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A Novel Continuous Enzyme Coupled Colorimetric Assay for Phospholipase A2 and its Application in the Determination of Catalytic Activity of Oil-Body–Associated Oleosin Protein

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Abstract

The present work reports a novel, continuous, specific, and colorimetric coupled method for the assay of phospholipase A2 (PLA2), which involves use of acyl-CoA synthetase (ACS) as coupling enzyme and Dilinoleoyl Phosphatidylcholine (DLPC) as substrate. This method is based on the principle that free fatty acids (FFA) produced by PLA2 are acted upon by ACS, which requires Coenzyme A (CoA) as co-substrate. The PLA2 activity was measured in terms of amount of CoA utilized in the coupled reaction, determined indirectly by measuring unreacted CoA using 5,5’-dithiobis (2-nitrobenzoate). The PLA2 assay was in agreement with Michaelis–Menten equation (Vm = 11.9 nmole. min−1 mg−1, Km = 5.3 nmole, Vm/Km = 2.2 min−1 mg−1). This method provides a true measure of PLA2 in comparison to other available methods, wherein PL analogs were used rather than the natural PL used in our study. The embedded advantages of this method would have wider acceptability and applicability.

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All data generated or analyzed during this study are included in this published article.

References

  • Abousalham A, Verger R (2000) Egg yolk lipoproteins as substrates for lipases. Biochem Biophys Acta 1485:56–62

    CAS  PubMed  Google Scholar 

  • Blanchard SG, Harris CQ, Parks DJ (1994) A fluorescence-based assay for human type II phospholipase A2. Anal Biochem 222:435–440

    Article  CAS  PubMed  Google Scholar 

  • Cao J, Yan P, Zhou Y, Zhou X, Sun Z, Zhu XQ (2021) Clinical utility of the serum level of lipoprotein-related phospholipase A2 in acute ischemic stroke with cerebral artery stenosis. Front Neurol 12:642483. https://doi.org/10.3389/fneur.2021.642483

    Article  PubMed  PubMed Central  Google Scholar 

  • Cirino G (1998) Multiple controls in inflammation. Extracellular and intracellular phospholipase A2, inducible and constitutive cycloxygenase, and inducible nitric oxide synthase. Biochem Pharmacol 55:105–111

    Article  CAS  PubMed  Google Scholar 

  • Egger D, Wehtje E, Adlercreutz P (1997) Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine. Biochem Biophys Acta 1343:76–84

    CAS  PubMed  Google Scholar 

  • Farooqui AA, Ong WY, Horrocks LA, Farooqui T (2000) Brain cytosolic phospholipase A2: localization, role, and involvement in neurological diseases. Neuroscientist 6:169–180

    Article  CAS  Google Scholar 

  • Farooqui AA, Wei-Yi O, Horrocks LA (2006) Inhibitors of brain phospholipase A2 activity: their neuropharmacological effects and therapeutic importance for the treatment of neurologic disorders. Pharmacol Rev 58:591–620

    Article  CAS  PubMed  Google Scholar 

  • Farooqui AA, Yang HC, Hirashima Y, Horrocks LA (1999) Determination of plasmalogen-selective phospholipase A2 activity by radiochemical and fluorometric assay procedures. Methods Mol Biol 109:39–47

    CAS  PubMed  Google Scholar 

  • Farooqui AA, Yang HC, Rosenberg TA, Horrocks LA (1997) Phospholipase A2 and its role in brain tissue. J Neurochem 69:889–901

    Article  CAS  PubMed  Google Scholar 

  • Fujita K, Murakami M, Yamashita F, Amemiya K, Kudo I (1996) Phospholipase D is involved in cytosolic phospholipase A2- dependent selective release of arachidonic acid by fmlP-stimulated rat neutrophils. FEBS Lett 395:293–298

    Article  CAS  PubMed  Google Scholar 

  • Groot PH, Scholte HR, Hulsmann WC (1976) Fatty acid activation: specificity, localization, and function. Adv Lipid Res 14:75–126

