Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties

Biochimie, 69 (1987) 223-230 © Soci&~ de Chimie biologique/Elsevier, Paris

223

Research article

Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties C.O. ECHETEBU, F.M. IFEM and Z.O. ECHETEBU

Department o f Biochemistry, University o f Nigeria, Nsukka, Nigeria (Received 8-10-1986, accepted after revision 11-2-1987)

Summary - Tyrosine aminotransferase, induced by dexamethasone in the liver of the rainbow lizard, Agama agama, was extracted under optimal conditions which yield the native undegraded enzyme; purified by heat treatment at 65°C, ammonium sulfate precipitation, chromatography on DEAE-Sephacel and Sephadex G-150-120 and then characterized. The enzyme was purified over 2000-fold to a specific activity of 2653 units/rag of protein. It had an optimum pH of 7.6 in potassium phosphate buffer, KTm yr: 1.0 mM; KmK~: 0.32 mM; Vmax: 1.33 nmol/min and a molecular weight of about 130000. It was inhibited by Lglutamate (competitively, Ki, 2.5 raM), and by metal ions Ca 2+, Mn 2+ , Zn 2+ , Hg 2+ and Ag2+ , but was unaffected by chelating agents and other divalent cations. Lizard hepatic cytosolic tyrosine aminotransferase was specific for t-tyrosine and o~-ketoglutarate as substrates sensitive to sulfhydryl inactivation and to protection from thermal lability by ~-ketoglutarate and pyridoxal phosphate.

tyrosine aminotransferase / rainbow lizard

R~sum~ - La tyrosine aminotransf~rase h~patique du l~zard arc-en-ciel A g a m a agama: purification et quelques propri~t~s. La tyrosine aminotransfdrase, dont la synth~se est inductible par la dexamethasone dans le foie de ldzard arc-en-ciel, Agama agama, a 6t6 extraite dans des conditions optimales qui pr6servent la forme native de l'enzyme; purifi6e par traitement thermique ~ 65°C, chromatographide sur DEAE-Sephacel et Sephadex G-150-120, et enfin caractdris6e. L'enzyme a dtd purifi6e par un facteur de plus de 2000 jusqu'~ une activit6 sp6cifique de 2653 unit6s/mg de prot6ine. I! a un p H optimum de 7.6 en tampon phosphate de potassium, KTmyr: 1.0 mM; I~KG: 0.32 mM; Vmax: 1.33 nmol/min et un poids moldculaire d'environ 130000. File est inhib6e par le L-glutamate (competitivement Ki; 2.5 mM) et par les ions metalliques Ca 2+, Mn 2+, Zn 2+, Hg 2+ et Ag 2+ mais n'est pas affect6e par les agents chdlateurs et les autres cations divalents. La tyrosine aminotransfe'rase cytosolique de foie de 16zard est sp~cifique des substrats, L-tyrosine et ~-c6toglutarate, sensible ~ l'inactivation sulfhydryle et est protdgd de la labilit6 thermique par l'~-c6toglutarate et le pyridoxal phosphate. tyrosine aminotransfdrase I Idzard arc-en-ciel

Abbreviations: TAT: tyrosine aminotransferase; DTT: 1,4-dithio-L-threitol; PLP: pyridoxal 5'-phosphate; ~-KG: ,,-ketoglutaric acid; PMSF: phenylmethyisulfonyl fluoride; K-EDTA: dipotassium salt of ethylenediaminetetraacetic acid; Na.EDTA: disodium salt of ethylenediaminetetraacetic acid.

224

C.O. Echetebu et al.

