CHAPTER II

PURIFICATION AND CHARACTERIZATION OF

L -ALANINE:4,5-DIOXOVALERA TE TRANSAMINASE

FROM RAT LIVER MITOCHONDRIA

- 60 -

SU.fvlMARY

Rat liver mitochondria has been found to contain

L-alanine:4,5~dioxovalerate transaminase which catalyzes

the formation of S-aminolevulinic acid via a transamination

reaction. The enzyme alanine:DOVA transaminase was

purified to apparent homogeneity with a 422-fold and

20% yield.

The purification procedure included protamine sulfate,

and bentonite precipitation, hydrophobic chromatography

on phenyl sepharose, DEAE cellulose-DE52 chromatography

and gel filtration. The purified enzyme had a specific

_activity of 59.5 unit/mg protein, and appeared homogenous

by gel electrophoresis. The molecular weight of the

native enzyme was estimated to be 232,000 + 3000 by gel

filtration. Under denaturing conditions, the dissociated

enzyme had a single subunit of approximately

Mr 41,000 + 2000, indicating the enzyme apparently is

composed of six identical subunits. The enzyme was heat

stable and had optimal activity at pH 6.9. The enzyme

is observed to be specific for L-alanine and 4,5-dioxovalerate

as its substrates. The Km values for L-alanine and

4,5-dioxovalerate ~ere 3.3 mM and 0.28 mM respectively.

The enzyme bound pyridoxal phosphate content was

estimated to be 10.6 nmole of PLP/rng of purified protein.

- 61 -

Alanine:DOVA transaminase activity was inhibited

by the sulfhydryl inhibitors i.e. p-chloromercuri benzene

sulfonate, n-ethylmaleimide and Iodoacetate, the reaction

product pyruvic acid and substrate analogs oe.-ketoglutaric

acid and methylglyoxal. Excess concentrations of

4,5-dioxovalerate and pyridoxal phosphate were also

found to be the inhibitors of alanine:DOVA transaminase.

INTRODUCTION

In the previous chapter, it has already been shown

that in the mammalian system, ·L-alanine: 4, 5-dioxovalerate

transaminase is an important enzyme which catalyzes the

formation of aminolevulinic acid. Recent reports (21,142)

indicate the existence of alanine:DOVA transaminase in

bovine liver and suggest its important role on heme

biosynthesis. The capacity of alanine:DOVA transaminase

to synthesize S-aminolev~linic acid, the first committed

precursor of heme appears to be far greater than the

capacity of ALA synthetase from the same source. However,

little is known about the role of this enzyme on the

process of heme biosynthesis.

On the other hand, the known fact is that the rate

limiting step in the hepatic heme biosynthesis is

normally ¥>A synthetase, the first enzyme of the pathway

(15,65}. The level of this enzyme is app~rently regulated.

- 62 -

by the concentrations of intracellular heme, and in

normal liver this mitochondrial enzyme is maintained at

low level. While heme may regulate hemeprotein synthesis,

and it regulates its own synthesis (65,68),which is

brought about by end product regulation of ALA synthetase.

Thus, it is apparent that ALA formation in the mammalian

system either by ALA synthetase or via alanine:DOVA

transaminase reaction is highly crucial for controlling

heme biosynthesis. Since nothing is known about the

nature of alanine: DOVA transaminase, our aim is to study

the role of alanine:DOVA transaminase on the regulation

of heme biosynthesis. Our ultimate goal of this

programme is to isolate mRNA specific for alanine:DOVA

transaminase by raising antibody against it, which in turn

can be used to precipitate specifically the ~olysomes

synthesizing nascent chains of this enzyme. As a part

of this programme, it is of importance to purify and

characterize the enzyme. Thus, in this chapter, the

results of purification and characterization of

alanine:DOVA transaminase has been presented.

RESULTS

0 All purification procedures were carried out at 4 C

unless stated otherwise. The mitochondria were isolated

from the liver of 48 hr fasted male wistar rats weighing

175-200 g.

