Search

Resume By means of a relatively simple procedure lysosomal enzymes from rat livers with 20% yield and a 50-fold enrichment were prepared. Density gradient centrifugation or tissue-preloading with triton VR 1339 is not necessary. Up to several liters of 50% liver homogenate may be used. Intact lysosomes are not obtained, because the crucial step in the tissue fractionation scheme is the selective release of lysosomal enzymes from a crude lysosome pellet by water extraction. The presence of neutral endopeptidases with high specific activities in lysosomes (estimated using azocasein and other proteins as substrates) is demonstrated.

Rat liver cathepsin B was tested for its peptide-bond specificity against bradykinin and the oxidized insulin A-chain. Bradykinin was shown to be resistant to the action of cathepsin B. One possible reason for this resistance is the proline content of the peptide and the discrimination against proline residues at three or four subsites of cathepsin B. Oxidized insulin A-chain was degraded by a peptidyl dipeptidase activity. Three dipeptides were cleaved from the C-terminal part of the insulin A-chain after having been incubated for 2 h (molar ration E:S = 1:2800) and six dipeptides were released after a longer digestion (10 h, E:S = 1:575).

If ageing is a problem of diminished adaptation, then studies of ageing changes of the intracellular protein catabolism might elucidate its more common regulatory phenomena. Therefore, last year we started studies on intracellular proteolysis in the aged organism.

Publisher Summary This chapter describes two types of purification methods for Cathepsin B, Cathepsin H, and Cathepsin L. Method I is applicable to large amounts of frozen tissues, whereas method II is used with flesh tissue and takes advantage of a 50-fold purification factor attainable by isolation of lysosomes: it has the further advantage of separating the enzymes from inhibitors that are present in the cytosol and plasma. In first purification method, cathepsins B and H are purified from human liver. Method II involves purification of Cathepsins B, H, and L from rat liver. Method I include: extraction, autolysis, and acetone fractionation and DEAE-cellulose chromatography. The pool of cathepsin B from DEAE-cellulose is further purified by covalent chromatography on a column of aminophenylmercuric acetate coupled to Sepharose. Method II include: homogenization and cell fractionation gel; chromatography on Sephadex G-75; CM-Sephadex chromatography; chromatography of cathepsin L on concanavalin A-Sepharose. Cathepsin B can be with BZ-DL-Arg-NPhNO2 or Bz-Arg-2-NNap as substrate, wheras, Cathepsin H can be assayed selectively by use of an unblocked substrate such as Leu-NNap, Arg-NNap, or Arg-NMec. Three synthetic substrates have been used for cathepsin L assay: Bz-Arg-NH2, Z-Lys-OPhNO2, and Z-Phe-Arg-NMec.

The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

Publisher Summary This chapter discusses protein catabolism in rat liver cells. The use of in vivo short- and long-lived proteins as substrates in vitro allows proving whether the conditions chosen in vitro might be a sufficient simulation of conditions in living cells, and also whether the proteinase used is important for the selective breakdown of short-lived substrate proteins. The protein degradation occuring in vitro in the microsomal fraction must not sufficiently simulate the in vivo process, because a selective autolytic and lysosomal degradation of the in vivo long-lived membrane proteins. Investigations of the primary steps of intracellular protein catabolism must include all organelles, especially the organelles in the microsomal fraction, the energy requirement, the possible role of microtubules and the distinction between basal and enhanced intracellular proteolysis.

The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz-Phe-X-CHN2, in which Phe promotes binding at S2 (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898–902]) while the amino acid X probes S1, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz-Phe-Tyr(O-t-Bu)CHN2 inactivates cathepsin L with a rate 2.5 × 104 greater than that for cathepsin B.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial 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% 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.

A characteristic of lysosomal cysteine proteinases is given by their kinetic constants with specific substrates, their sequence homology, and their reactivity with monospecific polyclonal antibodies.

At the first symposium on „Protein catabolism“ we mentioned our intentions to detect ail soluble proteinases in rat liver lysosomes, which are active at pH 6–7 under in vitro conditions.

Cathepsin L was capable of destroying rabbit muscle aldolase ( d -fructose-1,6-bisphosphate d -glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructase 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PP i and P i decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of P i . Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and P i . The thermostability of aldolase was also unaffected by these ligands. ATP and P i had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and P i was due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light micro-scopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Muller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.

A series of N-peptidyl-O-acyl hydroxylamines was synthesized and tested as inactivators of cysteine proteinases. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the lysosomal cathepsins, B, L and S, may be achieved. The most effective inhibitors display second-order rate constants of the inactivation in the range 10(5)-10(6) M-1.s-1. By contrast, the activity of the aminoendopeptidase cathepsin H is only negligibly affected by the N-terminal-protected peptidyl inhibitors.

