research article purification and characterization of a ... · research article purification and...

Research ArticlePurification and Characterization of a Novel IntracellularSucrase Enzyme of Leishmania donovani Promastigotes

Arpita Singh and Debjani Mandal

Division of Infectious Diseases and Immunology Indian Institute of Chemical Biology 4 Raja SC Mullick RoadJadavpur Kolkata West Bengal 700032 India

Correspondence should be addressed to Debjani Mandal mandaldebjaniyahoocom

Received 23 November 2015 Accepted 22 March 2016

Academic Editor Stefano Pascarella

Copyright copy 2016 A Singh and D Mandal This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The promastigote stage of Leishmania resides in the sand fly gut enriched with sugar molecules Recently we reported thatLeishmania donovani possesses a sucrose uptake system and a stable pool of intracellular sucrose metabolizing enzyme In thepresent study we purified the intracellular sucrase nearly to its homogeneity and compared it with the purified extracellularsucraseThe estimated size of intracellular sucrase issim112 kDa by gel filtration chromatography native PAGE and substrate stainingHowever in SDS-PAGE the protein is resolved at sim56 kDa indicating the possibility of a homodimer in its native stateThe kineticsof purified intracellular sucrase shows its higher substrate affinity with a 119870

119898of 161mM than the extracellular form having a 119870

119898

of 44mM The highly specific activity of intracellular sucrase towards sucrose is optimal at pH 60 and at 30∘C In this reportthe purification and characterization of intracellular sucrase provide evidence that sucrase enzyme exists at least in two differentforms in Leishmania donovani promastigotesThis intracellular sucrase may support further intracellular utilization of transportedsucrose

1 Introduction

Sucrose glucose and other hexosersquos are required for themaintenance of parasite redox balance and for generatingprecursors for DNA and RNA biosynthesis As a resultLeishmania donovani depends on carbohydrates to sustaintheir central carbon metabolism This parasite has the abilityto modify its biochemical machinery to adapt diverse micro-environment encountered within the host in order to guar-antee their survival

Sugar meal is important for the development of infec-tive forms of Leishmania sp and for its virulency [1 2]Considering the major metabolite constituents in sandfly gut[3 4] sucrose presumably is one of the preferred energysource where the division of the promastigotes takes placeTherefore it is important to understand how they utilize thissugar as well as the mechanism of sugar uptake inside thecell Recently we have reported the sucrose transport systemin Leishmania donovani promastigotes and the intracellular

sucrose splitting enzyme sucrase which may demonstratethe utilization of internalized sucrose [5] However sucroseinternalization and henceforth consumption is a relativelyunexplored area in Leishmania biology Leishmaniasis stillremains a major health concern of the 21st century through-out the world despite the sustained efforts to control thedisease over several decades Along with existing efforts ofdeveloping vaccines [6] and improveddrugs [7] it is necessaryto understand its physiology foremost and the inherentability of the parasite to adapt itself to a myriad of adverseenvironmental parameters

Our recent finding on the intracellular pool of sucraseenzyme as well as previous reports on secretary extracellularsucrase [5 8] prompted us to purify the intracellular sucraseHere we identified a sim112 kDa homodimer intracellularsucrase enzyme of Leishmania donovani promastigotes andcharacterized and compared it simultaneously with the puri-fiedsim71 kDa extracellular sucrase Further detailedmolecularcharacterization of the intracellular enzyme is important to

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2016 Article ID 7108261 8 pageshttpdxdoiorg10115520167108261

2 Biochemistry Research International

gain insight into its probable role in biochemical pathwayof the parasite and pathogenesis This understanding maycontribute knowledge towards antileishmanial drug design-ing

2 Materials and Methods

21 Materials Analytical grade reagents were used forexperimental purpose All the chemicals were purchasedfrom Sigma USA unless otherwise mentioned Brain HeartInfusion was obtained from Acumedia Manufacturers IncBaltimoreMDUSA andMedia 199 Penicillin-Streptomycinpowder were purchased from GIBCO USA

22 Strains The strain of L donovani used in this workMOHMIN1978UR6 was a clinical isolate from an Indianpatient with confirmedKala-azar collected in the year of 1978UR6 cells were maintained in solid blood agar media of pH74 and highly motile promastigotes were considered duringexperiments

23 Media and Culture Conditions

231 Solid BloodAgarMedia According to Kumar Saha et al[9] the cell line was maintained in solid blood agar media at22∘CThegrowth of promastigoteswasmeasured every 24 hrsof 72 hrs of growth period

232 Liquid Media 199 One liter of liquid medium wasprepared by adding 11 g ofmedia-199 power 10 FCS 22mMHepes and 100 units of Penicillin-Streptomycin in waterThepH of the media was adjusted to 74 and filter sterilized forfurther use

24 Preparation of Cell-Free Extract Cell-free extract wasprepared according to Singh and Mandal [5] Exponentiallygrowing promastigotes in liquid culture were harvestedwashed with PBS and suspended in lysis buffer containing(5mM Tris-HCl 05mM PMSF and 025mM EDTA pH74) The suspension incubated at ambient temperature wasmixed 10 times by vortexing 30 secs at 2min intervals Thiswas further sonicated at 15 pulses of 20 secs each with 1mininterval on ice The sonicated extract was adjusted to 50mMTris-HCl pH 74 (Buffer A) and ultracentrifuged at 1 times 105 gfor 1 hr at 4∘CThe supernatant considered as cell-free extractwas collected and stored at minus20∘C for further use

25 Purification Procedure

251 Enzyme Extraction The enzyme activity of the cell freeextract was considered as crude and its level of activity wastaken to be 100 for calculation of recovery

