Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine

1980 ◽  
Vol 58 (10) ◽  
pp. 898-912 ◽  
Author(s):  
David O. Tinker ◽  
Rosita Low ◽  
Maria Lucassen
Keyword(s):  
Equilibrium Constant ◽  
Quadratic Function ◽  
Lipid Binding ◽  
Reaction Products ◽  
Rapid Phase ◽  
Gel Phase ◽  
Desorption Equilibrium ◽  
Lipid Surface ◽  
Hydrolysis Of ◽  
Egg Pc

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 °C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97–106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis–Menten function dependent on the catalytic properties of the enzyme ([Formula: see text], [Formula: see text]), then level off to a slower rate determined by the desorption equilibrium constant [Formula: see text] and desorption rate constant [Formula: see text]. The relaxation time, τ, for the transition to the desorption-limited reaction is ~0.5 min; this large value of τ probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lyso-phosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decreased to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.

Kinetics of hydrolysis of dispersions of saturated phosphatidylcholines by Crotalus atrox phospholipase A2

1978 ◽  
Vol 56 (6) ◽  
pp. 552-558 ◽  
Author(s):  
David O. Tinker ◽  
A. David Purdon ◽  
Jane Wei ◽  
Eileen Mason
Keyword(s):  
Liquid Crystalline ◽  
Phase Reaction ◽  
Reaction Products ◽  
Crotalus Atrox ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Desorption Rate Constant ◽  
Rate Of Hydrolysis ◽  
Lag Period ◽  
Lipid Surface ◽  
Hydrolysis Of

Dispersions of lamellar phase dipalmitoyl phosphatidylcholine (DDPC) and dimyristoyl phosphatidylcholine (DMPC) in 0.01 M CaCl2 were subjected to hydrolysis by phospholipase A2 (EC 3.1.1.4) from Crotalus atrox venom. The reaction was followed continuously by titrating the released fatty acids. For hydrolysis of gel phase phosphatides, the steady-state initial velocities were hyperbolic functions of bulk lipid concentrations. At the 'pre-transition' temperature (34 °C for DPPC, 15 °C for DMPC), there was a large increase in the Michaelis parameter Vmax but no change in the parameter Km. A model was devised to account for these observations, in which the enzyme desorbs from the lipid surface after hydrolysis. The desorption rate constant is postulated to increase above the pretransition temperature.For hydrolysis of liquid crystalline phosphatides, the reaction consisted of a short initial burst of hydrolysis, a long 'lag' period of very slow reaction, followed by a dramatic increase in the reaction rate. Addition of 10 mol% lysolecithin or fatty acid abolished the 'lag' period. It was postulated that the enzyme adsorbs irreversibly to the surface of the liquid crystalline phase. Reaction products are postulated to stimulate desorption of enzyme from the surface. Thus, temperature-dependent changes in the rate of hydrolysis of dispersed phosphatidylcholines are attributed to changes in the rate of desorption of the enzyme from the lipid surface.

A Fluorescence Polarization Assay for Cyclic Nucleotide Phosphodiesterases

2002 ◽  
Vol 7 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Wei Huang ◽  
Yan Zhang ◽  
J. Richard Sportsman
Keyword(s):  
Cyclic Nucleotide ◽  
Fluorescence Polarization ◽  
Drug Targets ◽  
Second Messengers ◽  
Reaction Products ◽  
Cyclic Nucleotide Phosphodiesterases ◽  
Extracellular Signals ◽  
Fluorescence Polarization Assay ◽  
Hydrolysis Of ◽  
Nucleotide Phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.

Darstellung von o-Phenylen-bis(dibromphosphan) — Reaktionsprodukte von o-Phenylen-bis(dichlorphosphan) / Synthesis of o-Phenylene-bis(dibromophosphane) — Reaction Products of o-Phenylene-bis(dichlorophosphane)

1984 ◽  
Vol 39 (12) ◽  
pp. 1706-1710 ◽  
Author(s):  
H.-J. Wörz ◽  
E. Quien ◽  
H. P. Latscha
Keyword(s):  
Hydrogen Peroxide ◽  
Hydrogen Bromide ◽  
Equimolar Amount ◽  
Reaction Products ◽  
Large Excess ◽  
Cyclic Anhydride ◽  
Complete Hydrolysis ◽  
Hydrolysis Of

