title: Purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis: Induce apoptosis in human leukemia MOLT-4 cells.
abstract: Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this enzymatic drug is responsible for several life threatening side effects. This study describes the purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis. The purified enzyme exhibited molecular mass of approximately 205±3 kDa on native-PAGE and ̴34±1 kDa on SDS-PAGE, revealing that the enzyme is homohexamer. The isoelectric point of enzyme was 5.5, calculated by 2D-PAGE. Optimum activity of asparaginase was achieved at 40 °C and pH 7.5, which is close to the internal environment of the human body. Monovalent cations (Na(+) and K(+)) and reducing agents (2-mercaptoethanol and glutathione) has enhanced asparaginase activity. Whereas, divalent (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)), trivalent (Fe(3+)) cations and thiol group blocking agent (iodoacetamide) inhibited the enzyme activity significantly. In vitro serum and trypsin half life of asparaginase is almost 2 and 1.5 fold respectively, which is higher than commercial asparaginase. MTT assay results showed that the anticancer activity of purified asparaginase was comparable or higher than commercial E. coli asparaginase. Microscopic studies and cell cycle analysis suggested that purified enzyme induced apoptotic cell death in dose-dependent manner. Loss of mitochondrial membrane potential suggests that enzyme induces cell death via activation of intrinsic apoptotic pathway. Purified asparaginase was found to be nontoxic for human noncancerous FR-2 cells and human blood lymphocytes, which is a remarkable therapeutic feature.
authors.affiliation: Bacteriology Laboratory, Department of P. G. Studies and Research in Biological Science, Rani Durgavati University, Madhya Pradesh, India.
authors.forename: Islam
authors.initials: I
authors.lastname: Husain
authors.affiliation: Bacteriology Laboratory, Department of P. G. Studies and Research in Biological Science, Rani Durgavati University, Madhya Pradesh, India. Electronic address: anjoo1999@gmail.com.
authors.forename: Anjana
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Cancer Pharmacology Division, Indian Institute of Integrative Medicine (IIIM-CSIR), Jammu & Kashmir, India.
authors.forename: Suresh
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Cancer Pharmacology Division, Indian Institute of Integrative Medicine (IIIM-CSIR), Jammu & Kashmir, India.
authors.forename: Fayaz
authors.initials: F
authors.lastname: Malik
mesh_terms: D017209:Apoptosis / Q000502:physiology; D001215:Asparaginase / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005972:Glutaminase / Q000378:metabolism*; D006801:Humans; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D001215:Asparaginase; D005972:Glutaminase
keywords: Asparaginase; Cell cycle analysis; Glutaminase; MMP loss; MTT assay; Purification