Introduction Tyrosine aminotransferase (L-tyrosine" at-ketoglutarate aminotransferase; EC 2.6.1.5) is the first and rate-limiting enzyme of the major catabolic pathway of tyrosine [1,2]. The enzyme is induced by t y r o s i n e , glucocorticoids, dibutyryl3', 5'-cyclic nucleotides, glucagon and insulin [3]. D N A sequences coding for the rat enzyme have been cloned and isolated [4], and genetic and biochemical analyses of albino mutant mice [5] suggest that a control region located on chromosome 7 is required for its expression and induction. It has been postulated [6] that one gene product, tyrosine aminotransferase I, exists in the cytosol of intact rat fiver cells and in rat hepatoma cells grown in culture. Cathepsin T, a heat-labile, PMSF-sensitive lysosomal endoprotease, transforms by successive limited proteolysis, tyrosine aminotransferase I into forms II and III, which possess kinetic and immunological properties similar to those of form I. These forms are designated I - I I I in the order of their elution from hydroxyapatite columns. Form I is a dimer with subunit of 53 k D a ; form II comprises two unidentical subunits 53 kDa and 49 kDa, respectively, and form III has two subunits each of 49 kDa. In the presence of active cathepsin T, the resultant multiple forms of the enzyme may be confused, in hydroxyapatite chromatography and neutral electrophoresis, with the mitochondrial and cytosofic aspartate aminotransferases, which are pseudo-isoenzymes of tyrosine aminotransferase lOl. t:onversion of form i enzyme into forms ii and III in fiver extracts may be prevented by optimizing the conditions which inactivate cathepsin T [6]. Tyrosine aminotransferase has generated much interest in recent years because of its complex hormonal regulation [7], developmental regulation [8], its involvement in disorders of tyrosine metabolism, and its didactic utility [9]. It has been studied in a number of organisms [6,10-13], but since the enzyme has not been characterized in reptiles, we have attempted in this study to purify it from the rainbow lizard and characterize it under conditions of optimum yield.

Materials and methods Animals Rainbow lizards both male and female, average body weight 45 g, were caught within the Nsukka Campus of the University of Nigeria.

Chemicals Dithiothreitol (DTT) and materials for polyacrylamide gel electrophoresis were purchased from Aldrich Chemical Co., Gillingham, Dorset, England. Cytochrome c, bovine ~,-globulin, bovine serum albumin, pyruvate kinase, hexokinase, DEAE-Sephacel, Sephadex G-150-120, pyridoxal 5'-phosphate (PLP), at-ketoglutaric acid @t-KG), dexamethasone, phenylmethylsulfonyl fluoride (PMSF), all amino acids and their derivatives, iodoacetamide, p-chloromercuribenzoic acid, paminobenzoic acid, 3-(4,5-dimethylthiozolyl-2)-2,5-diphenyltetrazolium bromide, phenazine methosulfate and Nethylmaleimide were purchased from Sigma Chemical Co., St Louis, MO., U.S.A. Carbowax 20 m was bought from Raymond A. Lamb, London, U.K. MN-cellulose was from Machery and Nagel, Diii:en, Germany. All other chemicals were from BDH, Poole, England. Induction of tyrosine aminotransferase Fifty lizards were starved for 12 h and then injected intraperitoneally with dexamethasone (0.05 mg/100 g of body weight). The animals were sacrificed 8 h later, their livers removed and placed in 0.15 M KCI at -15°C. Enzyme extraction The livers were rinsed in cold 0.15 M KCI, blotted dry with filter paper, then homogenized in 4 volumes of buffer I (0.01 M Tris-HCl, pH 8.0, 1 mM K-EDTA, 0.1 M KCI, 1 mM DTT, 2 mM 0t-KG, 0.1 mM PMSF and 0.25 M sucrose) in a Virtis homogenizer (4 x 40 s bursts at full speed). The homogenate was centrifuged at 200 x g for 10 min. The supernatant was placed on ice and the residue was re-extracted three times with 10 ml of buffer I. The supernatants were pooled as the 'cytoplasmic fraction' and the residue discarded. The cytoplasmic fraction was then centrifuged at 46 000 x g for 60 min and the supernatant fraction conta_ining tyrosine aminotransferase reserved for further purification. All operations were performed at + 4°C. Enzyme purification The enzyme extract was brought quickly to 65°C and maintained at this temperature for 5 min wJ~h gentle stirring. It was then cooled to - 1 5 ° C and centrifuged at 16000 x g for 10 min at 4°C. The residue was discarded and the supernatant treated with solid ammonium sulfate to 70°70saturation. After gentle stirring for 60 min at 4°C, the precipitate was collected by centrifugation (20 min at 16 000 x g) and resuspended in minimal volume of buffer I. It was then dialyzed extensively against several changes of buffer II (0.01 M Tris-HC1, pH 8.0, 1 mM K-EDTA, 0.1 M KC1, 1 mM DTT and 2 mM 0t-KG). The dialyzed enzyme was applied to a column (2.5 x 12 cm) of DEAE-Sephacel previously equilibrated with buffer II, washed with 50 mls of buffer II and eluted with a gradient of 0.12-0.5 M KCI in the same buffer, fractions of 5 ml were collected and assayed for protein and enzyme activity. Fractions with tyrosine aminotransferase activity were pooled, dialyzed against 2 changes, 4 volumes of buffer II for 20 h, then concentrated with carbowax 20 m to a final volume of 12 ml. This volume was

