Comparative studies of two types of acid proteases from rat gastric mucosa and spleen

Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; these two types of acid proteases are quite different proteins existing in the individual tissues.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

Cathepsin E from rat neutrophils: its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0-3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by rho-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.     

Mannose 6-phosphate-independent targeting of lysosomal enzymes in I- cell disease B lymphoblasts

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1- phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6- phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.     

Immunochemical similarity between a gastric mucosa non-pepsin acid proteinase and neutrophil cathepsin E of the rat

Antiserum against a rat gastric mucosa non-pepsin acid proteinase precipitates rat neutrophil cathepsin E, with a precipitation curve essentially similar to that of the gastric enzyme. Taken together that the antiserum precipitates a cathepsin E-like acid proteinase from rat spleen (Muto, N., Yamamoto, M. and Tani, S. (1987) J. Biochem. (Tokyo) in press), the data indicate that the non-cathepsin D acid proteinases in rat neutrophils, gastric mucosa and spleen are immunochemically closely related. In contrast with the earlier data, cathepsin E from rabbit neutrophils exhibited a maximal activity at around pH 3.0-3.2 and preferred hemoglobin to albumin as substrate, which supports that the non-cathepsin D acid proteinases in the rat tissues are relevantly classified as cathepsin E.     

The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response.

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.     

Cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from mouse and guinea pig liver Golgi membranes during the acute phase response

1. Rat Gal beta 1-4GlcNAc alpha 2-6sialyltransferase (E.C. 2.4.99.1) is released from Golgi membranes by cleavage of a portion of the enzyme containing the active site from a membrane anchor; this effect was most dramatic during the acute phase response. The enzyme that cleaved sialyltransferase had the properties of cathepsin D was most active at pH 5.6 and was likely of lysosomal origin (Lammers and Jamieson, 1988). 2. The acute phase response of sialyltransferase in mouse and guinea pig was previously found to differ from that in the rat. Release of sialyltransferase from mouse and guinea pig Golgi membranes has now been studied in order to make a comparison with the rat system. 3. Maximum release of sialyltransferase from mouse and guinea pig Golgi occurred at pH 4.6 and 5.2, respectively; like the rat a cathepsin D-like proteinase was responsible for release of both enzymes. 4. Immunoblot analysis showed that membrane-bound rat and mouse sialyltransferase had Mr 49,000, whereas the guinea pig enzyme had Mr 42,000. The released form of the rat enzyme had Mr 42,000, but released forms of mouse and guinea pig enzymes had Mr 38,000 suggesting a different cleavage site for these two enzymes compared to the rat enzyme.     

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.     

Further studies on rat cathepsin E: subcellular localization and existence of the active subunit form

The subcellular localization of rat neutrophil cathepsin E was examined by a modification of the method of N. Borregaard et al. [(1983) J. Cell Biol. 97, 52-61]. When the postnuclear cavitate of rat neutrophils was subjected to density centrifugation on discontinuous Percoll gradients, three particulate bands, P1 (lowest; azurophil granule rich), P2 (middle; specific granule rich), and P3 (highest; plasma membrane rich), were segregated. A combined application of immunochemical and electrophoretic methods revealed a striking difference in subcellular localization between cathepsin E and cathepsin D: Cathepsin E was associated with P3 and soluble fractions, and cathepsin D was chiefly associated with P1 and P2 fractions. The results thus indicate that cathepsin E is a nonlysosomal acid proteinase in rat neutrophils. It was found that cathepsin E existed in two enzymatically active molecular forms, referred to as CE-I and CE-II, in rat neutrophil extracts. To examine the relationships between the two forms, cathepsin E was purified to homogeneity from rat gastric mucosae. The purified enzyme exhibited a single protein band of 43 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, but electrophoresis without SDS, followed by visualization of activity in the gel, revealed two activity bands corresponding to CE-II and CE-I in neutrophil extracts. Pretreatment of the enzyme with beta-mercaptoethanol or dithiothreitol resulted in an increase in CE-I activity with a concomitant decrease in CE-II activity on gels. Upon gel filtration, the molecular weights of CE-II and CE-I were estimated to be 98,000 and 51,000, respectively, strongly suggesting that they are the dimeric and monomeric forms of the cathepsin E subunit.     