- 63 -

Step 1 Preparation of Mitochondrial Extract : Rats

were sacrificed by cervical dislocation, the livers were

collected and cut into very small pieces with scissors.

Then livers were.washed thrice with cold saline and homo­

genized for 1 min in Waring blendor with 4 volumes of

0.25 M sucrose in 5 ~potassium phosphate buffer, pH 7.6.

The crude homogenate was centrifuged at 500 x g for 15 min

to sediment nuclei and debris. Then postnuclear fraction

was centrifuged at 10,000 x g for 10 min and the mito­

chondria pellet was washed two times with the same buffer.

The packed mitochondria were suspended finally in an

equal volume of 5 mM potassium phosphate buffer _containing

10% glycerol and freeze-thawed and sonicated for 1 min at

10 Khz, and centrifuged at 10,000 x g for 30 min.. The

pellet was washed twice with 0.5 volume of the same buffer

and supernatants were pooled.

Step II : Fractionation with Protamine Sulfate To the

above 100 ml of enzyme preparation, 20 ml of 2% (w/v)

protamine sulphate was added with stirring at 4°C. After

being left for 20 min the precipitate was removed by

centrifugation for 10 min at 10,000 x g. The enzyme

activity and protein were assayed in the supernatant, and

specific activity of alanine:DOVA transaminase was

· determined in this fraction which was 0.226/mg protein.

(Table IV) •

TABLE IV

PURIFICATION OF L-ALANINE:4,5-DIOXOVALERATE TRANSAMINASE FROM RAT LIVER MITOCHONDRIA

-----------------~------------------------------------------------Alanine:4,5-dioxovalerate transaminase

Fraction number Protein Total Specific Purifi- Yield

Mitochondrial extract

Protamine sulpl1.ate

Bentonite

Phenyl Sepharose CL-4B

DEAE cellulose DE-52

Sepharose-6B

activity activity cation

mg unitsa unit/mg fold

195.24 27.6 0.141 1

99.37 22.68 0.228 1.6

35.75 18.48 0.516 3.7

2.11 15.34 7.270 51.3

0.259 6.38 24.63 174.7

0.094 5.6 59.5 421.9

%

100

82.1

70.0

55.5

23.1

20.2

---7--------------------------------------------------------------

aone unit is the amount of enzyme that will catalyse the formation of 1 p mol of 6-arninolevulinic acid/30 min at

. 37°C.

- 64 -

Step III : Fractionation with Bentonite : Bentonite was

added to the resulting supernatant to 1.25% saturation.

and stirred for 15 min. The precipitated proteins were

removed by centrifugation at 10,000 x g for 10 min. The

specific activity of. transarninse in the supernatant was

0.526/rng protein (Table IV).

Step IV : Phenyl Sepharose CL-4B Column Chromatography

The enzyme solution was then passed through phenyl

sepharose column (2.5 x 11 ern) using 5 rnM potassium

phosphate buffer, pH 7. 6, containing 10% glycerol. The

enzyme did not bind to the hydrophobic support and

was eluted in the buffer wash (Fig. 7) . Th~ fractions

with activity were pooled and it was observed that the

specific activity was 7.24 unit/rng protein with 51.3 fold

purification (Table IV) .

Step V : DEAE Cellulose DE-52 Chromatography : The sample

from the above was applied to a column of DEAE cellulose

(4 x 7.5 ern), which was equilibrated previously with the

same buffer. The column was washed with 150 rnl of

buffer, then enzyme was eluted with a linear gradient of

KCl going from 0 to 0.25 M in the same buffer at the

flow rate of 36 rnl/hr. The total volume of the gradient

was 500 rnl and the enzyme was eluted approximately in

the middle of the gradient (Fig. 8). The fractions with

Fig. 7 : CHROMATOGRAPHIC PROFILE OF ALANINE:

DOVA TRANSAMINASE ON PHENYL-SEPHAROSE:

The post-bentonite treated enzyme was

applied to a column (2.5 x 11 em), which

was equilibrated with 5 mM potassium

phosphate buffer, pH 7.6 containing 10%

glycerol. The enzyme was eluted in the

buffer washo •-. --•, protein; o~o,

alanine:DOVA transaminase activity.

