Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacillus sp. both cleave glycosylphosphatidylinositols (GPIs). However, phosphatidylinositol, which is efficiently cleaved by PI-PLC, is a very poor substrate for GPI-PLC. We examined GPI-PLC substrate requirements using glycoinositol analogs of GPI components as potential inhibitors. Glucosaminyl (alpha 1-->6)-D-myo-inositol (GlcN(alpha 1-->6)Ins), GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate, GlcN(alpha 1-->6)-2-deoxy-Ins, and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate inhibited GPI-PLC. GlcN(alpha 1-->6)Ins was as effective as Man-(alpha 1-->4)GlcN(alpha 1-->6)Ins; we surmise that GlcN(alpha 1-->6)Ins is the crucial glycan motif for GPI-PLC recognition. Inhibition by GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC possess a GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate at the terminus of the residual glycan. The effectiveness of GlcN(alpha 1-->6)-2-deoxy-Ins indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required for substrate recognition, although it is probably essential for catalysis. GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol, unlike GlcN(alpha 1-->6)-2- deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins. Surprisingly, GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus PI-PLC, and GlcN(alpha 1-->6)Ins had no effect on the enzyme. However, both GlcN(alpha 1-->6)Ins 1-phosphate and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate were competitive inhibitors of PI-PLC. These observations suggest an important role for a phosphoryl group at the Ins 1-position in PI-PLC recognition of GPIs. Other studies indicate that abstraction of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC cleavage of GPIs. Furthermore, both GlcN(alpha 1-->6)-2-deoxy-Ins 1-phosphate and GlcN(alpha 1-->6)-2-deoxy-L- myo-inositol inhibited PI-PLC without affecting GPI-PLC. Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes represent mechanistic subclasses of GPI phospholipases C, distinguishable by their sensitivity to GlcN(alpha 1-->6)Ins derivatives and aminoglycosides. Possible allosteric regulation of PI-PLC by GlcN(alpha 1-->6)Ins analogs is discussed.
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PMID:Glycan requirements of glycosylphosphatidylinositol phospholipase C from Trypanosoma brucei. Glucosaminylinositol derivatives inhibit phosphatidylinositol phospholipase C. 785 13

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.
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PMID:Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418. 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C. 866 28

The human thromboxane A(2) (TXA(2)) receptor (TP) is known to mediate platelet aggregation and vasoconstriction. The receptor predominantly interacts with the Gq protein, thereby activating phospholipase C and increasing the intracellular calcium level. In this study, we synthesized a 15-residue peptide corresponding to the C-terminal domain of the Gq protein alpha subunit (Galphaq-Ct peptide) and characterized its interaction with recombinant TP purified from a baculovirus expression system in the presence and absence of an agonist using fluorescence and NMR spectroscopic studies. With fluorescence binding assays, we demonstrated that the Galphaq-Ct peptide was bound to TP, in the absence of the agonist, with a K(d) value of approximately 17 muM. Interestingly, upon addition of the agonist, U46619, the Galphaq-Ct peptide's binding affinity for this activated TP was reduced, thereby increasing the K(d) value to approximately 240 muM. NMR experiments demonstrated that the TP-bound Galphaq-Ct peptide shows a different affinity and conformation, in the absence and presence of the agonist, U46619. This suggested there is the possibility of ligand-free constitutive TP signaling through Galpha binding. Thus, an HEK293 cell line that stably expresses human TP and lacks the ability to produce TXA(2) was created by gene transfer and G418 selection. In comparison with the control cells, the stable cell line showed significant Galpha-mediated ligand-free calcium signaling. The study indicates a promising new outlook for the examination of prostanoid receptor-G-protein interactions in greater detail using integrated NMR spectroscopy, the purified receptor, and the stable cell line.
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PMID:Structural and functional analysis of the C-terminus of Galphaq in complex with the human thromboxane A2 receptor provides evidence of constitutive activity. 2059 Jan 59