CHAPTER II

A RAPID PURFICATION METHOD AND PARTIAL CHARACTERIZATION OF L-ALANINE:

DO"' TRANSAMINASE FROM RAT KIDNEY MITOCHONDRIA

The reaction catalysed by ALA synthetase has been

considered the primary source of ALA in the animal system,

yeast and some bacteria. In tissues of several plants and

in certain algae, however, most of the ALA is produced in

the stroma of greening plastids from glutamate in reactions

requiring the participation of chloroplast glutamate

acceptor tRNA (116,117,119,120). In a number of other

studies on ALA generation in plants (122-133) and in several

bacteria (134-139), involvement of L-alanine: DOVA

transaminase has been suggested to be physiologically

relevant. Recently, Breu and Dornemann (142-147) proposed

that at least in green alga Scenedesmus obliquus, the

formation of ALA from glutamate takes place with the

involvement of glutamyl tRNA giving rise to the

intermediates glutamate-1-semialdehyde and 4,5-

dioxovalerate. Other reports from bovine, rat and chicken

tissqes have also implicated mitochondrial enzyme L-alanine:

DOVA transaminase in ALA generation (151-161). In the

previous chapter it bas been conclusively shown that in

mammalian system this enzyme resides in the mitochondrial

matrix which is the known site for ALA formation.

Other than presenting an amplified assay system for L­

alanine: DOVA transaminase we have also shown the occurrence

of this enzyme in mitochondria lacking parasitic protozoa ~

histolytica. Some of the studies from various laboratories

have indicated that the enzyme L-alanine: DOVA transaminase

70

activity is under feedback control by hemin since among all

the heme biosynthetic pathway intermediates only hemin

appears to inhibit the enzyme activity

(137,156,158,160,164). It is important to mention here that

he.dn has already been reported to inhibit the translocation

of ALA synthetase ( 62, 66, 34) • For translocation studies,

mitochondrial proteins synthesized in a cell-free

synthesizing ~ystem (e.g., rabbit reticulocyte/wheat germ

extract) can now be successfully imported in vitro under

well defined conditions. Therefore, a similar study will

elucidate whether there is a control mechanism at the level

of translocation of L-alanine: DOVA transaminase, or whether

the synthesis of ALA via this enzyme is exclusively

controlled at the enzyae activity level with hemin as the

end-product inhibitor. To explore such a possibility, the

enzyme L-alanine: DOVA transaminase has been purified to

homogeneity from rat kidney mitochondria by a new rapid

purification method and the antibody raised against a highly

purified preparation of this enzyme. Thus, in this chapter,

the results of purification and partial characterization of

L~alanine: DOVA transaJiinase from rat kidney mitochondria

have been presented.

POIUPICA".l''OR OF L-ALAIIJliB: 4, 5-DIOXOVALBRA'.rE ".l'RAHSAIUHASB

Among the organs examined, the specific activity of the

enzyme L-alanine: DOVA transminase was highest in kidney

71

followed by liver. Therefore, the enzyme was purified from

the kidney mitochondria of adult male Wistar rats by a new

three-step procedure outlined below. All the steps involved

in enzyme purification were carried out at 0-4°C unless

stated otherwise. The results of the enzyme purification

are summarized in Table IV.