Rainbow lizard hepatic tyrosine aminotransferase split into two aliquots of 6 ml and each passed down a Sephadex G-150-120 column (2.5 x 45 cm) equilibrated with buffer II and eluted with the same buffer at a flow rate of 14 ml/h. 2 ml fractions were collected in 10070v/v glycerol and assayed for protein content and enzyme activity. Fractions containing tyrosine aminotransferase activity were pooled, concentrated with carbowax 20 m and stored at - 2 0 ° C as a 50070 solution in glycerol.

225

aminotransferase activity, the gels were first washed with 3 mM potassium ferricyanide for 5 min and then stained by the method of Gelehrter et al. [16].

Molecular weight determination The relative molecular weight was determined on a column of Sephadex G-100 (2.5 x 45 cm) as described by Andrews [17], using pyruvate kinase, y-globulin, hexokinase, bovine serum albumin and cytochrome c as markers.

Enzyme assay Tyrosine aminotransferase activity was assayed by a modification of the method of Diamondstone [14]. Assay mixtures (total volume 1 ml) contained 4 mM L-tyrosine, 10 mM ot-KG, 0.05 mM PLP, in 0.2 M potassium phosphate buffer pH 7.5 and 0.1 ml of the enzyme. The mixture was incubated at 37°C for 30 min and the reaction stopped with 0.2 ml of 10 N NaOH, and rapid mixing. After 30 min, the absorbance at 331 nm (E= 19900 M-lcm-l) was determined. A complete reaction mixture to which 0.2 ml 10 N NaOH was added before the enzyme, served as control. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 nmol of p-hydroxyphenyl pyruvate/min under the conditions of the assay.

Protein measurement Protein was determined by the method of Lowry [15] using bovine serum albumin as the standard.

Polyacrylamide gel electrophoresis Electrophoresis under non-denaturing conditions was carried out by a modification of the method of Gelehrter et ai. [16], using 5.50/0 acrylamide gels (0.6 x 7.5 cm) cast in 0.6 x 10 cm glass tubes. The purified enzyme (5 ml) was concentrated with carbowax 20 m to 200 ~l at 4°C and 50 bd of the concentrate was mixed with 20/~l of 40°70 (w/v) sucrose containing 0.2070 bromophenol blue and applied to the gels. After electrophoresis at 3.5 mA/tube at 50-100 V for 4 h, the gels were stained for either protein or enzyme activity. For protein, the gels were fixed and stained with 0.25070 (w/v) amidoschwartz in 50070 methanol, 9070 acetic acid. To demonstrate tyrosine

Results and Discussion Purification A summary of the purification of dexamethasoneinduced hepatic tyrosine aminotransferase from the rainbow lizard is shown in Table I. The enzyme was purified over 2000-fold to a specific activity of 2653 units/rag of protein. Advantage was taken of the properties of the enzyme from other sources [10,1 l, 18,19] to optimize the yield of the undegraded native enzyme. Thus, the extraction buffer contained, among other things, DTT, a-KG, P L P , PMSF and sucrose, and was alkaline (pH 8.0). The presence of DTT, PLP, 0~-KG and sucrose stabilized the enzyme; alkalinity of the buffer, presence of KCI and P M S F inhibited the activity o f the lysosomal cathepsin T, a heat-labile endoprotease that degrades and converts the native enzyme (form I) into forms II and III [6,19, 20]. The conditions were such that would prevent or suppress the conversion of the native enzyme into forms II or III, even though mechanical ,,12,,vL1~,~. of the tissue, --wm~:n'-might lead to the release of the said endoprotease, was used. Treatment at 65°C then eliminated most unwanted proteins as well as the converting factor. After a m m o n i u m sulfate precipitation and dialysis, the enzyme was fractionated by D E A E -

Table I. Summary of the purification of hepatic tyrosine aminotransferase from the rainbow lizard Agama agama. Steps

Total protein (rag)

Total activity (units)

Specific activity (units/mg of protein)

46000 x g supernatant Heated (65°C for 5 min) Ammonium sulfate and dialysis DEAE-Sephacel chromatography Sephadex G-150-120 gel filtration

7236 929

9380 7130

1.3 7.7

1 6

464

5940

12.8

10

63.3

23.80

5000

210.0

162

53.3

1.24

3290

2056.5

35.0

2653

Purification (fold)

Yield (070) 100 76

C.O. Echetebu et al.

