Characterization of an extracellular asparaginase of

0 downloads 0 Views 808KB Size Report
Abstract: An asparaginase specific for L-asparagine was purified from Rhodosporidium toruloides ... EC 3.5.1.1) catalysing the deamidation of L-asparagine are.
316

Characterization of an extracellular asparaginase of Rhodosporidium toruloides CBSI 4 exhibiting unique physicochemical properties M.S. Ramakrishnan and Richard Joseph

Abstract: An asparaginase specific for L-asparagine was purified from Rhodosporidium toruloides CBS14 to apparent M for L-asparagine and 6.45 x homogeneity. The enzyme was associated with L-glutaminase activity (K,, 1.43 x M for L-glutamine). The enzyme was found to be a homodimer with a subunit molecular mass of 87 kDa. Chemical modification of tryptophan residues significantly reduced both L-asparaginase and L-glutaminase activities of the enzyme, which was prevented by the presence of either of the substrates L-asparagine or L-glutamine. The pH and temperature optima for both activities were 6.35 and 37°C. The enzyme was serologically identical with the asparaginases of some of the other Rhodotorula and Rhodosporidiurn yeasts but was distinct from the asparaginase of Saccharomyces cerevisiae. Key words: asparaginase, glutaminase, Rhodosporidium toruloides. RCsurne : Une asparaginase spCcifique de la L-asparagine a CtC purifiCe B l'homogCnCit6 apparente B partir du Rhodosporidium M pour la L-asparagine et 6,45 x toruloides CBS14. L'enzyme Ctait associCe h l'activitk de la L-glutaminase (K,, 1,43 x M pour la L-glutamine). L'enzyme a CtC reconnue comme un homodimkre ayant une sous-unit6 de masse molCculaire de 87 kDa. La modification chimique des rCsidus du tryptophane rkduisait significativement les activitCs L-asparaginase et L-glutaminase de l'enzyme et qui se trouvait inhibCe par l'un ou l'autre des deux substrats L-asparagine ou L-glutamine. Le pH et la tempCrature optimums de ces deux activitCs Ctaient 6,35 et 37OC. L'enzyme Ctait ~Crologiquementidentique B des aspariginases d'autres levures du genre Rhodotorula et Rhodosporidium, mais elle Ctait distincte de l'asparaginase de Saccharomyces cerevisiae. Mots c l h : asparaginase, glutaminase, Rhodosporidium toruloides. [Traduit par la rCdaction]

Introduction The L-ass~araginases (L-as~aragine amidoh~drO1ase EC 3.5.1.1) catalysing the deamidation of L-asparagine are widely distributed in nature, in microbes, plants, and animals. Although in most microbes asparaginase possibly serves as a catabolic enzyme, it may have a more specialized biological role as in the case of the ascomycetous fungus Leptosphaeria rnichotii, where asparaginase activity was shown to correlate with the asexual sporulation rhythm (Jerebzoff-~uintinand Jerebzoff 1980; Jerebzoff and Jerebzoff-Quintin 1984). The microbial asparaginases have been studied especially for their application as therapeutic agents in the treatment of certain types of human cancers (Gallagher et al. 1989). Since high substrate affinity is essential for effective antineoplastic activity, much effort has gone into characterization of microbial asparaginases with respect to substrate specificity and affinity Received August 16, 1995. Revision received November 22, 1995. Accepted December 6, 1995.

M.S. Rarnakrishnan and R. J0seph.l Department of Microbiology, Central Food Technological Research Institute, Mysore-570 013, India. Author to whom all correspondence should be addressed.

(Wriston and Yellin 1973). The high affinity asparaginases characterized from members of Enterobacteriaceae (including Escherichia coli) display a mass of about 100-140 kDa, are tetrarneric, and show a minor activity towards glutamine (Wade 1980). The asparaginases of Grampositive bacteria and yeasts studied so far have indicated, in towards substrate and were general, far less of antineoplastic activity (Wriston 1985).ofthe yeast asparaginases, those of Saccharomyces cerevisiae (Dunlop et a1.1978) and Candida utilis (Sakamoto et al. 1 9 7 7 ~are ) perhaps the only ones that have been studied in detail. Saccharomyces cerevisiae was found to produce two asparaginases, a c~tosolicconstitutive L-asparaginase I, specific for Lasparaghe, and a periplasmic asparaginase II under control of nitrogen catabolic repression, having equal activities on both L- and D-asparagine. Candida utilis synthesized a single asparaginase secreted into the extracellular fluid and is active on both L- and D-asparagine. Both these asparaginases were highly mannosylated and devoid of any glutaminase activity. During studies on the nitrogen metabolism of the Rhodotorula and Rhodosporidium yeasts, an inducible extracellular asparaginase under multisubstrate induction (asparagine, aspartate, glutamine, and glutamate could induce the enzyme) was detected. The present report describes detailed studies on