STEP I: PRBPARATIOH OF IIITOCIIOHDIUAL IIA'ftUX

The procedure followed for kidney mitochondrial matrix

preparation was dependent on the present finding that L­

alanine: DOVA transa.inase was a soluble/non-membranous

component of ti'Ie . mitochondrial matrix. Kidneys from 15

adult rats were cleaned, minced and washed with several

changes of 'isolation .adium' containing 220 mM D-mannitol,

70 mM sucrose and 2 mH Hepes buffer at pH 7.4. A suspension

in the ratio of 1:3 with isolation mediWI was made and

homogenised in a glass-teflon homogeniser. The homogenate

was further diluted in the ratio of 1:3 and centrifuged at

600g , for 15 minutes. The supernatant was collected and

centrifuged again at 6900g for 15 minutes to pellet the

mi~ochondria. The mitochondrial layer was washed twice,

gently muddled with ainimal volume of isolation medium

(about 1/10th of the pellet volume) and adjusted to give 100

mg of mitochondrial protein/ml. A solution of 1. 6 mg

digitonin/10 mg mitochondrial protein was prepared in

isolation medium and added dropwise with stirring such that

the volume of the added digitonin was equal to the volume of

the mitochondrial suspension. After stirring for 15 minutes

72

T8ble IV: PURIFICATIU. OF L-Al.AIII•: 4.5-DIOXOYALEIATE TIUIISAIIIIASE FRGI IAT I:IDIIE\' IIITOC-IA.

Purification step

Mitochondrial matrix

L·alanine·sepharose 4B

Sepharose 68

Total protein

(lllg)

43.89

1.78

0.72

Total activity (units)

70.94

29.99

26.57

Specific activity units/1119 protein

1.62

16.85

36.90

Purification fold

10.4

22.8

Yield CX)

100

42.3

37.5

on ice the suspension was diluted with 3 volumes of

isolation medium, and centrifuged at lO,OOOg for 15 minutes.

The mitoplasts as pellet (inner membrane along with matrix)

were gently suspended and washed once with isolation medium.

Whenever required, the release of intermembrane space marker

enzyme adenylate kinase served as an indicator in evaluating

the purity of the mitoplast fraction. Mitoplasts from

diqitonin treated mitochondria were adjusted to give protein

concentration of 30 mg/ml. From a stock solution of 20mg/ml

lubrol WX was added a volume to give a final concentration

of 0.16 mg lubrolfmg of mitoplast protein. The mixture was

allowed to stand on ice for 15 minutes and then diluted by

addition of 3 volumes of isolation medium. The resulting

preparation was centrifuged at 144,000g for one hour and the

supernatant from this run was recovered as the mitochondrial

matrix.

STEP :n:: L-.ALMfDfB-S£liiiAROSB 48 COLUIOI aiROIIA'l'OGRAP

The mitochondrial matrix fraction obtained from the '

previous step was dialysed overnight against 10 mM potassium

phosphate buffer pH 7.6, containing 10% glycerol

(purification buffer) and applied to L-alanine-sepharose 48

affinity column (2.0x8.0cm) previously equilibrated with the

same buffer. The colUlllll was washed with the purification

buffer till oo280 of the flow-through approached zero. The

enzyme was eluted with 0-0.35 M KCl linear gradient prepared

in a total volume of 300 ml of the purification buffer.

Fractions of 6.0 ml size were collected at a flow-rate of 36

73

ml/hour. Figure 9 is a typical elution profile from this

step of purification.· The fractions havinq the hiqhest

enzyme activity were pooled, concentrated and dialysed

aqainst enzyme purification buffer which also contained

0.02% sodium azide to prevent microbial qrowth in the enzyme

samples.

STEP :I:I:I: SEPIIAROSB-68 COLUIOI aiROIIA'J.'OGRAPIIY

The enzyme sample from the second step was concentrated

to about 2 ml and loaded onto the sepharose-6B column (2.4 x

65. o em) previously equilibrated with purification buffer

containinq 0.02% sodium azide. The enzyme was collected as

10 minutes fractions at a flow-rate of 16 ml/hour. The

eluates were collected from 90 ml onwards after the void

volume (100 ml). The purity of the active fractions was

checked by 12.5% SDS-PAGE (Figure 10). The fractions bavinq

purified enzyme were pooled, concentrated and dialysed

aqainst purification buffer. The pure enzyme preparation ~

was stored at -2o0 c.

