Phospholipases defined

The biological effects of histamine (Table 15.1) are mediated via three receptor subtypes, HI, H2 and H3 that are linked to G protein but activate different cell-signalling systems. The histamine HI receptor is associated with the phospholipase C-catalysed formation of inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). The H2-receptor is coupled to adenylyl cyclase, increasing the production of cAMP. The cellular messenger system involved in H3-receptor activation has not been fully defined, but it may couple to N-type Ca2+-channels. The genes encoding for HI and H2 receptors have been cloned. A mutation of the human H2 receptor has been linked to schizophrenia. 

As mentioned in an earlier section, there are no chemical reagents that can establish with any certainty the specific position of attachment of an acyl ester on a diacylphosphatidylcholine or any other phosphoglyceride. The same is true in any attempt to establish the stereochemical conformation of phosphatidylcholine, whether sn-1 or sn-3, by strictly chemical means. The solution to this dilemma is to use synthetic phosphoglycerides of defined structure as well as phospholipase A2 to establish the correct stereospecificity of the latter enzyme. Certainly, this is reminiscent of the chicken or egg argument, yet it does work as will be explained below. 

A thoughtful and detailed outline of the isolation, purification and characterization of the bacterial phospholipases C is given by Waite (1987). Procedures similar to those mentioned above for the mammalian enzymes were also useful in defining the chemistry of the bacterial phospholipases C. 

In order to conduct research studies on this novel lipid mediator, it is necessary to have available a sufficient quantity of purified, well-defined material. This has not been an easy task, but an approach to preparation of sphingosine-l-P in milligram amounts was outlined by Van Veldhoven et al. (1989). In this procedure, a commercial preparation of sphingosylphospho-choline (which contains a free amine group due to removal of a fatty acyl residue from initial substrate, sphingomyelin) is incubated with phospholipase D in an ammonium acetate buffer at pH 8.0 for 1 hr. An insoluble reaction product is collected and subjected to purification by dissolution in water at room temperature followed by cooling to 4°C. A precipitate forms again and is collected and treated in a similar manner as above except that acetone is used as the solvent. 

Calculation One phospholipase unit is defined as the quantity of enzyme that produces 1 microequivalent of free fatty acid per min under the conditions of the test. Determine the rate, R, of titrant consumption during 0 to 3 min of the reaction. 

Golgi apparatus, the nucleus, and the plasma membrane (Donaldson and Klausner, 1994). The identification of ARFs as activators of an isoform of phospholipase D (Brown etal., 1993 Cockcroft eta/., 1994 /Vlassenburg ef a/., 1994 Hammond et a/., 1995), which appears to be present (at least in part) in the Golgi apparatus (Ktistakis etal., 1995), has helped to define further the function of ARF in cells. Investigations into the role of ARFs in these processes have employed recombinant wild-type and mutant ARF constructs. Thus, the assays discussed here have become commonly used to measure native ARFs, and to assess the integrity of recombinant proteins and the effects of mutations on their activity. 

Hammond SM, Altshuller YM, Sung T-C, et al. (1995) Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. In J. Biol. Chem. 270 29640-29643. 

Toxins in general are potent poisons. Nevertheless, the selectivity of action of some of these toxins means they have been harnessed in medical therapeutics (and even more widely in experimental pharmacology and physiology). Toxins that have been, or still are. us in medicine include atropine, botulinum toxin, cardiac glycosides, coichidne, eserine, hyoscine, picrotoxin, morphine, ouabain, strychnine, veratridine, vinca alkaloids and many more. All these work by an action at a defined molecular site, whether ion channel, neurotransmitter receptor, enzyme, pump or intracellular organelle. Those toxins that work at nonneuronal, or not specifically at neuronal sites (e.g. cholera toxin, pertussis toxin, cardiac glycosides, phospholipases) are discussed under TOXINS. 

Phospholipases are very versatile enzymes which allow the transformation of inexpensive natural products into highly valuable compounds like specific structurally defined phospholipids, organic monophosphates or diphosphates and DAG with the natural absolute configuration. Of particular synthetic utility is PLD from bacterial sources which is able to effect the phosphoryl transfer in a water-containing biphasic system. PLD shows a wide substrate specificity for both the polar head and the alcohol acceptors as well as for the lipophilic part of the molecule. The enzyme behaves like a generic phosphodiesterase with broad substrate specificity and high transphosphatidylation ability. The molecular basis of this behavior should become clear by inspection of the three-dimensional structure and comparison with other phosphoric acid ester hydrolytic enzymes. The crystal structure of this enzyme has not been elucidated. The potential of the many different PLD from plants which show peculiar substrate specificity should allow one to expand the synthetic utility to the hydrolysis-synthesis of natural and unnatural phosphatidylinositols. 

Sapirstein A, Bonventre JV. 2000. Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts. Biochim Biophys Acta 1488 139-148. 

Abstract The cannabinoid receptor family currently includes two types CBi, characterized in neuronal cells and brain, and CB2, characterized in immune cells and tissues. CBi and CB2 receptors are members of the superfamily of seven-transmembrane-spanning (7-TM) receptors, having a protein structure defined by an array of seven membrane-spanning helices with intervening intracellular loops and a C-terminal domain that can associate with G proteins. Cannabinoid receptors are associated with G proteins of the Gi/o family (Gil,2 and 3, and Gol and 2). Signal transduction via Gi inhibits adenylyl cyclase in most tissues and cells, although signaling via Gs stimulates adenylyl cyclase in some experimental models. Evidence exists for cannabinoid receptor-mediated Ca fluxes and stimulation of phospholipases A and C. Stimulation of CBi and CB2 cannabinoid... 

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