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0–3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5–3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.     

Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

A catalytically active high-Mr form of human cathepsin B from sputum.

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the formation of mannose 6-phosphate residues. To identify this protein determinant, we constructed chimeric molecules between two aspartyl proteases: cathepsin D, a lysosomal enzyme, and pepsinogen, a secretory protein. When expressed in Xenopus oocytes, the oligosaccharides of cathepsin D were efficiently phosphorylated, whereas the oligosaccharides of a glycosylated form of pepsinogen were not phosphorylated. The combined substitution of two noncontinuous sequences of cathepsin D (lysine 203 and amino acids 265-292) into the analogous positions of glycopepsinogen resulted in phosphorylation of the oligosaccharides of the expressed chimeric molecule. These two sequences are in direct apposition on the surface of the molecule, indicating that amino acids from different regions come together in three-dimensional space to form this recognition domain. Other regions of cathepsin D were identified that may be components of a more extensive recognition marker.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Proteolysis of poly(ADPribose) polymerase by a pyrophosphate- and nucleotide-stimulated system dependent on two different classes of proteinase

We have identified a system in human lymphocytes which proteolytically cleaves poly(ADPribose) polymerase to specific fragments of molecular weight 96 000, 79 000 and 62 000-60 000. This proteolytic processing is dependent on two different classes of proteinase. One of these proteinases is a serine proteinase, since the processing is inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor and diisopropylfluorophosphate, the other is a cathepsin D-like proteinase, since processing is also inhibited by pepstatin A. The processing that occurs in permeabilized cells can be simulated in vitro by treating purified poly(ADPribose) polymerase with trypsin, but not by treating the polymerase with cathepsin D. Since processing at the cellular level is blocked by inhibitors of either of the two proteinases, but only trypsin could cleave the purified polymerase, this suggests that in the cell the action of the cathepsin D-like proteinase is a prerequisite for cleavage of poly(ADPribose) polymerase by the serine proteinase. Thus, a pathway involving sequential action of these proteinases may exist. Proteolysis in permeabilized human lymphocytes is stimulated by nucleotides containing a pyrophosphate group, such as 5',5'-P1,P4-tetraphosphate and ATP, or by pyrophosphate itself. In contrast, nucleotides containing only a single phosphate, such as AMP and cyclic AMP, or inorganic sodium phosphate, do not show this stimulation of proteolysis. These results suggest that a pyrophosphate linkage is the minimum molecular requirement for stimulation of proteolytic processing of poly(ADPribose) polymerase. Proteolytic processing of poly(ADPribose) polymerase is independent of ADPribosylation. Following proteolysis, specific fragments of the polymerase, particularly the 62 000-60 000 molecular weight fragment(s), are still capable of being ADPribosylated.     

Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestica midgut

Musca domestica larval midgut display in cells and luminal contents a proteolytic activity with a pH optimum of 3.0–3.5. This activity is abolished by pepstatin and is insensitive to soybean trypsin inhibitor and to sulfhydryl proteinase inhibitors. The acid proteinase occurs in multiple forms with M_r values in the range 40,000–80,000 and with pI values of about 5.5. The proteinase inactivates at 60°C according to apparent first-order kinetics and Lineweaver-Burk plots of its activity against albumin concentration are rectilinear, suggesting that the multiple forms have similar properties. The proteinase reacts slowly with diazoacetylnorleucine plus CuSO₄, is stable in alkaline media, is inhibited by dithiothreitol, hydrolyses hemoglobin better than albumin and is virtually not active upon synthetic substrates for pepsin. These properties are similar to those of cathepsin D. The specific activity of the acid proteinase determined by titration with pepstatin is 680 units/mg of proteinase and the K_D of the pepstatin-proteinase complex is 1.5 nM at 30°C. The acid proteinase occurs mainly in midgut subcellular fractions characterized by a high specific activity of molybdate-inhibited acid phosphatase and a large number of secretory-like vesicles. It is proposed that the M. domestica midgut acid proteinase is a cathepsin D-like proteinase evolved to function in luminal contents. The lack of ATP activation of the midgut enzyme supports this hypothesis, since ATP is thought to regulate cathepsin D-proteolysis inside lysosomes.     

Cathepsin L. A new proteinase from rat-liver lysosomes.

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.