Biomaterial Database

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. 1991

S L Hazen, and L A Zupan, and R H Weiss, and D P Getman, and R W Gross

Molecular and Cellular Cardiovascular Biochemistry Section, Washington University School of Medicine, St. Louis, Missouri 63110.

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D009206	Myocardium	The muscle tissue of the HEART. It is composed of striated, involuntary muscle cells (MYOCYTES, CARDIAC) connected to form the contractile pump to generate blood flow.	Muscle, Cardiac,Muscle, Heart,Cardiac Muscle,Myocardia,Cardiac Muscles,Heart Muscle,Heart Muscles,Muscles, Cardiac,Muscles, Heart
D009281	Naphthalenes	Two-ring crystalline hydrocarbons isolated from coal tar. They are used as intermediates in chemical synthesis, as insect repellents, fungicides, lubricants, preservatives, and, formerly, as topical antiseptics.
D010741	Phospholipases A	Phospholipases that hydrolyze one of the acyl groups of phosphoglycerides or glycerophosphatidates.
D011753	Pyrones	Keto-pyrans.
D002118	Calcium	A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.	Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D003600	Cytosol	Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components.	Cytosols
D004285	Dogs	The domestic dog, Canis familiaris, comprising about 400 breeds, of the carnivore family CANIDAE. They are worldwide in distribution and live in association with people. (Walker's Mammals of the World, 5th ed, p1065)	Canis familiaris,Dog
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs