Cathepsin L

Abstract

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondria1 pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex.2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 -6.1 ascertained by analytical isoelectric focusing.3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 -7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30 -40 0 4 at pH 7.0.5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor.6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.Knowledge of the proteolytic enzymes in the cell is one of the preconditions in studying the molecular mechanism of intracellular protein breakdown. All organelles contain proteolytic activity in various amounts [l]. The lysosomes, in particular, are rich in proteinases. A cell fraction enriched in lysosomes showed at pH 3-4 as well as at pH 6-7 the highest activity hydrolyzing cell-derived proteins in comparison to all other cell organelles [l -61. From this finding it was to be concluded, that the lysosomes certainly play an important role in the overall process of intracellular proteolysis also at physiological pH. A wide variety of lysosomal proteinases has been investigated : cathepsin A (lysosomal carboxypeptidase A) [7-131, cathepsin B1 [7,12-201, cathepsin B2 (lysosomal carboxypeptidase B) [7,12 -14,20-221, cathepsin C (dipeptidyl aminopeptidase I) [7,12,13, Dedicated to Professor Horst Hanson on the occasion of his 65th birthday.Ahbveviation. Leu-CH2C1, 1 -chloro-3-amino-5-methyl-~-hexan-2-one; Tos-Lys-CH2C1, 7-amino-l-chloro-3-tosylamido-~-heptan-2-one ; Tos-Phe-CHzC1, l-chlor0-4-phenyl-3-tosylamido-~-butan-2-one; Bz-L-Arg-NHZ, or-N-benzoyl-L-arginine amide.Enzymes. Cathepsin L (EC 3.4.22.-); cathepsin B1 and B2 (EC 3.4.22.1); cathepsinc (EC 3.4.14.1); cathespin D (EC 3.4.23.5). 18,23,24],cathepsin D [6,7,12,13,23,25-311, cathepsin E [7,13] In studies on the proteolytic activity in a lysosomal extract we succeeded in separating cathepsins BI, C and D by gel filtration on Sephadex G-75. With cytosol proteins as well as with azocasein as substrates at pH 6 and 7, the main part of proteolytic activity was shown to be present in the protein fraction with molecular weights between 20000 and 30000 [2,43]. We could show that in addition to cathepsin B1, the...

Active center differences between cathepsins L and B: The S1 binding region

“…The protease, cathepsin L, initially isolated from rat liver lysosomes [2] is typically a 2-chain protease in its mature form [3]. It appears to be the most active of the lysosomal cysteine proteases, which include cathepsin B, H and possibly others [4].…”

“…These inhibitors have now been examined with cathepsin L in the hope of obtaining similar information and perhaps finding useful differences in reactivity of the two proteases. Cathepsin L was isolated from rat liver [2] and stored as the mercury salt [4]. The stock enzyme was 54pM by titration; 1 : 100 dilutions, activated with thiol-containing buffer [4] at 25°C for about 10 min, were used to prepare reaction mixtures with [E] = 1.1 nM and inhibitor at 25°C.…”

Multifunctional anti‐angiogenic activity of the cyclic peroxide ano‐2 with antitumor activity

“…25,28 To inhibit contaminating cathepsin B enzyme activity, 5 M CA-074, a specified cathepsin B inhibitor, was added to the reaction mixture.…”

Purification and properties of a thiol protease from rat liver nuclei

“…Cathepsin H also hydrolyzed BzArgNan and shows the maximal activity at pH6.0, differing from the optimal pH of the nuclear thiol protease [15]. Cathepsin L has an acidic PI (5.8 -6.1) and a smaller molecular weight (22 000 -24000) [16,17]. Furthermore, when the lysosomal extract from rat liver was applied to chromatofocusing under the same conditions as those described above, most of the activities for hydrolyzing BzArgNan and BzArg NH, and protein were eluted by 1 M NaCl after the pH gradient (datanot shown).…”