252 Ammonium Sulfate Precipitation Solid ammoniumsulfate was added into the cell-free extract first to 33saturation and then to 75 saturation The pellet holdingmajor sucrase activity was finally suspended in Buffer Acontaining protease inhibitor and immediately considered forfurther purification

253 Size-Exclusion Chromatography (SEC) Following am-monium sulfate concentration the resuspended sample(active fraction) was loaded onto a Sephacryl S-200 column(120 times 1 cm) preequilibrated with Buffer A The proteinswere eluted with Buffer A at a flow rate of 22mLhr Proteinfractionstube containing the major activity of sucrase werepooled for further steps of purification

254 Ion-Exchange Chromatography (IEC) A column (20times 2 cm) was packed with CM-Cellulose matrix swollenovernight at room temperature to have a bed volume of 10times 2 cm The pooled active enzyme fractions from S-200 col-umn were passed through CM-Cellulose (cation exchanger)columnpreequilibratedwithBufferA for further purificationThe flow-through containing the major sucrase activity waspooled immediately and subjected to DEAE Sephadex (anionexchanger) column of same bed volume preequilibrated withBuffer A The bound protein containing the enzyme fractionwas eluted with a salt gradient of 0ndash02M NaCl The pooledfraction from DEAE Sephadex was passed through S-200column (120 times 1 cm) again to equilibrate the semipurifiedenzyme sample with 20mM potassium-phosphate buffer pH74 for Hydroxyapatite batch adsorption

255 Hydroxyapatite Batch Adsorption Hydroxyapatite [10]matrix was equilibrated with 20mM potassium-phosphatebuffer at pH74Thepooled fraction containingmajor sucraseactivity from S-200 column was subjected to Hydroxyapatitebatch adsorption and allowed the enzyme to mix properlyby occasional stirring After the matrix settled down theunabsorbed content mostly purified enzyme was collectedby a low spin centrifugation at 4∘C The supernatant wasfound to contain gt95 purified enzyme

26 Protein Estimation Modified Lowry method was usedfor protein estimation [11] and BSA was taken as a standard

27 Gel Electrophoresis and Activity Staining Polyacrylamidegel electrophoresis (PAGE) under native and denaturingcondition was performed according to Laemmlirsquos discontin-uous Tris-glycine buffer system [12] with little modificationDuring activity staining the gel was sliced into two halveseach half bearing identical samples One part of 55 nativegel containing the purified intracellular sucrase enzyme wasincubated in 50mM Tris-HCl with 100mM sucrose for 2 hrsat 30∘C followed by a wash with distilled water [13] The gelwas then immersed in 1M of iodoacetamide for 15min atroom temperature Following the wash with double distilledwater the gel was incubated in 05N NaOH containing 2235-Triphenyl Tetrazolium Chloride (TTC) for 15min ina boiling water bath until a diffuse pink background colordevelops After proper distaining with 75 acetic acid aphoto was taken immediately as the color developed persistsvery shortly The other gel part was subsequently stainedin coomassie R-250 for the corresponding protein bandidentification

28 Purification of Extracellular Sucrase Promastigote cellsgrew in liquid culturemedia for 66-67 hrs were pelleted down

Biochemistry Research International 3

Table 1 Purification of intracellular sucrase the table shows the list of different steps taken during the purification processThe total enzymeactivity the amount of protein recovered and the specific activity of the sucrase enzyme were estimated from each step of the purificationprocedure to calculate the increased fold of purification

Steps of purification Total activity nmoles ofglucose formedmin

Total protein 120583g Specific activity nmoles ofglucosemgmin

Fold purification

Crude extract 2084 790002 2637 1

Size-exclusion chromatography 1075 11078 9703 367

IEC-CM-cellulose 85436 3628 23554 893

IEC-DEAE Sephadex 45908 1250 367 1392

SEC-Sephacryl S-200 15464 3454 447712 16978Hydrophobic chromatography(HA batch absorption) 10386 11816 878977 33332

by centrifugation and the cell-free media was lyophilizedto semidryness It was reconstituted in 50mM Potassium-Phosphate buffer pH 74 and concentrated to sim15mL byusing a centricon membrane filter YM 10 Following thesame purification procedure of intracellular sucrase thefiltrate was allowed to pass through the preequilibrated S-200 column Subsequent ion-exchange chromatography andHydroxyapatite batch adsorption steps were performed toobtain purified extracellular enzyme

29 Enzyme Assays With slight modification to Messer andDahlqvistrsquos method [14] sucrase activity was estimated Theenzyme reaction was initiated by adding the enzyme inthe assay mixture (50mM sodium acetate pH 5560) inpresence of 4mM of substrate sucrose and the reactionstopped by heat inactivation after 30min of incubation at30∘C The colorimetric estimation of glucose was taken at505 nm One unit of enzyme activity is defined as the amountof enzyme that hydrolyzes sucrose to produce 1 mole ofglucose at 30∘C

210 Optimum Temperature and Thermostability The opti-mum temperature for the enzyme was evaluated by mea-suring sucrase activity in 50mM sodium acetate buffer(pH 55ndash60) at different temperature Thermostability wasdetermined by preincubating the purified enzyme for 30minat a range of temperature (4ndash60∘C) prior to the standardactivity assay

211 Optimum pH Purified enzyme activity was assayed in50mM of four different buffering agents glycine-HCl (pH20-30) acetate (pH 40ndash60) phosphate (pH 60-70) andTris-HCl (pH 70-80) in order to record the pH profile underthe standard experimental conditions

