Optimum Ph For Sucrase Activity
Biochem Res Int. 2016; 2016: 7108261.
Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes
Arpita Singh
Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4 Raja Due south.C. Mullick Route, Jadavpur, Kolkata, West Bengal 700032, India
Debjani Mandal
Division of Infectious Diseases and Immunology, Indian Plant of Chemical Biology, four Raja S.C. Mullick Road, Jadavpur, Kolkata, Westward Bengal 700032, Republic of india
Received 2015 Nov 23; Accepted 2016 Mar 22.
Abstruse
The promastigote stage of Leishmania resides in the sand wing gut, enriched with sugar molecules. Recently we reported that Leishmania donovani possesses a sucrose uptake system and a stable pool of intracellular sucrose metabolizing enzyme. In the present study, we purified the intracellular sucrase nearly to its homogeneity and compared it with the purified extracellular sucrase. The estimated size of intracellular sucrase is ~112 kDa by gel filtration chromatography, native PAGE, and substrate staining. All the same, in SDS-PAGE, the protein is resolved at ~56 kDa, indicating the possibility of a homodimer in its native state. The kinetics of purified intracellular sucrase shows its higher substrate affinity with a K m of i.61 mM than the extracellular form having a Thousand m of 4.4 mM. The highly specific action of intracellular sucrase towards sucrose is optimal at pH 6.0 and at 30°C. In this report the purification and label of intracellular sucrase provide evidence that sucrase enzyme exists at least in ii different forms in Leishmania donovani promastigotes. This intracellular sucrase may support farther intracellular utilization of transported sucrose.
one. Introduction
Sucrose, glucose, and other hexose's are required for the maintenance of parasite redox balance and for generating precursors for Dna and RNA biosynthesis. As a outcome Leishmania donovani depends on carbohydrates to sustain their cardinal carbon metabolism. This parasite has the ability to modify its biochemical machinery to adapt diverse microenvironment, encountered within the host in guild to guarantee their survival.
Saccharide meal is of import for the evolution of infective forms of Leishmania sp. and for its virulency [1, 2]. Considering the major metabolite constituents in sandfly gut [3, 4], sucrose presumably is ane of the preferred free energy source where the division of the promastigotes takes identify. Therefore it is important to empathise how they use this sugar likewise as the mechanism of sugar uptake inside the cell. Recently we have reported the sucrose ship organization in Leishmania donovani promastigotes and the intracellular sucrose splitting enzyme sucrase, which may demonstrate the utilization of internalized sucrose [five]. However, sucrose internalization and henceforth consumption is a relatively unexplored area in Leishmania biology. Leishmaniasis notwithstanding remains a major health concern of the 21st century throughout the world, despite the sustained efforts to control the disease over several decades. Along with existing efforts of developing vaccines [6] and improved drugs [seven] it is necessary to understand its physiology foremost and the inherent power of the parasite to suit itself to a myriad of adverse ecology parameters.
Our recent finding on the intracellular puddle of sucrase enzyme, as well equally previous reports on secretary extracellular sucrase [5, viii], prompted us to purify the intracellular sucrase. Here we identified a ~112 kDa homodimer intracellular sucrase enzyme of Leishmania donovani promastigotes and characterized and compared it simultaneously with the purified ~71 kDa extracellular sucrase. Further detailed molecular label of the intracellular enzyme is of import to gain insight into its probable role in biochemical pathway of the parasite and pathogenesis. This understanding may contribute cognition towards antileishmanial drug designing.
2. Materials and Methods
two.1. Materials
Analytical class reagents were used for experimental purpose. All the chemicals were purchased from Sigma, USA, unless otherwise mentioned. Brain Heart Infusion was obtained from Acumedia Manufacturers Inc. Baltimore, MD, United states of america, and Media 199, Penicillin-Streptomycin powder, were purchased from GIBCO, U.s..
