Limited proteolysis and 'in vitro' mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity.
Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.
Whiting
P
P
Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, U.K.
Gee
N S
NS
Potter
J
J
Howell
S
S
Ragan
C I
CI
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002872:Chromosome Deletion; D010450:Endopeptidases; D000939:Epitopes / Q000032:analysis; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D010450:Endopeptidases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase