Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.     

Cathepsin D. Characteristics of immunoinhibition and the confirmation of a role in cartilage breakdown

1. Antisera were raised against lysosomal cathepsin D of man, chicken and rabbit. 2. The antisera were found to be specific and potent inhibitors of cathepsin D activity. 3. The immunological nature of the inhibition was established. 4. The inhibitory effect was studied by varying pH, antiserum/enzyme ratio, time of incubation, concentration of components and order of mixing, and by using purified antibody and univalent antibody fragments. 5. The specificities of the antisera were examined with respect to other enzymes, isoenzymes of cathepsin D and cathepsin D from different organs. 6. The antisera prevented the action of cathepsin D on isolated proteoglycans and on cartilage. 7. The antisera produced up to 90% inhibition of the autolysis of cartilage from chicks and rabbits, indicating that cathepsin D is the enzyme mainly responsible for the breakdown of proteoglycans in this system.     

Effect of estrogen on lysosomal enzyme activities in rat heart

The activities per microgram DNA of five lysosomal enzymes [cathepsin D, cathepsin B, beta-N-acetylglucosaminidase (beta-NAG), beta-glucuronidase, and acid phosphatase] were measured in homogenates of female and male rat (Sprague-Dawley) hearts. Female rats were studied during stages of the estrous cycle and at 3 weeks after ovariectomy. Three-week-postovariectomized female rats and intact male rats were injected subcutaneously with 17 beta-estradiol-3-benzoate. Lysosomal enzyme activities in the male rat heart were more responsive to exogenous estradiol than were activities in the female rat heart. Cathepsin B, beta-NAG, and beta-glucuronidase were increased dramatically in the male rat heart upon short-term administration of estrogen (4 days). In both female and male rat hearts, activities of two lysosomal proteinases, cathepsins B and D, were reduced significantly (approximately 50%) by extended administration of estrogen for 10 days.     

Effects of protein deficiency on selective elicitation of lysosomal enzymes in rat peritoneal macrophages

Young albino rats were fedad libitum 4, 8 or 20 % (control) protein diet for 1–4 weeks. Total activities of some of the lysosomal enzymes, namely, acid phosphatase, aryl sulphatase, Β-glucuronidase and cathepsin D, were determined in resident and protease-peptone elicited peritoneal macrophages. Total cell number, protein content and the lysosomal enzyme activities were increased significantly in protease-peptone elicited macrophages; though at a lower rate in 4 % protein-fed group compared to control ones. However, the rate of induction of the tested hydrolases was selective and their response to the stimulant varied widely. Similarly, response of each enzyme to low protein diet also varied. Thus, at 4 weeks, cathepsin D and Β-glucuronidase activities, expressed per total number of elicited macrophages were reduced by 45 and 60 %, respectively, in 4 % protein-fed animals. These results indicate that the metabolic events related to lysosomal function in macrophages, are affected by dietary restriction of proteins     

Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.     

Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson''s disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson''s disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson''s disease and other neurodegenerative disorders.     

Premalignant Variations in Extracellular Matrix Composition in Chemically Induced Hepatocellular Carcinoma in Rats

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.     

Suppression of the 'uncovering' of mannose-6-phosphate residues in lysosomal enzymes in the presence of NH4Cl

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.     

A comparative study of two kinds of cathepsin D-type proteinases from rat gastric mucosa

The localization of cathepsin D-like acid proteinase in the rat stomach and other tissues was studied, and its biochemical properties were compared with those of rat gastric cathepsin D (EC 3.4.23.5). Cathepsin D-like acid proteinase existed overwhelmingly in the mucosal layer and was hardly detected in the gastric juice. Its subcellular distribution profile was very similar to that of acid phosphatase, but not to that of pepsinogen. This proteinase-like enzyme activity was also found in rat splenic extract. These results strongly suggest that the proteinase is a lysosomal enzyme. In addition, cathepsin D-like acid proteinase demonstrated an in vitro transition of molecular species during storage at -30 degrees C. Although this molecular change was distinctive in ion-exchange column chromatography and susceptibility to some enzyme inhibitors, it was not accompanied by a significant decrease in molecular weight. To compare cathepsin D-like acid proteinase with ordinary cathepsin D, gastric cathepsin D was newly purified to apparent homogeneity in polyacrylamide gel electrophoresis. Its biochemical properties demonstrate that this is a true cathepsin D in rat gastric mucosa. Moreover, this cathepsin D activity was not abolished by treatment with antiserum specific to cathepsin D-like acid proteinase or pepsinogen. From these results, we can conclude that the proteinase is a lysosomal acid proteinase different from newly purified gastric cathepsin D.     

