Purification and molecular properties of rabbit lung indolamine N-methyltransferase

Publisher Website
Google Scholar
Abstract
Indoleamine N-methyltransferase (INMT) was purified to an apparent homogeneity from rabbit lung, and some of its catalytic and physicochemical properties were examined. The enzyme is a monomeric protein with a MW of 31,500 .+-. 1000, a molecular Stokes radius of 21.5 .ANG. and a diffusion coefficient of 8.7 .times. 10-7 cm2/s. The frictional ratio of the native enzyme (1.05) suggests that the shape of the molecule is nearly spherical. Denaturation experiments performed with increasing concentrations of guanidine hydrochloride (Gdn.cntdot.HCl) at neutral pH indicated that the active site of the enzyme was destroyed by a structural rearrangement of the protein molecule without a large change in its size and shape. The final state reached in 6.0 M Gdn .cntdot. HCl seemed to correspond to a disulfide cross-linked randomly coiled polypeptide. Full normalization of the fluorescent parameter was attained only in the presence of 0.1 M .beta.-mercaptoethanol. A structural rearrangement was observed upon acidification of INMT from pH 7.0 to 2.0. At pH 4.5, most of the peptide backbone appeared to be unorganized, but further acidification to pH 2.0 produced a reorganization of protein structure which became able to bind 8-anilino-1-naphthalenesulfonate. The enzyme structure apparently results from the close packing of organized regions joined by structureless segments.