2.2. Strains
The strain of L. donovani used in this work, MOHM/IN/1978/UR6, was a clinical isolate from an Indian patient with confirmed Kala-azar collected in the twelvemonth of 1978. UR6 cells were maintained in solid claret agar media of pH 7.4 and highly motile promastigotes were considered during experiments.
two.3. Media and Culture Atmospheric condition
2.three.1. Solid Blood Agar Media
According to Kumar Saha et al. [9] the cell line was maintained in solid claret agar media at 22°C. The growth of promastigotes was measured every 24 hrs of 72 hrs of growth period.
2.3.two. Liquid Media 199
One liter of liquid medium was prepared by adding 11 m of media-199 power, ten% FCS, 22 mM Hepes, and 100 units of Penicillin-Streptomycin in water. The pH of the media was adjusted to vii.4 and filter sterilized for farther use.
2.4. Preparation of Cell-Free Excerpt
Cell-free extract was prepared according to Singh and Mandal [5]. Exponentially growing promastigotes in liquid civilisation were harvested, washed with PBS, and suspended in lysis buffer containing (5 mM Tris-HCl, 0.5 mM PMSF, and 0.25 mM EDTA, pH seven.4). The suspension incubated at ambient temperature was mixed 10 times past vortexing 30 secs at 2 min intervals. This was further sonicated at fifteen pulses of 20 secs each with one min interval on ice. The sonicated extract was adjusted to 50 mM Tris-HCl, pH 7.iv (Buffer A), and ultracentrifuged at 1 × 10five m for ane hr at 4°C. The supernatant considered as cell-free extract was collected and stored at −20°C for further apply.
two.5. Purification Process
2.five.1. Enzyme Extraction
The enzyme action of the cell free excerpt was considered every bit crude and its level of activeness was taken to be 100% for calculation of recovery.
2.5.two. Ammonium Sulfate Precipitation
Solid ammonium sulfate was added into the cell-free excerpt, beginning to 33% saturation and then to 75% saturation. The pellet belongings major sucrase activeness was finally suspended in Buffer A containing protease inhibitor and immediately considered for further purification.
2.5.3. Size-Exclusion Chromatography (SEC)
Post-obit ammonium sulfate concentration the resuspended sample (active fraction) was loaded onto a Sephacryl S-200 cavalcade (120 × 1 cm) preequilibrated with Buffer A. The proteins were eluted with Buffer A at a flow charge per unit of 22 mL/hr. Protein fractions/tube containing the major activity of sucrase were pooled for further steps of purification.
2.5.iv. Ion-Exchange Chromatography (IEC)
A cavalcade (20 × 2 cm) was packed with CM-Cellulose matrix, swollen overnight at room temperature to have a bed volume of ten × 2 cm. The pooled agile enzyme fractions from South-200 cavalcade were passed through CM-Cellulose (cation exchanger) column preequilibrated with Buffer A for farther purification. The period-through containing the major sucrase action was pooled immediately and subjected to DEAE Sephadex (anion exchanger) column of same bed volume preequilibrated with Buffer A. The bound protein containing the enzyme fraction was eluted with a salt gradient of 0–0.2 M NaCl. The pooled fraction from DEAE Sephadex was passed through S-200 column (120 × 1 cm) over again to equilibrate the semipurified enzyme sample with 20 mM potassium-phosphate buffer pH 7.four for Hydroxyapatite batch adsorption.
2.5.five. Hydroxyapatite Batch Adsorption
Hydroxyapatite [x] matrix was equilibrated with xx mM potassium-phosphate buffer at pH 7.4. The pooled fraction containing major sucrase activity from Due south-200 column was subjected to Hydroxyapatite batch adsorption and allowed the enzyme to mix properly past occasional stirring. After the matrix settled down, the unabsorbed content, mostly purified enzyme, was collected by a depression spin centrifugation at 4°C. The supernatant was establish to comprise >95% purified enzyme.
ii.vi. Protein Estimation
Modified Lowry method was used for protein estimation [eleven] and BSA was taken as a standard.