    Article  CAS  PubMed  Google Scholar 

  • Groot PHE, Scholte HR, Hulsmann WC (1976) Fatty acid activation: specificity, localization, and function. Adv in Lipid Res 14:75–126

    Article  CAS  Google Scholar 

  • Huang Z, Laliberte F, Tremblay NM, Weech PK, Street IP (1994) A continuous fluorescence-based assay for the human highmolecular- weight cytosolic phospholipase A2. Anal Biochem 222:110–115

    Article  CAS  PubMed  Google Scholar 

  • Ichihara K, Shibasaki Y (1991) An enzyme-coupled assay for acyl-CoA Synthetase. J Lipid Res Note on Methodol 32:1709–1712

    Article  CAS  Google Scholar 

  • Jain MK, Rogers J, Hendrickson HS, Berg OG (1993) The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent. Biochemistry 32:8360–8367

    Article  CAS  PubMed  Google Scholar 

  • Jimenez M, Cabanes J, Gandıa-Herrero F, Escribano J, Garcıa-Carmona F, Perez-Gilabert M (2003) A continuous spectrophotometric assay for phospholipase A2 activity. Anal Biochem 319:131–137

    Article  CAS  PubMed  Google Scholar 

  • Jingkun Qu, Xixi Z, Jizhao W, Chao L, Yuchen S, Hui C, Jianlin L (2018) Plasma phospholipase A2 activity may serve as a novel diagnostic biomarker for the diagnosis of breast cancer. Oncol Lett 15:5236–5242

    Google Scholar 

  • Kumari S, Memba LJ, Dahuja A, Vinutha T, Saha S, Sachdev A (2016) Elucidation of the role of oleosin in off-flavour generation in soymeal through supercritical CO2 and biotic elicitor treatments. Food Chem 205:264–271

    Article  CAS  PubMed  Google Scholar 

  • Lopez-Bernal A, Newman GE, Phizackerley PJ, Bryan-Greewood G, Keeling JW (1992) Human placental phospholipase a2 activity in term and preterm labour. European J Obstetrics & Gynecol Reprod Biol 43:185–192

    Article  CAS  Google Scholar 

  • Nisha D, Nam KW, Robert WL (2002) Lp-PLA2: an emerging biomarker of coronary heart disease. Expert Rev Mol Diagn 2:17–22

    Article  Google Scholar 

  • Pekka M, Jukka LYA, Paavo KKJ (1998) Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme. Biochemistry 37:12051–12057

    Article  Google Scholar 

  • Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Meth Enzymol Methods in Enzymology 91:49–60

    Article  CAS  Google Scholar 

  • Salman M, Rottem S (1995) The cell membrane of mycoplasma penetrans: lipid composition and phospholipase a1 activity. Biochem Biophys Acta 1235:369–377

    Article  PubMed  Google Scholar 

  • Samanta U, Bahnson BJ (2008) Crystal structure of human plasma plateletactivating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis. J Biol Chem 283:31617–31624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schaloske RH, Dennis EA (2006) The phospholipase A2 superfamily and its group numbering system. Biochem Biophys Acta 1761:1246–1259

    CAS  PubMed  Google Scholar 

  • Sharifpour S, Fakhraee S, Behjatmanesh-Ardakani R (2020) Insights into the mechanism of inhibition of phospholipase A2 by resveratrol: an extensive molecular dynamics simulation and binding free energy calculation. J Mol Graphics and Modelling 100:107649

    Article  CAS  Google Scholar 

  • Shinohara H, Ishida H, Amabe EJ, NagataWakano YTY (1992) Phospholipase A2 in rat gingival tissue. J Periodontal Res 27:528–533

    Article  CAS  PubMed  Google Scholar 

  • Siniavin A, Grinkina S, Osipov A, Starkov V, Tsetlin V, Utkin Y (2022) Anti-hiv activity of snake venom phospholipase A2s: updates for new enzymes and different virus strains. Int J Mol Sci 23:1610. https://doi.org/10.3390/ijms23031610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Six DA, Dennis EA (2000) The expanding superfamily of phospholipase A2 enzymes:classifcation and characterization. Biochem Biophys Acta 1488:1–19

