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Three low molecular mass cysteine proteinase inhibitors were purified from a bovine skeletal muscle crude extract using a three-step procedure. The crude extract was first subjected to gel filtration on a Sephadex G100 column which separated five active fractions (F-I to F-V). Three papain inhibitors, P1, P2 and P3, were fractionated from the F-V fraction by chromatofocalisation on a poly buffer exchanger column. Purification was completed by chromatography on a Mono Q column. After SDS-PAGE, the three inhibitors showed only one band with an M(r) of 14,300. P1, P2 and P3 appeared to be highly resistant to temperature (40-90 degrees C), pH (3-10), reducing agents (5-50 mM) and to be specific for cysteine proteinases since no activity was detected against either serine or aspartyl proteinases. Although to a varying extent, P1, P2 and P3 inhibited papain, cathepsin B and cathepsin L. Analysis of the peptide mixtures of these inhibitors by RP-HPLC after hydrolysis with CNBr or aspartly endoproteinase N together with their amino acid composition revealed that P1, P2 and P3 cysteine proteinase inhibitors are isoforms of the same protein. As their N-terminal ends were blocked, partial sequence of some of these peptides was determined. Computer search in protein identification resources did not reveal any homology of these sequences with proteinase inhibitors of known primary structure. In contrast, they matched well with different parts of the total sequence of a fatty acid binding protein isolated from bovine heart. This homology was supported by the ability of these inhibitors to bind long chain fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)