2.7. Gel Electrophoresis and Activity Staining
Polyacrylamide gel electrophoresis (Folio) under native and denaturing condition was performed according to Laemmli'south discontinuous Tris-glycine buffer system [12] with little modification. During activity staining the gel was sliced into 2 halves, each one-half bearing identical samples. 1 part of 5.v% native gel containing the purified intracellular sucrase enzyme was incubated in 50 mM Tris-HCl with 100 mM sucrose for 2 hrs at 30°C followed by a wash with distilled water [13]. The gel was then immersed in ane Grand of iodoacetamide for fifteen min at room temperature. Post-obit the wash with double distilled water, the gel was incubated in 0.v Due north NaOH containing two% 2,three,5-Triphenyl Tetrazolium Chloride (TTC) for xv min in a boiling water bathroom until a diffuse pinkish background color develops. Afterwards proper distaining with 7.five% acetic acid, a photograph was taken immediately as the color adult persists very soon. The other gel function was subsequently stained in coomassie R-250 for the corresponding protein ring identification.
two.8. Purification of Extracellular Sucrase
Promastigote cells grew in liquid culture media for 66-67 hrs were pelleted down by centrifugation and the cell-gratuitous media was lyophilized to semidryness. It was reconstituted in 50 mM Potassium-Phosphate buffer, pH 7.4, and concentrated to ~1.v mL by using a centricon membrane filter YM 10. Following the same purification procedure of intracellular sucrase, the filtrate was allowed to laissez passer through the preequilibrated Due south-200 column. Subsequent ion-commutation chromatography and Hydroxyapatite batch adsorption steps were performed to obtain purified extracellular enzyme.
2.9. Enzyme Assays
With slight modification to Messer and Dahlqvist'southward method [14] sucrase activity was estimated. The enzyme reaction was initiated by adding the enzyme in the analysis mixture (l mM sodium acetate, pH 5.5/6.0) in presence of four mM of substrate sucrose and the reaction stopped by oestrus inactivation after 30 min of incubation at xxx°C. The colorimetric estimation of glucose was taken at 505 nm. One unit of enzyme activeness is defined as the amount of enzyme that hydrolyzes sucrose to produce 1 mole of glucose at xxx°C.
ii.x. Optimum Temperature and Thermostability
The optimum temperature for the enzyme was evaluated by measuring sucrase activity in fifty mM sodium acetate buffer (pH 5.v–6.0) at different temperature. Thermostability was determined past preincubating the purified enzyme for 30 min at a range of temperature (4–60°C) prior to the standard activity assay.
two.11. Optimum pH
Purified enzyme activity was assayed in 50 mM of four different buffering agents, glycine-HCl (pH ii.0-3.0), acetate (pH 4.0–half dozen.0), phosphate (pH 6.0-vii.0), and Tris-HCl (pH 7.0-8.0), in order to record the pH profile under the standard experimental conditions.
2.12. Effect of Metallic Ions on Intracellular Sucrase Activity
The metal ions outcome on enzyme activity was determined after preincubating the purified enzyme with various metal ions such as ZnCltwo, HgCl2, CaCl2, KCl, AgNOiii, FeSO4, MgSO4, MnSO4, CuSO4, CoSO4, and NiSOiv, one at a time at desired concentration for 10 min at 30°C. Post-obit standard assay condition enzyme activity was measured and expressed equally percent of control (without metallic ion).
3. Results
In the present study both the intra- and extracellular enzymes were simultaneously purified to characterize the intracellular sucrase enzyme in comparison to extracellular one. The molecular size of intracellular sucrase was confirmed by size-exclusion chromatography and native gel analysis followed past activity staining.
3.one. Molecular Weight Conclusion
3.1.one. Size-Exclusion Chromatography
Size-exclusion chromatography of cell-free excerpt followed by ammonium sulfate atmospheric precipitation shows intracellular sucrase activity (Figure i) and the molecular mass of the poly peptide estimated from gel filtration chromatography was approximately 112 kDa (Effigy i inset). The active fractions from S-200 column were pooled and passed through different steps (Table 1) to get pure active protein. The entire purification steps yield almost 330-fold the purified intracellular sucrase.