O'lfiBil Bl"l'BC'l':IvB ALTBRHAT:IVB S'l'BPS Df PDR:IP:ICA'l'J:Oif

DEAE-cellulose DE-52 (ion exchanqe) coluJIJl

chromatoqraphy was quite effective. The enzyme which bound

to the matrix could be eluted with a linear KCl qradient (0-

0.3 M) at a flow-rate of 20 ml/hour. The enzyme eluted over

a broad ranqe with a broad peak around the middle of the

qradient ranqe.

74

•

~ ·- -i.,

'I

I 0.12

~ c

c ~ E ~. ~ c < ..

0 - · .r-co

...... N 0.4-::.. ~

ClJ -c - · u 3 ~ c 0 0.3 0 0

-< .D ttl c

<.... ,_

0 ttl Ill 3 ...,

.D 0.2 .::e. <t

0.1 I~ VI c 3

4 8 12 16 20 24 28 32 36 40 44 ::J c

FRACTION NUMBER VI ttl

Figure 9: L-Alanine-sepharose affinity chromatography of kidne y mitochondrial L-alanine:DOVA transaminase. The mitochondrial matrix fraction was applied to the affinity column and washed with purification buffer, pH 7 .6. The enzyme was eluted using gradient of 0 - 0.35 KCl (300 ml) in purification buffer at a flow rate of 36 rnljhour. Fraction size was 6 ml. Protein profile of the enzyme active fractions ( 4 to 18) by 12. 5% SDS-PAGE is shown above the graph and the enzyme band is marked with an arrow.

kDa

-94

- 67 - -43

- -30

- 20 .1

-1 4 .4

Figure 10: Sepharose - 6B column chromatography. The purity of the fractions having activity for L-alanine: DOVA transaminase was checked by 12.5% SDS-PAGE. About 20 Ml of the sample from each fraction was electrophoresed and the active fractions having the purified enzyme were poo l ed. The position of the enzyme band is marked with an arrow.

Phenylsepharose CL-4B column has also been utilized.

The e~zyme does not bind to the column and is eluted in the

buffer wash. Chromatography on hydroxyapatite and protamine

sulphate or heat treatment of the protein samples proved to

be less effective. Of the several combinations the three-

step purification procedure described above proved to be

most outstanding in terms of enzyme yield and rapidity. It

resulted in a purification of approximately 23-fold with an

overall recovery of 37.5%. Other combinations requiring

about five steps do result into a purified preparation of

the enzyme but the overall yield never exceeded 20%.

HOMOGENEITY

The purity of the enzyme was checked by 7.5%

polyacrylamide gel electrophoresis (PAGE) at pH 8.8. Figure

12 shows that the kidney mitochondrial L-alanine: DOVA

transaminase was apparently homogeneous as evidenced by a

single band in the gel. The enzyme also yields a single

band on two-dimensional electrophoresis gel and silver

stained SDS-PAGE gels (Figure 11,16), thereby confirming the

purity of the enzyme and efficacy of the purification

method.

CHARACTERISTICS OF L-.ALAifiHE: DOVA TRANSAMINASE

ENZYME STABILITY

The stability of the enzyme is enhanced in the presence

of 10% glycerol and can be stored on ice for a few weeks.

The enzyme in purified state or in form of mitochondrial

75

1 2 3 4 kOa

-94 -67 -43

-30

-20.1

-14.1

Figure 11: SDS-PAGE pattern of proteins obtained dur i ng the different purification steps. Lane 1, mitochondrial matrix; lane 2, after L-alanine sepharose chromatography; lane 3, after gel filtration; lane 4, standard marker proteins. Equal amounts of enzyme activity from each purification step was electrophoresed on a 12. 5% SDS polyacrylamide gel and visualised by silver staining.

+

Figure 12: Polyacrylamide gel electrophoresis of the purified L-alanine: DOVA transaminase. 10 Mg of the purified enzyme was subjected to electrophoresis at pH 8. 9 in 7 . 5% gel

matrix crude can be stored at -20°C for nearly one year

without signi~icant loss of enzyme activity. Frequent

freezing and thawing follows inactivation of the enzyme.