226 ~

12-

_~1o. x

centrifugation (105000×g) [10, 11, 13, 14], it is possible to obtain dexamethasone-induced hepatic tyrosine aminotransferase in high yield in its undegraded native form. The native form may be further highly purified by heat treatment, ion-exchange chromatography and gel filtration.

30,%

E ~

~o-

.~ 1.0-

[

6-

i4

"

Characterizat'on o f the enzyme

0.4--0.3

== 2-

Effect of temperature L:=O

4O

6O

,oo

'

Fig. 1. D E A E - S e p h a c e l chromatography of tyrosine aminotransferase from rainbow lizard liver. Enzyme solution (42 ml) was applied to the column (2.5 x 12 cm), washed with 50 ml of buffer II, and eluted with a gradient of 0.12-0.5 M KCI in the same buffer. Fractions (5 ml) were collected starting from the point of application of the sample. Peak I is aspartate aminotransferase, peak II is tyrosine aminotransferase. Enzyme activity: e - e ; protein: ~ and KC1: - - .

Sephacel chromatography. Two peaks of activity were eluted (Fig. 1); the first and minor peak (fractions 13-19) was identical L-aspartate aminotransferase (EC 2.6.1.1) which is known to transaminate L-tyrosine [6,19,21]. The second and major peak was eluted in fractions 76-86. Fractions 77-83 were pooled and purified further by gel filtration on Sephadex G-150-120. Only one peak of activity was found, corresponding to fractions 40-68. Fractions 41-65 were pooled, concentrated with carbowax and stored as the purified enz~ane. Most workers further purify this enzyme with chromatography on hydroxyapatite [6, 10, 11,20]. The three (forms I, II and III) peaks separable by this technique have been attributed to partial cleavage of tyrosine aminotransferase by cathepsin T [6,19, 22]. We were unable to purify our enzyme on hydroxyapatite columns because the batches we prepared using the method of Tiselius et al. [23] had extremely slow flow rates. It was therefore uncertain as to whether or not our purified enzyme contained forms II and III in addition to form I. However, electrophoresis under non-denaturing conditions showed only one band of protein and enzyme activity, indicating either that forms II and III of the enzyme if present, comigrated with form I [6] or that the enzyme is highly purified and homogeneous [19]. It appears that by adequately controliing the extraction buffer; suppressing the activity of cathepsin T; removing particulate fractions from the homogenate (46 000 x g) without recourse to ultra-

The effect of temperature on the stability of the lizard enzyme was tested by preincubating the enzyme at 37.5 and 46°C (the preferred and critical temperatures of the animal, respectively) and at 50, 60 and 70°C. Samples were taken at time zero, and at regular time intervals up to 60 min and assayed for remaining activity. The results presented in Fig. 2 show that the enzyme could withstand temperatures as high as 60°C for 60 min, and still retain about 44°70 of its original activity. At 70°C, however, the enzyme lost about 7007o of its activity within the first 10 rain, and retained only 20070 after 60 min. Thermal treatment at 65°C during enzyme purification led to a loss of 25070 of the original activity, but 5 min at 60°C after purification led to a loss of about 3607o. It seems that the absence of pyridoxal phosphate in the final product or loss of some stabilizing factor(s) during purification led to increased thermal lability of the purified enzyme. Thus PLP or other factor(s) is (are) essential for stabilizing the enzyme against thermal inactivation.

"N 6

F ,ol

"G 30 .j

201-

o

,;

3'o

do 6'o

Time, rain Fig, 2. Effect of heat on the stability of tyrosine aminotransferase. Enzyme samples were heated at various temperatures, aliquots were taken at regular intervals and assayed for residual activity. Temperatures: 37.5°C: o-o; 46°C: 50°C: B--II; 60°C: @-0 ; and 70°C: o-o.