Size-exclusion chromatography of intracellular sucrase. The ammonium sulfate saturated sample protein was run on a Sephacryl (Due south-200) column, preequilibrated with Buffer A. The protein absorbance measured at 280 nm (-◆-) was plotted on primary centrality. The secondary y-axis shows the intracellular sucrase action (-■-) of the corresponding fractions. The molecular weight of the native enzyme was determined to be ~112 kDa from the Due south-200 scale graph (Figure one inset). The ↑ arrow indicates the top of intracellular sucrase elution volume and the molecular weight (MW) of the poly peptide. The molecular weight markers used were as follows: amylase MW = 200,000; alcohol dehydrogenase MW = 150,000; hemoglobin MW = 64,500; carbonic anhydrase MW = 29,000; and cytochrome C MW = 12,400.
Table 1
Purification of intracellular sucrase: the tabular array shows the list of unlike steps, taken during the purification procedure. The total enzyme activity, the amount of poly peptide recovered, and the specific activity of the sucrase enzyme were estimated from each step of the purification procedure to summate the increased fold of purification.
| Steps of purification | Total activity nmoles of glucose formed/min | Total protein µg | Specific activity nmoles of glucose/mg/min | Fold purification |
|---|---|---|---|---|
| Crude extract | 2084 | 790002 | 26.37 | 1 |
| Size-exclusion chromatography | 1075 | 11078 | 97.03 | 3.67 |
| IEC-CM-cellulose | 854.36 | 3628 | 235.54 | 8.93 |
| IEC-DEAE Sephadex | 459.08 | 1250 | 367 | thirteen.92 |
| SEC-Sephacryl South-200 | 154.64 | 34.54 | 4477.12 | 169.78 |
| Hydrophobic chromatography (HA batch absorption) | 103.86 | 11.816 | 8789.77 | 333.32 |
three.1.ii. Relative Mobility
Presence of a single band in SDS-Folio (Figure 2(a)) reveals the purity of the enzyme. The estimated size of the protein is nearly 56 kDa equally calculated from the relative mobility and the molecular weight of the known marker protein (Figure 2(b)). The 56 kDa protein size is near half the size estimated from gel filtration chromatography (112 kDa. Effigy 1 inset), which may denote the homodimerization of the enzyme sucrase in its native land.
Gel electrophoretic assay and action staining of purified intracellular sucrase. (a) Electrophoresis (10% SDS-Page) was done in a discontinuous buffer organisation. Lane 1: molecular mass standards indicated on the left are as follows: galactosidase (116 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa); lane 2 shows the purified intracellular sucrase protein band of almost ~56 kDa in size. (b) x% SDS-Page standard curve plotted with logarithm of the molecular weight confronting relative mobility. The pointer indicates the log of molecular weight of intracellular sucrase subunit in x% SDS-Folio. (c) Substrate staining of purified intracellular sucrase in native gel; the arrow indicates the action ring in native gel. (d) Coomassie stained purified intracellular sucrase in native gel. Lane 1 represents the proteins galactosidase (116 kDa) and phosphorylase B (97.4 kDa) as molecular weight marker and lane 2 shows the native form of purified intracellular sucrase.
3.ane.3. Activity Staining
A confirmatory test for identifying the enzyme is to locate the enzymatic activity in the polyacrylamide gel. Thus, to confirm the position of intracellular sucrase amidst electrophoretically separated proteins, a modified approach of Gabriel and Wang [13] was used for substrate staining of the native intracellular sucrase. Purified protein shows a deep pink violet band, representing the active protein (Figure ii(c)). The subsequent unmarried ring in coomassie stain corresponding to the substrate stain (Figure two(d)) confirms its approximate size of 112 kDa. This result corroborates with the protein size estimated from the gel filtration chromatography.
three.2. Enzyme Kinetics of Crude and Purified Intracellular Sucrase
The intracellular sucrase action was measured at different substrate concentration. The range of substrate concentration varied from 0.05–10 mM to 0.4–v.0 mM, respectively, for estimating the rough and purified intracellular sucrase enzyme activity. The Lineweaver-Burk plot of the rough intracellular sucrase enzyme activity shows the K chiliad of ~6.six mM and the 5 max of ~125 nmoles/min/mg (Figure three(a)), while the purified intracellular enzyme has more affinity towards the substrate as calculated from the Lineweaver-Burk plot and the estimated K 1000 and V max are ~i.6 mM and ~190.five nmoles/min/mg, respectively (Effigy 3(b)).
