PHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C IS ACTIVATED ALLOSTERICALLY BY THE AMINOGLYCOSIDE G418 - XY-2-FLUORO-SCYLLO-INOSITOL-1-O-DODECYLPHOSPHONATE AND ITS ANALOGS INHIBIT GLYCOSYLPHOSPHATIDYLINOSITOL PHOSPHOLIPASE-C

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus c ereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dod ecylphosphonate) (Morris, J, C,, Ping-Sheng, L,, Shen, T, Y,, and Mens a-Wilmot, K, (1995) J, Biol, Chem, 270, 2517-2524), A set of novel flu orinated 2-deoxy-Ins-1-Q-dodecylphosphonates were tested against PI-PL C, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1 -O-dodecylphosphonate (VP-616L) (X(i(50)) = 0.09), 2-Deoxy-2-fluoro-my o-Ins-1-O-dodecylphosphonate and -deoxy-2,2-difluoro-myo-Ins-1-O-dodec ylphosphonate were 8.3 fold and 4.8-fold less effective, respectively, than VP-616L, Methyl -deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphona te was inactive, Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI, Imp lied in these observations are the following: (i) in powerful inhibito rs an active site residue probably interacts with the equatorially ori ented fluoro substituent; (ii) substrate recognition requires a negati ve charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanam ycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anc hor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- t o 4-fold, G418, which appears to act on the enzyme substrate complex, increased k(cat) and K-m 6.4-fold and 9.9-fold, respectively, PI-PLC w as 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 (X(i(50)) = 0. 00025) and GPI-specific phospholipase D (X(i(50)) = 0.00018), G418 fai led to activate PI-PLC when ConA was present, These observations indic ate that G418 is an allosteric activator of Bacillus cereus PI-PLC, Si nce G418 stimulates a purified enzyme that is not involved in aminogly coside metabolism, we propose that binding of aminoglycosides to cellu lar proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.