Experiments revealing lysosomal heterogeneity have been published during the past years from various laboratories (1).

Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.

The number of possible subsites of the rat liver cysteine proteinases cathepsin B and cathepsin H was determined in the N-terminal direction from the scissile bond. An elongation of the substrate peptide chain of up to four amino acid residues enhances the hydrolysis rate of both cathepsins. The greatest increase in activity was observed by elongation to the dipeptide substrate for cathepsin B and to the tetrapeptide substrate for cathepsin H. Both proteinases discriminate proline from their subsites S1 and S2, but accept it well in S3. A quantitative distinction between the endopeptidase and the peptidyl dipeptidase activity of cathepsin B was feasible by using two model peptides: (Formula: see text) (Z = benzyloxycarbonyl; X = NH2 or OH; the arrow shows the cleavage site). Whereas the peptide acid, representing the peptidyl dipeptidase substrate, was hydrolysed by cathepsin B twice as fast as the peptide amide as an endopeptidase substrate, cathepsin H clearly had a preference for the amide substrate.

Beside the system of thiol-proteindisulfide oxidoreductases (TPO) described in Reinhardsbrunn which catalyzes the cleavage of disulfide groups of insulin, the liver disposes of a spectrum of proteolytic enzymes capable of degrading insulin and also glucagon at a wide range of hormone concentrations. This communication presents new results on the breakdown of 125l-insulin and -glucagon by the so-called insulin-specific proteinase (cytosol) and the lysosomal cathepsins B1, B3, and L.

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. (in a publication from the Cold Spring Harbor Laboratory, New York) for naming new proteinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KC1 washes), the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 M NaC1/0.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25 000-30 000.

Monospecific antisera against cathepsins B, D, and H were used to immunolocalize these proteinases in neural structures of rat brain. Cathepsins B and D were found to be largely co-localized in nerve cells. Cathepsin H could not be identified by use of immunocytochemistry.

Properties of cathepsin L from rat liver lysosomes were compared with those of a similar enzyme, cathepsin S from beef spleen. Major characteristics of cathepsin L are the high activity against Z-Phe-Arg-methylcoumarylamide and sensitivity to the fast reacting irreversible inhibitor Z-Phe-Phe-diazomethane. In contrast, cathepsin S hydrolyzes Z-Phe-Arg-methylcoumarylamide only slowly and Z-Phe-Phe-diazomethane cannot be regarded as a potent inhibitor of this enzyme. The differences in the substrate specificity of cathepsin L from rat liver and cathepsin S from beef spleen are discussed in comparison with the substrate specificity of cathepsin B from rat and human liver and beef spleen.

Publisher Summary Intracellular protein degradation is necessary for changes in the composition of enzymes in all cells. Only in rapidly growing cells is the continual degradation of proteins less necessary because the cells can outgrow their enzyme complement by diluting the preexisting enzymes. The auto-proteolysis of cytosol is extremely weak in comparison to that of the other cell organelles. The proteinases within these cell organelles are more specific, and they hydrolyze only special proteins, such as the group-specific proteinases of mitochondria. Cathepsins L, B, and H are all thiolproteinases. The three enzymes differ from one another precisely in their specificity of splitting substrates. Cathepsin H is an endopeptidase that hydrolyzes proteins and synthetic low-molecular weight substrates, esters, and amides. This enzyme also hydrolyzes aminopeptidase substrates. In contrast to the other two cathepsins, cathepsin L particularly hydrolyzes proteins but almost no synthetic substrates. Cathepsin L has a greater importance in protein degradation than cathepsin B and cathepsin H.

From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated 'proteinase I', which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of 'proteinase I' is similar to alpha-macroglobulin. Immunoelectrophoretic analyses of 'proteinase I' with antisera to rat alpha 1-macroglobulin, alpha 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex of alpha 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar 'proteinase' has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against 'proteinase I'. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of alpha-macroglobulin and cysteine proteinases.

It is a great honour and pleasure for the group at Halle and for myself to participate in this Second Symposium on Intracellular Catabolism as invited guests and speakers of the Yugoslav Committee. I wish to thank Dr. Turk and coworkers for their kind invitation to read the opening lecture of this Second Symposium on „Intracellular Protein Catabolism“. I hope to meet you all again at Reinhardsbrunn Castle in May 1977. For I would like to invite you on behalf of the Biochemical Society of the GDR and the University of Halle to our III. Symposium of „Intracellular Protein Catabolism“, which should be held on the occasion of the hundredth anniversary of Emil Abderhalden’s birth.

In 1968 we proposed a hypothetical mechanism for the selectivity of intracellular protein catabolism: different degrees of exposure of superficial hydrophobic areas of substrate proteins could cause different affinities for lysosomal membranes and/or proteolytic enzymes (Naturwissenschaften 55, 211 (1968).