212 Effect of Metal Ions on Intracellular Sucrase Activity Themetal ions effect on enzyme activity was determined afterpreincubating the purified enzyme with various metal ionssuch as ZnCl

2 HgCl

2 CaCl

2 KCl AgNO

3 FeSO

4 MgSO

4

MnSO4 CuSO

4 CoSO

4 and NiSO

4 one at a time at desired

concentration for 10min at 30∘C Following standard assaycondition enzyme activity was measured and expressed aspercentage of control (without metal ion)

3 Results

In the present study both the intra- and extracellular enzymeswere simultaneously purified to characterize the intracellularsucrase enzyme in comparison to extracellular one Themolecular size of intracellular sucrase was confirmed by size-exclusion chromatography and native gel analysis followed byactivity staining

31 Molecular Weight Determination

311 Size-Exclusion Chromatography Size-exclusion chro-matography of cell-free extract followed by ammoniumsulfate precipitation shows intracellular sucrase activity(Figure 1) and the molecular mass of the protein esti-mated from gel filtration chromatography was approximately112 kDa (Figure 1 inset) The active fractions from S-200column were pooled and passed through different steps(Table 1) to get pure active protein The entire purificationsteps yield nearly 330-fold the purified intracellular sucrase

312 Relative Mobility Presence of a single band in SDS-PAGE (Figure 2(a)) reveals the purity of the enzyme Theestimated size of the protein is nearly 56 kDa as calculatedfrom the relative mobility and the molecular weight of theknown marker protein (Figure 2(b)) The 56 kDa proteinsize is nearly half the size estimated from gel filtrationchromatography (112 kDa Figure 1 inset) which may denotethe homodimerization of the enzyme sucrase in its nativestate

313 Activity Staining A confirmatory test for identifyingthe enzyme is to locate the enzymatic activity in the poly-acrylamide gel Thus to confirm the position of intracellu-lar sucrase amidst electrophoretically separated proteins amodified approach of Gabriel and Wang [13] was used forsubstrate staining of the native intracellular sucrase Purifiedprotein shows a deep pinkish violet band representing theactive protein (Figure 2(c)) The subsequent single bandin coomassie stain corresponding to the substrate stain(Figure 2(d)) confirms its approximate size of 112 kDa Thisresult corroborates with the protein size estimated from thegel filtration chromatography


Abso

rban

ce at

280

nm

5 10 15 20 250Fraction number

0

5

10

15

20

25

30

35

Sucr

ase a

ctiv

ity (n

mol

esm

gm

in)

0

1

2

3

4

5

6

7

8

9

40 50 60 70 80 90 10030Elution volume (mL)

3

35

4

45

5

55 1 23

45

log M

W

Figure 1 Size-exclusion chromatography of intracellular sucraseThe ammonium sulfate saturated sample protein was run on aSephacryl (S-200) column preequilibrated with Buffer A Theprotein absorbance measured at 280 nm (-X-) was plotted onprimary axis The secondary 119910-axis shows the intracellular sucraseactivity (-◼-) of the corresponding fractions The molecular weightof the native enzyme was determined to be sim112 kDa from the S-200calibration graph (Figure 1 inset) The uarr arrow indicates the peak ofintracellular sucrase elution volume and themolecularweight (MW)of the protein The molecular weight markers used were as followsamylase MW = 200000 alcohol dehydrogenase MW = 150000hemoglobin MW = 64500 carbonic anhydrase MW = 29000 andcytochrome C MW = 12400

32 Enzyme Kinetics of Crude and Purified IntracellularSucrase The intracellular sucrase activity was measured atdifferent substrate concentrationThe range of substrate con-centration varied from 005ndash10mM to 04ndash50mM respec-tively for estimating the crude and purified intracellularsucrase enzyme activity The Lineweaver-Burk plot of thecrude intracellular sucrase enzyme activity shows the 119870

119898of

sim66mM and the119881max of sim125 nmolesminmg (Figure 3(a))while the purified intracellular enzyme has more affinitytowards the substrate as calculated from the Lineweaver-Burk plot and the estimated 119870

119898and 119881max are sim16mM and

sim1905 nmolesminmg respectively (Figure 3(b))

33 Substrate Specificity Different disaccharides were usedto check the substrate specificity of the purified intracellularenzyme The enzyme was incubated in presence of differentsubstrate namely raffinose melibiose maltose trehalose

and palatinose and the corresponding enzyme activity wasmeasured according toMesser and Dahlqvist [14] in compar-isonwith sucrose as a control (100)The substrate specificityof the Leishmania intracellular sucrase is highly specific innature as it was unable to hydrolyze any of the substratesmentioned above except sucrose and partially raffinose at aconcentration range from 1 to 10mm (data not shown)

34 Molecular Mass and Kinetics of Extracellular SucraseMolecular mass of extracellular sucrase determined by size-exclusion chromatography and SDS-PAGE (data not shown)was sim7079 kDa For kinetic study enzyme activity of thepurified extracellular sucrase was estimated by incubatingwith varying substrate concentration (0125ndash8mM) at 30∘Cfor 30min The double reciprocal plot of the velocity of thereaction and the substrate shows the 119870

119898of purified extra-

cellular sucrase as sim44mM which corroborates the reporton purified extracellular sucrase of Leishmania by Gontijoet al [15] The result illustrates that the purified extracell-ular sucrase has nearly three times reduced substrate affinitythan that of intracellular sucrase enzyme

Purification and the subsequent kinetic studies of thepurified enzyme further confirmed that at least two differentforms of the enzyme sucrase exist in L donovani promastig-otes

