and 9.3 m&r for glyoxylat,e. Substrate inhibition was observed for hydroxypyruvate at eonccntrations greater than 3.3 rnli and for glyoxylatc at concentrations greater than 5.7 rn>i. The specificity of t,hc amino group donor was investigated with both hydroxypyruvate and glyoxylate (Table II). Of the amino arids investigated, only L-alanine gave a

TRANSAMINASE

763

significant rate with either cu-keto acid. The rate with serine as amino donor was only 5.5y0 of the rate obtained with alanine when glyoxylate was used and no activity was observed when glycine was used with hydroxypyruvate as the a-keto acid substrate. Reversibility

of the Reaction

The hydroxypyruvat8c: L-alanine transaminase activity of the enzyme is readily reversible although the rate obtained with serine and pyruvate as substrates (reverse direction) is much lower than that with hydroxypyruvate and alanine as substrates (forward direction). On the other hand, in the case of the glyoxylatc: I,-alaninc t,ransaminase activity of the enzyme, WC were unable to detect any rate in the reverse direction, i.e., starting with glycine and pyruvate as substrates, even though substrates were used at five times t’he concentrations in t’hc standard assay and incubations were carried out for I hr. Several investigators have reported similar results for other transaminases that utilize glyoxylate as a substrate (23-2.5) ; in each case, although the enzymes were very active in the forward direction, no transamination was observed with glycine and the appropriat’c a-k&o acid as substrates in the reverse direction.

Various compounds related t’o scrine metabolism via the phosphorylatcd and nonphosphorylated pathways were tested as inhibitors of the t’ransaminase. No inhibit’ion of either the hydroxypyruvate or glyoxylate act,ivity was found when the following compounds were included in the st’andard assay at a final concentration of 5 mnr, n-glyccratc, 3-phosphoglycerate, %phosphoglyccrate,, and phosphohydroxypyrw phosphoserine, vat’e. Preincubation of the enzyme for I hr at 37°C in the standard incubation system with either 1 rnlr p-chloromercuribenzoate or X-ethylmaleimide failed t’o produce inhibition of the enzyme indicating that reactive sulfhydryl groups arc not involved in the catalysis. Since Kopelovich et al. (26) has dcmonstrated that u-glyceraldt,hydr-X-phosph~~te

704

FELD

AND

SALLACTI

8 A

7 Amino

donor

6

wKet0 Hydroxypyruvate

I

I

2

4

6 I

Glyoxylote

I a

,

I

I

IO

12

14

(md)

SO

Glyoxyla~c I‘.‘ :o 100 0 0 0 0 5.5

“/I 100 0 0 0 0

I,-Alanine L-Glutamine L-Glutamate L-Threonine L-Aspartate L-Serinc Glycine fl-Alanine

I

acicl

0 0

‘& Ten micromoles bated with 5 pmoles under standard assay active amino acids described in Materials specificities are based as 100~~. Each assay

0

of amino donor were incuof a-keto acid and enzyme conditions. Assay for radioformed was carried out as and Methods. Amino donor on the rate with L-alanine cont,ained 0.11 IU of enzyme.

6

60-1

50-

I/,

40

-

30

7

2

4

6

8

IO

12

14

FIG. 7. Competitive studies using the two 01keto acid substrates. A, double reciprocal plot of velocity versus glyoxylate concentration in the presence of fixed concentrations of hydroxypyruvate; (0-O) no hydroxypyruvate; (A--A) 0.5 pmoles; and (H--m) 5.0 pmoles of hydroxypyruvate. Each assay contained 0.75 IU of enzyme. B, double reciprocal plot of velocity versus hydroxypyruvate concentration in the presence of fixed concentrations of glyoxylate; (a---@) no glyoxylate; (A---A) 0.5 pmoles; and (+w) 5.0pmoles of glyoxylate. Each assay contained 1 IU of enzyme. Specific activities of the radioactive substrates were HPA, 59,000 cpm/pmole; glyoxylate, 31,000 cpm/gmole. Experimental conditions are described in Materials and Methods.

inhibited the isozymes of aspartatc aminotransferase by forming a Schiff base with an E-amino group of lysine at the active site, th(l cffccts of this compound on the hydroxypyruvate: L-alanincr transaminase were invcstigated. n-Glyccraldehyde-s-phosphate at, a concentration of 1.5 mnI inhibited the enzyme 18%. Prcincubation under the same conditions wit’h 1 m&I glycolaldehydc, which has also been sho\vn to inhibit L-glutaminc:u-fructose-li-phosphate aminotransfcrase (27), gave 4S0J0inhibition. The most effect,ivc carbon\-1 compound tested IV:~S glyoxal. (ilyoxal has bwn shown to react, prefercnt,ially wit’h lysine and argininc residues (28). This compound at a concent’ration of 50 PM produced 6870 inhibition of both the hydroxypyruvate and glyoxylate activities after a SOmin prcincubation at 37°C. I>ISCUSSION

