Phospholipase C, hydrolysis

Figure 25.1. Proposed biosynthetic pathway of castor oil. Heavy arrows show the key enzyme steps driving ricinoleate into acylglycerols. Two arrows with solid bars show a complete block. Two dashed arrows show the phospholipase C hydrolysis which can be targeted to block the incorporation of non-hydroxyl fatty acids into triacylglycerols to increase presumably the content of ricinoleate in transgenic seed oils.

In traditional approaches, the fatty acyl profile is obtained after release of the sn-1 and sn-2 fatty acids from phospholipids via alkaline or phospholipase C hydrolysis, followed by analysis by GC or GC/MS of the fatty acid components released [17]. 

Figure 8.8. Separation by high temperature GC of the trimethylsilyl ether derivatives of diacylglycerols, prepared by phospholipase C hydrolysis from the phosphatidylcholines of rat liver [646], The column was a 10 m x 0.25 mm glass capillary coated with SP-2330 , and was temperature-programmed from 190°C to 250 C at 20 C/min, then was held isothermally at 250°C. Splitless injection was used with hydrogen as the carrier gas. A few only of the major peaks are identified here for illustrative purposes. (Reproduced by kind permission of the authors and of the Canadian Journal of Biochemistry and Cell Biology, and redrawn from the original paper).

Covalent regulation. Following occupation and activation of the M2 acetyl choline receptors, phospholipase C (PLC), is activated and both inositol (l,4,5)-trisphosphate (IP3), and diacylglycerol (DAG), are formed by hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2). 

Two possible pathways for the biosynthesis of 2-AG have been proposed (1) a phospholipase C (PLC) hydrolysis of membrane phospholipids followed by a second hydrolysis of the resulting 1,2-diacylglycerol by diacylglycerol lipase or (2) a phospholipase Ai (PLA,) activity that generates a lysophospholipid, which in turn is hydrolyzed to 2-AG by lysophospholipase C (Fig. 5) (Piomelli, 1998). Alternative pathways may also exist from either triacylglycerols by a neutral lipase activity or lysophosphatidic acid by a dephosphorylase. The fact that PLC and diacylglycerol lipase inhibitors inhibit 2-AG formation in cortical neurons supports the contention that 2-AG is, at least predominantly, biosynthesized by the PLC pathway (Stella, 1997). However, a mixed pathway may also be plausible. 

Inositol triphosphate (IP3)-gated channels are also associated with membrane-bound receptors for hormones and neurotransmitters. In this case, binding of a given substance to its receptor causes activation of another membrane-bound protein, phospholipase C. This enzyme promotes hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to IP3. The IP3 then diffuses to the sarcoplasmic reticulum and opens its calcium channels to release Ca++ ions from this intracellular storage site. 

Phorbol esters are promoters that interact with cellular receptors and activate protein kinase C. Usually protein kinase C is activated by Ca++ and diacylglycerol, both of which result from the hydrolysis of phosphoinositides catalyzed by phospholipase C. Phospholipase C is normally activated by several different growth factors. Thus phorbol esters bypass a tightly regulated step in the control of cell growth. Since protein kinase C phosphorylates various proteins, it is not known how this activity participates in establishing a cancerous line of cells. 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

Muscarinic receptor activation causes inhibition of adenylyl cyclase, stimulation of phospholipase C and regulation of ion channels. Many types of neuron and effector cell respond to muscarinic receptor stimulation. Despite the diversity of responses that ensue, the initial event that follows ligand binding to the muscarinic receptor is, in all cases, the interaction of the receptor with a G protein. Depending on the nature of the G protein and the available effectors, the receptor-G-protein interaction can initiate any of several early biochemical events. Common responses elicited by muscarinic receptor occupation are inhibition of adenylyl cyclase, stimulation of phos-phoinositide hydrolysis and regulation of potassium or other ion channels [47] (Fig. 11-10). The particular receptor subtypes eliciting those responses are discussed below. (See also Chs 20 and 21.)... 

Berstein, G., Blank, J. L., Smrcka, A. V. etal. Reconstitution of agonist stimulated phosphatidylinositol 4-5 bisphosphate hydrolysis using purified mi muscarinic receptor, Gq/11, and phospholipase C-Pl. /. Biol. Chem. 267 8081-8088, 1992. 

The family of heterotrimeric G proteins is involved in transmembrane signaling in the nervous system, with certain exceptions. The exceptions are instances of synaptic transmission mediated via receptors that contain intrinsic enzymatic activity, such as tyrosine kinase or guanylyl cyclase, or via receptors that form ion channels (see Ch. 10). Heterotrimeric G proteins were first identified, named and characterized by Alfred Gilman, Martin Rodbell and others close to 20 years ago. They consist of three distinct subunits, a, (3 and y. These proteins couple the activation of diverse types of plasmalemma receptor to a variety of intracellular processes. In fact, most types of neurotransmitter and peptide hormone receptor, as well as many cytokine and chemokine receptors, fall into a superfamily of structurally related molecules, termed G-protein-coupled receptors. These receptors are named for the role of G proteins in mediating the varied biological effects of the receptors (see Ch. 10). Consequently, numerous effector proteins are influenced by these heterotrimeric G proteins ion channels adenylyl cyclase phosphodiesterase (PDE) phosphoinositide-specific phospholipase C (PI-PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phospholipase A2 (PLA2), which catalyzes the hydrolysis of membrane phospholipids to yield arachidonic acid. In addition, these G proteins have been implicated in... 

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 Group I mGluRs are coupled to Gaq proteins and stimulation of phospholipase C (PLC). Stimulation of phosphoinositide hydrolysis increases the formation of the... 

Several zinc enzymes that catalyse the hydrolysis of phosphoesters have catalytic sites, which contain three metal ions in close proximity (3-7 A from each other). These include (Figure 12.11) alkaline phosphatase, phospholipase C and nuclease PI. In phospholipase C and nuclease PI, which hydrolyse phosphatidylcholine and single-stranded RNA (or DNA), respectively, all three metal ions are Zn2+. However, the third Zn2+ ion is not directly associated with the dizinc unit. In phospholipase C, the Zn-Zn distance in the dizinc centre is 3.3 A, whereas the third Zn is 4.7 and 6.0 A from the other two Zn2+ ions. All three Zn2+ ions are penta-coordinate. Alkaline phosphatase, which is a non-specific phos-phomonoesterase, shows structural similarity to phospholipase C and PI nuclease however,... 

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 6.8. Cyclic and non-cyclic inositol phosphates. Hydrolysis of phosphatidyl 4,5-bisphosphate (PIP2) by phospholipase C can generate cyclic and non-cyclic inositol phosphates.

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.

Nishio, K., Sugimoto, Y., Fujiwara, Y., Ohmoii, T Moiikage, T, Takeda, Y., and Saijo, N., 1992, Phospholipase C-mediated hydrolysis of phosphotidylcholine is activated by cis-diamminedichloroplatinum (II)- J- Clin. Invest. 89 1622-1628. 

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