Enzyme kinetics of crude and purified intracellular sucrase: intracellular sucrase was incubated in the assay mixture with varying range of substrate concentration (0.20–ten mM) at a fixed incubation fourth dimension to estimate the enzyme activity of rough (a) and purified (b) intracellular sucrase. The inset represents the Lineweaver-Burk plot of velocity and substrate concentration of crude and purified intracellular sucrase which shows K chiliad of 6.half dozen mM and 1.61 mM of sucrose, respectively. The results are the mean of three independent experiments (n = 3).
three.3. Substrate Specificity
Different disaccharides were used to check the substrate specificity of the purified intracellular enzyme. The enzyme was incubated in presence of different substrate, namely, raffinose, melibiose, maltose, trehalose, and palatinose, and the corresponding enzyme activity was measured according to Messer and Dahlqvist [14] in comparison with sucrose every bit a control (100%). The substrate specificity of the Leishmania intracellular sucrase is highly specific in nature, as it was unable to hydrolyze any of the substrates mentioned above except sucrose and partially raffinose at a concentration range from one to 10 mm (data non shown).
3.four. Molecular Mass and Kinetics of Extracellular Sucrase
Molecular mass of extracellular sucrase, determined by size-exclusion chromatography and SDS-PAGE (data not shown), was ~70.79 kDa. For kinetic study, enzyme activity of the purified extracellular sucrase was estimated by incubating with varying substrate concentration (0.125–eight mM) at thirty°C for xxx min. The double reciprocal plot of the velocity of the reaction and the substrate shows the K thousand of purified extracellular sucrase equally ~4.4 mM, which corroborates the report on purified extracellular sucrase of Leishmania by Gontijo et al. [xv]. The upshot illustrates that the purified extracellular sucrase has virtually three times reduced substrate affinity than that of intracellular sucrase enzyme.
Purification and the subsequent kinetic studies of the purified enzyme further confirmed that at least 2 dissimilar forms of the enzyme sucrase be in 50. donovani promastigotes.
3.five. Temperature Tolerance and pH Sensitivity of Intra- and Extracellular Sucrase
Intracellular sucrase is susceptible to higher temperatures and loses 50% of its activity with an increase of temperature above 45°C. On the other hand the extracellular sucrase is mostly stable upwardly to 50°C and has a wide range of temperature tolerance (Figure 4(a)). The intracellular sucrase shows ~20% of its maximum activeness at this temperature and so gets deactivated completely with further increase of temperature. Nonetheless both the purified enzymes showroom its maximum activity at 30°C (data non shown).
The temperature tolerance and optimum pH of intra- and extracellular sucrase: (a) the intra- and extracellular enzymes were preincubated for xxx min at dissimilar temperature (4–threescore°C) prior to estimating the enzyme activity under standard assay condition in 50 mM sodium acetate buffer at pH half dozen.0 and pH five.5, respectively. The enzyme activities of intracellular (-◆-) and extracellular sucrase (-●-) were plotted as percent of maximum action (100%) at different temperature. (b) The pH optimum was measured by performing activity analysis at different pH at a range of 02.75–8.0 as mentioned in Materials and Methods. The intracellular (-▲-) and extracellular (-◆-) sucrase activities were plotted as a percent of activeness versus pH, with maximum activity being 100%. Each point represents the mean of three different experiments.
