Thiol Cathepsins Research Articles

Thiol Cathepsins and Oxidative Modification of Stallion’s Seminal Plasma Proteins with Normal and Low Percentage of Live Spermatozoa Post Cryopreservation

Background: Stallion sperms are very sensitive to cryopreservation. Free radicals generated during cryopreservation lead to spermatozoa membrane damage, decline in their viability and change the activity of cysteine cathepsins. On the other hand, cathepsins may involve in the utilization of oxidatively modified proteins. The present study was conducted to assess cysteine cathepsins activity and level of oxidatively modified proteins in seminal plasma of stallions with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation Methods: A total of 33 breeding stallions comprising 6 stallions of the Budennovskaya breed, 22 Arabian breed, 4 Soviet Heavy-Draft and 1 Bashkir breed. We assessed standard quality parameters in fresh and cryopreserved sperm, as well as cysteine cathepsins B,L,H activity, the level of oxidatively modified proteins in stallions seminal plasma with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation. Result: The group with a low percentage of live spermatozoa was characterized by decreased sperm quality, increased level of aldehyde dinitrophenylhydrazones (p=0.045) and ketone dinitrophenylhydrazones (p=0.049) of a neutral nature, as well as increased activity of cathepsin L (p=0.029). Evaluation of activity of cathepsin L and the concentration of oxidatively modified proteins in the seminal plasma can serve as a marker in the selection of stallions for sperm cryopreservation.

PSXV-13 The activity of thiol cathepsins in seminal plasma of stallions with different percentages of viable sperm after freezing-thawing

Abstract The aim of this work was to study the activity of cathepsins B, L and H in the seminal plasma of stallions with normal (>25%, n = 11) and low (< 25%, n = 13) percentage of viable sperm after freezing-thawing. Sperm of 24 Arabian stallions aged from 5 to 20 years (12.1 ± 4.8 years on average) was collected during the breeding season (February-May). The activity of cathepsins in spermoplasm was studied by the spectrofluorometric method (System 3 Scanning Spectrofluorometry, Optical technology devices, inc. Elmstord, New York, 10523) by Barrett&Kirschke. The viability of the sperm was determined after its freezing-thawing. Sperm smears were eosin stained and live:dead-ratio was examined using an Olympus BX41 phase contrast microscope (Olympus Corporation, Japan). The significance of differences in the studied groups was determined using the Mann-Whitney U-test. There were no significant differences in the activity of catepsins B and L in the spermoplasm of stallions with normal and low percentages of viable spermatozoa after freezing-thawing. It was found that in the group of stallions with a normal percentage of viable spermatozoa after freezing-thawing, the activity of cathepsin H in the spermoplasm was significantly higher than in the group with a low percentage of viable spermatozoa (P = 0.0219). Free radicals formed during freezing-thawing of sperm can damage cell membranes, leading to loss of sperm viability. Thiol cathepsins are involved in the degradation of oxidatively modified proteins and, apparently, it is cathepsin H that is most actively involved in this process in the sperm of stallions. We assume that the low activity of cathepsin H in the seminal plasma of stallions with a low percentage of viable spermatozoa indicates a high involvement of this enzyme in the protection of sperm membranes from oxidative damage. The research was supported by the Russian Science Foundation grant No. 20-16-00101.

Immunotoxicity of Gallium Arsenide on Antigen Presentation: Comparative Study of Intratracheal and Intraperitoneal Exposure Routes