35 Temperature Tolerance and pH Sensitivity of Intra- andExtracellular Sucrase Intracellular sucrase is susceptible tohigher temperatures and loses 50 of its activity with anincrease of temperature above 45∘C On the other hand theextracellular sucrase is mostly stable up to 50∘C and has awide range of temperature tolerance (Figure 4(a)) The intra-cellular sucrase shows sim20 of its maximum activity at thistemperature then gets deactivated completely with furtherincrease of temperature However both the purified enzymesexhibit its maximum activity at 30∘C (data not shown)

Interestingly the intracellular sucrase shows nearly 80of its activity at a pH range from 45 to 70 (Figure 4(b))nevertheless the optimum enzyme activity occurs at pH 60(data not shown) In standard assay condition the maximumenzyme activity of purified extracellular sucrase appears atpH 55 (data not shown) although more than 80 of itsactivity was observed between pH 50 and 65 (Figure 4(b))

36 Effect of Metal Ion on Intracellular Sucrase Activity Theenzyme activity was compared in presence of various metalions at 1mM concentration as presented in Figure 5 Metalions such as ZnCl

2 AgNO

3 andHgCl

2strongly inhibited the

enzyme activity while a moderate inhibition of activity wasnoted by the sulfate of cobalt nickel and copper ions

4 Discussion

The parasites in the insect vector are exposed to an entirelydifferent microenvironment than their vertebrate host Leish-mania promastigotes possess sucrose transporter in theplasma membrane [5] which helps the internalization ofsucrose a major food constituent of the insect gut Toaddress the issue on further utilization of the accumulated


Ovalbumin

Carbonic anhydrase

Sucrase

Bovine serum albumin

Phosphorylase

(b) (c) (d)

(kDa) 1 2

116

974

66

45

29

(a)

1 2

974

116120573-galactosidase

(kDa)

log10

MW

02 04 06 08 1044

45

46

47

48

49

5

51

52

Relative mobility (Rf)

Figure 2 Gel electrophoretic analysis and activity staining of purified intracellular sucrase (a) Electrophoresis (10 SDS-PAGE) was done ina discontinuous buffer system Lane 1 molecular mass standards indicated on the left are as follows galactosidase (116 kDa) phosphorylase B(974 kDa) bovine serum albumin (66 kDa) ovalbumin (45 kDa) carbonic anhydrase (29 kDa) lane 2 shows the purified intracellular sucraseprotein band of nearly sim56 kDa in size (b) 10 SDS-PAGE standard curve plotted with logarithm of the molecular weight against relativemobilityThe arrow indicates the log of molecular weight of intracellular sucrase subunit in 10 SDS-PAGE (c) Substrate staining of purifiedintracellular sucrase in native gel the arrow indicates the activity band in native gel (d) Coomassie stained purified intracellular sucrase innative gel Lane 1 represents the proteins galactosidase (116 kDa) and phosphorylase B (974 kDa) asmolecular weightmarker and lane 2 showsthe native form of purified intracellular sucrase

0

20

40

60

80

100

(nm

oles

of g

luco

sem

inm

g)

25 50 75 10000Sucrose (mM)

15 40 65 90 115 140minus10

1S

005

015

025

035

045

055

1V

(a)

1 2 3 4 50Sucrose (mM)

0

40

80

120

160

(nm

oles

of g

luco

sem

inm

g)

2000 05 10 15 25 30minus05minus10

1S

001

002

003

1V

(b)

Figure 3 Enzyme kinetics of crude and purified intracellular sucrase intracellular sucrase was incubated in the assay mixture with varyingrange of substrate concentration (020ndash10mM) at a fixed incubation time to estimate the enzyme activity of crude (a) and purified (b)intracellular sucraseThe inset represents the Lineweaver-Burk plot of velocity and substrate concentration of crude and purified intracellularsucrase which shows119870

119898of 66mM and 161mM of sucrose respectively The results are the mean of three independent experiments (119899 = 3)


20 40 60 800Temperature (∘C)

0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)

Figure 4 The temperature tolerance and optimum pH of intra- and extracellular sucrase (a) the intra- and extracellular enzymes werepreincubated for 30min at different temperature (4ndash60∘C) prior to estimating the enzyme activity under standard assay condition in 50mMsodium acetate buffer at pH 60 and pH 55 respectively The enzyme activities of intracellular (-X-) and extracellular sucrase (-e-) wereplotted as percentage of maximum activity (100) at different temperature (b)The pH optimum was measured by performing activity assayat different pH at a range of 0275ndash80 asmentioned inMaterials andMethodsThe intracellular (-998771-) and extracellular (-X-) sucrase activitieswere plotted as a percentage of activity versus pH with maximum activity being 100 Each point represents the mean of three differentexperiments

KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)

Figure 5 Effect of metal ion on the intracellular sucrase enzymeactivity The purified enzyme was preincubated with metal ions for10min at room temperature and the activity assay was performedin 50mM sodium acetate buffer at pH 60 The enzyme activity inabsence of metal ions was considered to be 100 and compared therelative activity of the enzyme in presence of differentmetal ionsThevalue presented corresponds to the mean value of three replicates

sucrose in L donovani promastigotes we focus on the sucrosemetabolizing enzyme sucrase Recently we reported [5] thatthemajority of enzyme remains inside the cell as intracellularsucrase and the rest is secreted as extracellular sucraseThese sucrase enzymes are constitutive in nature the specificactivity of the enzyme remains the same in the presence orabsence of external pressure (ie sucrose) in the media The119870