NATIVE AND SUBUNIT MOLECULAR WEIGHT

The apparent native molecular weight of the enzyme was

found to be 210 kDa by 4-30% gradient polyacrylamide gel

electrophoresis (Figure 13). This is in close agreement to

estimation by sepharose 6B gel filtration where it was

estimated to be 225 kDa. The subunit molecular weight of L-

alanine: DOVA transaminase was determined by SDS-PAGE under

denaturing condition which showed a well defined single band

with a relative molecular weight of 50,000 (Figure 14). L-

Alanine: DOVA transaminase is thus a homotetramer contrary

to earlier reports (154,156) in which it was reported to be

homohexamer. Such errors in molecular weight determination

are frequently encountered when the unknown and standard

marker proteins are compared for molecular weight

determination in separate tube gels. Therefore, in the ..

present study the relative subunit molecular weight was

accurately estimated by electrophoresing L-alanine: DOVA

transaminase along with standard marker proteins on a 12.5%

SDS-PAGE gel.

PRESENCE OF INTRA CHAIN DISULPHIDE BOND ( S)

SDS-polyacrylamide gel electrophoresis of L-alanine:

DOVA transaminase in reduc~d and non-reduced (i.e., in

76

6.0

5.8 Thyroglobulin

1- 1 2 :r: L':J w 5.6 3 -0::: -<! 5.4 _j

:J L-alanine:OOVA transaminase • LJ ( 210,000 ) w _j 5.2 0 Lactat e dehydrogenase L:

L':J -0 5.0 _j

4 8 ~ I I

0.2 0.4 0.6 0.8 1.0 Rf

Figure 13: Native molecular weight determination of L­alanine: DOVA transaminase by polyacrylamide gradient gel electrophoresis. Position of various native proteins of known molecular weight electrophoresed in a gradient polyacrylamide gel ( 4 3 0%) are shown in lane 2. Lane 1 shows the position of L-a l anine: DOVA transaminase at 210 kDa determined by plotting the Rf values of the calibration kit proteins vs the logarithms of their corresponding molecular weights. Calibration kit proteins were run as described under 'materials and methods' and i ncluded thyroglobulin {669 kDa), ferritin (440 kDa), catalase {232 kDa, lactate dehydrogenase ( 140 kDa ) and albumin (67 k Da ) .

f­

I C)

w ~ a: <!: _J

::J u w _J 4 ~ 2 X 10

4

Phosphorylase b

L-a lanine: OOVA transaminase 150,000) Ovalbum in

1x10 ~--~~--_.--~--~--~--~--~--~--~----0.2 0.4 0.6 0.8 1.0

Figure 14: Subunit molecular weight determination of L­alanine: DOVA transaminase by SDS-PAGE. About 2 ~g of pure L-alanine: DOVA transaminase was electrophoresed on a 12.5% SDS polyacrylamide gel along with the standard marker proteins. The graph is a plot of relative mobility vs log molecular weight of the standard marker proteins used for determination of subunit molecular weight of L-alanine: DOVA transaminase. Pharmacia standard marker proteins used are phosphorylase (94 kDa), bovine serum albumin (67 kDa) ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lactalbumin {14.4 kDa).

presence or absence of ~-mercaptoethanol) conditions

displays that in relation to reduced state under non-reduced

state the enzyme invariably shows up the band below 50 kDa

position along the 43 kDa posit i on (Figure 15). Such an

increase in mobility of the protein in non-reduced form

manifests the presence of intrachain disulphide bond(s)

(230). Such a possibility was also indicated later by the

presence of cysteine residues in this protein (Table V) .

ISOELECTRIC POINT (pi)

In the first instance when two-dimensional gel

electrophoresis analysis was performed with broad range

ampholines (pH 3-10) the protein spot appeared slightly

towards the acidic pH in between 5-6. Since this

preliminary observation with broad range ampholines

suggested its approximate pi between 5-6 we further repeated

the two-dimensional analysis with narrow range ampholines pH

5-7 (1.6% of pH 5-7 and 0.4% of pH 3-10). The results

indicated that the enzyme L-alanine: DOVA transaminase is an

acidic protein having a pi value of 5.0±0.1 (Figure 16).

