Purification and some properties of cathepsin B from rabbit skeletal muscle

Abstract

Cathepsin B was purified from rabbit skeletal muscle by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocellulose, peptide-conjugated Sepharose, DEAE-Toyopearl and Sephadex G-100. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis. The enzyme did not abolish the Ca sensitivity of the ATPase activity of myofibrils. The molecular mass of the enzyme was found to be 27 kDa on gel filtration and SDS/polyacrylamide gel electrophoresis. The optimum pH for the hydrolysis of N"-benzoyl-DL-arginine-P-naphthylamide was 6.5. The enzyme was stable in the range of pH 4.5 -5.5. Tetrathionate reacted with thiol groups of the enzyme reversibly so that it stabilized the enzyme. The enzyme was strongly inhibited by iodoacetate, HgC12, antipain, leupeptin, N"-p-tosyl-L-lysine chloromethane and L-tosylphenylalanylchloromethane, but not by pepstatin or trypsin inhibitor.The purification of muscle cathepsin B has already been reported by Schwartz and Bird [I] and Hirao et al. [2]. Schwartz and Bird [l] partially purified the enzyme from rat skeletal muscle and showed that it could degrade myosin. Hirao et al. [2] purified the enzyme to the electrophoretically homogeneous state from monkey skeletal muscle and found that it could degrade myosin and actin. However, in these studies it was not determined at which step of the respective purification process cathepsin B was separated from cathepsin L, which was shown to be present in a crude extract of cathepsin B and to be similar in several properties with cathepsin B in our previous study [3], or whether or not cathepsin L was really excluded from the purified cathepsin B preparation. Therefore, in the present study we established a new method for obtaining rabbit skeletal muscle cathepsin B completely free from cathepsin L, and investigated its properties. MATERIALS AND METHODS MaterialsThe rabbit muscle (longissimus dorsi) was obtained from the carcasses immediately after slaughter and, after removal of fat and connective tissue, it was minced with a meat chopper. Bovine serum albumin, ovalbumin, chymotrypsinogen and cytochrome c were purchased from Boehringer Mannheim Correspondence zo A. Okitani,

Mechanism Involved in the Improvement of Meat Taste during Postmortem Aging.

“…Mechanism of the increase in peptides In the muscle, there are lysosomal cathepsins B (Okitani et al, 1988), D (Suzuki & Fujimaki, 1968;Fukushima et al, 1971) (Okitani et al, 1985) and L (Okitani et al, 1980b) which are most active in an acidic pH region, sarcoplasmic p-and m-calpains (Dayton et al, 1976(Dayton et al, , 1981Ishiura et al, 1978), which are activated by Ca2+ and exhibit most activities in a neutral pH region, and a proteasome (Dahlmann et al, 1985;Driscoll & Goldberg, 1989;Matsuishi & Okitani, 1997) dependent on ATP for activation. It is reported that cathepsins B, D and L degrade myofibrillar proteins, while cathepsin H scarcely causes hydrolysis ofthese proteins (Okitani et al, 1980b;Ouali et al, 1987).…”

“…It is reported that cathepsins B, D and L degrade myofibrillar proteins, while cathepsin H scarcely causes hydrolysis ofthese proteins (Okitani et al, 1980b;Ouali et al, 1987). Zeece et al (1986) and Lisa et al ( 1995) reported that calpain is involved in the degradation of myofibrillar proteins during the postmortem storage of meat.…”

Comparative evaluation of the quality changes in Squid (Ommastrephes bartrami) during flake and slurry ice storage

“…The main changes in myofibrillar are reported to occur in myosin light-chain but actin and actinin also degrade during storage period (Careche et al, 1998;Kjaersgard et al, 2006;Schubring, 2005). Numerous studies attributed the degradation of myofibrillar to proteases such as cathepsins, as well as calcium-dependent proteases (Okitani et al, 1980;Zhang et al, 2015). In several marine species, cathepsins B, D, and L were considered as the enzymes playing the most important role in postmortem muscle softening (Ladrat et al, 2003).…”

A Judgment on Postmortem Aging inLongissimus DorsiBased on a Peptide Substrate Library

“…Of the proteases involved in postmortem aging, calpains, 9) cathepsins, 10,11) and aminopeptidases 12,13) have been purified, but the purification of proteases requires considerable effort and cost. Furthermore, because postmortem aging of meat is caused by the combined effects of proteases, it is not practical to determine the degree of postmortem aging using only one isolated protease.…”