119898of intracellular sucrase in cell-free extract and the purified

form differs markedly from each other Reduced affinity ofthe crude enzyme towards the substrate may occur by theinterference of cytosolic inhibitory factors

The distinctive characteristic properties of the enzymesucrase suggest the possibility of having two forms of enzymein Leishmania promastigotes asim71 kDamonomer extracellu-lar sucrase and asim56 kDahomodimer of intracellular sucraseSo there is a probability that the monomer of intracellularsucrase with an active catalytic sitemay be posttranslationallymodified to become extracellular sucrase [8] However todetermine whether each monomer is catalytically active ornot further studies need to be doneThe significant differenceobserved in the kinetics between the two forms of the sucrasemay be due to differing accessibility and efficiency of catalyticsites

It has been established that the two forms of invertaseextracellular and intracellular one in Saccharomyces cere-visiae do not differ much in 119870

119898or velocity yet pH stability

changes [16] According to Wallis et al [17] Aspergillus nigersecretes two fructofuranosidases and both may be dimersin their natural conformation considering the protein sizein SDS-PAGE and native state The enzymes have affinitytowards sucrose and to some extent to raffinose howevertheir affinity varies with other substrates In Leishmania thewide range of temperature tolerance of extracellular sucraseis probably to remain functional in outside temperatures incontrast the intracellular sucrase has slight temperature toler-ance (Figure 4(a)) Strong inhibition of intracellular enzymeactivity with thiol modifying reagents like 551015840-dithiobis-2-nitrobenzoic acid (DTNB) N-ethylmaleimide (NEM) and


p-chloromercuribenzoate (PCMB) suggests that the activesite of the enzyme may possess ndashSH moieties (data notshown) This finding corroborates the report on purified b-fructofuranosidase of B infantis [18]

The enzymatic characterization and preliminary dataon the N terminal sequence of the purified intracellularsucrase show its considerable homology with glycosidase thebacterial 120573-fructofuranosidase class of enzyme [19] underthe broader heading glycosidase (Singh and Mandal unpub-lished data) This supports the findings of bacterial nature inmany of the enzymes which lies in the metabolic machineryof Leishmania [20] The 120573-fructofuranosidases belong to theglycosyl hydrolasersquos family of 32 proteins and catalyze thehydrolysis of sucrose to glucose and fructose Literature sur-vey suggests that the bifidobacterial 120573-fructofuranosidasesalso have activity against the longer chained substrates such asraftilose raftiline and inulin [21] The phylogenetic analysisof the related intracellular fructofuranosidases includes alarge group of sucrose-6P-hydrolases of which all are physi-cally linked with genes encoding sucrose transport proteinsof the PTS [20] Very recently Lyda et al [22] discoveredthe secretory invertase (LdINV) gene of Leishmania pro-mastigotes and also identified a beta-fructofuranosidase-likegene during the homology search of LdINV which encodesa 120 kDa protein To validate our preliminary results furtherstudy is necessary to identify the gene encoding intracellularsucrase

The uptake and subsequent metabolism of glucose inTrypanosomatidae is an example of an adjustment leadingto maximum energy efficiency [23] However it varies fromspecies to species as L donovani is confronted with widelyvarying conditions in the sandfly gut and strives for internalhomeostasis even at the expense of energy Thus it mayhappen that two different metabolic strategies represent twoopposing trends the capability of uptake of sucrose and itsutilization by hydrolyzing sucrose to glucose and fructoseare the efficient adaptation at the expense of short termflexibility and on the other hand the ability to rapidly adaptto environmental changes at the expense of energy

Interestingly very recently Dirkx et al [24] reportedthat the flagellated protozoan Trichomonas vaginalis genomecontains nearly 11 putative sucrose transporters and a putativeszlig-fructofuranosidase (invertase) Thus the machinery forboth uptake and cleavage of intracellular sucrose appearsto be present in the protozoa as the cell lysates retaininvertase activity It is likely that the most recent commonancestor of T vaginalis was a gut-dwelling protist where thecapacity to utilize fructose containing compounds might beadvantageous [25]

Representatives of the Kinetoplastids spread over a vari-ety of different environments and in due time many ofthem became parasites of insects leeches major vertebrateslineages and even plants Where Kinetoplastids evolved todigenetic parasites involving two different hosts and ofteneven different host tissues they had to find means forefficient adaptation of their metabolism at highly differentenvironments encountered This implied the developmentof mechanisms to regulate differentially the expression oftheir metabolism in different life cycle stagesThus metabolic

flexibility must have been a highly selective advantage duringthe different stages of this evolutionary scenario Trypanoso-matids apparently have a considerable number of plants-liketraits and several plants-like genes encoding homologs ofproteins found in either chloroplast or the cytosol of plantsand algae In fact elegant studies had proved earlier thatmanyof the genes in different Trypanosomatids are orthologues[26] Thus Leishmania a Trypanosomatid parasite belongingto the order Kinetoplastida together with Euglenoids isunder Euglenozoa In this backdrop thus it is not surprisingto observe that Leishmania possesses an efficient sucrosetransporter and metabolizing machinery Recent report onintracellular invertase BfrA of Lmajor [27] further supportsour hypothesis on the participation of intracellular sucraseof L donovani promastigotes in the sucrose metabolismpathway Our limited findings are a partial endeavor toexplore this very intriguing field of metabolite Concludingremarks can only be that future knowledge of the role of thissucrase enzyme in the physiology and life cycle of the parasitecan lead to the opening up ofmany avenues towards the betterperception of parasitic biology and hence containment of thisdreaded disease