N-TmunNAL SEQUENCING

Attempts undertaken to sequence the N-terminal end of

the L-alanine: DOVA transaminase suggested that this protein

cannot be sequenced by Edman degradation method because of a

blocked N-terminal amino acid residue. Evidence has already

been presented that about 80% of the soluble proteins in

mammalian cells have these blocks due to acetylated N-

77

kDa

94 67 43-

30-

20.1-

14.4-

1 2 3

Figure 15: Presence of intrachain disulph i de bond(s). About 2pg of L-alanine:DOVA transaminase was electrophorese, in a 12. 5% SDS-PAGE gel in presence (lane 2 ) and absenc (lane 3) of ~ -mercaptoethanol. Lane 1 shows the standar• marker proteins. The protein bands were visualised b · silver staining.

pH gradient(IEF}

Vl 0 Vl

t

0

Ln

I

® Ln

.J-1

kOa

-94 3: 0

-67 r rn

~so n c -43 r )> :::0

-30 ~ rn C1 I

-20.1 ~

14.4

Figure 16: Two dimensional electrophoresis of purified rat kidney mitochondrial L-alanine: DOVA transaminase. For the first d i mension, 15 ug of the purified protein was applied to isoelectric focusing gel as described in 'materials and methods' and then electrophoresed over a 12.5% SDS-PAGE gel to achieve final separation. Proteins were visualized by coomassie brilliant blue staining.

terminal amino acids {231). Of these about 41% have an N-

terminal acetylated serine and another 2% an acetylated

threonine . Therefore, deblocking of these possibly existing

blocked N-terminal residues was attempted according to the

recent procedure described by Wellner et al. (216).

Sequencing results after deblocking showed that though the

protein was being sequenced from the N-terminal end (?), the

background was too high for the sequence to be read. This

difficulty is reported particularly with the proteins that

are rich in serine and threonine as was the case with this

protein.

GLYCOPROTEIC NATURE

Preliminary study with PAS stain suggested that L­

alanine: DOVA transaminase may be a glycoprotein.

Subsequently, its glycoprotein nature has been confirmed

with concanavalin A binding as shown in Figure 17. It is

seen that concanavalin A has reactivity towards L-alanine:

DOVA 'transaminase and fibrinogen (positive control) but not

towards ferritin (negative control).

THERMAL STABILITY

Figure 18 shows the thermal stability of L-alanine:

DOVA transaminase. The enzyme was maintained at 65°C for

different lengths of time either in absence or presence of

one of its substrates and then assayed in standard

conditions. The enzyme was found to be heat-stable, but

78

1 2 3 kOo

-43

-30

-20.1

-14.4

Figure 17: Binding of concanavalin A to L-alanine: DOVA transaminase. About 15 ~g of fibrinogen as positive control (lane 1), 15 pg of ferritin as negative control (lane 2) and 2 )lg of L-alanine : DOVA transaminase were electrophoresed on 12.5% SDS-PAGE and electrophoretically transferred onto the nitrocellulose sheet. The sheet was rinsed in PBS and incubated for 30 minutes with 1% periodate treated BSA followed by incubation with 50 ~gjml concanavalin A for one hour. The concanavalin A was visualised as described in 'materials and methods'.

...--0 -0 ....__..

>-........

> ........ u d

(:::}

> ........ d (:::}

0::

100

80

60

40

5 10 20 30 40 50

Time (min)

Figure 18: Thermostability of kidney mitochondrial L-alanine: DOVA transaminase. Tubes containing equal amounts of L­alanine: DOVA transaminase were maintained at 65°C for different lengths of time either in absence or presence of only one of its substrates. Following incubation periods the enzyme activity was measured under standard conditions in presence of both the substrates. The graph is a plot of relative enzyme activity remaining under such conditions vs time. Thermostability of the enzyme: in absence of both the substrates ( ---o---o--- ) 1 in presence of DOVA only ( ~ ) 1 and in presence of L-al a nine only (~).