Gallium arsenide (GaAs) is a semiconductor utilized in electronics and computer industries. GaAs exposure of animals causes local inflammation and systemic immune suppression. Mice were administered 2 to 200 mg/kg GaAs. On day 5, intratracheal instillation increased lung weights in a dose-dependent manner and induced pulmonary inflammation exemplified by mononuclear cell infiltration and mild epithelial hyperplasia. No fibrosis, pneumocyte hyperplasia, proteinosis, or bronchial epithelial damage was observed in the lungs. Splenic cellularity and composition were unaffected. GaAs' effect on antigen presentation by macrophages was similar after intratracheal and intraperitoneal exposure, although the lowest observable adverse effect levels differed. Macrophages from the exposure site displayed an enhanced ability to activate an antigen-specific CD4+ helper T-cell hybridoma compared with vehicle controls, whereas splenic macrophages were defective in this function. The chemical's impact on peritoneal macrophages depended on the exposure route. GaAs exposure augmented thiol cathepsins B and L activities in macrophages from the exposure site, but decreased proteolytic activities in splenic macrophages. Alveolar macrophages had increased expression of major histocompatibility complex (MHC) Class II molecules, whereas MHC Class II expression on splenic and peritoneal macrophages was unaffected. Modified thiol cathepsin activities statistically correlated with altered efficiency of antigen presentation, whereas MHC Class II expression did not. Our study is the first one to examine the functional capability of alveolar macrophages after intratracheal GaAs instillation. Therefore, thiol cathepsins may be potential target molecules by which GaAs exposure modulates antigen presentation.

Gallium arsenide exposure impairs splenic B cell accessory function

Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4 + T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.

Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B–specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B–specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

Determination of the mRNA sequence of cathepsin Y, a cysteine endopeptidase from rat spleen, 1 and confirmation of its ubiquitous expression.

A cysteine endopeptidase from rat spleen was purified, characterized and its gene cloned. This enzyme was originally recognized by its action of producing kinin-potentiating peptide from a plasma protein. We named it cathepsin Y due to its localization, acidic pH optimum and the presence of the same set of active site amino acids as in other thiol cathepsins. Here we show the total sequence of the mRNA obtained by means of TaKaRa 5' Full RACE Core Set and complete the previously reported sequence. This completion of the mRNA sequence resulted in the omission of the strangely attached C-terminal peptide from cathepsin Y.

Delta 9-tetrahydrocannabinol selectively increases aspartyl cathepsin D proteolytic activity and impairs lysozyme processing by macrophages

Delta 9-tetrahydrocannabinol (THC) causes an antigen-dependent defect in the ability of macrophages to activate helper T cells, and this drug-induced impairment is mediated through the peripheral CB2 receptor. Various requirements for the processing of the antigen, lysozyme, were examined to determine where along the pathway THC exerts its influence. A THC-exposed macrophage hybridoma inefficiently stimulated interleukin-2 secretion by a helper T cell hybridoma in response to native lysozyme and its reduced form, suggesting that disulfide bond reduction was unaffected. Cell surface expression of major histocompatibility complex class II molecules was normal on THC-exposed macrophages. The drug-exposed macrophages also competently presented a lysozyme peptide to the T cells, indicating that the class II molecules were functional. The proteolytic activity of two thiol cathepsins was unaltered, but aspartyl cathepsin D activity was significantly increased in THC-exposed macrophages. Thus, selective up-regulation of aspartyl cathepsin activity accompanied the deficiency in lysozyme processing and may contribute, at least in part, to the antigen-dependent processing defect in THC-exposed macrophages.

Gallium Arsenide Modulates Proteolytic Cathepsin Activities and Antigen Processing by Macrophages

Gallium arsenide (GaAs) is a semiconductor utilized in the electronics industry. Chemical exposure of animals causes a local inflammatory reaction, but systemic immunosuppression. Mice were administered i.p. 200 mg/kg GaAs crystals or latex beads, or vehicle. Five days after exposure, splenic macrophages were defective, whereas thioglycolate-elicited peritoneal macrophages (PEC) were more efficient in processing the Ag, pigeon cytochrome c, than vehicle control macrophages. Various aspects of the MHC class II Ag-processing pathway were examined. Both macrophage populations normally presented a peptide fragment to the CD4+ T cells. Surface MHC class II expression on the PEC was up-regulated, but splenic cells had normal MHC class II expression. PEC had elevated levels of glutathione and cysteine, major physiologic reducing thiols. However, the cysteine content of splenic macrophages was diminished. Proteolytic activities of aspartyl cathepsin D, and thiol cathepsins B and L were decreased significantly in splenic macrophages. On the other hand, thiol cathepsin activities were increased selectively in PEC. Latex bead-exposed PEC were not more potent APC, and their thiol cathepsin activities were unchanged, indicating that phagocytosis and nonspecific irritation were not responsible. The phenotype of PEC directly exposed to GaAs mirrored cytokine-activated macrophages, in contrast to splenic macrophages from a distant site. Therefore, GaAs exposure differentially modulated cathepsin activities in splenic macrophages and PEC, which correlated with their Ag-processing efficiency. Perhaps such distinct alterations may contribute to the local inflammation and systemic immunotoxicity caused by chemical exposure.