Can. J. Microbiol. 42: 316-325 (1996). Printed in Canada / Imprim6 au Canada

L

Ramakrishnan and Joseph

the characterization of a purified preparation of the asparaginase of Rhodosporidium toruloides CBS14 that indicated specificity for only L-asparagine but not for D-asparagine. Strikingly this enzyme had an associated glutaminase activity and data has been provided to suggest that both the activities are located in the same enzyme.

Materials and methods Organisms Rhodosporidium toruloides CBS14, Rhodotorula glutinis NCYC59, Rhodosporidium toruloides ATCC10788, Rhodotorula rubra MTCC248 (IMTECH, Chandigarh, India), and the locally isolated Rhodotorula glutinis (=gracilis) ATCC90950 were used. Serratia marcescens MTCC97 and Saccharomyces cerevisiae X2180 m m 2 mutant (gift from Dr. C. E. Ballou, University of California, Berkeley, Calif.) were also used.

Preparation of crude enzyme For production of asparaginase, Rhodotorula and Rhodosporidium yeasts were cultured in the medium containing 20 g ~-tnannitol,5 g L-asparagine, 1.2 g K2HP04, 6 g KH2P04, 0.5 g KC1, 0.01 g MgS04. 7H20, and 1 g yeast extract, in 1 L of distilled water (pH 6.0). The inoculum was developed by overnight growth in the same medium and added at 10% v/v level (initial OD, 0.07) to 200 mL of medium in 1-L flasks. After 12-14 h of growth in a rotary shaker (150 rpm) at 30°C, to a final OD of 1.5, the cells were harvested by centrifugation (6000 rpm for 20 min at 4OC) and acetone dried by passing ice-cold acetone through the cells. Around 6-8 g of acetonedried cells were obtained after growth of cells in 2 L of medium. Roughly 2 g of acetone-dried cells were ground with 15 g glass beads (diameter, 100-250 ym) at 4OC in 25 mM sodium phosphate buffer (pH 7) using a pestle and mortar. A protease inhibitor cocktail was added to a final concentration of 2 mM PMSF, 2 mM EDTA, and 1 mM benzamidine to the buffers. This crude extract was separated from the cell debris and glass beads by centrifugation at 4OC at 16 000 rprn for 40 min. Serratia marcescens MTCC97 was cultured in trypticase soy agar (Rowley and Wriston 1967), and then scraped, resuspended in 50 mM Tris-HC1 - 0.9% saline buffer (pH 7.6), and sonicated (Braun sonicator) at 75-100 W for 10 min with intermittent bursts of 1 min and a gap of 30 s each, with continuous cooling in an ice bath, for preparation of the crude extract. Cell debris was removed by centrifugation at 25 000 rpm for 30 min. Saccharomyces cerevisiae was cultured under nitrogen starvation conditions for the production of asparaginase (Roon et al. 1982). The crude extract of the enzyme was prepared by grinding the cells with glass beads as before with inclusion of the protease inhibitor cocktail.

Enzyme purification All steps in the purification protocol were carried out at 4OC, with the protease inhibitor cocktail included in all buffers. The crude enzyme extract was concentrated by taking it in a dialysis bag and keeping it buried in dry Sephadex G-200 overnight. It was then loaded onto a PBE94 ion exchanger (20 x 0.8 cm), previously equilibrated with 25 mM imidazole buffer (pH 7.4) and eluted with 1:lO diluted Polybuffer 74, pH adjusted to 4 at a flow rate of 30 mL/h. The fraction size was 2.5 mL. The asparaginase activity came in the flow through without binding to the column. The active fractions were pooled and the enzyme was eluted at 60-90% ammonium sulphate saturation. This precipitate dissolved in a small volume of sodium phosphate buffer (50 mM, pH 7) was subjected to gel filtration on a Sepharose 6B column (140 x 0.8 cm) previously equilibrated with the same buffer. At a flow rate of 20 mL/h, fractions of 3 rnL were

