Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle.
A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially 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-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.
Hori
S
S
Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033. horishin@tmin.ac.jp.
Ohtani
S
S
Hori
C
C
Nokihara
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008040:Genetic Linkage; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D009137:Muscular Dystrophy, Animal / Q000201:enzymology* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012492:Salts; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014960:X Chromosome
D006023:Glycoproteins; D012333:RNA, Messenger; D012492:Salts; D012697:Serine Endopeptidases; C112964:myonase