Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

Olson, Erin; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

doi:10.1074/jbc.a109.070797

Cited by 3 publications

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“…The activity measurement assay relies on the shift in the ADIFAB fluorescence, excited at 386 nm, from 432 to 505 nm upon binding of ADIFAB to the hydrolysis products that partition into the aqueous phase from the bilayer 15 . The ADIFAB fluorescence response, quantified by the generalized polarization, GP, given by 16,17 …”

Section: Methodsmentioning

confidence: 99%

Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Singh

Ranganathan

2014

J. Phys. Chem. B

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Phospholipase A2 (PLA2) enzymes catalyze hydrolysis of phospholipids in membranes. Elucidation of the kinetics of interfacial enzymatic activity is best accomplished by investigating the interface substrate concentration dependence of the activity, for which appropriate diluents are required. PLA2 is stereo selective toward the L_enantiomers of phospholipids. A novel approach employing D_phospholipids as diluents to perform surface dilution kinetic studies of PLA2 is presented. Activity of bee-venom PLA2 at mixed L+D_DPPC (dipalmitoylphosphatidylcholine) bilayer interfaces was measured as functions of substrate L_DPPC mole fraction and vesicle concentration, using a sensitive fluorescence assay. A model for interface enzymatic activity based on the three-step kinetic scheme of: (i) binding of PLA2 to the bilayer interface; (ii) binding of a lipid to PLA2 at the interface; and (iii) hydrolysis, was applied to the hydrolysis data. Activity profiles showed that D_enantiomers also bind to the enzyme but resist hydrolysis. Activity dependences on vesicle and substrate concentrations could be disentangled, bringing resolution to an outstanding problem in membrane hydrolysis, of separating the effects of the three steps. Individual values of the kinetic parameters of the model including the vesicle-PLA2 equilibrium dissociation constant of step (i), interface Michaelis-Menten-Henri constant for L and D_DPPC of step (ii), and the rate constant for interface hydrolysis, step (iii) were obtained as solutions to equations resulting from fitting the model to the data.

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Section: Methodsmentioning

confidence: 99%

Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Singh

Ranganathan

2014

J. Phys. Chem. B

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“…2010). Many isoforms are associated with dynamic physiological and pathological processes including inflammation and apoptosis (Olson et al. 2010).…”

Section: Secretory Pla2 Is Involved In Neuroinflammationmentioning

confidence: 99%

Phospholipases A₂ and neural membrane dynamics: implications for Alzheimer’s disease

Lee

Simonyi

Sun

et al. 2011

Journal of Neurochemistry

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J. Neurochem. (2011) 116, 813–819. Abstract Phospholipases A2 (PLA2s) are essential enzymes in cells. They are not only responsible for maintaining the structural organization of cell membranes, but also play a pivotal role in the regulation of cell functions. Activation of PLA2s results in the release of fatty acids and lysophospholipids, products that are lipid mediators and compounds capable of altering membrane microdomains and physical properties. Although not fully understood, recent studies have linked aberrant PLA2 activity to oxidative signaling pathways involving NADPH oxidase that underlie the pathophysiology of a number of neurodegenerative diseases. In this paper, we review studies describing the involvement of cytosolic PLA2 in oxidative signaling pathways leading to neuronal impairment and activation of glial cell inflammatory responses. In addition, this review also includes information on the role of cytosolic PLA2 and exogenous secretory PLA2 on membrane physical properties, dynamics, and membrane proteins. Unraveling the mechanisms that regulate specific types of PLA2s and their effects on membrane dynamics are important prerequisites towards understanding their roles in the pathophysiology of Alzheimer’s disease, and in the development of novel therapeutics to retard progression of the disease.

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“…The excitation was at 386 nm. The fl uorescence response to the presence of binding entities represented by the generalized polarization (GP) defi ned by ( 12,13 ) 505 432 505 432 I I GP= I +I was calculated where I 505 and I 432 are the fl uorescence emission intensities at 505 nm and 432 nm respectively. The GP is a normalized representation of the concentration of the binding species.…”

Section: Methodsmentioning

confidence: 99%

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Singh

Ranganathan

2012

Journal of Lipid Research

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is a measure of the concentration of FA present ( 2, 3 ). This property, useful for detection of low levels of FAs, led to the development of a sensitive fl uorescence assay for phospholipase A 2 (PLA 2 ) activity ( 4 ).ADIFAB also binds lysophospholipids, anionic and some zwitterionic detergents. This fact, although intrinsically useful for quantitating not just FAs but lysophosphatidylcholines (LPCs) as well, calls into question the earlier PLA 2 assay that took into account only FA binding. The goals of this work were to establish the quantitative and linear nature of ADIFAB fl uorescence response to FA and LPC, develop quantitative assays for detecting both FA and LPC in aqueous solutions as well as for continuous real time monitoring of PLA 2 hydrolysis at membrane interfaces expressing activity in absolute units of M of product/minute/mg of enzyme, and extend the applicability of the ADIFAB PLA 2 assay to more lipids and lipid bilayers.Phospholipase catalyzed hydrolysis of phospholipids at membrane interfaces produces FAs and lysophospholipids ( 5 ). The rate of hydrolysis depends on membrane physicochemical properties like membrane curvature, lipid composition, lipid chain length, and headgroup charge ( 6, 7 ). It is typically quite low at membrane interfaces and can be well below the precision levels of the standard pH-Stat method ( 8 ). The importance of PLA 2 activity to several physiological functions and membrane biochemistry demands sensitive methods not only to detect membrane hydrolysis but also to quantitatively and continuously monitor PLA 2 activity in real time in absolute units ( 9, 10 ). In this context, the fl uorescence response of the probe ADIFAB to the presence of hydrolysis products offers just such a method ( 1 ).The ADIFAB fl uorescence assay, fi rst reported by Richieri and Kleinfeld, relies on the shift in fl uorescence Acrylodan-labeled rat-intestinal fatty acid binding protein (ADIFAB) exhibits a fl uorescence emission peak at 432 nm when excited by light of wavelength 386 nm ( 1 ). The emission peak shifts to 505 nm upon binding an FA. The ADIFAB fl uorescence response depends on the type of FA. The ratio of the intensities at 505 nm to that at 432 nm

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Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

Abstract: The wrong image was inadvertently printed. The correct Fig. 1 is shown below.

Cited by 3 publications

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Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Phospholipases A₂ and neural membrane dynamics: implications for Alzheimer’s disease

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

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Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

Abstract: The wrong image was inadvertently printed. The correct Fig. 1 is shown below.

Cited by 3 publications

References 0 publications

Surface Dilution Kinetics of Phospholipase A2 Catalyzed Lipid-Bilayer Hydrolysis

Surface Dilution Kinetics of Phospholipase A2 Catalyzed Lipid-Bilayer Hydrolysis

Phospholipases A2 and neural membrane dynamics: implications for Alzheimer’s disease

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Contact Info

Product

Resources

About

Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Surface Dilution Kinetics of Phospholipase A₂ Catalyzed Lipid-Bilayer Hydrolysis

Phospholipases A₂ and neural membrane dynamics: implications for Alzheimer’s disease