1. Introduction The enzymatic capacity of proteinases from rat skeletal muscle with optimum activity in the alkaline pH range increases during several hormone-mediated protein catabolic conditions like starvation [ 1,2], insulin deficiency [l-3], testosterone deficiency [4], glucocorticoid treatment [l] as well as during muscle dystrophy [4,6,7]. With the obvious correlation between enhancement of enzyme activity and muscle protein breakdown, we assume that this proteinase system is involved in pathological muscle wasting. Alkaline proteinases from rat skeletal muscle have been isolated, purified and characterized by several groups. The ‘muscle alkaline protease’ (MAP) isolated from the myofibrillar fraction has been characterized in [8]. The properties of this chymotrypsin-like enzyme are very similar to those of the alkaline pro- teinase [9] designated ‘group specific proteinase’ (GSP), and shown to immunologically crossreact with the mast cell chymase I [lo]. In our laboratory, a chymotrypsin-like proteinase called ‘cytoplasmic proteinase’ (CP) has been isolated from the post-myofibrillar fraction of rat skeletal muscle, a proteolytic activity shown to be similar to both of the above enzymes [ 11,121. Here, immunological methods were used to estab- lish whether the 3 chymotrypsin-like enzymes, MAP, GSP and CP, are identical. 2. Experimental For the isolation of enzyme, pooled hindleg skele- tal muscles from male Wistar rats -200 g body wt were used. The ‘muscle alkaline proteinase’ was iso- lated as in [8,13]. The ‘group-specific protease’ was purified as in [ 141 but omitting the last crystallization step. The ‘cytosolic proteinase’ was prepared as in [ 111. The purity of all 3 enzyme preparations was assessed by SDS-polyacrylamide gel electrophoresis [ 151. Analysis of gels revealed a single protein band with each purified proteinase. Antisera to CP were raised in rabbits by intradermal injection of 0.1 mg enzyme protein suspended in complete Freund’s adjuvants followed by an intrave- nous booster injection (0.1 mg enzyme) after 4 weeks. One week later the animals were bled. The IgG frac- tion was isolated from the antiserum by DEAE-Affigel blue chromatography (BioRad Labs., Mtinchen) on a 2.5 X 20 cm column by the procedure described by the manufacturer. The isolated IgG fraction was further purified by gel filtration on a 2 X 100 cm col- umn of Ultrogel AcA 34 (LKB, Bromma) equilibrated with PBS-buffer (0.15 M NaCl/O.Ol M sodium phos- phate (pH 7.4)). Using this procedure complete sepa- ration of the serum proteinase inhibitory activity, the serum proteolytic activity and the IgG fraction was achieved. Immuno-inhibition tests were performed by incu- bation of 0.5 ml of proteinase solution with 0.5 ml anti-CP-IgG solution. Buffers used to dilute the anti- serum were 1 .l M kJ/6 mM NazSzOz (pH S.O), 0.25 M K-phosphate (pH 8.0) and 1 M KC1/50 mM Tris-HCl (pH 8.5) when testing MAP, GSP and CP, respectively. After a 60 mm incubation at 37”C, the antigen-antibody mixture was centrifuged for 10 mm at 45 000 X g. Residual proteolytic activity was determined by incubation of 0.2 ml supernatant fraction with 0.05 ml 3% azocasein solution for 60 min at 37’C as in [ 161. Proteolytic activity is given as % of the control containing proteinase solution and the corresponding buffer solution.