Rainbow lizard hepatic tyrosine aminotransferase Protection from thermal denaturation by PLP:, =KG and L-tyrosine Tyrosine aminotransferase was dialyzed for 18 h in buffer II (from which =-KG was omitted) to remove =-KG from the enzyme preparation. Dialyzed enzyme solutions (3 ml) were incubated at 70°C in the presence of PLP, =-KG or L-tyrosine. Samples of 0.5 ml were removed and residual activity measured under standard conditions. A plot of the remaining activity versus time of incubation (Fig. 3) shows that PLP + =-KG protected the

227

enzyme better than PLP alone, PLP + L-tyrosine showed no protective effect whatsoever. These results show that PLP and =-KG together protect the enzyme from thermal lability, especially during the heat stage of enzyme purification.

Optimum p H The enzyme was assayed in 0.2 M K-phosphate buffer at various pH values between pH 6 and 8. Optimum activity was found to be at pH 7.6 (data not presented).

Effect of metal ions and chelating agents Tyrosine aminotransferase was assayed in the presence of metal ions: Mn 2+, Co 2+, Cu 2+, Ca 2+, Mg 2+, Zn 2+, Ag 2 + , Hg2 + and chelators K-citrate, K-tartrate, K-EDTA and Na-EDTA added in 1 mM final concentration to standard assay mixtures. The results shown in Table II indicate that the chelating agents had no effect on the activity of the enzyme, but Na-EDTA seemed to inactivate it. The enzyme was appreciably inhibited by the metals Co 2+, Mn 2+, A g 2 + Hg2 + and Zn 2+, but was indifferent to CU 2+, Ca 2+ and Mg 2+.

IOOq 80

60 :z-

~ 4o ~I 30

i%

•

5

'

I0

,,

15

I

20

Time of Incubolion, min

Fig. 3. Protection from thermal denaturation of tyrosine aminotransferase by PLP, =-ketoglutarate and L-tyrosine. Enzyme was treated as described in the text and aliquots were mixed with: 0.5 mM PLP + 10 mM = - K G : o - o ; 0.5 mM PLP + 2 mM =-KG:~-A ; 5 mM PLP only: H ; 0.5 mM PLP + 0.5 mM L-tyrosine: 0 - ~ ; and finally, alone: o-o. They were then incubated at 70°C. Aliquots were taken at the indicated times and assayed for residual activity.

Table II. Effect of metal ions a1~d chelating agents on the activity of lizard liver tyrosine aminotransferase.

Addition

Concentration Activity (raM) Units % of control

None (control) Cu 2+ 1 Ca 2+ 1 Co 2+ 1 Mn 2+ 1 Mg 2+ 1 Zn 2+ 1 Hg 2+ 1 Ag 2+ 1 K-Citrate 10

8.50 7.53 8.17 2.09 4.08 7.64 5.13 4.27 4.82 8.48

K-Tartrate K-EDTA Na-EDTA

8.48 8.38 3.86

10 10 10

100 88.79 96.34 24.69 48.14 90.08 60.48 50.39 56.77 I00 100 98.74 69.08

Substrate specificity Various analogues of =-KG and tyrosine were used to determine the substrate specificity of the enzyme. Standard assay conditions were used except that Ltyrosine or =-KG was replaced quantitatively by the acceptor or donor analogues, respectively. Qualitative thin-layer chromatography was used to confirm the result. After incubation, the assay mixtures were boiled for 5 rain and i00 btl aliquots of each were spotted on microscope slides coated with MN 300 cellulose. Chromatograms were developed in a micro-stain tank with isopropanol: formic acid-water (40:2-10). Spots were detected with ninhydrin-collidine (1 g of ninhydrin in anhydrous ethanol, 700 ml; 2,4,6-collidine, 29 ml and acetic acid, 210 ml) [25]. A spot corresponding to glutamic acid was found with only the control mixture to which =-KG and L-tyrosine were added. Very faint spots corresponding to glutamic acid were also found with DL-tyrosine (Table III). Results from thin-layer chromatography support the observation that tyrosine aminotransferase is specific for =-KG and L-tyrosine because, although the standard assay method indicated some transamination with L-tryptophan, 3,4-dihydroxyphenylalanine, adrenalin and pyruvic acid, chromatography showed no formation of glutamate, and therefore those transaminations were regarded as 'false'. The lizard enzyme in this respect is likened to the dog enzyme

[13].