A

B

Figure 19: Ouchterlony double-immunodiffusion analysis with antiserum raised against rat kidney mitochondrial L-alanine: DOVA transaminase. Well A and P contain antiserum and pre immune serum respectively. Figure A: well 1 are loaded with purified mitochondrial L-alanine: DOVA transaminase and well 2 has kidney mitochondrial crude. Well 3 contains kidney tissue homogenate wher eas well 4 has bovine serum albumin. Figure B: well 1 are loaded with purified kidney mitochondrial L-alanine: DOVA transaminase. Well 2 contains liver mitochondrial crude and well 3 has liver tissue homogenate. Well 4 contains bovine serum albumin.

when heated for 40 minutes at 65°c lost about 80% of its

enzyme activity both in absence or presence of DOVA .. On the

other hand, thermal stability of L-alanine: DOVA

transaminase in presence of its substrate L-alanine reduced

significantly and lost 90% of its enzyme activity within 10

minutes. such a loss in enzyme activity was not observed in

the presence of other amino acids (L-glycine, D-alanine)

which do not serve as substrates for this enzyme (163).

x.miiOLOGICAL COMPAIUSOII OF LIVER AHD IO:DREY L-AI.Un:R: DOVA '.rRAJISAIIIIfASE

Results of ouchterlony double diffusion analysis using

polyclonal antibody raised in rabbit against kidney

mitochondrial L-alanine: DOVA transaminase are shown in

Figure 19. When the antibody was allowed to react with

kidney crude homogenate, kidney mitochondrial crude and

purified L-alanine: OOVA transaminase a single precipitin

line was formed. No such precipitin lines were seen with

preimmune sera. This antibody was also found to cross-react

with'· the rat liver mitochondrial L-alanine: DOVA

transaminase hence proving that the enzyme present in

miochondria of these organs are immunologically identical.

Dl:SCUSS:ION

,,

The present study reports a new three-step purification

method of rat kidney mitochondrial L-alanine: DOVA

transaminase. The procedure employed here is relatively

much rapid than the ones reported earlier from bovine, rat

and chicken 1 i ver. The enzyme has been purified 23-fold

79

from the mitochondrial matrix to apparent homogeneity with a

high yield of 37.5%. Improvement in the initial step of the

procedure was accomplished with the establishment of the

submitochondrial position of this enzyme to the

mitochondrial matrix. The mitochondrial fraction treated

with digitonin helps to eliminate some of the non­

mitochondrial organelles under conditions that solubilized a

significant fraction of the outer mitochondrial membrane.

Lubrol, used for the preparation of mitochondrial matrix is

reported to inhibit soae of the mitochondrial enzymes (204)

but does not inhibit L-alanine: DOVA transaminase. Thus,

utilizing these two nonionic detergents a significantly

clean mitochondrial matrix fraction could be prepared. Use

of L-alanine- sepharose affinity chromatography served as a

potent tool, and ion exchange (DEAE-cellulose)

chromatography commonly used by earlier researchers could be

safely eliminated. We observed that the use of an ion

exchange column before affinity chromatography did not have .

an appreciable improva.ent on the enzyme purity but rather

resulted in a drop in the enzyme yield. Lastly, gel

filtration on sepharose 6B column yielded the purified

enzyme. Gel filtration was preferred· as the last step

precisely because the purity of the active fractions

(eluates) could also be checked by SDS-PAGE before pooling

them. A significant proportion of enzyme during the

chromatographic steps is lost in assaying the eluates.

Using amplified assay for L-alanine: DOVA transaminase these

losses could be significantly lowered *bus contributing in

80

improving the overall enzyme yield. During the processes of

purification we have also tried some other procedures which

included protamine sulphate treatment, heat treatment, as

well as phenyl sepharose, hydroxyapatite and DEAE-cellulose

column chromatography. But these procedures were not as

effective so as to be included in the purification scheme.