The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues.

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.

Regulation of proteinase levels in the nematode Caenorhabditis elegans. Preferential depression by acute or chronic starvation

Acute starvation of the wild-type of the nematode Caenorhabditis elegans depresses the level of cathepsin D by 65% within 4-8 h and the level of the thiol cathepsins Ce1 and Ce2 to about the same extent after 24 h. There is no parallel loss of lysosomal beta-glucosidase or beta-hexosaminidase activities. In strains which are chronically starved as a result of mutations which compromise feeding behaviour (unc-52) or nutrient uptake into the intestinal cells (daf-4), cathepsin D levels are decreased to about 15% of the level in fully fed wild-type animals. We suggest that the decline in the cathepsin D level results from autodigestion when alternative protein substrates are depleted in the lysosomes.

A novel cathepsin from the mussel ( Perna perna) (Linne)

1. 1. Proteases from the digestive gland of the mussel Perna perna were subjected to an isolation procedure similar to that used for mammalian thiol cathepsins. 2. 2. A protease was purified by ammonium sulphate fractionation, molecular exclusion chromatography and ion exchange chromatography on CM-Sephadex. 3. 3. The enzyme was activated by thiol-reducing agents and metal chelators, inhibited by iodoacetamide, N-ethylmaleimide, divalent mercury ions and TPCK, at high concentration, and is therefore apparently a thiol protease. It is not affected by leupeptin or pepstatin. 4. 4. The enzyme has a MW of 21,000 and digests azocasein, with an acidic pH optimum, but is not active against either l-arginine β-naphthylamide or l-arginyl- l-arginine β-naphthylamide.

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.

Metabolic acidosis stimulates protein degradation in rat muscle by a glucocorticoid-dependent mechanism.

Metabolic acidosis is associated with enhanced renal ammonia-genesis which is regulated, in part, by glucocorticoids. The interaction between glucocorticoids and chronic metabolic acidosis on nitrogen utilization and muscle protein metabolism is unknown. In rats pair-fed by gavage, we found that chronic acidosis stunted growth and caused a 43% increase in urinary nitrogen and an 87% increase in urinary corticosterone. Net protein degradation in incubated epitrochlearis muscles from chronically acidotic rats was stimulated at all concentrations of insulin from 0 to 10(4) microU/ml. This effect of acidosis persisted despite supplementation of the media with amino acids with or without insulin, indomethacin, and inhibitors of lysosomal thiol cathepsins. Acidosis did not change protein synthesis; hence, the increase in net protein degradation was caused by stimulation of proteolysis. Acidosis did not increase glutamine production in muscle. The protein catabolic effect of acidosis required glucocorticoids; protein degradation was stimulated in muscle of acidotic, adrenalectomized rats only if they were treated with dexamethasone. Moreover, when nonacidotic animals were given 3 micrograms/100 g of body weight dexamethasone twice a day, muscle protein degradation was increased if the muscles were simply incubated in acidified media. We conclude that chronic metabolic acidosis depresses nitrogen utilization and increases glucocorticoid production. The combination of increased glucocorticoids and acidosis stimulates muscle proteolysis but does not affect protein synthesis. These changes in muscle protein metabolism may play a role in the defense against acidosis by providing amino acid nitrogen to support the glutamine production necessary for renal ammoniagenesis.

