1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-m latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membranebound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.
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...
1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N ('collagenolytic cathepsin'). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.
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