Interestingly the intracellular sucrase shows nigh 80% of its activity at a pH range from 4.5 to vii.0 (Figure 4(b)); still, the optimum enzyme activity occurs at pH 6.0 (data not shown). In standard assay condition the maximum enzyme activity of purified extracellular sucrase appears at pH v.v (data not shown) although more than fourscore% of its activity was observed between pH 5.0 and 6.5 (Effigy 4(b)).
3.6. Effect of Metal Ion on Intracellular Sucrase Activeness
The enzyme activity was compared in presence of various metallic ions at i mM concentration as presented in Figure 5. Metal ions such as ZnCl2, AgNOthree, and HgCl2 strongly inhibited the enzyme activeness, while a moderate inhibition of activity was noted by the sulfate of cobalt, nickel, and copper ions.
Effect of metal ion on the intracellular sucrase enzyme activity. The purified enzyme was preincubated with metallic ions for 10 min at room temperature and the activity assay was performed in 50 mM sodium acetate buffer at pH half-dozen.0. The enzyme activity in absenteeism of metallic ions was considered to be 100% and compared the relative activeness of the enzyme in presence of unlike metal ions. The value presented corresponds to the mean value of three replicates.
4. Give-and-take
The parasites in the insect vector are exposed to an entirely different microenvironment than their vertebrate host. Leishmania promastigotes possess sucrose transporter in the plasma membrane [5], which helps the internalization of sucrose, a major food elective of the insect gut. To address the outcome on farther utilization of the accumulated sucrose in Fifty. donovani promastigotes we focus on the sucrose metabolizing enzyme sucrase. Recently we reported [v] that the majority of enzyme remains within the cell every bit intracellular sucrase and the remainder is secreted equally extracellular sucrase. These sucrase enzymes are constitutive in nature; the specific activity of the enzyme remains the same in the presence or absence of external pressure (i.e., sucrose) in the media. The K one thousand of intracellular sucrase in cell-costless excerpt and the purified form differs markedly from each other. Reduced analogousness of the rough enzyme towards the substrate may occur by the interference of cytosolic inhibitory factors.
The distinctive characteristic properties of the enzyme sucrase advise the possibility of having 2 forms of enzyme in Leishmania promastigotes, a ~71 kDa monomer extracellular sucrase and a ~56 kDa homodimer of intracellular sucrase. So there is a probability that the monomer of intracellular sucrase with an agile catalytic site may be posttranslationally modified to go extracellular sucrase [viii]. All the same, to determine whether each monomer is catalytically active or not, further studies need to exist done. The pregnant difference observed in the kinetics betwixt the two forms of the sucrase may be due to differing accessibility and efficiency of catalytic sites.
It has been established that the two forms of invertase extracellular and intracellular one in Saccharomyces cerevisiae, do non differ much in K thou or velocity, even so pH stability changes [16]. According to Wallis et al. [17] Aspergillus niger secretes two fructofuranosidases and both may be dimers in their natural conformation considering the protein size in SDS-PAGE and native state. The enzymes take affinity towards sucrose and to some extent to raffinose; nevertheless, their analogousness varies with other substrates. In Leishmania the wide range of temperature tolerance of extracellular sucrase is probably to remain functional in outside temperatures; in contrast the intracellular sucrase has slight temperature tolerance (Effigy four(a)). Potent inhibition of intracellular enzyme activity with thiol modifying reagents like 5,5′-dithiobis-ii-nitrobenzoic acid (DTNB), N-ethylmaleimide (NEM), and p-chloromercuribenzoate (PCMB) suggests that the active site of the enzyme may possess –SH moieties (data not shown). This finding corroborates the report on purified b-fructofuranosidase of B. infantis [18].