Lysosomal sequestration of cytosolic enzymes and lysosomal thiol cathepsins

Cytoplasmic proteins are degraded with different half-lives in vivo. Large parts of proteins are believed to be degraded primarily in autophagic vacuoles-lysosomal system. However, the mechanism by which cell proteins are delivered to lysosomes and whether such a process might be selective for certain cell proteins are still unresolved. We examined the mechanism of autophagy with isolated autophagic vacuoles. Administration of leupeptin, a inhibitor of lysosomal thiol proteinases, induced the accumulation of numerous autophagic vacuoles in the liver. Highly purified preparation of autophagic vacuoles was isolated by Percoll density gradient equilibrium fractionation of crude lysosomal fractions. When cytosolic enzyme activities in autophagic vacuoles were measured, tyrosine aminotransferase and tryptophan oxygenase with short half-lives, and lactic dehydrogenase and aspartate aminotransferase with long half-lives were detected at similar ratios of enzymes in autophagic vacuoles/cytosol. During the time that cathepsin B plus L activities in autophagic vacuoles are inhibited by the injection of leupeptin, cytosolic enzymes are being accumulated in autophagic vacuoles suggesting that leupeptin blocks intralysosomal proteolysis, and that cytosolic enzymes are sequestered continuously into autophagosomes. Administration of glucocorticoid, which induces the synthesis of tyrosine aminotransferase, tryptophan oxygenase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Dietary manipulation and administration of insulin, which inhibit the formation of autophagic vacuoles, suppressed completely the accumulation of autophagic vacuoles in liver by administration of leupeptin. Results indicate that there is no selective uptake of cytosolic enzymes into autophagosome. When distribution of lysosomal cathepsin B and L in liver, which are inhibited strongly by leupeptin, was examined immunohistochemically, cathepsin L is found only in hepatocytes, but cathepsin B is localized in sinusoidal cells rather than in hepatocytes, suggesting that cathepsin L plays a most important role in intralysosomal proteolysis in hepatocytes.

Degradation of [6-3H]- and [1-14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver.

We studied the degradation and metabolism of radioactive glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver. Removal of 130 micrograms of the protein from the perfusate occurred with a T 1/2 of 10.3 min. Radioactivity associated initially with the microsomal fraction of the tissue homogenate and was then transferred to the lysosomes where hydrolysis of N-acetylglucosamine residues took place. Radioactivity left the lysosomal-rich fraction of the homogenate with a T 1/2 of 50 min. Final accumulation of radioactivity was in the supernatant and greater than 80% of this material was found to be UDP-N-acetylhexosamines. In those studies, 10 mumol of unlabeled glucosamine had been added to the perfusate to expand the intracellular pool of UDP-GlcNAc and prevent further metabolism of any radioactive N-acetylglucosamine released by the lysosomes. When this procedure was not done, greater than one-third of the lysosomally derived N-acetylglucosamine was reused by the liver to form new radioactive glycoproteins secreted into the perfusate. Overall hydrolysis of N-acetylglucosamine from the glycoprotein substrate was greater than 80% during 2 h of perfusion. However, leupeptin, a thiol cathepsin inhibitor, decreased the release of amino sugar by 50%, even though this compound had no effect on the activity of any lysosomal glycosidase in vitro. Large glycopeptides of molecular weight 35,000 accumulated in the lysosomes due to leupeptin. From these studies, we conclude that efficient digestion of the carbohydrate component of this glycoprotein in situ requires the concerted activity of both lysosomal glycosidases and proteases.