C.O. Echetebu et al.

228

Table HI. Substrate specificity of lizard liver tyrosine aminotransferase. Amino acids or tyrosine analogues

Apparent transamination (070 of control)

Formation of L-glutamic acid (TLC)

Control: L-tyrosine + 0t-KG

100

4-++

Pyruvic acid Oxaiacetic acid D-Tyrosine DL-Tyrosine L-Tryptophan L-Alanine L-Phenylalanine p-Aminobenzoic acid 3,4-Dihydroxyphenylaianine 3-Amino-L-tyrosine Adrenalin Aspartic acid Histidine

~-KG analogues 2 0 tyrosine analogues 0 11 5 0 15 1 15 5 24 0 0

+ +-+ + -

The ratio of molar concentrations of analogue to ~t-ketoglutarate or L-tyrosinewas kept at 1. + + + : strong; +_: weak; - : no formation of L-glutamate detectable by thin-layer chromatography.

Effect o f sulfhydryl compounds on enzyme activity The enzyme preparation contained 1 mM DTT. When it was preincubated for 20 min at 0°C with 1 m M each of the sulfhydryl reagents: iodacetamide, p-chloromercuribenzoate and N-ethylmaleimide, or with 1 mM of the mercaptans, 2-mercaptoethanoi, UH, L-cysteine and reduced glutathione, the enzyme was generally inhibited as compared with the control which contained only 1 m M DTT (Table IV). This amount of DTT in the

buffer seemed to protect the enzyme from inactivation b y the sulfhydryl reagents and to cause inhibition by the mercaptans, which in this assay would be in excess of 1 m M by the presence of DTT. DTT was removed by dialyzing the enzyme for 24 h at 4°C in 20 volumes of buffer II (from which lJl

I

W~S

UIIIlLLI~U.)

;411U L I I ~ d b b d ~

W~I~

z~;V~;~I.~u.

The results (Table IV) show that in the absence of added mercaptans the enzyme was much less active than with 1 mM DTT, and was completely inac-

Table IV. Effect of sulfhydryl reagents and mercaptans on lizard liver tyrosine aminotransferase. Assay conditions

Enzyme contains 1 mM DTT

Enzyme dialyzed to ecmove DTT

Additions (1 mM)

Activity (units)

Relative activity (070 of control)

Activity (u--,h~)

Relative activity (°70 of control)

None (cqntrol) 2-Mercdpto ethanol Dithiothreitol L-Cysteine-hydrochloride Reduced glutathione p-Chloromercuribenzoate Iodoacetamide N-Ethylmaleimide

8.00 5.07 6.10 7.39 6.60 5~98 6.22 4.84

100 63.4 76; 3 92.1 82.5 74.7 77.8 60.5

3.75 4.46 4.69 4.20 3.74 0.00 0.00 0.00

100 119 125 112 I00 0.00 0.00 0.00

Rainbow lizard hepatic tyrosine aminotransferase

229

tivated by iodoacetamide, p-chloromercuribenzoate and N-ethylmaleimide. Enzyme activity was however enhanced in the presence of the mercaptans and L-cysteine, but was unaffected by the presence of reduced glutathione. When the dialyzed enzyme was preincubated at 0°C for 20 min with 1 mM concentrations of the thiol reagents, then 5 rain with 1 mM of the mercaptans at 37°C and assayed for remaining activity, only very little reversal of inactivation was achieved. The fact that tyrosine aminotransferase could be inactivated by thiol reagents; that mercaptans could protect the enzyme from such inactivation and may even reverse them, suggests that the enzyme contains sulfhydryl group(s). Valeriote et al. [24] had found earlier that the enzyme from rat liver contains about 15.7 sulfhydryl groups and 28 cysteic acid radicals/molecule of the enzyme.

