Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium.
We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D005260:Female; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009215:Myometrium / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature
D000199:Actins; D046911:Macromolecular Substances; D000205:Actomyosin; D010450:Endopeptidases; D008666:Metalloendopeptidases; D009218:Myosins