Soluble phospholipases

Wymann D, Akdis CA, Blesken T, Akdis M, Crameri R, Blaser K Enzymatic activity of soluble phospholipase A2 does not affect specific IgE, IgG4 and cytokine responses in bee sting allergy. Clin Exp Allergy 1998 28 839-849. 

Saffer LD, Schwartzman JD A soluble phospholipase of Toxoplasma gondii associated with host cell penetration. J Protozool 1991 38 454-458. 

Van der Wiele, F. C., Atsma, W., Roelofsen, B., van Linde, M., Binsbergen, J. V., Radvanyi, F Raykova, D., Slotboom, A. J., and de Haas, G. H. (1988). Site-speciBc E-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating membrane-bound form. Biochemistry 27, 1683-1688. 

Roberts et al.40 used bolaform phosphatidylcholine as a probe of water soluble phospholipase catalysis. These bolaphiles (Figure 14) contain two phosphatidylcholines, as the ionic head groups permitting the evaluation of the proposal that two phosphatidylcholines are required for phospholipase activity. Phospholipase activity was measured using micelles formed from these bolaphiles and phosphatidylcholine containing amphiphiles. Increased membrane stability of these bolaform... 

Biochemical characteristics of purified PLD are alike to most of the plant soluble phospholipase D (TABLB II) concerning the pH optimum, pi and mol. wt. (denaturing conditions) and differes in native mol.wt. and higher concentration of activators is demanded. In the contrast with results published for PLD from Castor bean endosperm (5), rape seed PLD seems to be a monomeric protein. 

Further studies (2) revealed that a soluble phospholipase activity in potato leaves could be stimulated to the same degree (30-50%) by either calmodulin or protein kinase (-t-ATP). Two other plant enzymes, quinate NAD+ oxidoreductase (3) and isofloridoside-phosphate synthase (4), have also been shown to be similarly stimulated by both calmodulin and protein phosphorylation. However, the latter enzyme was also shown to be stimulated to an even greater degree by proteolysis with trypsin or chymotrypsin (4). This study was undertaken to investigate whether the phospholipase activity in potato leaves may also respond to proteolytic activation. 

The phospholipase activities in tonoplast were studied by following the hydrolysis of added sn-2-[ 4c]linoleyl-PC to tonoplast vesicles in the presence of 0.1% Triton X-100. The results presented table I indicated that 90 to 95% of radioactivity lost from PG was recovered in lyso-PC and phosphatidic acid (PA). The production of radioactive PA appeared to be Ca +-dependent and was stimulated at acidic pH as it was previously described [1,2]. The release of radioactive lyso-PC was, however, the same irrespective of the pH and the addition of EDTA or divalent ions. Almost no radioactivity was recovered in free fatty acids (FFA) in the different assays, but almost all the radioactivity was detected in FFA in the control after addition of bee venom phospholipase A2 (Table I). The vacuolar sap did not reveal any significant soluble phospholipase activity. 

Heat-labile soluble toxin Exoenzymes (phospholipases, hyaluronidase) Vegetative bacterial cells... 

The other activity associated with transmembrane receptors is phospholipase C. Phosphatidyl inositol is a membrane phospholipid that after phosphorylation on the head group is found in the membrane as a phos-photidylinostitol bis phosphate. Phospholipase C cleaves this into a membrane associated diacylglycerol (the lipid part) and inositol trisphosphate (IP3, the soluble part). Both play a later role in elevating the level of the second messenger, Ca2+. 

A bacterial phosphatidylinositol specific phospholipase C (PI-PLC) had been available for many years before it was demonstrated to strip a number of membrane-bound proteins from eukaryotic cell surfaces [1], Such proteins are anchored by a PI moiety in which the 6 position of inositol is glycosidically linked to glucosamine, which in turn is bonded to a polymannan backbone (Fig. 3-10). The polysaccharide chain is joined to the carboxyl terminal of the anchored protein via amide linkage to ethanolamine phosphate. The presence of a free NH2 group in the glucosamine residue makes the structure labile to nitrous acid. Bacterial PI-PLC hydrolyzes the bond between DAG and phosphati-dylinositols, releasing the water-soluble protein polysac charide-inositol phosphate moiety. These proteins are tethered by glycosylphosphatidylinositol (GPI) anchors. 