0·30 1· 50 >-.,_;.--> 1-0·25 1·2 5 u <!

w l/)

I 1·00 <! z -~ <! E U)

c: 0·75 z

I 0 <! 0:: co I-N

<! o· so <! > 0 0

w 0·2 5 z

z <! _J

0 <! 0 20 40 60 80 100 120 14 0

ELUTION VOLUME,ml

i I

Fig. 8 DEAE cellulose DE-52 chromatography

of rat L-alanine:4,5-dioxovalerate

transaminase. The buffer wash from

the preceeding phenyl. sepharose

chromatographic step was applied to

a column (4 x 7.5 ern} of DEAE cellulose

DE-52. The column was washed with

150 rnl of buffer. The enzyme was eluted

using a linear gradient of 0-0.25 M KCl

(500 ml) in potassium phosphate buffer,

pH 7.6, containing 10% glycerol. Flow

rate, 36 rnl/hr. Fraction size, 4 rnl.

0

0.100 0.10 0 I "'"' I 0.075 E

0.075 -::> ->-

E t--

c >

~ 0.050 0.050 t-u

N <(

<( UJ 2 >-

'0.025 0.025 N z w

o--~~~--~~~~----~----~--~ 0 20 40 60 80 100 120

FRACTION NUMBER

- 65 -

highest activity were pooled in dialysis tube

(processed) and concentrated with polyethylene glycol

20,000. The procedure gave a purification fold of

175 and yield of 23% (Table IV).

Step VI : Sepharose 6B Gelfiltration Chromatography :

The concentrated alanine:DOVA transaminase preparation

was applied to a column (2.0 x 3.7 em) of sepharose-6B,

equilibrated with same buffer, and the flow rate was

maintained 16 ml/hr. The active fractions were pooled

and centrifuged. This procedure resulted in a purifica-

tion of approximately 422-fold with an overall recovery

of 20.2% (Table IV). '

Homogeneity

In polyacrylamide gel electrophoresis of the purified

transaminase fractions resulted only one defined band

showing the homogeneity of the enzyme (Fig. 9a). Moreover,

densitometric scanning of the gel also showed single peak

(Fig. 9b) .

Properties and Characterization of L-Alanine:4,5-Dioxo-

velerate Transaminase

Effect of enzyme concentration When transaminase

activity was plotted against enzyme concentrations, straight

line was obtained, indicating that the reaction rate of

Fig. 9a

Fig. 9b :.

Polyacrylamide disc gel electrophoresis

of the purified L-alanine:4,5-dioxo­

valerate transaminase. The purified

enzyme preparation (protein 30 pg)

was subjected to electrophoresis at

pH 8.9 in 7% gel.

Densitometric scanning of the gel.

The purified enzyme preparation

(protein 50 Jlg) was subjected to

electrophoresis at pH 8.9 in 7% gel •

•

9a

... ·--

-- - _I

2·5 9b

2·0

E c 1-5

0

~

w u 1·0 z <{ CD 0::: 0 t/)

CD 0·5 <!

0 2 4 8

LENGTH(Cm)

- 66 -

the enzyme is directly proportional to enzyme concentra­

tion (Fig. 10) .

Effect of time : The rate of reaction of alanine:DOVA

transaminase was linear with the time upto 90 mins

(Fig. 11) .

Effect of pH Fig. 12 shows the effect of pH on the

formation of ALA by alanine:DOVA transaminase. The

enzyme has optimal activity at pH 6.9 in 50 mM potassium

phosphate buffer, with a broad peak exhibiting 90% activity

at pH 6.3 and at 7.4. The enzyme was virtually inactive

when 0.1 M Tris HCl was used as the assay buffer.

