A calcium(2+) ion-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme

Abstract

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.

Calcium activated neutral protease from human skeletal muscle

“…The result that human CANP is composed of two subunits of 80 k and 30 k is consistent with that reported by Dayton et al [3,4] for the pig muscle enzyme, although the latter is not completely pure. We also purified rabbit and monkey muscle enzymes and found that both enzymes are composed of two subunits of 80 k and 30 k (to be published).…”

“…Since Ca2+ activated neutral protease (CANP) specifically removes the Z-band, which is~~~ant in rn~t~~g the rnyo~b~~ structure, CANP is thought to play an important role in the initial step of myofibrillar protein degradation [ 1 J. CANP was first identified by Huston and Krebs [2] as a kiiase-activating factor and was later partially purified from pig muscle by Dayton et al [3,4]. They reported that CANP is composed of two subunits of molecular weights of 80 000 and 30 000 [3,4]. Recently we purified CANP for the first time from chicken muscle and found that the protease is a monomer of molecular weight of 80 000 [5].…”

Increased activity of intramuscular proteases in the hyperthyroid state

“…0.03 M KCl, 5 mM neutralized EDTA and 20 mM Na-phosphate buffer (pH 7.0), and centrifuged at 8000 X g for 30 min. The supematant was acidified to pH 4.7 by adding ElsevierlNorth-Holland Biomedical Press 1 N acetic acid, an essential step for separating the endogenous protease inhibitor from CANP [6,7,15]. The precipitate was suspended in equal volume of 0.4 M Tris-HCl buffer (pH 7.4) and 1 mM EDTA, and centrifuged at 100 000 X g for 1 h. The resultant supernatant was used for the assay of CANP, following the method in [7].…”

“…We suggest that thyroid hormones may regulate myofibrillar protein degradation by altering the amount of proteases in skeletal muscle and not in cardiac muscle, and that the activities of endogenous proteases, which degrade either contractile proteins (i.e., myosin and actin) or the regulatory proteins, (i.e., tropomyosin and troponin) [6,7,11], were increased in the skeletal muscle, resulting in muscle atrophy or muscle weakness.…”

“…Here we report increased activities in hyperthyroid skeletal muscle of calcium-activated neutral protease (CANP), cathepsin B and D. CANE' is included in the cytosol fraction [5], is activated by Ca*' at neutral pH [6,7] and is inhibited with leupeptin or antipain [8]. Both cathepsin B and D are contiined in the lysosome fraction [9,10] and are active at acidic pH [9-111. These proteases are important, since CANP has been shown to degrade both tropomyosin and troponin [6-81 and cathepsin B and D have been demonstrated to destroy both myosin and actin among myofibrillar proteins [ 111.…”