collected. The active fractions were pooled, but the enzyme was found to lose activity rapidly. This could be overcome by maintaining the pH at 9 and with the addition of 250 mM glycine (stabilization buffer, 50 mM Tris 250 mM glycine (pH 9)). The purified enzyme when lyophilized to dxyness in the presence of the stabilization buffer retained about 90% of activity when stored at -lO°C for 3 months). For convenience of handling and further processing, the enzyme preparation was divided into aliquots and lyophilized to dryness. Each aliquot was separately electrophoresed on a nondenaturing alkaline preparative p~lyacrylamideelectrophoresis gel and then electroeluted. The electro~horesiswas carried out by the standard method (Davis 1964), except using a 5% continuous gel. Before loading the enzyme, it was extensively dialysed, rapidly against 50 mM Tris-HC1 buffer (pH 7) to remove excess salt. A sample of 1.5 mL containing 2000 U of enzyme activity was loaded and electrophoresed at 60 V at 4OC for 12 h. After the run, the enzyme protein was identified by performing a zymogram on a cut strip of the gel. For the development of zymograms, the gel was flooded with the reaction mixture containing 10 mL of 100 mM L-asparagine (10 mL of 100 mM L-glutamine for glutaminase), 10 mL of 1 M hydroxylammonium sulphate (neutralized with 1 M KOH and pH adjusted to 6.3), 30 rnL of 50 mM sodium phosphate buffer (pH 6.3), and 50 mL of distilled water and incubated at 37OC for 30 min. The gels were developed by flooding with ferric chloride reagent (5% FeC13, 10% trichloroacetic acid, 0.66 M HC1). Asparaginase and glutaminase gave a brown coloured band because of the complex formed between ferric chloride and the respective products P-aspartyl hydroxamate and y-glutamyl hydroxamate (Mesas et al. 1990). The region of the unstained gel corresponding to asparaginase activity was cut out separately, sliced into small pieces, and in a dialysis bag containing 50 mM Tris - 250 mM glycine buffer (pH 9), placed in a horizontal electrophoresis tank containing the same buffer. The electroelution of asparaginase was carried out at 4OC for 24 h at 80 V with three changes of the buffer in the dialysis bag. The buffer contents containing the enzyme were pooled, and lyophilized and dialysed extensively against stabilization buffer before characterization of the enzyme was carried out. Table 1 gives the data obtained in the different steps of enzyme purification.

Estimation of protein Protein was generally estimated by the difference of absorptions at 215 and 225 nm (Waddell 1956).

Assay of asparaginase and glutaminase Asparaginase and glutaminase assays were performed using the Paspartyl hydroxamately-glutamyl hydroxamate method (Drainas et al. 1977). The reaction mixture in a total volume of 4 mL contained 10 mM L-asparagine or L-glutamine, 100 mM hydroxylammonium sulphate (adjusted to pH 6.3 with 100 mM KOH), 15 mM sodium phosphate buffer (pH 6.3), and the enzyme. This was incubated at 37°C for 30 min. The enzyme reaction products P-aspartyl hydroxamate and y-glutamyl hydroxamate were developed using ferric chloride reagent (see above) and the brown-colored complex formed with P-aspartyl hydroxamate was measured at 500 nm and that with yglutamyl hydroxamate was measured at 540 nm. Controls were run by adding the enzyme after the addition of the ferric chloride reagent at the end of the incubation period. Authentic P-aspartyl hydroxamate and y-glutamyl hydroxamate (Sigma) were employed as standards. A unit of asparaginase or glutaminase is defined as the amount of enzyme that causes the formation of 1 nmol of P-aspartyl hydroxamate or y-glutamyl hydroxamatelmin. The specific activity is expressed as the units of asparaginase or glutaminase per milligram protein in solution. Asparaginase and glutaminase were also assayed using Nesseler's reagent (Imada et al. 1973), wherein 1 IU of L-asparaginase or Lglutaminase was the amount of enzyme that liberated 1 ymol of ammonia in 1 min.

318

Can. J. Microbial. Vol. 42, 1996 m

s

Determination of pI

.y

s0 . E5- $:z g 2

Purified enzyme was loaded onto a chromatofocusing PBE94 ionexchanger column (20 x 0.8 cm) equilibrated with 0.025 M triethanolamine buffer (pH 9.4) and eluted with 1: 10 diluted Polybuffer 96, pH adjusted to 6. At a flow rate of 20 mL/h, 2-mL fractions were collected.