Substrate specificity studies : The specificity of the

alanine:DOVA transaminase with regard to the amino donor

was examined by replacing alanine with other amino

donors (Table V). The enzyme is specific for alanine and

is sterospecific for L-isomer. Studies on the specificity

of amino acceptor- other than OOVA were technically .

difficult because the product of transamination was not

ALA.

Effect of substrate concentration : The rate of reaction

was determined with varying concentration of one substrate

at a time keeping the other constant. As shown in

Fig. 13a and l3b, the Km values were determined

Fig. 10 Relationship between alanine:

4,5-dioxovalerate transaminase

activity and amount of protein

used (purified enzyme) •

:r ·( ">-

.!.:.) t-

~- ... > 60 ...,:: ' t-

(_)

<{

w 50 tf)

<{ ,-...

z c 2 E 40 <{ U)O

z (Y')

<{ ' . - 30 0:: 0 t-

E <{ c >- 20 0 0

w z 10 --z· <{ __J

<{ 0 0 2 3 4 5 6

ALANINE DOVA TRANSAMIN-ASE. CONCENTRATION ()Jg)

Fig. 11 Relationship between alanine:

4, 5.-dioxovalerate transaminase

activity and duration of

incubation. Fixed amount of

enzyme was used for each assay.

>- 3 0 t-

> t-

~ ~25 ClJ w .......

Lf) 0 ~

~ 0.20 - en ~ E <( lf)........_

z -15 <( 0

a: E t-:J:.. <( '-"'1 0 > 0 0

w z z <( _J

5

<( 0~--~--~--~----~--~--~--~~--+---~--~ 0 20 40 60 80 10 0

TIME (min)

Fig. 12 Effect of pH: L-alanine:

4,5-dioxovalerate transaminase

activity was determined as

described under experimental

procedures, over the pH range

from 6 • 3 to 7 • 9 •

,....... c

18 ·-C1J -0 L a. 0'1 16 E -:::> ->-t- 14 -> t-u <( 12 w 2 >-N z 10 w

01' 6 7 8

pH

TABLE V

COMPARISON OF AMINODONORS FOR L-ALANINE:4,5-DIOXO­VALERATE TRANSAMINASE*

Amino donor % of enzyme activity

L-Alanine 100

DL-Alanine 59.8

D-Alanine 0

Arginine 0

L-glutamic acid 0

Glycine 2.3

Phenylalanine 12.6

*The enzyme was assay.ed under standard conditions. The concentration of all amino donors was 20 mM.

- 67 -

by plating Lineweaver Burk plots (1/v against 1/substrate)

according to the method of Dixon and Webb (173). The

enzyme demonstrated Michaelis-Menton kinetics for both

substrates. The apparent Km value for L~alanine was 3.3 mM

and the apparent Km for 4,5-dioxovaleric acid was 0.28 mM.

A double reciprocal plots of the initial velocity (v)

against DOVA concentration in a series of fixed concen­

tration of alanine yielded a set of parallel lines (Fig.l3a).

Similarly, plots of 1/alanine against a 1/v at varying DOVA

generated a series of parallel lines (Fig. 13b). Thus,

Lineweaver Burk plots generated a series of parallel lines

indicating ping-pong reaction mechanism, which is character­

istic of transaminases.

Molecular weight determination The elution volume of

alanine:4,5-dioxovalerate transaminase in a calibrated

Sepharose-6B column is as described in Materials and

Methods, corresponds to a apparent molecular weight of

232,000 ~ 3000 (Fig. 14). Disc gel electrophoresis of

the purified enzyme in presence of SDS showed a very

defined single band proving the monomeric nature of the

protein. Migration of transaminase in SDS-polyacrylamide

gel electrophoresis was compared to the migration of the

marker proteins and thereby molecular weight was found

to be approximately 41,000 + 2000 (Fig. 15). These

Fig. 13a Lineweaver-Burk double reciprocal

plots of initial velocity of

alanine:4,5-dioxovalerate transa­

minase activity against alanine

concentration at a series of fixed

DOVA concentrations.