The procedure reported here results in apof hy-

proximately a 37-Ffold purification droxypyruvate: L-alaninc transaminasc

from

rabbit liver. Polyacrylamidc gel electrophoresis of the enzyme preparat,ion gave a single, but rather broad, protein band; however, eith(lr

staining

of

the

intact,

gels for cnxy-

matic activit),, or analyzing individual slices of t’hc gc’l, demonstrated that protein and enzymatic activity were coincident. The fact that SDS-clectrophoresis of the enzyme gave one major protein band wit’h one small minor contaminant suggests t’hat’ the enzyme has bran purified cxtensivcly. The results of gel tilt’ration studies and SDS-clcctrophorcsis indicate that the c’nzymcbhas a molecular weight of approximately 41,000 and consists of a single polypcptidc chain. The purified enzyme catalyzed the transamination of both hydroxypyruvate and glyoxylatc with L-alaninc serving as the preferred amino donor for both a-k&o acid substrates. On the basis of t,he fact that the two activitiw arc not rcsolwd during the purificat’ion prowdurc or by clwt,rophoresis on polyacrylamidc or in isoelwtric focusing expcrimmts, it, would appear that a single protein cat’alyzes the two transamination reactions. Further support for this conclusion was obtained in kimtic expcrimcnts in which glyoxylatc and hydroxypyruvat,c> wrc sho\vn to be competit’iw subst’ratos. Although t,hc the rate of reaction of the glyoxylatc:~alanine activity is greater than that with hydroxypyruvate (approx t\vofold under standard assay conditions), the latter compound has a lower apparent’ Ii-,,, (1.6 mlr) than dots glgoxylato (9.3 mAI). Scvcral transaminasesthat utilize glyoxylatcbas a subst’ratc have been purified from mammalian liver; thcsc include glyoxylat’e: L-alanincx transaminasc from human liver (25) and glyoxylato: L-glutamate transaminasc from human (23) and rat (24) liver. Of thaw, the propertiw of the human liver glyoxylatc: L-alanine transaminasc most rescmble those established in the prewnt stud& for thr rabbit liver cnzymc’. Both (‘nzymes use L-alaninc as the prcfcrred amino donor but thr activit,y with serinr diffcw in that this amino acid was 84% as c+fwtiw as alaniw at pH X.4 for the human enzyme whereas it was ml\- 5.7;I, as cffwtiw for the rabbit liver cnzymc. The human liver PIIzyme binds pyridoxal phosphate loosely as dots thr rabbit liwr cxnzyrnclas evidenced b\. the results obtaincbd with DEAEScphadcx chromatography. On the other hand, although the human live cbnzymcutilizes hy-

droxypyruvatc: as wil as glyoxylatt>, the maximal velocity with the latter substrate is 16 times that nith hydroxyq>wvate whereas in the present studies the dlffcrcnce is approximately twofold. The relative afTinitics of the two a-k&o acid substrates \\-(Ire not reported for the human liver enzyme mcl thus it remains to be wtablished whether the differences observed with the rabbit liver enzyme hold for the human liver enzyme as well. With respect to the glyoxylate:L-glutamate transaminases, t’he enzymes purified from human (23) and rat (24) liver were active lvith alanine but glutamate was the preferred amino donor. In both cases,no data are available with respect, t’o the utilization of hydroxypyruvate as an altcrnatc, C+k~to acid substrate. The one common propert? of all of the above transaminases that utilize glyoxylatc is the lack of reversibi1it.y of the reaction. As found in the present studies with thr rabbit liver enzyme, no reaction could be demonstrated when glycinc and t#he appropriate cr-kcto acid were utilized as substrates. With respect to the physiological role of hydroxypyruvate: L-alaninc transaminasc, dict’ary and hormonal stud& haw shown that the level of activity of this cnz\.mc: is increawd under conditions of glucowogc~nesis (2, 6). Serine is kno\vn to be a gluconcogcnic amino acid; howwer, the route by \vhich the carbon chain of this amino acid enters the gluconcogenic pathway is cquivocal. Evidence has been prewntcd that the pathway involves the initial conversion of sprint: to pyruvate via the action of swine dchydratase (cf. 29, 30). On the other hand, liver pclrfusion stud&, in which PEP carboxykinase is inhibited by quinolinatc, have dcmonstratcd a marked reduction in the conversion of the carbon chain of [Y’Jpyruvate or [14C]alaninc into glucose but had little effect cm that observed with [14C]scxrine (31, 32). One possible explanation for th(w rcwlts would be the conversion of sorim: to 2-P-glyccrate by the following route: serinc t-f hydroxypyruvatc H u-glywratcb H 2-P-glycerate via the action of the hydroxypyruvate: L-alanino transaminaw, I)~glywrate dehydrogenasc (X3), and wgl\.wrato kinase (8)) respect,ivcly.

7Hi

FISLD

AND

E‘urthcr evidence for the functional role of Dhe abovc pathway comw from the study of a rare genetic disordrr, L-glyceric aciduria (34). This disease is characterized by the excretion of large amounts of L-glycerato in urine. The cnzymc dcfcct,, as demonstrated by Williams and Smith in studies with lcukocytcs (34), is in u-glycerate dchydrogenaw and t)hesc invcstigatjors postulate that, I,-glycrrate is produced by the reduction of hydroxypyruvatc by the action of lactate dchydrogenasc. To our knowledge, the on11 known sources of hydroxypyruvattx in mammals arc its formation by transamination of swine and by the oxidation of n-glywratc~. I’atir~nts \\-ith the disease excrete from 225 to M8 mg of I,-glycerato in urine in a 24 hr period whewas t)hls compound is undctectablc in t’hr uriw of normal individuals. The large cxcrction of L-glycrratc> in this diwasc suggests that in the normal individual, wit’h a functional u-glyccrate dehydrogenase, there is a subst’antial flow of carbon through the above pathway.

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9.

34.

ACKNOWLEDGMENT The authors skillful technical this work.

thank Mr. assistance

Edward Kmiotek in certa,in phases

for of

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