L-tyrosine in 1 ml (total volume) incubation mixtures containing 0.1 mM PLP and 0.1 ml enzyme in 0.1 M potassium phosphate buffer pH 7.6. Data obtained were analyzed by a primary double reciprocal and a secondary plot of slopes and intercepts (Fig. 4A and B). Kinetic parameters evaluated from Fig. 4B indicate that the reptilian enzyme has Km Tyr= 1 mM; Km'KG= 0.32 mM and Vmax= 1.33 nmol.min -1. These parameters are comparable to those from other sources [9,11, 24,27].

Kinetic studies

7

10

c E

-

A

. u

Effect of substrate concentration In the presence of saturating concentrations of PLP, tyrosine aminotransferase may be regarded as a two-substrate enzyme system. Given that KmVLP=1.6X 10 -5 mM for tyrosine aminotransferase [26], it was postulated that the enzyme would be saturated in the presence of 0.1 mM PLP, and the system would then reduce to a two-substrate one. The concentration of o~-KG was varied from 0.2-2mM at fixed concentrations (0.2-2 mM) of

6

U) m

O E c

q

'-

2

o

I .

0

I

I

I

I

,l

1

2

3

/4

5

1/[c¢-KG],

mM -1

10 e-

~o

6

I/[a-KG], mU'~

i ~q

2 J

I

0 I/[Tyrosirm], mM "1

Fig. 4. Effect of substrate concentration on tyrosine aminotransferase activity. A. Double reciprocal plots of aketoglutarate concentrations with fixed (0.2-2 raM) concentrations of L-tyrosine: 0.2 mM: H ; 0.4 taM: ~ 1.0 mM: 1-1 ; and 2.0 raM: o-o. B. Secondary plots from A: intercepts: H ; slope :a-a; for the tyrosine concentrations. Enzyme: 0.1 ml; PLP: O.1 mM; DTT: 1 mM.

5

I

I

15

[Glutamate],

25 mM

Fig. 5. Effect of L-glutamate concentrations on tyrosine aminotransferase activity. A. Double reciprocal plots of various 0t-ketoglutarate concentrations with fixed (0-25 mM) concentrations of L-glutamate: 0 m M : H ; 5 mM:Av-a; 10 mM: H ; 12.5 mM: o-o; and 25 mM: zs-A.B. Slope of plots from A. Enzyme: 0.1 ml; PLP: 0.1 mM; L-tyrosine: 0.4 mM; and DTT: 1 mM.

230

C.O. Echetebu et at.

2

400 I

'_o

200

3

I00

4

50

5 6

zo 6

%/% Fig. 6. Molecular weight determination for l;zard tyrosine aminotransferase. This was done by the method of Andrews [17] on a column (2.5 ×45 cm) of Sephadex G-100, using: (1) pyruvate kinase: Mr = 237 000, (2) bovine y-globulin: Mr= 160000; (3) lizard tyrosine aminotransferase; (4) yeast hexokinase: M r=96000; (5) bovine serum albumin: Mr = 67 000; and (6) cytochrome c = Mr = 13000 as markers.

7 8 9 10 11 12

The effect o f product (L-glutamate) concentration on the forward reaction was studied by adding fixed amounts ( 0 - 2 5 raM) of L-glutamate to varying concentrations of =-KG, whilst keeping the concentrations of tyrosine and P L P constant. Plots (Fig. 5A and B) indicate that L-glutamate inhibits the reaction competitively with a K i value of 2.5 raM. Reaction with the second product, p hydroxyphenylpyruvate, was not studied because the c o m p o u n d was unavailable to us. The results presented in this section would be consistent with the findings o f Rosenberg and Litwack [28], if we observed o u i n : u H l p C- -t x' "t i"v c. . . . l. i.a. t.!n_.c. a. u1_m n competitive inhibition when =-KG was varied in the presence of fLxed concentrations of L-glutamate.

13 14 15 16 17 18 19 20 21

Molecular weight Native tyrosine aminotransferase is dimeric and has a molecular weight of about 85 0 0 0 - 1 1 5 ¢00 [ 1 0 12, 29]. The molecular weight o f the enzy~ne from lizard liver was estimated to be about 130000 by gel filtration (Fig. 6). This value is slightly higher than those from other sources, which m~y indicate that the lizard enzyme has a higher molecular weight than tyrosine aminotransferase f~'om other sources, or that the enzyme is aggregated to other proteins or small molecules.

22 23 24 25 ~6 27

References 1

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28 29

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