The enzymatic characterization and preliminary information on the N terminal sequence of the purified intracellular sucrase bear witness its considerable homology with glycosidase, the bacterial β-fructofuranosidase class of enzyme [19], nether the broader heading glycosidase (Singh and Mandal, unpublished data). This supports the findings of bacterial nature in many of the enzymes, which lies in the metabolic machinery of Leishmania [20]. The β-fructofuranosidases belong to the glycosyl hydrolase'due south family unit of 32 proteins and catalyze the hydrolysis of sucrose to glucose and fructose. Literature survey suggests that the bifidobacterial β-fructofuranosidases also take activity against the longer chained substrates such as raftilose, raftiline, and inulin [21]. The phylogenetic assay of the related intracellular fructofuranosidases includes a big grouping of sucrose-6P-hydrolases, of which all are physically linked with genes encoding sucrose transport proteins of the PTS [20]. Very recently Lyda et al. [22] discovered the secretory invertase (LdINV) gene of Leishmania promastigotes and too identified a beta-fructofuranosidase-like gene, during the homology search of LdINV, which encodes a 120 kDa protein. To validate our preliminary results farther study is necessary to identify the gene encoding intracellular sucrase.
The uptake and subsequent metabolism of glucose in Trypanosomatidae is an example of an adjustment leading to maximum energy efficiency [23]. Yet, it varies from species to species as L. donovani is confronted with widely varying conditions in the sandfly gut and strives for internal homeostasis even at the expense of free energy. Thus information technology may happen that two different metabolic strategies represent two opposing trends: the capability of uptake of sucrose and its utilization by hydrolyzing sucrose to glucose and fructose are the efficient adaptation at the expense of short term flexibility and on the other hand the power to rapidly adapt to environmental changes at the expense of free energy.
Interestingly very recently Dirkx et al. [24] reported that the flagellated protozoan Trichomonas vaginalis genome contains nearly 11 putative sucrose transporters and a putative ß-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of intracellular sucrose appears to be present in the protozoa equally the cell lysates retain invertase activity. It is likely that the virtually recent common ancestor of T. vaginalis was a gut-dwelling protist, where the capacity to apply fructose containing compounds might be advantageous [25].
Representatives of the Kinetoplastids spread over a variety of different environments and, in due time, many of them became parasites of insects, leeches, major vertebrates lineages, and fifty-fifty plants. Where Kinetoplastids evolved to digenetic parasites, involving two different hosts and often even unlike host tissues, they had to find means for efficient adaptation of their metabolism at highly unlike environments encountered. This implied the development of mechanisms to regulate differentially the expression of their metabolism in different life cycle stages. Thus metabolic flexibility must take been a highly selective advantage during the dissimilar stages of this evolutionary scenario. Trypanosomatids plainly take a considerable number of plants-like traits and several plants-like genes encoding homologs of proteins found in either chloroplast or the cytosol of plants and algae. In fact, elegant studies had proved before that many of the genes in different Trypanosomatids are orthologues [26]. Thus Leishmania, a Trypanosomatid parasite belonging to the order Kinetoplastida, together with Euglenoids, is under Euglenozoa. In this properties, thus, it is not surprising to observe that Leishmania possesses an efficient sucrose transporter and metabolizing machinery. Recent report on intracellular invertase BfrA, of Fifty. major [27], further supports our hypothesis on the participation of intracellular sucrase of L. donovani promastigotes in the sucrose metabolism pathway. Our limited findings are a fractional try to explore this very intriguing field of metabolite. Concluding remarks can only exist that future noesis of the role of this sucrase enzyme in the physiology and life cycle of the parasite can lead to the opening upwards of many avenues towards the better perception of parasitic biology and hence containment of this dreaded illness.
Acknowledgments
The authors thank Professor Due south. Roy, old Director of IICB, Kolkata, India, for supporting this work and Dr. Tanmoy Mukherjee (IICB, Kolkata, India) for helpful suggestions and comments on designing experiments. They are thankful to Dr. Ashis Chowdhury (USUHS, Maryland, The states) for carefully editing the paper.
Disclosure
The present Address for Arpita Singh is University of Virginia, Department of Medicine, Infectious Diseases, Charlottesville, VA 22904-4101, USA. Corresponding address for Debjani Mandal is Uniformed Services University of Health Scientific discipline, Department of Biochemistry and Molecular Biological science, School of Medicine, Bethesda, Doctor 20814, USA.
Competing Interests
The authors declare that they have no competing interests.
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Optimum Ph For Sucrase Activity,
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