Disclosure

The present Address for Arpita Singh is University of Vir-ginia Department of Medicine Infectious Diseases Char-lottesville VA 22904-4101 USA Corresponding address forDebjani Mandal is Uniformed Services University of HealthScience Department of Biochemistry andMolecular BiologySchool of Medicine Bethesda MD 20814 USA

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thank Professor S Roy former Director of IICBKolkata India for supporting this work and Dr TanmoyMukherjee (IICB Kolkata India) for helpful suggestions andcomments on designing experiments They are thankful toDrAshisChowdhury (USUHSMarylandUSA) for carefullyediting the paper

References

[1] D Rodrıguez-Contreras and S M Landfear ldquoMetabolicchanges in glucose transporter-deficient Leishmania mexicanaand parasite virulencerdquoThe Journal of Biological Chemistry vol281 no 29 pp 20068ndash20076 2006

[2] S M Landfear ldquoGlucose transporters in parasitic protozoardquoMethods in Molecular Biology vol 637 pp 245ndash262 2010

[3] R S Bray ldquoLeishmania mexicana mexicana attachment anduptake of promastigotes to and by macrophages in vitrordquo Jour-nal of Protozoology vol 30 no 2 pp 314ndash322 1983

[4] Y Schlein ldquoSandfly diet and Leishmaniardquo Parasitology Todayvol 2 no 6 pp 175ndash177 1986


[5] A Singh and D Mandal ldquoA novel sucroseH+ symport systemand an intracellular sucrase in Leishmania donovanirdquo Interna-tional Journal for Parasitology vol 41 no 8 pp 817ndash826 2011

[6] E Handman ldquoLeishmaniasis current status of vaccine develop-mentrdquo Clinical Microbiology Reviews vol 14 no 2 pp 229ndash2432001

[7] F R Opperdoes and P A M Michels ldquoEnzymes of carbohyd-rate metabolism as potential drug targetsrdquo International Journalfor Parasitology vol 31 no 5-6 pp 482ndash490 2001

[8] J J Blum and F R Opperdoes ldquoSecretion of sucrase by Leish-mania donovanirdquo The Journal of Eukaryotic Microbiology vol41 no 3 pp 228ndash231 1994

[9] A Kumar Saha T Mukherjee and A Bhaduri ldquoMechanism ofaction of amphotericin B on Leishmania donovani promastig-otesrdquoMolecular and Biochemical Parasitology vol 19 no 3 pp195ndash200 1986

[10] T Kawasaki ldquoHydroxyapatite as a liquid chromatographicpackingrdquo Journal of Chromatography A vol 544 pp 147ndash1841991

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquoThe Jour-nal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[12] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[13] O Gabriel and S-F Wang ldquoDetermination of enzymatic activ-ity in polyacrylamide gels I Enzymes catalyzing the conversionof nonreducing substrates to reducing productsrdquo AnalyticalBiochemistry vol 27 no 3 pp 545ndash554 1969

[14] MMesser andA Dahlqvist ldquoA one-step ultramicromethod forthe assay of intestinal disaccharidasesrdquo Analytical Biochemistryvol 14 no 3 pp 376ndash392 1966

[15] N F Gontijo M N Melo E B Riani S Almeida-Silva and ML Mares-Guia ldquoGlycosidases in Leishmania and their impor-tance for Leishmania in phlebotomine sandflies with specialreference to purification and characterization of a sucraserdquoExperimental Parasitology vol 83 no 1 pp 117ndash124 1996

[16] F P Chavez L Rodriguez J Dıaz J M Delgado and J ACremata ldquoPurification and characterization of an invertasefrom Candida utilis comparison with natural and recombinantyeast invertasesrdquo Journal of Biotechnology vol 53 no 1 pp 67ndash74 1997

[17] G L F Wallis I F W Hemming and J F Peberdy ldquoSecretionof two 120573-fructofuranosidases by Aspergillus niger growing insucroserdquo Archives of Biochemistry and Biophysics vol 345 no2 pp 214ndash222 1997

[18] M Warchol S Perrin J P Grill et al ldquoCharacterization ofa purified b-fructofuranosidase from Bifidobacterium infantisATCC 15697rdquo Letters in Applied Microbiology vol 35 pp 462ndash467 2002

[19] S M Ryan G F Fitzgerald and D van Sinderen ldquoTranscrip-tional regulation and characterization of a novel 120573-fructofura-nosidase-encoding gene fromBifidobacteriumbreveUCC2003rdquoApplied and EnvironmentalMicrobiology vol 71 no 7 pp 3475ndash3482 2005

[20] B Kullin V RAbratt and S J Reid ldquoA functional analysis of theBifidobacterium longum cscA and scrP genes in sucrose utili-zationrdquo Applied Microbiology and Biotechnology vol 72 no 5pp 975ndash981 2006

[21] M Rossi C Corradini A Amaretti et al ldquoFermentation offructooligosaccharides and inulin by bifidobacteria a compara-tive study of pure and fecal culturesrdquoApplied and EnvironmentalMicrobiology vol 71 no 10 pp 6150ndash6158 2005

[22] T A Lyda M B Joshi J F Andersen et al ldquoA unique highlyconserved secretory invertase is differentially expressed bypromastigote developmental forms of all species of the humanpathogen Leishmaniardquo Molecular and Cellular Biochemistryvol 404 no 1-2 pp 53ndash77 2015

[23] VHannaert F Bringaud F R Opperdoes and P AMMichelsldquoEvolution of energy metabolism and its compartmentation inKinetoplastidardquoKinetoplastid Biology and Disease vol 2 article11 2003

