Phospholipase hydrolysis, products

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

Ktistakis, N.T., Delon, C., Manifava, M., Wood, E., Ganley, I. and Sugars, J.M., 2003, Phospholipase D1 and potential targets of its hydrolysis product, phosphatidic acid. Biochem. Soc. Trans. 31 94-97. 

The available methods are suitable only for fractionation according to the basic moieties, whereas phosphatides differing in the lipid moieties are not separated from each other. Compounds which contain differing functional groups in the lipid moiety can be indirectly separated from each other using the following procedure [174] the enzyme phospholipase C from bacteria [Clostridium perfringens or Bacillus cereus) splits off phosphoryl ethanolamine and phosphoryl choline from the phosphatides. The lipophilic hydrolysis products can then be separated as acetyl derivatives (I, II, III) on adsorbent layers. 

The D-a,/8-diglycerides are the naturally occurring diglycerides, found as hydrolysis products of phosphatidyl choline acted upon by phospholipase C (chapter IV, 2) or as intermediates in the biosynthesis of a number of glycero-phosphohpids and triglycerides. 

The experiments of Haverkate and van Deenen (1964) indicate that phosphatidyl glycerol is broken down by the same enzymes that degrade lecithin. Phospholipases A, B, C (from Bacillus cereus, hut not from Clostridium perfringens) and D all act upon phosphatidyl glycerol with the formation of the expected hydrolysis products. 

Thus, one can expect that, due to their different composition in acyl lipids, the two monolayers will be assigned different roles in the thylakoid membrane function. One of the approaches to test this hypothesis has been to use the acyl lipid depletion technique in which special precautions have to be taken to remove all hydrolysis products (free fatty acids and lysoderivatives) which could alter membrane function [6]. Recent evidence concerning the role of phospholipids in sustaining the uncoupled non-cyclic electron flow activity is that several distinct populations of PG and PC have to be considered [3,6]. A first one, which is easily accessible to phospholipase A2, supports only about 15% of the activity. A second phospholipid population which is less accessible to phospholipase A2 sustains the remaining 85% of the activity. Finally, a third population of phospholipids does not seem to be involved in the uncoupled non-cyclic electron flow activity. These results and several other reports using mainly reconstitution procedures [4,5] point to the fact that acyl lipids may sustain the photosynthetic membrane function. 

Figure 4. Hydrolysis of a lipid monolayer by phospholipase A2 (schematic) (A) monolayer in the ase transition region with solid-analogous Upd domains in a liquid-analogous matrix mixed with SR-DPPE (B) injection of FlTC-labeled phospholipase A2 (Q speciHc recognition of the lipids by the enzyme (D) hydrolysis of the lipid domains and accumulation of the hydrolysis products in the monolayer aggregation of the enzyme. PC = fdios diatidylcholine.

In order to ascertain that the "Ijarrier" properties of thylalcoids are preserved during PLA2 treatment, we have studied their osmotic response in sorbitol solutions (Fig. 4). It can lae seen that under all eaperimental conditions, the mean packed volumes of thylakoids vary linearly with the reciprocal of sorbitol concentration. Phospholipase A2-treated thylakoids have a larger volume after 60 min at 2 C (i.e., when only those phospholipids localized in the outer monolayer are hydrolyzed) and also after 120 min at 20 C (i.e., when all phospholipids are destroyed). These changes can be attributed to the hydrolysis products only since an... 

Figure 1A-B. Envelope membrane vesicules (150 pg proteins for phospholipase C from Bacillus cereus PLC and 240 pg proteins for the lipase from Rhizopus arrhizus LRa) were incubated for various times with (A) PLC ( 0.6U for 210 pi of reaction medium) —> low [ ] > 8U for the remaining volume of reaction medium -> high [ ] (see arrow) or (B) LRa ( 7U for 480 pi of reaction medium) -> low [ ] > SOU for the remaining volume of reaction medium -> high [ ] (see arrow) before lipid extraction. A Time course of the PC (a) and PG (a) hydrolysis with PLC. B Time course of the MGDG ( ) and DGDG (a) hydrolysis with the LRa. The hydrolysis products (a) LDG (O) LMG (0) FFA are also shown in the graph.