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

Growth hormone modulates the degradative capacity of muscle nucleases but not of cathepsin D in post-weaning mice

We determined whether recombinant human growth hormone (rhGH) administration might modulate the enzyme degradative capacity of the muscle lysosomal system and influence muscle growth. Muscle cathepsin D, acid RNase and DNase II activities are determined in the gastrocnemius muscle of rhGH-treated post-weaning female BALB/c mice. Linear regressions were used to analyze the relationships of each enzyme with their respective substrate. GH induced a depletion-recovery response of muscle growth through a mechanism which is similar to catch-up growth. In these conditions, cathepsin D activity decreased with age in all animals (GH: 40%; saline: 79%), showing a substantial developmental decline that could reflect changes in the rate of protein breakdown. However, the degradative capacity of cathepsin D was paradoxically unmodified in rhGH-mice compared with saline mice (according to the enzyme vs. substrate linear regression slope), in spite of the increase in enzyme activity elicited by GH. This suggests that the muscle protein breakdown is not increased by GH-treatment in post-weaning mice. The enhancement of muscle protein deposition as indicated by the augmented muscle cell size (protein:DNA ratio) of rhGH-mice (increased 178% from 25 to 50 days) vs. saline, can be attributed to a higher muscle K(RNA). In contrast, acid RNase and DNase II activities directly participate in muscle RNA and DNA degradation. Both nucleases were inhibited by GH treatment (a decrease of 48% and 63%, respectively, vs. saline at 50 days). The decrease in RNase activity suggests an inverse relation between the rate of protein synthesis (high) and acid RNase activity (low), leading to spare muscle RNA for synthesizing protein during catch-up growth. Also, low DNase II activity could contribute to inhibiting of muscle DNA degradation, facilitating muscle growth. Thus, GH seems to act as a direct modulator of the degradative capacity of skeletal muscle nucleases but not of cathepsin D, influencing DNA and RNA degradation during the depletion-recovery response to GH of gastrocnemius muscle in female post-weaning mice.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats

Changes in activities of a new proteinase cathepsin T as well as some other lysosomal acid proteinases and hydrolases were examined in liver homogenate from rats treated with a single hepatotoxic dose of carbon tetrachloride. The most striking changes were several-fold increases of liver cathepsin T and D activities over their levels in untreated rats 3 days after administration of the agent to rats. Increase of cathepsin T was greater than that of cathepsin D at all doses of the hepatotoxin examined. The activities of N alpha-benzoyl-DL-arginine 2-naphthylamide hydrolase, acid phosphatase, beta-galactosidase and beta-glucuronidase in poisoned rat liver were unchanged or only slightly increased. Cathepsin T and D activities were less enhanced in mitochondrial lysosomal fractions than in the homogenate, and were greatly elevated in the supernatant fractions of liver from the treated rats. As judged from the molecular weights, the elevated activities of cathepsins T and D in the treated rat liver could be attributable to the two cathepsins themselves and not to other proteinases. Administration to rats of other hepatotoxic agents, thioacetamide and dimethylnitrosamine, also induced the elevation of the two cathepsin activities in liver, but on partial hepatectomy the activities of liver cathepsins T and D did not show such marked increases. Nonparenchymal liver cell fractions were responsible for almost all the increased activities of liver cathepsins T and D. It is possible that cathepsins T and D play a role in the heterolytic breakdown of hepatocyte molecules following CCl4 poisoning.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articulage were examined in detail in 7 joints within the age rangee 21 to 72 years. The results of preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented.Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in unixial compression perpendicular to the articular surface.The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres.At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was not significantly reduced by the action of cathepsin D.In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue.Biochemical analysis of the incubation media, and the specimens, reveled that a large proportion of the proteoglycans was released from the cartilage by each of the freeze enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase relesed between 5.3 and 27.8 per cent of the collagen after 24 h.     

Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro

Procathepsins B and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39,000 by immunoblot analysis. The proenzymes were then purified to remove the mature enzymes by concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased: the activities of cathepsins B and L after 36 h incubation were 60 and 210 times those of the controls, respectively. Immunoblot analysis showed that after 36 h incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increases of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin, which is a potent inhibitor of aspartic proteases. The results strongly suggested that a processing protease for procathepsins B and L might be cathepsin D, a major lysosomal aspartic protease. Indeed, lysosomal cathepsin D could convert microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B and L seem first to be synthesized as enzymatically inactive forms in endoplasmic reticulum and successively may be converted into active forms by cathepsin D in lysosomal compartments.     