[24] M Dirkx M P Boyer P Pradhan A Brittingham and W AWilson ldquoExpression and characterization of a 120573-fructofurano-sidase from the parasitic protist Trichomonas vaginalisrdquo BMCBiochemistry vol 15 article 12 2014

[25] JM Carlton R PHirt J C Silva et al ldquoDraft genome sequenceof the sexually transmitted pathogen Trichomonas vaginalisrdquoScience vol 315 no 5809 pp 207ndash212 2007

[26] N M El-Sayed P J Myler G Blandin et al ldquoComparativegenomics of trypanosomatid parasitic protozoardquo Science vol309 no 5733 pp 404ndash435 2005

[27] S Belaz T Rattier P Lafite et al ldquoIdentification biochemicalcharacterization and in-vivo expression of the intracellularinvertase BfrA from the pathogenic parasite LeishmaniamajorrdquoCarbohydrate Research vol 415 pp 31ndash38 2015

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


gain insight into its probable role in biochemical pathwayof the parasite and pathogenesis This understanding maycontribute knowledge towards antileishmanial drug design-ing

2 Materials and Methods

21 Materials Analytical grade reagents were used forexperimental purpose All the chemicals were purchasedfrom Sigma USA unless otherwise mentioned Brain HeartInfusion was obtained from Acumedia Manufacturers IncBaltimoreMDUSA andMedia 199 Penicillin-Streptomycinpowder were purchased from GIBCO USA

22 Strains The strain of L donovani used in this workMOHMIN1978UR6 was a clinical isolate from an Indianpatient with confirmedKala-azar collected in the year of 1978UR6 cells were maintained in solid blood agar media of pH74 and highly motile promastigotes were considered duringexperiments

23 Media and Culture Conditions

231 Solid BloodAgarMedia According to Kumar Saha et al[9] the cell line was maintained in solid blood agar media at22∘CThegrowth of promastigoteswasmeasured every 24 hrsof 72 hrs of growth period

232 Liquid Media 199 One liter of liquid medium wasprepared by adding 11 g ofmedia-199 power 10 FCS 22mMHepes and 100 units of Penicillin-Streptomycin in waterThepH of the media was adjusted to 74 and filter sterilized forfurther use

24 Preparation of Cell-Free Extract Cell-free extract wasprepared according to Singh and Mandal [5] Exponentiallygrowing promastigotes in liquid culture were harvestedwashed with PBS and suspended in lysis buffer containing(5mM Tris-HCl 05mM PMSF and 025mM EDTA pH74) The suspension incubated at ambient temperature wasmixed 10 times by vortexing 30 secs at 2min intervals Thiswas further sonicated at 15 pulses of 20 secs each with 1mininterval on ice The sonicated extract was adjusted to 50mMTris-HCl pH 74 (Buffer A) and ultracentrifuged at 1 times 105 gfor 1 hr at 4∘CThe supernatant considered as cell-free extractwas collected and stored at minus20∘C for further use

25 Purification Procedure

251 Enzyme Extraction The enzyme activity of the cell freeextract was considered as crude and its level of activity wastaken to be 100 for calculation of recovery

252 Ammonium Sulfate Precipitation Solid ammoniumsulfate was added into the cell-free extract first to 33saturation and then to 75 saturation The pellet holdingmajor sucrase activity was finally suspended in Buffer Acontaining protease inhibitor and immediately considered forfurther purification

253 Size-Exclusion Chromatography (SEC) Following am-monium sulfate concentration the resuspended sample(active fraction) was loaded onto a Sephacryl S-200 column(120 times 1 cm) preequilibrated with Buffer A The proteinswere eluted with Buffer A at a flow rate of 22mLhr Proteinfractionstube containing the major activity of sucrase werepooled for further steps of purification

254 Ion-Exchange Chromatography (IEC) A column (20times 2 cm) was packed with CM-Cellulose matrix swollenovernight at room temperature to have a bed volume of 10times 2 cm The pooled active enzyme fractions from S-200 col-umn were passed through CM-Cellulose (cation exchanger)columnpreequilibratedwithBufferA for further purificationThe flow-through containing the major sucrase activity waspooled immediately and subjected to DEAE Sephadex (anionexchanger) column of same bed volume preequilibrated withBuffer A The bound protein containing the enzyme fractionwas eluted with a salt gradient of 0ndash02M NaCl The pooledfraction from DEAE Sephadex was passed through S-200column (120 times 1 cm) again to equilibrate the semipurifiedenzyme sample with 20mM potassium-phosphate buffer pH74 for Hydroxyapatite batch adsorption

255 Hydroxyapatite Batch Adsorption Hydroxyapatite [10]matrix was equilibrated with 20mM potassium-phosphatebuffer at pH74Thepooled fraction containingmajor sucraseactivity from S-200 column was subjected to Hydroxyapatitebatch adsorption and allowed the enzyme to mix properlyby occasional stirring After the matrix settled down theunabsorbed content mostly purified enzyme was collectedby a low spin centrifugation at 4∘C The supernatant wasfound to contain gt95 purified enzyme