8

6 -1-(j')

~ 4 ::>

-1> 2

2 4 6 8

1 10-4 mM ALANINE

10

DOVA 0.~ mM DOVA 0.2 mM DOVA 0.4mM

~

Fig. 13b Lineweaver-Burk double reciprocal

plots of initial velocity of

alanine:4,5-dioxovalerate transa­

minase activity against DOVA

concentration at a series of fixed

alanine concentrations.

10

8

6

-1_;, 0\ 4

-E .......

':J --1> 2

2 4 6

1 -3 --10· mM DOVA

ALANINE 1 mM

ALANINE 2mM

ALANINE 4mM

8 10

Fig. 14 Determination of the molecular

weight of purified alanine:

4, 5-dioxovaler·ate transaminase

in native form. Sepharose-6B

(2,0 x 37 em) column was calibrated

with 1 mg of Ferritin (440,000),

2 mg of Thyroglobulin (669,000),

and Aldolase (158,000). 5 mg of

catalase (232,000) and 1 mg of

enzyme was applied.

0.6

0.5

0.4

> 0 0.3 ~

0.2

0.1

L-Ata nine DOVA transaminase ~ o Catalase

o~------~---L-----L----~~-1 00000 500000 1X10

6

LOG MOLECULAR WEIGHT

~ig. 15 Molecular weight determination

of alanine:DOVA transaminase by

SDS-polyacrylamide gel electro­

phoresis: 10 pg of each standard

and the enzyme were treated as

described in "Materials and

Methods". Calculation of mobili­

ties of protein standards,

electrophoresed simultaneously are

expressed relative to that of

bromophenol blue dye front.

10 5 Phosphorylase b

t-I (!)

w 5 X 104 3 Ovalbumin

0::: L-Alanine DOVA transaminase <{

~

Carbonic anhydrous :::> u w ~

0 2 Xl04 Trypsin inhibitor ~

~ Lactalbumin 0

....J

Rf

- 68 -

results indicate that the rat liver alanine:DOVA

transaminase contains six identical subunits.

Stability The purified enzyme was stable in 50 mM

potassium phosphate buffer, pH 7.6, containing 10% glycerol 0 .

and could be stored at 4 C for at least 1 week without any

significant loss of activity. It is stable at -20°C for

atleast a month without any significant loss o-f its

activity and at -70°C the enzyme was stable for at least

2-3 months. Freezing and thawing did not affect the

enzyme activity significantly. The purified enzyme

is heat stable, but when heated for 30 min at 65°C

lost 88% of its activity (Fig. 16).

Inhibitors of alanine:DOVA transaminase : The effect of

several known inhibitors on alanine:DOVA transaminase were

studied. Among sulfhydryl reactive agents p-chloromercuric

benzene sulfonate was particularly effective inhibitors

at micromolar concentrations. Iodoacetate was effective

inhibitor when employed at millimolar concentrations,

whereas N-ethyl-maleimide was the least effective

inhibitor (Table W\11. The other comp<:mnds viz. pyruvate '•'\i

.the reaction product and substrate analogs i.e • ..c-ketoglutaric

acid,methylglyoxal were also found to be as inhibitors

(Table VII). The enzyme activity was markedly decreased

by the addition of pyridoxal phosphate to the incubation

Fig. 16 : Thermal stability of L-alanine:

DOVA transaminase. The

purified enzyme was maintained

at 65°C for different lengths

of time before assay of the

enzyme.

!

_9.0 c ClJ -0 !.... a. Ol

E

2.s.o >­t-

> t­u <(

UJ 2 >­N z w o~----~----J------L----~------~-

0 10 20 30 40 50

TIME

TABLE VI

EFFECT'OF SULPHYDRYL INHIBITORS ON L-ALANINE: 4,5-DIOXOVALERATE TRANSAMINASE ACTIVITY*

Inhibitor

P-Chloromercuric­benzene sulfonate

N-Ethylmaleimide

Iodoacetate

cocentration ( mH )

0.01 0.02 0.05

0.5 1.0 2.0

0.1 0.5 1.0 2.0

Specific activity pmol ALA formed/mg protein/30 min

1.41

1.07 (24) 0.11 ( 9 2) 0 (100)

1. 37 (3) 1. 33 ( 6) 1.27 ( 11)

1.27 (11) 1.11 ( 31) 0.55 (62) 0.22 (85)

-----------------------------------------------------------Numbers in the parenthesis are percent inhibition.

