PURIFICATION AND CHARACTERIZATION OF MYONASE FROM X-CHROMOSOME LINKEDMUSCULAR DYSTROPHIC MOUSE SKELETAL-MUSCLE

A chymotrypsin-like proteinase, designated myonase, was successfully p urified to homogeneity from X-chromosome linked muscular dystrophic mo use skeletal muscle by affinity chromatography on agarose conjugated w ith lima bean trypsin inhibitor as ligand. The molecular mass of the p urified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,1 87 Da by mass spectrometry. The native enzyme is a single chain molecu le and a monomeric protein without sugar side-chains, The nucleotide s equence of myonase mRNA is similar to mouse mast cell proteinase 4 (MM CP-4) cDNA. This is the first report of a native enzyme whose amino ac id sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotryps in-like activities and hydrolyzes the amide bonds of synthetic substra tes having Tyr and Phe residues at the P-1 position, Myonase is most a ctive at pH 9 and at high concentration of salts, Myonase preferential ly hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-H is9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of a myloid beta-protein, Myonase is completely inhibited by such serine pr oteinase inhibitors as chymostatin, diisopropylfluorophosphate and phe nylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chlorom ethyl ketone, p-tosyl-l-lysine chloromethyl ketone, pepstatin, E-64, E DTA, and o-phenanthroline. It is also inhibited by lima bean trypsin i nhibitor, soy bean trypsin inhibitor, and human plasma alpha(1)-antich ymotrysin. These properties match those of chymase, but unlike chymase , myonase does not interact with heparin in the regulation of its acti vity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.