Phospholipase substrate specificity

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],... 

Selected entries from Methods in Enzymology [vol, page(s)] Detergent-resistant phospholipase Ai from Escherichia coll membranes, 197, 309 phospholipase Ai activity of guinea pig pancreatic lipase, 197, 316 purification of rat kidney lysosomal phospholipase Ai, 197, 325 purification and substrate specificity of rat hepatic lipase, 197, 331 human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase, 197, 339 phospholipase activity of milk lipoprotein lipase, 197, 345. 

Rehfeldt, W., K. Resch, and M. Goppelt-Struebe. 1993. Cytosolic phospholipase A2 from human monocytic cells Characterization of substrate specificity and Ca2+-dependent membrane association. Biochem J 293 255. 

In a study designed to investigate the structural features of a phospho-glyceride interaction with a bacterial phospholipase C, El-Sayed et al. (1985), reported that the carbonyl group and its closely related environment are most important. A more detailed treatment of the substrate specificity of this enzyme can be found in an excellent review by Massing and Eibl (1994). 

Phosphodiesterase (Hydrolysis) Activity. A rather broad substrate specificity is exhibited by the purified phospholipase D (phosphodiesterase activity). It can attack phosphatidylcholine, phosphatidylethanolamine, phospha-tidylserine, and phosphatidylglycerol. In most cases, Ca2+ was an activator, but variable results were obtained on the positive influence of diethyl ether on the catalytic activity of different sources of this enzyme. Usually the optimum pH was in the range from 5.0 to 7.0. Mammalian phospholipase D, containing both the phosphodiesterase and transphosphatidylase activities, exhibited a broad-range substrate specificity similar to that of the plant enzyme. However, the mammalian enzyme showed a dependency for the presence of oleic acid in the reaction system (Kobayashi and Kanfer, 1991). 

AC, adenylyl cyclase PhL C, unless denoted otherwise, phospholipase C with specificity for phospha-tidylinositol bisphosphate PhL A , phospholipase A2 (substrate specificity unknown) PhtdChol, phosphatidylcholine. 

In addition, a reciprocal interaction between the two phases occurs, in that PKC exerts important feedback effects during the sustained phase of the response. Since activation of PKC leads to an enhancement of calcium efflux and a reduction in the calcium concentration within the submembrane domain, PKC essentially limits its own activity. Furthermore, in adrenal glomerulosa and cultured vascular smooth muscle cells, PKC activation during the sustained phase acts as a feedback modulator of the initial transducing events. The artificial activation of PKC with phorbol esters prevents these initial events (the calcium transient and PIP2 hydrolysis) [59,60] yet at least in cultured smooth muscle cells PI hydrolysis is not blocked [30]. Thus, PKC activation may result in a shift in the substrate specificity of phospholipase C. 

This shift in the substrate specificity of phospholipase C may persist for some time after the removal of agonist and may contribute to the phenomenon of cellular memory in the adrenal. When glomerulosa cells are sequentially exposed to All (20 minutes)-, no agonist (10 minutes), and again All, the character of the response elicited by the second addition of All differs dramatically from that elicited by the first the cell seems to remember its prior exposure to All [29], Although this second addition of All induces a smaller calcium transient, the rate of aldosterone secretion increases more rapidly and reaches a higher plateau value than is seen in response to the first exposure to the hormone. This result suggests either that the second addition of All elicits a smaller increase in 1,4,5-IP3 as a result of an altered... 

The examples provided in this Chapter demonstrate that directed evolution resembles a very useful tool to create enzyme activities hardly accessible by means of rational protein design (Table 14.1). Even if the desired substrate specificity is known from other biocatalysts - e.g. phospholipase A1 activity - the advantage of the directed evolution approach resides in the already achieved functional expression of a particular protein. Thus bottlenecks arising from the identification of enzymes by traditional screening and cultivation methods can be circumvented. In addition, directed evolution can dramatically reduce the time required for the provision of a suitable tailor-made enzyme, also because cloning and functional expression of the biocatalyst has already been achieved. 

Jain, M. K., Rogers, J. (1989). Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode. Biochim. Biophys. Acta 1003,91—97. 

Phosphatidylcholine specific phospholipase C (PLC) can be used for the hydrolysis of PC (and PX) affording natural diacyl glycerol (DAG ) and chohne phosphate (CP) (or corresponding organic phosphate, OP). Transesterification has not been observed with this enzyme. The substrate specificity is less broad than desirable for the synthesis of OP that is otherwise... 

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. 

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. 

Two PLAs have been purified from Escherichia coli based on their differential sensitivity to treatment with detergents [2]. A detergent-insensitive enzyme is localized in the outer membrane, whereas a detergent-sensitive enzyme is found on the cytoplasmic membrane and in soluble fractions. The outer membrane enzyme, known as outer membrane phospholipase A, has broad substrate specificity and demonstrates PLA, PLAj, lysophospholipase A, and lysophospholipase Aj activity as well as activity for hydrolyzing monoacylglycerols and diacylglycerols. The crystal structure allows a more detailed discussion of an integral membrane phospholipase [12]. 

A. Masayama, T. Takahashi, K. Tsukada, S. Nishikawa, R. Takahashi, M. Adachi, K. Koga, A. Suzuki, T. Yamane, H. Nakano, and Y. Iwasaki Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis. Chembiochem 9 (2008) 974-981. 

The high substrate specificity of some enzymes can be used as the basis for highly selective determinations of organic phosphorus species. For example, Amini (2001) determined phosphatidyl choline using coimmobilized phospholipase C, alkaline phosphatase and choline oxidase (Fig. 1.2a). A stoichiometric product of this sequence is hydrogen peroxide, and this was determined using the luminol chemiluminescence reaction, using the flow injection manifold shown (Fig. 1.2b) to provide rapid, sensitive and reproducible indirect measurement of phosphatidyl choline (Amini,... 

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. 

Saccomani et al. [85] treated a vesicular (K + H )-ATPase preparation from pig gastric mucosa with phospholipase Aj, resulting in a breakdown of 50% of the phospholipids. This treatment also results in partial loss of ATPase activity, but the residual activity is still 25% of the original activity. The K -stimulated phosphatase activity is not affected by this treatment. Schrijen et al. [86] used two phospholipases with shght difference in substrate specificity, alone and in combination. With each of these phospholipases approx. 50% of the phospholipids could be hydrolysed resulting in a 50% loss in enzyme activity. When the two phospholipases were used successively 70% of the phospholipids were hydrolysed and the loss of activity was also 70%. This represents a striking parallelism between residual phospholipid content and ATPase activity. In this case the K -stimulated phosphatase activity... 

Billah MM, Lapetina EG, Cuatrecasas P (1980) Phospholipase A2 and phospholipase C activities of platelets. Differential substrate specificity, Ca requirement, pH dependence, and cellular localization. J Biol Chem 255 10227-10231... 

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