Substrate availability phospholipases

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

COX-1 and COX-2 are the targets of the nonsteroidal anti-inflammatory drugs (NSAID, see Chapter 28). Indirectely the effect of these enzymes is also inhibited by corticosteroids (see also Chapter 26) through a decrease of the availibility of its substrate arachidonic acid by inhibition of phospholipase A2. Relatively new drugs, known as COX-2 selective inhibitors or coxibs (celecoxib, rofecoxib and others), are used as specific inhibitors of COX-2. 

The lipases and phospholipase Aj differ from classic esterases in that their natural substrates are insoluble in water and their activity is maximal only when the enzyme is adsorbed to the oif/water interface. Therefore a special treatment of the enzyme kinetics of these enzymes is imposed. The term substrate concentre thm becomes different, as only the substrate present in the interface is available for the enzyme. Consequently, the interface itself becomes the substrate. While in homogeneous svsims. the enzymarlr wngk snare is in three dimensions- and... 

It is dear that the interconnection between the activities of the different lipolytic enzymes, where the first enzyme modifies the physicochemical state of the lipid substrate in such a way that it becomes available to another enzyme, is not only of prime importance for lipid digestion, but also results in a broad synergism between gastric lipase, colipase, pancreatic lipase, phospholipase As. calcium, caiboxylester Lipase, bite salts, and substrate interraecK tes [55,62-64]. 

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. 

Haas et al. (162) have studied enzymatic phosphatidylcholine hydrolysis in organic solvents by examining selected commercially available lipases. Enzymatic hydrolysis of oat and soy lecithins, and its effect on the functional properties of lecithin, was investigated by Aura et al. (163). The phospholipase used was most effective at low enzyme and substrate concentrations. 

In the body, enzymes are compartmentalized and function under highly restricted conditions. Some enzymes (e.g., proteinases) are not substrate-specific. When present in active form in an inappropriate part of the body, they act indiscriminately and cause considerable damage to the tissue. Inhibitors inactivate these enzymes at sites where their action is not desired. Proteinase inhibitors, which are themselves proteins, are widely distributed in intracellular and extracellular fluids. Protein inhibitors of enzymes other than proteinases are relatively rare. Such inhibitors are available for a-amylases, deoxyribonuclease I, phospholipase A, and protein kinases. 

The requirement of certain phospholipids for the activity of an enzyme can be investigated by means of specific phospholipases [84. The degree of breakdown of the various phospholipids depends on the substrate specificity of the phospholipase, but also on the accessibility of the phospholipids. Phospholipids located on the inside of vesicles or in close connection with membrane proteins may not be available to the phospholipases. 

As shown in Table 1, lAP-substrate G-proteins having a-subunits of Mr=40,000 have been purified from rat brain (tentatively referred to as Go in Table 1 (32) and from HL-60 cells that had been differentiated to neutrophils by dimethylsulfoxide [referred to as Ghl (33)]. They differ from G-proteins previously purified, since neither of them interact with any of the antibodies currently available for the G-proteins, i.e., those raised against purified a-subunits of Gi and Go and purified py. It remains to be determined whether these new lAP substrates with apparently identical molecular weights are really identical with each other. Nor is any decisive information available for the physiological role of Go, Go and Ghl, although the latter one is a candidate of the G-protein acting as transducer between the chemotactic peptide receptors and phospholipase C in neutrophils. This is because activation of the phospholipase by the chemotactic peptide in neutrophils was abolished by prior treatment of the cells with lAP (20-22, 34). A similar lAP-substrate G-protein has been partially purified firom sea urchin eggs (35). 

Leukotriene biosynthesis depends upon the availability of arachidonic acid (8) as the free carboxylic acid as the 5-LOX substrate, which typically requires the action of cytosolic phospholipase to release arachidonic acid (8) from membrane phospholipids [27]. The name leukotriene was conceived to capture two unique attributes of these molecules. The first relates to those white blood cells derived from the bone marrow that have the capacity to synthesize this class of eicosanoids, for example, the polymorph nuclear leukocyte. The last part of the name refers to the unique chemical structure, a conjugated triene retained within these eicosanoids [29,30]. The first step for the leukotriene biosynthesis is the insertion of molecular oxygen at position 5 of arachidonic add (8) to produce 5-HPETE (17) that can be converted to leukotriene (18) by the second catalytic activity... 

---