When the receptor interacts with its associated G protein, the conformation of the guanine-nucleotide-binding site is altered. The subunits then dissociate, and a phosphatidylinositol-specific phospholipase C (PI-PLC) is activated [5]. The subsequent hydrolysis of phosphatidylinositol bisphosphate then produces inositol triphosphate (IP3) and diacylglycerol (DAG), which are known to be secondary messengers. For example, the water soluble IP3 is released into the cell where its ultimate targets are the calcium storage organelles from which Ca2+ is released [3]. The presence of DAG in cells is known to activate the cellular enzyme protein kinase C (PKC) [6, 7], which phosphorylates a number of cellular... 

The acid-soluble SH-groups in platelets are mainly those of glutathione (GSH). GSH is a cofactor for enzymes such as peroxidase. If feverfew is able to interfere with this cofactor, enzyme function may be impaired. One pathway that may be affected in this way is the metabolism of arachidonic acid (Figure 6.1). In the presence of feverfew extract an increase was found in lipoxygenase product formation and impaired conversion of HPETE to HETE, for which GSH is a cofactor [52]. Inhibition of the liberation of [ " C]arachidonic acid from phospholipids was also found [53], which implies impairment of phospholipase A2 activity and for which SH-groups are thought to be important. 

Fig. 3. Mechanisms of vasocontraction and vasorelaxation in endothelial and smooth muscle cells. COX cyclooxygenase, eNOS endothelial nitric oxide synthase, HO-1 heme oxygenase-1, EET epoxyeicosatrienoic acid, EDHF endothelium-derived hyperpolariz-ing factor, PGI2 prostaglandin I2, NO nitric oxide, CO carbon monoxide, PLC phospholipase C, IP3 inositol 1,4,5-trisphosphate, DAG diacylglycerol, ER/SR endo-plasmic/sarcoplasmic reticulum, AC adenylyl cyclase, cAMP cyclic adenosine monophosphate, sGC soluble guanylyl cyclase, cGMP cyclic guanosine monophosphate.

Figure 14-3. Signaling through protein kinase C (PKC). Activated phospholipase C cleaves the inositol phospholipid PIP2 to form both soluble (IP3) and membrane-associated (DAG) second messengers. DAG recruits PKC to the membrane, where binding of calcium ions to PKC fully activates it. To accomplish this, IP3 promotes a transient increase of intracellular concentration by binding to a receptor on the endoplasmic reticulum, which opens a channel allowing release of stored calcium ions. PIP2, phosphatidylinositol 4,5-bisphosphate DAG, diacylglycerol PLC, phospholipase C IP3, inositol trisphosphate.

I agree with Professor McConnell that phospholipid phase transitions may play a role in controlling the activity of a membrane-bound enzyme. However, the case cited is somewhat ambiguous, since porcine phospholipase A2 is a soluble enzyme acting on a phospholipid surface. The major effect of the phase transition in this case is to alter the nature of the substrate rather than the intrinsic catalytic activity of the enzyme. 

Phospholipase C hydrolyzes the bond between glycerol and phosphate in phosphatidylinositol 4,5-bisphos-phate, releasing two products inositol 1,4,5-trisphos-phate (IP3), which is water-soluble, and diacylglycerol, which remains associated with the plasma membrane. IP3 triggers release of Ca2+ from the endoplasmic reticulum, and the combination of diacylglycerol and elevated cytosolic Ca2+ activates the enzyme protein kinase C. 

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

Many of the proteins of membranes are enzymes. For example, the entire electron transport system of mitochondria (Chapter 18) is embedded in membranes and a number of highly lipid-soluble enzymes have been isolated. Examples are phosphatidylseiine decarboxylase, which converts phosphatidylserine to phosphatidylethanolamine in biosynthesis of the latter, and isoprenoid alcohol phosphokinase, which participates in bacterial cell wall synthesis (Chapter 20). A number of ectoenzymes are present predominantly on the outsides of cell membranes.329 Enzymes such as phospholipases (Chapter 12), which are present on membrane surfaces, often are relatively inactive when removed from the lipid environment but are active in the presence of phospholipid bilay-ers.330 33 The distribution of lipid chain lengths as well as the cholesterol content of the membrane can affect enzymatic activities.332... 

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