HPLC is the most common technique applied to the determination of the chemical composition of lecithin. Normal phase HPLC is convenient for the determination of the major constituents (i.e., phosphatidylcholine, phosphatidylethanolamine, etc), as described in Chapter 7. P NMR is also suitable for this analysis, as discussed in Chapter 14. The biochemical literature contains many enzymatic methods, mainly for specific determination of phosphatidylcholine and its hydrolysis product, choline (32). For instance, phosphatidylcholine can be hydrolyzed by phospholipase C to a diacylglycerol and the phosphate ester of choline, which itself can be hydrolyzed by alkaline phosphatase to form choline and phosphate ion. Alternatively, action of phospholipase D on phosphatidylcholine yields phosphatidic acid and choline. These methods are not applied to analysis of the commercial lecithin used as a surfactant. 

Figure 6.7. Phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C. Occupancy of receptors (R) results in exchange of bound GDP for GTP on the a-subunit of a het-erotrimeric G-protein. The a-subunit then dissociates from the fi- and y-subunits and activates phospholipase (PLC). This enzyme is calcium dependent and, upon activation, can hydrolyse phosphatidylinositol 4,5-bisphosphate (PIP2). The products of this hydrolysis are inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which is released into the cytoplasm, and diacylglycerol (DAG), which remains in the membrane. The DAG is an activator of protein kinase C, which moves from the cytoplasm to the membrane, where it forms a quaternary complex with DAG and Ca2+. 

Figure 6.9. Pathways of inositol phosphate metabolism. Ins 1,4,5-P3, generated via the hydrolysis of phosphatidyl 4,5-bisphosphate by phospholipase C, can be metabolised by a kinase (to generate Ins 1,3,4,5-P4) or via a phosphatase (to yield Ins 1,4-P2). These products can be metabolised further to produce inositol, which itself may be sequentially phosphory-lated to regenerate phosphatidylinositol 4,5-bisphosphate.

Figure 6.16. Hydrolysis of phosphatidylcholine by phospholipase A2. This reaction yields two important products arachidonic acid and lyso-PAF.

The excitatoiy amino acids (EAA), glutamate and aspartate, are the principal excitatory neurotransmitters in the brain. They are released by neurons in several distinct anatomical pathways, such as corticofugal projections, but their distribution is practically ubiquitous in the central nervous system. There are both metabotropic and ionotropic EAA receptors. The metabotropic receptors bind glutamate and are labeled mGluRl to mGluRB. They are coupled via G-proteins to phosphoinositide hydrolysis, phospholipase D, and cAMP production. Ionotropic EAA receptors have been divided into three subtypes /V-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA), and kainate receptors (Nakanishi 1992). 

The phosphorylated phospholipid, phosphatidylinositol bisphosphate, is present in cell membranes. On hydrolysis by a phospholipase, it produces two products, inositol trisphosphate and diacylglycerol (Figure 11.25), as follows ... 

A detailed analysis of the effect of mixed monolayers of 15 and DMPC on the activity of phospholipase A2 was reported by Grainger et al. [53]. Monolayers composed of different ratios of DMPC and either 15 or primarily poly 5 were characterized by Langmuir isotherms and isobars. The phospholipse-A2-mediated hydrolysis of selected monolayer compositions was usefully employed to ascertain the effectiveness of the enzyme. Both 15 and polyl5 were resistant to hydrolysis. The DMPC hydrolysis was sensitive to its molecular environment in a manner that suggests the phase separation of the polyl5 from DMPC. Phospholipase A2 activity is known to be sensitive to the concentration of the hydrolytic products, i.e. the fatty acid and lysophospholipid. The effect of these reaction products of the activity of phospholipase A2 on mixed monolayers of nonpolymerizable lipids is the subject of a series of interesting studies which are beyond the scope of this review. Ahlers et al. reviewed some of this research [54],... 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

---