Since it is now established that L-alanine: DOVA

transaminase is a component of the mitochondrial matrix, and

affinity chromatography as well as amplified enzyme assay

has proved effective, the procedure may also be applied to

other mammalian tissues, for these properties are commonly

shared by other enzyme sources as well.

Using gradient polyacrylamide gel electrophoresis the

native molecular weight of the enzyme was estimated to be

210 kDa. The subunit molecular weight of the enzyme

determined by SDS-PAGE under denaturing condition was 50

kDa·. Thus, the enzyme was found to be a homotetramer as in

the ,case of bovine and rat liver. SDS-polyacrylamide gel

electrophoresis of L-alanine: DOVA transaminase under

reduced and non-reduced conditions exhibited the presence of

intrachain disulphide bonds. Also the enzyme was found to

be acidic in nature with an isoelectric point of 5.0 ± 0.1.

Our attempts to sequence the N-terminal residue by Edman's

degradation method were not successful for the reason that

the protein has a blocked N-terminal end.

Our preliminary study with PAS stain suggested that L­

alanine: DOVA transaminase may be a glycoprotein. Following

81

this its glycoprotein nature was confirmed by its reactivity

towards concanavalin A. A general survey of mitochondrial

proteins by Ades (232) demonstrated that about 14% of the

mitochondrial matrix proteins are glycosylated and bind to

concanavalin A which is specific for mannose

oligosaccharides (233). To our knowledge L-alanine: DOVA

transaminase is perhaps the first protein to be identified

as a glycoprqtein from the mitochondrial matrix. 'The

potential presence of glycoproteins in mitochondria has

important implications with respect to mechanisms of

biosynthesis, maturation and assembly of mitochondrial

components' Ades, 1990 (232).

The thermal stability of the enzyme was examined at

65°C over a time period of 60 minutes and then enzyme

activity measured under standard conditions. When the

enzyme was incubated for 40 min at 65°C, it lost 80% of its

activity at high te.perature. Then our interest was to

examine whether any of the two substrates alone can confer

thermostability to the enzyme. Surprisingly, none of the

two substrates alone enhanced the enzyme stability at 65°C.

on the contrary, one of the substrates, L-alanine rather

decreased the enzyme stability at 65°c and the enzyme lost

90% of its activity within 10 minutes. The second substrate

DOVA alone did not influence thermostability. At present we

have no adequate explanation for the observations made here.

But it may be hypothesized that during the double

displacement (ping-pong) reactions, L-alanine binds to the

82

catalytic site of the enzyme molecule. As the second

substrate DOVA is not· available to accept the functional

group from the enzyme a positive charge is imparted onto the

enzyme molecule, which may induce conformational changes.

Since the charges are known to influence the conformation,

as well as thermostability of the proteins (234), occurrence

" of similar phenomena may be responsible for the

thermolability of this enzyme at high temperature. It is to

be noted that such alterations were not observed with other

amino acids (D-alanine and L-glycine) which do not serve as

substrate for the enzyme. This enzyme might be an

interesting model in order to investigate the yet unclear

molecular basis for protein thermal stability.

Finally, after fulfilling the present task of

purification we have raised the polyclonal antibody against

the homogeneous preparation of L-alanine: DOVA transaminase.

We have shown that_the antibody raised against kidney

mitochondrial enzyme cross reacts with the one from liver .

mitochondria proving that the enzyme present in the

mitochondria of these two organs are immunologically

identical. Since antibody is an indispensable tool to

examine the translocation of proteins across membranes, the

same will be utilized to study the translocation of L­

alanine: DOVA transa.Jli.nase into rat kidney mitochondria.

So, as part of our programme, we present the work related

to translocation of L-alanine: DOVA transaminase in the

forthcoming chapter.

83