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Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis

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Abstract

The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.

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Acknowledgments

S.M. acknowledges the Research fellowship in science for meritorious students (RFSMS), U.G.C., New Delhi. Partial financial supports from the DST-PURSE, UGC-CAS, UGC-UPE, DBT-BHU-ISLS to RM are sincerely acknowledged. Authors thank Prof. Nagendra Kumar, Professor in English, IIT, Roorkee, and Prof. S. P. Shukla, Professor in Zoology (Retd.), for their valuable time and help in editing our manuscript. We are thankful to DBT-BHU-ISLS for doing MALDI-MS/MS analysis, the Department of Zoology, BHU for providing SEM facility. Technical supports during SEM studies by Prof. Dinesh Kumar and Mr. Gaurav Srivastava are appreciated. We also appreciate AIIMS, New Delhi for extending TEM facility for this study.

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  1. Biochemistry and Molecular Biology Lab, Department of Zoology, Banaras Hindu University, Varanasi, 221005, India

    Suman Mishra & Rajnikant Mishra

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  1. Suman Mishra
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  2. Rajnikant Mishra
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Correspondence to Rajnikant Mishra.

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Mishra, S., Mishra, R. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis . Mol Biol Rep 43, 359–369 (2016). https://doi.org/10.1007/s11033-016-3965-3

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