Spontaneous degradation of insect myosin during metamorphic cell death. Conditions for proteolysis

Abstract

Department of Biology, Molloy College, Rockville Centre, NY 11570, U.S.A. The intersegmental muscles of the Sphingiid moth, Manduca sexta, degenerate completely within 48 hr after the emergence of the adult. Although lysosomal proteases are present in the tissue, the mechanisms for initiation and control of lysis are not known. Conditions for in vitro proteolysis were therefore investigated. A 70,000 dalton fragment of myosin, seen in spontaneously-degenerating muscle, was generated occasionally in vitro, at pH 6.5. M. sexta intersegmental muscle myosin, with an initial subunit size of 220,000 daltons, was shown to degrade spontaneously in vitro to 170,000 daltons, at pH 6.5 and to 127,000 daltons, at pH 8.5. Exogenous addition of Ca 2+ or ATP did not affect these results. Cathepsins B and D were present in high quantities during times of active lysis and were likewise unaffected by Ca 2+. Native or iodinated (Bolton-Hunter) insect myosin was cleaved to 130,000 daltons by rat liver cathepsin B and to 145,000 daltons by rat liver cathepsin D. Our interpretation is that a protease or proteases acting at or near neutral pH initiates the degradation of myosin, and that in vitro and potentially in vivo, lysosomal cathepsins terminate the digestion.