Estrogen Receptor in Avian Embryo and Adult Liver: Estrogen Receptor Activation and Dissociation Kinetics of Estradiol, Ethynylestradiol, and Moxestrol*

Estrogen receptor-enriched fractions from cockerel and embryo liver cytosol were obtained by precipitation at 0--35% ammonium sulfate saturation. At low ionic strength conditions, the embryo receptor sediments in sucrose density gradients mainly as an 8S entity. The cockerel receptor, however, sediments at 4.9S, unless an inhibitor of thiol proteases, iodoacetate, is included in the isolation medium. Moreover, the affinity of the cockerel receptor for moxestrol is doubled in the presence of iodoacetate and equals that of the embryo receptor (Kd = 1.4--1.8 x 10(-10) M). We conclude that thiol cathepsins from cockerel liver cause proteolytic degradation of the estrogen receptor. At temperatures between 0--28 C, the dissociations of moxestrol, ethynylestradiol, and estradiol follow monophasic kinetics. At 28 C, the half-times for the moxestrol-, ethynylestradiol-, and estradiol-receptor complexes are 65, 11, and 6 min, respectively. The chaotropic salt, NaSCN, reduces 6-fold the half-time of the moxestrol-receptor complex at 28 C (t 1/2 = 10 min). In 0.15 M KCl, the estrogen-receptor complex from embryo liver sediments at 4S. Incubation at 28 C, before the sucrose gradient analysis at 0 C, does not change the sedimentation coefficient. Sucrose gradient centrifugation carried out at 20 C results in the transformation to a 5S form of the moxestrol- as well as the estradiol-receptor complex. Our findings suggest that the estrogen receptors in the cytosol of avian liver and mammalian reproductive tissues are very similar. From both tissues, the native 8--10S form is extracted, and the receptors are transformed into the 5S form upon incubation with hormone at 20--30 C.

Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells.

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.

Are lysosomes involved in hexose transport regulation? Turnover of hexose carriers and the activity of thiol cathepsins are arrested by cyanate and ammonia.

The cycloheximide-related loss of transport activity (manifested as a decrease in Vmax for transport) in cultured Nil hamster fibroblasts was blocked by the addition of carbamoyl phosphate, cyanate (a product of spontaneous phosphate elimination from carbamoyl phosphate), or ammonium salts to the culture medium. Acid proteases capable of hydrolyzing alpha-N-benzoyl-D,L-arginine-beta-naphthylamine (cathepsins B1, H, and L) were also inhibited in situ by ammonia and cyanate. The inactivation of these cathepsins by ammonia was irreversible and probably was related to the increase in the intralysosomal pH known to be caused by an accumulation of ammonia in the lysosomes. The inhibition of the cathepsin activity by cyanate in situ (and in cell-free extracts) was completely reversible and blocked irreversible inhibition of the cathepsin(s) by N-ethylmaleimide. The inactivation of the cathepsins caused by cyanate was deduced to be the result of reversible blocking of sulfhydryl groups essential to the thiol cathepsin activity. The concomitant inhibition of thiol cathepsins and hexose carrier inactivation provided further evidence for the involvement of lysosomal proteases in at least part of the mechanism that regulates the rate of hexose transport in animal cells.

Degradation of apolipoprotein B of low density lipoprotein by cultured bovine smooth muscle cells: Accumulation of intermediates in the presence of protease inhibitors

The incubation of bovine aortic smooth muscle cells with 125I-labeled low density lipoprotein in the presence of protease inhibitors resulted in the significant intracellular accumulation of intact apolipoprotein B, as well as a number of high molecular weight degradation intermediates. This effect was brought about both by leupeptin, a specific inhibitor of thiol proteases (40% inhibition of degradation), as well as by the more general lysosomotrophic inhibitors, chloroquine and NH 4Cl. Qualitatively identical spectra of degradation intermediates were formed in the presence of chloroquine and NH 4Cl as determined by autoradiography of SDS-polyacrylamide gel electrophoretic fractions, ranging from the apolipoprotein B band ( M r = 340 000) to bands with molecular weights of less than 14 000. The effect of NH 4Cl was reversible and release of inhibition resulted in the sequential loss of intermediates from the cells; those species having higher molecular weights disappearing first. Inhibition by leupeptin was associated with the accumulation of degradation products in the lower molecular weight range only ( M r ⩽ 72000). These results provide evidence that apolipoprotein B proteolysis progresses in distinct stages via specific breakdown products and suggest that the thiol cathepsins become more active later in the degradation pathway.