. .

*The purified enzyme was preincubated at 37°C for 5 min with the inhibitors and the reaction was started immediately by adding substrates. Conditions for the assay were the same as described in the "Materials and Methods".

TABLE VII

INHIBITORS OF L-ALANINE:4,5-DIOXOVALERATE TRANSAMINASE*

Inhibitor

Reaction product Pyruvic acid

Substrate analogs methylglyoxal

oC-ketoglutaric acid

Concentration ( mM }

0.05 0.10 0.25 .0. 50 l.oo· 2.00

0.25 0.50 1.00

5.00 15.00 30.00

Specific activity pmol ALA formed/mg . protein/30 min

1.000

0.912 (9} 0.·821 (18) 0.569 (43) 0.370 (63) 0.192 (81)

( 10 0)

0.613 (39) 0.403 ( 60} . 0.185 (82}

0.833 ( 1 7) 0.759 (24) 0.500 (50)

Numbers in the parenthesis are percent inhibition.

*The purified enzyme was preincubated at 37oc for 5 min with the inhibitors and the reaction was started by additing the substrates. Assay conditions were same as mentioned in "Materials and Methods".

- 69 ...:.

mixture (Table VIII). As it was evident from our studies

excess concentration of dioxovalerate inhibited the enzyme

activity (Table VIII).

Pyridoxal phosphate studies : Pyridoxal phosphate functions

at the active site of a number of enzymes. Usually it is

expected to find this cofactor associated with transaminase.

The homogeneous enzyme preparation appeared to have a pale

yellow colour. The enzyme bound pyridoxal phosphate in

the purified enzyme was 10.6 nmole of PLP/mg of purified

protein. It was not possible to enhance the enzyme

activity by the addition of py-ridoxal phosphate at any

stage during enzyme assay. It supports our observation

that the cofactor is tightly bound.

DISCUSSION

The report describes the purification and properties

of alanine:OOVA transaminase of rat liver mitochondria.

This is the first report on the purification using a new

procedure to apparent homogeneity as demonstrated by

polyacrylamide gel electrophoresis. The DOVA transaminase

was purified 422-fold with a recovery of 20%. The final

enzyme preparation has a specific activity of 59.5 u/mg.

protein.

The initial purification steps, fractionation with

protamine sulfate, which is a polycation, are especially

TABLE VIII

INHIBITION OF L-ALANINE:4,5-DIOXOVALERATE TRANSAMINASE BY PYRIDOXAL PHOSPHATE AND 4,5-DIOXOVALERIC ACID*

-------~-----------------------------------------------------

Inhibitor

Control

PLP

2.5 mM

5.0 mM

DOVA

1.5 ml\'1

2.5 mM

4.0 mM

Specific Activity <p mol./30 min/mg protein)

18.63

4.11 (78)

2.03 (89)

13.91 (29)

11.88 (41)

5.77 (70)

Numbers in the parentheses are percent inhibition.

*The purified enzyme was preincubated at 37°C for 5 min with the inhibitors and the reaction was started by additing the substrates. Assay conditions were same as mentioned in "I>laterials and Methods.

- 70 -

useful in the removal of nucleic acids because of the

large negative charges of such compounds make complex

quite efficient. Bentonite, which is a clay, precipitates

the proteins by adsorption depending on its concentration,

and provided-a 3.7 fold purification with a good recovery.

