Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes

Biochemistry Research International, Apr 2016

The promastigote stage of Leishmania resides in the sand fly 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. However, 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 of 1.61 mM than the extracellular form having a of 4.4 mM. The highly specific activity of intracellular sucrase towards sucrose is optimal at pH 6.0 and at 30°C. In this report the purification and characterization of intracellular sucrase provide evidence that sucrase enzyme exists at least in two different forms in Leishmania donovani promastigotes. This intracellular sucrase may support further intracellular utilization of transported sucrose.

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Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes

Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes Arpita Singh and Debjani Mandal Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, West Bengal 700032, India Received 23 November 2015; Accepted 22 March 2016 Academic Editor: Stefano Pascarella Copyright © 2016 Arpita Singh and Debjani Mandal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The promastigote stage of Leishmania resides in the sand fly 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. However, 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 of 1.61 mM than the extracellular form having a of 4.4 mM. The highly specific activity of intracellular sucrase towards sucrose is optimal at pH 6.0 and at 30°C. In this report the purification and characterization of intracellular sucrase provide evidence that sucrase enzyme exists at least in two different forms in Leishmania donovani promastigotes. This intracellular sucrase may support further intracellular utilization of transported sucrose. 1. 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 result Leishmania donovani depends on carbohydrates to sustain their central carbon metabolism. This parasite has the ability to modify its biochemical machinery to adapt diverse microenvironment, encountered within the host in order to guarantee their survival. Sugar meal is important for the development 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 one of the preferred energy source where the division of the promastigotes takes place. Therefore it is important to understand how they utilize this sugar as well as the mechanism of sugar uptake inside the cell. Recently we have reported the sucrose transport system in Leishmania donovani promastigotes and the intracellular sucrose splitting enzyme sucrase, which may demonstrate the utilization of internalized sucrose [5]. However, sucrose internalization and henceforth consumption is a relatively unexplored area in Leishmania biology. Leishmaniasis still 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 [7] it is necessary to understand its physiology foremost and the inherent ability of the parasite to adapt itself to a myriad of adverse environmental parameters. Our recent finding on the intracellular pool of sucrase enzyme, as well as previous reports on secretary extracellular sucrase [5, 8], 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 characterization of the intracellular enzyme is important to gain insight into its probable role in biochemical pathway of the parasite and pathogenesis. This understanding may contribute knowledge towards antileishmanial drug designing. 2. Materials and Methods2.1. Materials Analytical grade 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, USA, and Media 199, Penicillin-Streptomycin powder, were purchased from GIBCO, USA. 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 year of 1978. UR6 cells were maintained in solid blood agar media of pH 7.4 and highly motile promastigotes were considered during experiments. 2.3. Media and Culture Conditions2.3.1. Solid Blood Agar Media According to Kumar Saha et al. [9] the cell line was maintained in solid blood agar media at 22°C. The growth of promastigotes was measured every 24 hrs of 72 hrs of growth period. 2.3.2. Liquid (...truncated)


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Arpita Singh, Debjani Mandal. Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes, Biochemistry Research International, 2016, 2016, DOI: 10.1155/2016/7108261