o-Phenylene-bis(dibromophosphane) (1) is prepared by reaction of P ,P,P′,P′-tetrakis(dim ethylamino)-o-phenylenediphosphane with hydrogen bromide in ether. The reaction of o-phenylene- bis(dichlorophosphane) (2) with CH3OH in ether yields o-phenylene-bis(phosphonousacid- dimethylester) (3). The Michaelis-Arbuzov conversion of 3, either by heat or by catalytic amounts of CH3I in toluene, yields ophenylene-bis(methylphosphinic-acid-methylester) (4). The reduction of 2 must be carried out with a large excess of LiAlH4 (1:4) in ether to give o-phenylenediphosphane (5). The cyclic anhydride (6) of P.P′-dichloro-o-phenylenediphosphonous acid is formed when 2 is hydrolyzed in ether with an equimolar amount of water. Complete hydrolysis of 2 with an excess of water gives o-phenylenediphosphonous acid (7). With hydrogen peroxide o-phenylenediphosphonic acid (8) can be isolated.

Equilibrium Constant of the Galactokinase Reaction and Free Energy of Hydrolysis of Adenosine Triphosphate

Nature ◽  
1959 ◽  
Vol 184 (4703) ◽  
pp. 1925-1927 ◽  
Author(s):  
M. R. ATKINSON ◽  
ELEANOR JCHNSON ◽  
R. K. MORTON
Keyword(s):  
Free Energy ◽  
Equilibrium Constant ◽  
Adenosine Triphosphate ◽  
Hydrolysis Of

scholarly journals Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1

Biochemistry ◽  
2018 ◽  
Vol 57 (12) ◽  
pp. 1842-1846 ◽  
Author(s):  
Andrew N. Bigley ◽  
Tamari Narindoshvili ◽  
Dao Feng Xiang ◽  
Frank M. Raushel
Keyword(s):  
Reaction Products ◽  
Hydrolysis Of

The acid-catalysed hydrolysis of α-methylallyl acetate in aqueous solution

1968 ◽  
Vol 21 (7) ◽  
pp. 1727
Author(s):  
RA Fredlein ◽  
I Lauder
Keyword(s):  
Aqueous Solution ◽  
Reaction Products ◽  
Arrhenius Parameters ◽  
Crotyl Alcohol ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Tracer Experiments

The kinetics of the acid-catalysed hydrolysis of a-methylallyl acetate in aqueous solution have been studied over the range 30-100�. Oxygen-18 tracer experiments reveal the mechanism to be solely Aac2 and the Arrhenius parameters are consistent with this conclusion. Crotyl alcohol is observed in the reaction products. The formation of rearranged alcohol is explained by allylic isomerization of the α-methylallyl alcohol produced by the hydrolysis.

scholarly journals Structure of lipoprotein lipase in complex with GPIHBP1

2019 ◽  
Vol 116 (21) ◽  
pp. 10360-10365 ◽  
Author(s):  
Rishi Arora ◽  
Amitabh V. Nimonkar ◽  
Daniel Baird ◽  
Chunhua Wang ◽  
Chun-Hao Chiu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Lipoprotein Lipase ◽  
Structural Information ◽  
Density Lipoprotein ◽  
Lipid Binding ◽  
Structural Basis ◽  
Maturation Factor ◽  
Lipase Maturation Factor 1 ◽  
Hydrolysis Of

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5–3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Delayed cotyledon senescence following treatment with a cytokinin; an effect at the level of membranes

1980 ◽  
Vol 58 (16) ◽  
pp. 1797-1803 ◽  
Author(s):  
M. L. Gilbert ◽  
J. E. Thompson ◽  
E. B. Dumbroff
Keyword(s):  
Weight Loss ◽  
Phaseolus Vulgaris ◽  
Liquid Crystalline ◽  
Seedling Development ◽  
Hydrolysis Products ◽  
Microsomal Membranes ◽  
Gel Phase ◽  
Storage Cells ◽  
Hydrolysis Of ◽  
Cotyledon Senescence

Application of 10−4 M benzyladenine to Phaseolus vulgaris germinated under etiolating conditions markedly delayed the onset of cotyledon senescence. Weight loss was curtailed, hydrolysis of starch and protein reserves was delayed, and the rate at which hydrolysis products were translocated out of the cotyledons was reduced in treated plants. Microsomal membranes of cotyledons from control seedlings acquired increasing proportions of gel phase lipid as senescence of the tissue intensified. The resulting mixture of liquid-crystalline and gel phase lipid within the membrane matrix renders the membranes leaky and may partially contribute to metabolite translocation out of the cotyledon storage cells during seedling development. This prospect is supported by the observation that in benzyladenine-treated plants the onset of gel phase lipid, and hence of membrane leakiness, was delayed in a manner that corresponded temporally with the decreased rate of metabolite translocation out of the cotyledons.

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