.~ 2 u s"m ym ymTmqm % + .

&

2 27 Y-md

9

cd

L g,

-

"

Molecular mass determination by gel filtration

7

The native molecular mass of asparaginase was determined by gel filtration using a calibrated column (140 x 0.8 cm) of Sepharose 6B. The column was equilibrated with 50 mM sodium phosphate buffer (pH 7). The column was run at a flow rate of 20 mL/h and 2-mL fractions were collected. Void volume was determined using Blue dextran. The calibration of the column was done with standard proteins: thyroglobulin (669 OOO), carmin (260 OOO), bovine serum albumin (BSA, 66 OOO), and carbonic anhydrase (29 000).

0 ~ 3 o ~ m ~ z r - c ~ m N

2 * "

3

u

g

'?c.?O\w

m

-

mm

S . r o 0 c b s x clm

g z > x

WmmN* QNPWW m - o m m

U3

N ~ N - +

a m - N W ONWOW - a m 2

Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)

U3

2

This was canied out according to the method of Laemmli (1970) by using a 12.5% separating gel and protein bands were visualized by staining with silver diamine reagent (Dunn 1990). For molecular mass determination, standard protein markers were run simultaneously with the unknown sample. Markers used were BSA (66 OOO), egg albumin (45 OOO), glyceraldehyde-3-phosphate dehydrogenase (36 OOO), carbonic anhydrase (29 OOO), and trypsin inhibitor (20 100).

2

Analysis of reaction products of asparaginase and glutaminase by paper chromatography

N

2 $.

%$mmm-")

8 -" - 2 7 % .$ a o x

g 2

" W

3 2

9

~ t m m m o~

3.23 G & z g z

g

9 x

~

2% z .+ > x

.9

2 2

c

~

~

s z

mmC\J\D* m30\

1

se,G 0 5 E F k-

2

m

~

Determination of effect of metal salts, activators, and inhibitors on enzyme activity

m m

m

N

0;+,?0! d + mNwpO

2

5-

All the compounds were tested at 1 mM level except urea at 2.25 M, P-mercaptoethanol at 2 rnM, and diethyl pyrocarbonate at 5 rnM levels. Enzyme was preincubated with these compounds for 10 min at room temperature prior to determination of enzyme activity. Except for thep-chloromercuribenzoate, diethyl pyrocarbonate and N-acetylimidazole treatments that were performed at pH 7 in 50 mM sodium phosphate buffer, all the other effector treatments were carried out at pH 9 in 50 mM Tris - 250 rnM glycine buffer. The enzyme activities in these studies were determined by the P-aspartylly-glutamyl hydroxamate method.

I

In

'E .c)

3

1

M

a

s

$-

a

0

3aE3 w m m A w 1

3sg m >

N

i

m

3

- -

.

"

The reaction mixture comprising 0.1 mL of 0.04 M L-asparagine or L-glutamine, 0.1 mL of 50 mM sodium phosphate buffer (pH 6.3), 0.1 mL of enzyme preparation, and distilled water to a total volume of 0.4 mL was incubated at 37°C for 4 h. The reaction mixture was analyzed 0by paper chromatography using the phenol-water (4:l) solvent system. The chromatogram was developed by spraying with 0.5% ninhydrin in acetone. The chromatogram was dried at 100°C until the spots developed.

u

e,

3 2

*9 0

Preparation of polyclonal antibody against purified asparaginase and Ouchterlony double diffusion Rabbits were immunized with 250 pg proteinlkg body weight by intramuscular injections administered every 14 days. The polyclonal antiserum separated from the blood 1 week after the fourth injection was used as the antibody. Ouchterlony double diffusion was done using agarose gels (2 mm thick) with 0.8% agarose in 50 mM TrisHC1 buffer plus 0.9% saline (pH 7.6) containing 0.1% sodium azide as the preservative. Double diffusion was carried out at 25°C for 48 h.

c

.-0 C)

cd

o

3a e,

55

.B

z

22

m

4

= P 8

UE E D

g

.

c 3 ~ 0

m Fc so c sg. Eu E m.c E S Z 2

2E

a 2,

d

3"

DmU