Phospholipase specificity

Phospholipases specific for one of the bonds in a phospholipid can be used to generate simpler compounds for subsequent analysis. 

DSC has been used to study the individual protein components of biological membranes of relatively simply protein composition and the interaction of several of these components with lipids and with other proteins. The red blood cell membrane, which has been most intensively studied, exhibits five discrete protein transitions, each of which has been assigned to a specific membrane protein. The response of each of these thermal transitions to variations in temperature and pH as well as to treatment with proteases, phospholipases, specific labelling reagents, and modifiers and inhibitors of selected membrane activities, has provided much useful information on the interactions and functions of these components in the intact erythrocyte membrane (46-49). Similar approaches have been applied to the bovine rod outer segment membrane (50) and to the spinach chloroplast thylakoid membrane (51). 

PH domains consist of about 120 amino acid residues. They do not interact with other proteins, but associate with specific polyphosphoinositides. Consequently, PH domains appear to be important for localizing target proteins to the plasma membrane. Examples of PH domain-containing proteins include phospholipase C andpl20/RasGAP (Fig. 1). 

Activation of Mi, M3, and M5 mAChRs does not only lead to the generation of IP3 followed by the mobilization of intracellular Ca2+, but also results in the stimulation of phospholipase A2, phospholipase D, and various tyrosine kinases. Similarly, M2 and M4 receptor activation does not only mediate the inhibition of adenylyl cyclase, but also induces other biochemical responses including augmentation of phospholipase A2 activity. Moreover, the stimulation of different mAChR subtypes is also linked to the activation of different classes of mitogen-activated protein kinases (MAP kinases), resulting in specific effects on gene expression and cell growth or differentiation. 

Rhee SG, Bae YS (1997) Regulation of phosphoinositide-specific phospholipase Isozymes. J Biol Chem 272 15045-15048... 

Figure 10. The G-protein cascades in smooth muscle catalyze the exchange GDP for GTP on G-protein. Following the binding of GTP, the trimeric G-protein splits into an a-GTP part and a P-y part. The a-GTP part ordinarily then combines with its specific apoenzyme to constitute the active enzyme. For the activation of the contractile activation path, the enzyme is phospholipase C and the second messenger products are IP3 and DAG. The IP3 in the myoplasm binds to Ca channels in the SR membrane, opening them. Other second messengers include the inhibitors of contractile activity, cGMP and cAMP.

A number of allergens from both honey bee and vespid venoms have been cloned and expressed by either Escherichia coli or baculovirus-infected insect cells (table 1) phospholipase Aj [20], hyaluronidase [21], acid phosphatase [13] and Api m6 [14] from honey bee venom, as well as antigen 5 [22], phospholipase A and hyaluronidase [23] from vespid venom, and dipeptidylpeptidases from both bee and Vespula venoms [15, 16]. Their reactivity with human-specific IgE antibodies to the respective allergens has been documented [11-16, 22, 23] and their specificity is superior... 

In studies with specific phospholipases the asymmetry in the composition of the lipid bilayer was also suggested [102,162]. The requirement of specific phospholipids, which are essential for enzyme activity, however, has not been established. For example, Saccomani et al. [102] demonstrated that readdition of various phospholipids, after phospholipase A2 treatment, results in a restoration of the K -ATPase activity. On the other hand, Nandi et al. [161] observed a restoration of the K -ATPase activity with addition of phosphatidylcholine and not with phosphatidyl-... 

Weber, A., Schroder, H., Thalberg, K. and Marz, L. (1987) Specific interactions of IgE antibodies with a carbohydrate epitope of honey-bee venom phospholipase-A2. Allergy 42, 464-470. 

S. G., McLaughlin, S., Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-beta 1, -gamma 1, and -delta 1, Biochemistry 1994, 33, 3032-3037. 

FRET probes have not only been generated to measure the phospholipase activity but to study its substrate specificity as well. Several substrates of PLA2 with a variety of head groups and labeled with a BODIPY dye and a Dabcyl quencher were created by Rose et al. and tested against different PLAs in cells to determine substrate specificity and intracellular localization [137], The specificity of PLA2 isoforms towards the number of double bonds in the sn2 position was evaluated with a small series of PENN derivatives. It was demonstrated that the cytosolic type V PLA2 preferred substrates with a single double bond [138],... 

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

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