Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle

Abstract

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

Electron microscopy and image analysis of the multicatalytic proteinase

“…A neutral proteinase has been identified in a variety of eukaryotic cells and has been designated 'multicatalytic proteinase' (MCP) since it appears to contain more than one active site for catalysis of peptide-bond cleavage [1][2][3]. The latent enzyme which can be activated by SDS and fatty acids [1,4,5], by phospholipids [6] as well as by polylysine [3] is localized in the cytoplasm [3], in the cell nucleus [7] or in both compartments [8].…”

“…The enzyme purified from rat skeletal muscle has a molecular mass of 650 kDa [2]. A 19 S ribonucleoprotein (RNP) with a similar subunit pattern has been identified in a number of eukaryotic cells as reviewed in [9].…”

RNA degrading activity is tightly associated with the multicatalytic proteinase, ingensin

“…la, only one band was detected in the non-denatured gel. And SDS gel elctrophoresis of the purified protease demonstrated its multisubunit structure, which was the same as that of the multicatalytic proteinase, ingensin, from other sources [2][3][4][5]11] (fig.lb). These results indicated that the enzyme sample was highly purified, i.e.…”

“…The multicatalytic proteinase, ingensin, is a cytosolic protease which has an unusually high molecular mass, 650-750 kDa, and is composed of multiple subunits of 25-35 kDa [1][2][3][4][5]. One of the important characteristics of the enzyme is the multiplicity of its activity.…”

Cloning and sequence analysis of pituitary cDNA encoding the β‐subunit of Xenopus proteasome

“…This particle with a sedimentation coefficient of approximately 20 S (molecular mass about 750 kDa) is composed of a characteristic set of at least 13 non-identical polypeptide subunits with molecular masses between 20 and 35 kDa [2][3][4]. The individual subunits may each have a unique catalytic property, i.e.…”