26 Protein Estimation Modified Lowry method was usedfor protein estimation [11] and BSA was taken as a standard

27 Gel Electrophoresis and Activity Staining Polyacrylamidegel electrophoresis (PAGE) under native and denaturingcondition was performed according to Laemmlirsquos discontin-uous Tris-glycine buffer system [12] with little modificationDuring activity staining the gel was sliced into two halveseach half bearing identical samples One part of 55 nativegel containing the purified intracellular sucrase enzyme wasincubated in 50mM Tris-HCl with 100mM sucrose for 2 hrsat 30∘C followed by a wash with distilled water [13] The gelwas then immersed in 1M of iodoacetamide for 15min atroom temperature Following the wash with double distilledwater the gel was incubated in 05N NaOH containing 2235-Triphenyl Tetrazolium Chloride (TTC) for 15min ina boiling water bath until a diffuse pink background colordevelops After proper distaining with 75 acetic acid aphoto was taken immediately as the color developed persistsvery shortly The other gel part was subsequently stainedin coomassie R-250 for the corresponding protein bandidentification

28 Purification of Extracellular Sucrase Promastigote cellsgrew in liquid culturemedia for 66-67 hrs were pelleted down





Fold purification

Crude extract 2084 790002 2637 1


IEC-CM-cellulose 85436 3628 23554 893








2 HgCl

2 CaCl

2 KCl AgNO

3 FeSO

4 MgSO

4

MnSO4 CuSO

4 CoSO

4 and NiSO



3 Results







Abso

rban

ce at

280

nm


0

5

10

15

20

25

30

35

Sucr

ase a

ctiv

ity (n

mol

esm

gm

in)

0

1

2

3

4

5

6

7

8

9


3

35

4

45

5

55 1 23

45

log M

W



119898of













2 AgNO

3 andHgCl



4 Discussion



Ovalbumin

Carbonic anhydrase

Sucrase


Phosphorylase

(b) (c) (d)

(kDa) 1 2

116

974

66

45

29

(a)

1 2

974


(kDa)

log10

MW

02 04 06 08 1044

45

46

47

48

49

5

51

52



0

20

40

60

80

100

(nm

oles

of g

luco

sem

inm

g)

25 50 75 10000Sucrose (mM)

15 40 65 90 115 140minus10

1S

005

015

025

035

045

055

1V

(a)


0

40

80

120

160

(nm

oles

of g

luco

sem

inm

g)

2000 05 10 15 25 30minus05minus10

1S

001

002

003

1V

(b)





0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)


KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)
















Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Fold purification

Crude extract 2084 790002 2637 1


IEC-CM-cellulose 85436 3628 23554 893








2 HgCl

2 CaCl

2 KCl AgNO

3 FeSO

4 MgSO

4

MnSO4 CuSO

4 CoSO

4 and NiSO



3 Results







Abso

rban

ce at

280

nm


0

5

10

15

20

25

30

35

Sucr

ase a

ctiv

ity (n

mol

esm

gm

in)

0

1

2

3

4

5

6

7

8

9


3

35

4

45

5

55 1 23

45

log M

W



119898of













2 AgNO

3 andHgCl



4 Discussion



Ovalbumin

Carbonic anhydrase

Sucrase


Phosphorylase

(b) (c) (d)

(kDa) 1 2

116

974

66

45

29

(a)

1 2

974


(kDa)

log10

MW

02 04 06 08 1044

45

46

47

48

49

5

51

52



0

20

40

60

80

100

(nm

oles

of g

luco

sem

inm

g)

25 50 75 10000Sucrose (mM)

15 40 65 90 115 140minus10

1S

005

015

025

035

045

055

1V

(a)


0

40

80

120

160

(nm

oles

of g

luco

sem

inm

g)

2000 05 10 15 25 30minus05minus10

1S

001

002

003

1V

(b)





0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)


KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)
















Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abso

rban

ce at

280

nm


0

5

10

15

20

25

30

35

Sucr

ase a

ctiv

ity (n

mol

esm

gm

in)

0

1

2

3

4

5

6

7

8

9


3

35

4

45

5

55 1 23

45

log M

W



119898of













2 AgNO

3 andHgCl



4 Discussion



Ovalbumin

Carbonic anhydrase

Sucrase


Phosphorylase

(b) (c) (d)

(kDa) 1 2

116

974

66

45

29

(a)

1 2

974


(kDa)

log10

MW

02 04 06 08 1044

45

46

47

48

49

5

51

52



0

20

40

60

80

100

(nm

oles

of g

luco

sem

inm

g)

25 50 75 10000Sucrose (mM)

15 40 65 90 115 140minus10

1S

005

015

025

035

045

055

1V

(a)


0

40

80

120

160

(nm

oles

of g

luco

sem

inm

g)

2000 05 10 15 25 30minus05minus10

1S

001

002

003

1V

(b)





0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)


KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)
















Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ovalbumin

Carbonic anhydrase

Sucrase


Phosphorylase

(b) (c) (d)

(kDa) 1 2

116

974

66

45

29

(a)

1 2

974


(kDa)

log10

MW

02 04 06 08 1044

45

46

47

48

49

5

51

52



0

20

40

60

80

100

(nm

oles

of g

luco

sem

inm

g)

25 50 75 10000Sucrose (mM)

15 40 65 90 115 140minus10

1S

005

015

025

035

045

055

1V

(a)


0

40

80

120

160

(nm

oles

of g

luco

sem

inm

g)

2000 05 10 15 25 30minus05minus10

1S

001

002

003

1V

(b)





0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)


KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)
















Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

20

40

60

80

100

120

of e

nzym

e act

ivity

(a)minus20

0

20

40

60

80

100

120

o

f enz

yme a

ctiv

ity

2 4 6 8 100pH

(b)


KCl

Non

e

CoS

O4

CuSO

4

MnS

O4

NiS

O4

MgS

O4

HgC

l 2

AgN

O3

CaCl

2

FeSO

4

ZnSO

4

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

of c

ontro

l)
















Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Disclosure


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article purification and characterization of a ... · research article purification and...

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