    CAS  PubMed  Google Scholar 

  • Tan NG, Tan CS (1988) Acidimetric assay for phospholipase A using egg yolk suspension as substrate. Anal Biochem 170:282–288

    Article  CAS  PubMed  Google Scholar 

  • Tan TL, Goh YY (2017) The role of group IIA secretory phospholipase A2 (sPLA2-IIA) as a biomarker for the diagnosis of sepsis and bacterial infection in adults—a systematic review. PLoS ONE 12–7:e0180554

    Article  Google Scholar 

  • Vasil A, Silviya S, Hristo K, Mariana M, Svetla P, Ivayla P (2014) Effect of some divalent metal ions on enzymatic activity of secreted phospholipase A2 (sPLA2) isolated from Bulgarian Vipera ammodytes meridionalis. Biotechnol Biotechnol Equip 27(5):4181–4185

    Google Scholar 

  • Yu L, Ternanasky RJ, Crisologo JF, Chang J, Baker BL, Coutts SM (1998) Carbonothioate phospholipids as substrate for a spectrophotometric assay of phospholipase A2. Anal Biochem 265:35–41

    Article  CAS  PubMed  Google Scholar 

  • Zamudio FZ, Conde R, Arevalo C, Becerril B, Martin BM, Valdivia HH, Possani LD (1997) The mechanism of inhibition of ryanodine receptor channels by imperatoxin I, a heterodimeric protein from the scorpion Pandinus imperator. J Biol Chem 272:11886–11894

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors sincerely thank Indian Agricultural Research Institute (IARI) and Indian Council of Agricultural Research (ICAR) for providing financial support under institutional project.

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Authors and Affiliations

  1. Division of Biochemistry, ICAR - Indian Agricultural Research Institute (IARI), New Delhi - 110012, India

    Sweta Kumari, Om Prakash Gupta, Sandeep Kumar, Minnu Sasi, S. R Arpitha, D. Amirtham, Vinutha Thimmegowda, Veda Krishnan, Archana Sachdev & Anil Dahuja

  2. Indian Institute of Wheat and Barley Research (IIWBR), Karnal, 132 001, India

    Om Prakash Gupta

  3. Dr. BR Ambedkar Center for Biomedical Research (ACBR), University of Delhi (North Campus), Delhi, 110007, India

    Chandra Bhushan Mishra

  4. College of Pharmacy, Sookmyng Women’s University, Seoul, South Korea

    Chandra Bhushan Mishra

  5. ICAR - Indian Agricultural Statistics Research Institute (ICAR-IASRI), Library Avenue, Pusa Campus, New Delhi - 110012, India

    Rajeev Ranjan Kumar

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  1. Sweta Kumari
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  2. Om Prakash Gupta
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Corresponding authors

Correspondence to Sweta Kumari or Anil Dahuja.

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Conflict of Interest

Sweta Kumari declares that she has no conflict of interest. Om Prakash Gupta declares that he has no conflict of interest. Sandeep Kumar declares that he has no conflict of interest. Minnu Sasi declares that she has no conflict of interest. Arpitha S R declares that she has no conflict of interest. D Amirtham declares that she has no conflict of interest. Chandra Bhushan Mishra declares that he has no conflict of interest. Vinutha T. declares that she has no conflict of interest. Veda K. declares that she has no conflict of interest. Archana Sachdev declares that she has no conflict of interest. Rajeev Ranjan Kumar declares that he has no conflict of interest. Anil Dahuja declares that she has no conflict of interest.

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Kumari, S., Gupta, O.P., Kumar, S. et al. A Novel Continuous Enzyme Coupled Colorimetric Assay for Phospholipase A2 and its Application in the Determination of Catalytic Activity of Oil-Body–Associated Oleosin Protein. Food Anal. Methods 15, 2155–2162 (2022). https://doi.org/10.1007/s12161-022-02284-5

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