Two-step mechanism of myofibrillar protein degradation in acute plasmocid-induced muscle necrosis

Acute muscle necrosis was induced in rats by intramuscular injection of plasmocid, a known myotoxic agent. A single injection of 5 mg/ml plasmocid produced massive fiber necrosis with extensive phagocytosis. Plasmocid administration led to a preferential decrease of alpha-actinin with preservation of other structural proteins within 3 h after injection, and large increases (2-7-fold) in the activities of acid hydrolases, cathepsins B and L, cathepsin D and alpha-galactosidase within 48 h after injection. The plasmocid-induced stimulation of alpha-actinin loss seen at 3 h, when no increases of acid hydrolases occurred, could be inhibited by a cysteine protease inhibitor, Ep-475 (E-64-c), and EGTA. On the other hand, increased lysosomal enzyme activity seemed to have a close correlation with the appearance of invading mononuclear cells, probably macrophages, and not muscle lysosomes. These observations suggest that a two step mechanism of protein degradation (nonlysosomal and lysosomal processes) possibly occurs in plasmocid-induced muscle degradation and macrophages can serve as a main endogenous reservoir of proteases in pathological states.     

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.     

Toxic effects of different concentrations of dimethoate on lysosomal enzymes of female albino rats.

The effect of three different concentrations of dimethoate on the activity of certain lysosomal enzymes, viz. beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin B and cathepsin D in serum, skin, liver, kidney and spleen and the stability of liver and kidney lysosomes was studied in female albino rats. The activity of beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin D was found to increase in serum and tissues in higher concentration (2.25 mg/100 g body weight) of dimethoate treated rats. A significant increase in the rate of release of beta-glucuronidase was found in the liver and kidney of higher concentration of dimethoate treated rats compared to controls. The results demonstrate that the activity of lysosomal enzymes increased in higher concentration of dimethoate treated rats than the lower concentration (0.56 mg/100 g body weight) of dimethoate treated rats.     

The dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats

We performed a longitudinal study for 20 weeks on spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR) to determine the relationship between peptide metabolism and the age-dependent increase in blood pressure. In both SHR and WKR, the plasma level of aminopeptidase A (AP-A) clearly showed an age-dependent decrease. The plasma level of aminopeptidase B paralleled that of AP-A in WKR, but such an age-dependency was not observed in SHR, thus showing a dissociation between the two aminopeptidases. With age in both strains, the level of angiotensin-converting enzyme tended to decrease, while that of kallikrein activity tended to increase. In addition to these findings, a multivariate study testing the relationship of blood pressure to these enzyme activities, as well as to plasma levels of angiotensin I and renin activity, suggested abnormalities in the networks of proteolytic enzymes and in the peptide metabolism surrounding the renin-angiotensin system in SHR. These abnormalities may play some important roles in pathophysiological mechanisms of hypertension in SHR.     

Cysteine proteinase plays a key role for the initiation of yolk digestion during development of Xenopus laevis

In electrophoretic analyses, extracts of Xenopus laevis neurulae exhibited activities digesting yolk proteins maximally at pH4.8. These activities were completely inhibited by a mixture of pepstatin A and Z-Phe-Phe-CHN₂, thus being identifiable as cathepsin D and cysteine proteinase. The electrophoretic profiles of yolk proteins cleaved by embryonic extracts changed at gastrula stages; the profile before stage 13 was the same as that given by cathepsin D treatment and the profile at stage 13 was a combination of the profile given by cathepsin D treatment and that given by cysteine proteinase treatment. Quantitative measurement of enzyme activities showed that the cathepsin D activity that was preserved from the beginning of development increased from stages 13 to 25 and decreased thereafter, whereas the cysteine proteinase activity appeared at stage 13, gradually increased until stage 35 and strongly increased thereafter. Immunoblot analyses showed that the 43 kDa form of cathepsin D was processed to its 36 kDa form, presumably by cysteine proteinase. This change can explain the increase of cathepsin D activity at stage 13 and thereafter. Immunofluorescent staining with the antibody against cysteine proteinase occurred in mesodermal and ectodermal cells other than neural ones at stages 13–24, and in the endodermal cells at stages 24–36. Faint staining in the neural ectoderm persisted from stages 18 to 36. Immunoelectron microscope observation showed that what stained was the superficial layer of yolk platelets. All these results indicate that cysteine proteinase plays a key role in the initiation of yolk digestion during embryonic development.