290s Biochemical Society Transactions ( 1 992) 20 Bovine Inositol Monophosphatase; Observation of the Modification of a Cysteine Residue Using Protein Fluorescence.

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PETER J. GREASLEY, MICHAEL G. GORE, 'C. IAN RAGAN, 'MICHAEL R. KNOWLES, 'NICHOLAS S. GEE and 'GEORGE McALLISTER. Department of Biochemistry, University of Southampton, Bassett Crescent East, Southampton and 'Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex. Intracellular responses to extracellular stimuli are mediated by secondary messengers. One such mechanism utilizes the hydrolysis of phosphatidylinositol4,5- bis phosphate (PIP,) by phosphoinositidase C to generate the secondary messengers 1,2- diacylglycerol and inositol 1,4,5- trisphosphate (IP, ). IP, mediates signal transduction by stimulating the release of calcium from intracellular stores. The regeneration of the membrane located PIP, is dependent upon the supply of inositol which is produced by sequential dephosphorylation of IP, [ l ] and by de novo production of inositol from glucose 6-phosphate [2]. Both of these pathways are dependent on inositol monophosphatase activity [3]. Under normal conditions dietary inositol is available to replenish intracellular levels. This does not occur in brain tissue since transport of inositol across the blood - brain barrier is slow [4]. As a result of this the enzyme inositol monophosphatase plays an essential role in the maintenance of suitable levels of inositol in the brain. Inositol monophosphatase is a homodimer with a subunit molecular weight of 30,000 Da [3]. The enzyme has an essential requirement for the divalent cation magnesium and is inhibited uncompetitively by Li, ions (51 which is believed to trap a covalent phospho-enzyme intermediate [6]. The enzymes from human, bovine and rat brain show 79% homology [7] and recent chemical modification studies have suggested that there is an argimne residue in the active site of the enzyme [8]. We have shown that the bovine enzyme reacts with various thiol reagents in a differential manner. Modification with DTNB leads to inactivation of the enzyme with the release of approximately 2 mol equivalents of NTB'. which increases to 5.5 in the presence of 0.1% SDS; reaction with N-ethyl maleimide (NEM) lead to a loss of enzyme activity [9]. DTNB modification of enzyme previously modified by NEM generates virtually no (0.05 mol equivalents) production of NTB2- under non-denaturing conditions. In contrast prior modification by iodoacetic acid (IAA) (which does not inactivate the enzyme) leads to the production of one mol equivalent of NTB2- upon DTNB modification (Fig 1). When the enzyme is reacted with NEM in the presence of inositol 1-phosphate and Li' ions only one mol equivalent of NEM is incorporated into the enzyme and no activity is lost. We conclude therefore that there are two equivalents of cysteine per subunit of enzyme that are reactive to NEM or DTNB and that one of these lies at or close to the active site. of the enzyme. We have identified the residue only modified by NEM in the absence of substrate and Li' ions as Cys 218. A mutant enzyme with a Cys 218 M a replacement shows full activity which is not sensitive to NEM. The modification by NEM can be followed using a change in protein fluorescence which decreases by approximately 15%. This change proceeds at the same rate as the modification of Cys 218 by NEM and does not occur if modification by NEM is attempted in the presence of substrate and Li' ions. Reaction

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P Fig. 1. Modification of Inositol monophosphalase by DTNB The stoichiometry of h'TB2- releasc from a solution of 6pM enzyme in O.OSM TrisHCI buffer, pH 8.0 under the following conditions (A) in the prexnce of 0.IS SDS. (B) no additions, (C) enzyme previously modified by 5mM iodoacetic acid. (D) enzyme previously modified by ImM NEM. (E)in the prescncc of IOmM inositol I-phosphate and IOmM LiCI. (F)mutated enzyme Cys 218 Ala. ( G ) mutated enzyme Cys 218 Ala in the presence of IOmM inositol I-phosphate and IOmM LiCI. (H) mutated enzyme Cys 218 Ala previously reacted with 5mM iodoacctic aad.

of Cys 218 Ala enzyme with NEM does not result in significant fluorescence changes. We have used chemical modification, protein sequencing and site directed mutagenesis studies to identify the source of this fluorescence signal change as being Trp 219 in the linear sequence. This residue is sensitive to the ionisation of Cys 218 and the pH induced fluorescence change of Trp 219 has been used to show that the pK of this residue is about pH 8.2.

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Shears, S.B. (1989) Biochem. J., 260 313 - 324 Eisenberg F. Jr. J. Mol. Chem. (1967). 242 1375 - 1382 Gee N.S., Ragan C.I., Watling K.J., Aspley S., Jackson R.G., Reid G.R., Gani D. and Shute J.K. Biochem J (1988), 249 883 - 889 Spector R. and Lorenzo A.V. Am. J. Physiol. (1975). 228 1510- 1518 Hallcher L.M. and Sherman W.R. J. Bid. Chem. (1980). 255 10986- 10991 Shute J.K., Baker R., Billington D.C. and Gani D. J. Chem. SOC.Chem. Commun., 626 -628 McAllister G., Whiting P.J., Hammond E.A., Knowles M.R., Atack J.R., Bailey F.J., Maigetler R. and Ragan C.I. Biochem. J. Submitted Jackson R.G., Gee N.S. and Ragan C.I. Biochem. J. (1989), 264 419 -422 Knowles M.R., Gee N.S., McAllister G.. Ragan C.I., Greasley P.J. and Gore M.G. Biochem. J. Submitted 1991.

Bovine inositol monophosphatase; observation of the modification of a cysteine residue using protein fluorescence.

290s Biochemical Society Transactions ( 1 992) 20 Bovine Inositol Monophosphatase; Observation of the Modification of a Cysteine Residue Using Protein...
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