The affinity of phospholipase A2 for the interface of the substrate and analogs

Jain, Mahendra Kumar; DeHaas, G.H.; Marecek, James F.; Ramírez, Fausto

doi:10.1016/0005-2736(86)90544-4

Cited by 45 publications

(42 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Dissociation constants ranging from 30 nM to 10 mM have been reported for the interaction of pancreatic PLA 2 s with various phospholipid surfaces. 19,67,68,71,72 The number of lipids that constitute a binding site for group I/II PLA 2 s varied between 28 and 48. 32,67,68,72-74 The value of Nz37 determined here agrees with these data.…”

Section: Discussionmentioning

confidence: 99%

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

Qin

Pande

Nemec

et al. 2004

Journal of Molecular Biology

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

Qin

Pande

Nemec

et al. 2004

Journal of Molecular Biology

View full text Add to dashboard Cite

“…However, a neutral diluent has little affinity for the catalytic site of the E or the E* forms of the enzyme. In this definition, it is implied that binding of PLA2 to the interface is a distinctly different and independent step from the binding of a ligand to the active site of free or bound PLA2 (Jain et al, 1986d). A neutral diluent is neither a substrate nor an inhibitor of PLA2, but it changes the surface concentration (mole fraction) of other species that participate in the catalytic cycle at the interface.…”

Section: Discussionmentioning

confidence: 99%

“…5 University of Uppsala. was monitored in the presence of competitive inhibitors (Jain et al, 1986d and interpreted in terms of KMs, Kp, and K, values.…”

mentioning

confidence: 99%

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Jain¹,

Bao²,

Rogers³

et al. 1991

Biochemistry

Self Cite

120

253

View full text Add to dashboard Cite

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.

show abstract

“…Therefore, the membrane properties play an important role in PLA 2 -catalyzed membrane leakage. It has been reported that the negatively charged lipid-water interface seems to be essential for the efficient binding and activation of PLA 2 [21,22] and for increasing PLA 2 activity [23,24]. Therefore, we systematically studied the effect of the liposome composition on PLA 2 hydrolytic action using the liposome column.…”

Section: Effect Of Ph Incubation Time and Liposome Composition On Pmentioning

confidence: 99%

Effect of lipid composition on phospholipase A₂‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

et al. 2003

View full text Add to dashboard Cite

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.

show abstract

The affinity of phospholipase A2 for the interface of the substrate and analogs

Cited by 45 publications

References 14 publications

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Effect of lipid composition on phospholipase A₂‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Contact Info

Product

Resources

About

The affinity of phospholipase A2 for the interface of the substrate and analogs

Cited by 45 publications

References 14 publications

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-mode Orientation of the Enzyme at the Membrane Surface

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Effect of lipid composition on phospholipase A2‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Contact Info

Product

Resources

About

Effect of lipid composition on phospholipase A₂‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column