Purification and some properties of an alkaline proteinase from rat skeletal muscle

Abstract

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.

Effects of chymostatin and other proteinase inhibitors on protein breakdown and proteolytic activities in muscle

“…Rat skeletal-muscle homogenates exhibit appreciable proteolytic activity at alkaline-pH values (Koszalka & Miller, 1960). Several groups have proposed that this alkaline activity, which may consist of one or more enzymes, plays a role in breakdown of myofibrillar protein (Noguchi & Kandatsu, 1971;Mayer et al, 1974;Dahlmann & Reinauer, 1978). The so-called 'group-specific proteinase' in skeletal muscle resembles this activity as well (Banno et al, 1975).…”

Pancreatic exocrine insufficiency in LXRβ −/− mice is associated with a reduction in aquaporin-1 expression

“…After stopping the reaction by the addition of 0.5 ml of 8% trichloroacetic acid, the solution was centrifuged at 9,500 ϫ g for 5 min. The supernatant was subjected to absorbance measurement at 366 nm by using a spectrophotometer (Molecular Devices Spectra Max 250) at room temperature as described (34). All assays were done in triplicate.…”

Characteristics of an alkaline proteinase and exopeptidase from shrimp (Penaeus indicus) muscle

“…Similarly, Iwata et al (1973) demonstrated the molecular weight of carp alkaline proteinase as 780,000. In constrast, molecular mass of alkaline proteinase from mammalian sources seemed to vary from 30,800 (Dahlmann and Reinauer, 1978) to 120,000 (Dahlmann et al, 1981).…”

“…While some investigators (Lin and Lamer, 1980;Busconi et al, 1984) suggested that the enzyme is sulthydryl dependent, others showed it to be either a metallo (Kozlovskaya and Elyakova, 1975) or a serine proteinase (Kozlovskaya and Elyakova, 1975;Busconi et al, 1984). Differences also seemed to exist between mammalian and invertebrate enzyme (Eguchi and Kuriyama, 1983;Dahlmann and Reinauer, 1978).…”