An improvement in the purification of alanine:DOVA

transaminase was accomplished by exploitation of its

unique hydrophobic properties. The enzyme even in the

presence of high concentration of salt did not bind to the

phenyl sepharose, however, most of other hydrophobic

proteins were bound to this support, the enzyme was eluted

in the buffer wash. The hydrophobic column provided a

significant fold of purification. Further, su-bsequent steps

with DEAE cellulose DE-52 which is an anion exchange

chromatography, Sepharose-6B gel filtration gave a final

homogenous protein with a specific activity of 59.5 u/mg.

protein. The specific ac~ivity is higher than previously

reported values for homogenous liver alanine:DOVA transa­

minase (142). During ~&e processes of purification we

have also tried some other procedures to purify the

enzyme from the crude extract. These include ammonium

sulphate pre?ipitation, calcium phosphate gel chromato­

graphy and heat denaturation, which are frequently used

for all transaminases purification. Unfortunately none

of these procedures gave satisfactory results and

- 71 -

therefore, have not been included in the purification

scheme.

An overall yield of about 20% of alanine:DOVA

transaminase was obtained from rat liver Mitochondria.

This was significantly greater than that obtained for the

purified enzyme from bovine liver 0.753 units/mg. protein.

The polyacrylamide gel electrophoresis at pH 8.9

(Fig. 9a) confirms the purity of enzyme preparations and

further SDS-PAGE at pH 8.3 suggests (Fig. 15) that the

enzyme is monomeric in nature. The native enzyme of rat

had a molecular weight of 232,000 + 3000 and appeared to

be composed of identical subunits of M 41,000 ::!::_ 2_000 r

daltons. This data indicated that the enzyme was a

homohexamer. The molecular weight suggests that the

enzyme was isolated as an oligomer. Most known transa-

minases have molecular weights approximately 100,000

dal tons.

Alanine:DOVA transaminase has distinct substrate

specificity for both amino donor (L-alanine) and the

amino aceptor (4,5-dioxovalerate). Other amino donors

and aceptors had only a fraction of the reactivity of

these two compounds. None of the alternate amino donors

studied appeared to inhibit the enzyme, whereas the

alternate amino aceptors were inhibitory.

- 72 -

The enzyme had a pH optimum which was similar to

those reported by others (21,142) for bovine liver alanine:

DOVA transaminase.

The Km values found for rat alanine:DOVA transaminase

for alanine was 3.3 mM and 4,5-dioxovaleric acid was

0.28 mM. According to Varticoviski et al. (21) the bovine

liver had the apparent Km values 3.7 m!o!J. for alanine and

0.24 mM for 4,5-dioxovalerate. Whereas, Noguch and

Mori (140) reported Km values of 2.5 mM and 0.3 mM for

alanine and 4,5-dioxovalerate respectively.

A similar property of the rat hepatic alanine:DOVA

transaminase with bovine hepatic enzyme was the pale

yellow colour of the homogeneous preparation. Since a

pyridioxal derivative has been shown to be associated

with the bovine liver DOVA transaminase (21), our

findings further report the a:nount of enzyme bound pyridoxal

phosphate.

The inhibitory effect of sulfhydryl inhibitors, PLP

on the activity of alanine:DOVA transaminase have not

been shown ea~lier in bovine liver enzyme. However, the

inhibitory effect of DOVA being one of the substrates

has been reported in Rhodopseudomonas spheroides (174) ..

Similarly the substrate analogs -ketoglutaric acid and

methylglyoxal, pyrugic acid, a product has been observed

- 73 -

to be inhibitors of alanine:DOVA transaminase.

Therefore, after purification of alanine:DOVA

transaminase to homogeneity, we believe that it will be a

useful system to study the regulatory role of this enzyme

in heme biosynthesis and to examine whether drugs or

inducers of cytochrome P-450 have any effect on this

enzyme activity. In our laboratory, further work is

going on to raise antibody against alanine:DOVA transa­

minase in rabbit and by immunotitrating we are planning

to study the role of specific DOVA transaminase in

different erythropoietic conditions or hemeprotein

synthesis to enlighten its role on heme biosynthesis.