Phospholipase C mammalian

Nine cloned mammalian adenylylcyclases (ACs) can be activated by stimulatory a subunits (Gas) and several are modulated by inhibitory a subunits (Ckti) and/or Gp/y complexes. cAMP can activate the PKA, which in turn phosphorylates a wide range of substrates, such as the cAMP responsive element binding protein (CREB). When PICA translocates to the nucleus and phosphorylates CREB, the latter is stimulated to regulate gene transcription. There are three mammalian phospholipase C (PLC) isoforms families PLC-P, PLC-y and PLC-6. The first is activated by serpentine receptors, while the second is stimulated by RTKs. 

Write the reaction catalyzed by phospholipase C to produce the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Note that there are several forms of mammalian phospholipase C. 

This can be illustrated by known interactions between the cAMP and Ca2+ pathways. A first messenger that initially activates the cAMP pathway would be expected to exert secondary effects on the Ca2+ pathway at many levels via phosphorylation by PKA. First, Ca2+ channels and the inositol trisphosphate (IP3) receptor will be phosphorylated by PKA to modulate intracellular concentrations of Ca2+. Second, phospholipase C (PLC) is a substrate for PKA, and its phosphorylation modulates intracellular calcium concentrations, via the generation of IP3) as well as the activity of PKC, via the generation of DAG, and several types of CAMK. Similarly, the Ca2+ pathway exerts potent effects on the cAMP pathway, for example, by activating or inhibiting the various forms of adenylyl cyclase expressed in mammalian tissues (see Ch. 21). 

Phosphoinositase C (i.e. phosphoinositide-specific phospholipase C [PLC]) enzymes are found in the vast majority of mammalian cells. Molecular cloning of these enzymes, analysis of their predicted amino acid sequences and immunological cross-reactivity indicate that at least three major forms of the enzyme exist PLC-/I, -8 and -y. Each of these enzyme types is encoded by a distinct gene. More recent experiments using the polymerase chain reaction and molecular cloning have revealed even greater enzyme di-... 

In mammalian cells, there are multiple forms of enzymes within the same family, e.g., protein kinase C, phospholipase C, and caspase. A selective assay system would be instrumental in such cases to advance understanding of the respective role for each form within the same enzyme family. An example for design of a selective assay system for the superfamily of phospholipase A2 is provided in this chapter. Such a selective assay system may play a signihcant enabling role for PLA2 and other enzyme families in the discovery of inhibitors relevant to the treatment of pathologies involving those enzymes. 

Essentially the existence of this enzymatic activity was first established with certainty in the early 1940s in the filtrates of the bacterial organism Cl. perfringens (Cl. welchii) and has been detected subsequently in the filtrates of many other bacteria. Sloane-Stanley (1953) first presented evidence supportive of the presence of a phospholipase C in a mammalian cell, the guinea pig brain. Since that time, momentum in this field of study has increased exponentially with the exciting developments in the signal transduction field. In the latter system, stimulation of a cell leads to activation of a phospholipase C,... 

A thoughtful and detailed outline of the isolation, purification and characterization of the bacterial phospholipases C is given by Waite (1987). Procedures similar to those mentioned above for the mammalian enzymes were also useful in defining the chemistry of the bacterial phospholipases C. 

When attention is directed toward the phospholipase.C found in mammalian tissue, a rather unique and different substrate profile is evident. It appears that the most favored substrate status must be assigned to the inositol- containing phosphoglycerides, namely, phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). There is some evidence that the plasma membrane of certain mammalian cells contains a phospholipase C with high specificity for the bisphosphate, PIP2. The latter enzymatic interaction would be closely associated with the signal transduction pathway in mammalian cells. 

In contrast to the phospholipase C of bacterial origin, there appears to be a consensus that the enzyme from mammalian sources prefers Ca2+ at millimo-lar concentrations. Interestingly, heavy metal ions such as Hg2+ or Zn2+ are strong inhibitors of this source of enzyme and EGTA must be included in the reaction mixture to chelate these cations. 

Although the occurrence of analogs of PAF in mammalian cells was always considered a possibility, Mueller et al. (1984) first reported the presence of 1-0 long-chain acyl-2-acetyl-glycero-3-phosphocholine in rabbit neutrophils. Later, Tokumura et al. (1989) isolated a vasopressor active phosphatidylcholine fraction from bovine brain. Treatment of the fraction with phospholipase C yielded a diglyceride component which was converted to the t-butyldimethylsilyl derivative. Analysis of the latter by gas-liquid chroma-... 

Essen, L.-O., Perisic, O., Cheung, R., Katan, M., and Wilhams, R.L., 1996, Crystal structure of a mammalian phosphoinositide-specific phospholipase C. Nature 380 595-602. 

Kurokawa, M., Sato, K., Rissore, R.A., 2004, Mammalian fertilization from sperm factor to phospholipase C . Biol. Cell 96 37-45. 

---