http://www.scripps.edu/cravatt/pdf/Blankman2007.pdf

A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol

1.1. Discovery of the Phospholipase A2 Superfamily
Phospholipases represent one of the earliest enzyme activities to be identified and studied and the phospholipase A2 (PLA2) superfamily (see defining specificity1 in Figure 1) traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. The enzyme was first purified and characterized from cobra venom and later from rattlesnake venom. As protein sequencing methodologies advanced in the 1970’s, it became apparent that these enzymes had an unusually large number of cysteines (over 10% of the amino acids) and as secreted enzymes, that they were all in the form of disulfide bonds. It was further recognized that in the case of PLA2, cobras and rattlesnakes had six disulfides in common, but one disulfide bond is located in distinctly different locations. This led to the designation of Type 1 and Type 2 for cobras (old world snakes) and rattlesnakes (new world snakes), respectively.2 During that same period, studies on the porcine pancreatic digestive enzyme that hydrolyzes phospholipids led to the determination that this mammalian enzyme (and also the human pancreatic enzyme) had the same disulfide bonding pattern as cobras and hence the designation as IB with the cobra enzyme as IA.






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Figure 1


The specific reaction catalyzed by phospholipase A2 at the sn-2 position of the glycerol backbone is shown. X, any of a number of polar headgroups; R1, fatty acids, or alkyl, or alkenyl groups and R2, fatty acids or acyl moieties.

/pdf/phospholipase-a2-enzymes-physical-structure-biological-usiwyw5zej.pdf

Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.

The phospholipase A2 superfamily and its group numbering system

Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities

Current issues in organophosphate toxicology

Human Neuropathy Target Esterase Catalyzes Hydrolysis of Membrane Lipids

Membrane Association of and Critical Residues in the Catalytic Domain of Human Neuropathy Target Esterase

Molecular cloning of neuropathy target esterase (nte)

NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727-1216) of neuropathy target esterase. NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions. Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis. The pattern of digestion by proteinase K of NEST-phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium. Significant quantities of NEST were liberated from NEST-phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values. Treatment of NEST-phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers. Similarly, the inactivation of esterase activity in NEST-phospholipid complexes by radiation indicated that NEST monomers are catalytically active. The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.

Monomers of the catalytic domain of human neuropathy target esterase are active in the presence of phospholipid.

In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current-voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells.

Jane Atkins

Papers

Human Neuropathy Target Esterase Catalyzes Hydrolysis of Membrane Lipids

Membrane Association of and Critical Residues in the Catalytic Domain of Human Neuropathy Target Esterase

Molecular cloning of neuropathy target esterase (nte)

Monomers of the catalytic domain of human neuropathy target esterase are active in the presence of phospholipid.

The catalytic domain of human neuropathy target esterase mediates an organophosphate‐sensitive ionic conductance across liposome membranes