Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity.
Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0.