Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation.Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective.Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway.The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance.The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.     

Inhibition of cardiac proteolysis by colchicine. Selective effects on degradation of protein subclasses

1. The effect of colchicine (2.5 microM) on cardiac protein turnover was tested with foetal mouse hearts in organ culture. 2. Colchicine had no effect on protein synthesis, but inhibited total protein degradation by 12-18%. Lumicolchicine, which lacks colchicine's ability to disaggregate microtubules, but shares its non-specific effects, did not alter protein degradation. 3. The colchicine-induced inhibition of protein degradation was accompanied by significant changes in cardiac lysosomal enzyme activities and distribution. 4. Colchicine inhibited the degradation of organellar proteins, including mitochondrial cytochromes, more than that of cytosolic proteins. 5. Colchicine decreased the rate of myosin degradation and the rate of proteolysis of the total protein pool to a similar extent. Since the regulation of myosin degradation does not involve lysosomes, this suggests that colchicine affects non-lysosomal as well as lysosomal pathways. 6. Release of branched-chain amino acids from colchicine-treated hearts was disproportionately decreased, suggesting that colchicine increased their metabolism. 7. It is concluded that colchicine, via its actions on microtubules, exerts important inhibitory effects on cardiac proteolysis. Colchicine is especially inhibitory to the degradation of organellar proteins, including mitochondrial cytochromes. Its inhibitory effects may be mediated in part via lysosomal mechanisms, but non-lysosomal mechanisms are probably involved as well.     

Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.     

Distinction between major chloroquine-inhibitable and adrenergic-responsive pathways of protein degradation and their relation to tissue ATP content in the Langendorff isolated perfused rat heart.

In the Langendorff isolated perfused rat heart, 36% of total basal protein degradation was inhibited by the lysosomal inhibitor chloroquine (30 microM), after elimination of rapid turnover proteins during a 3 h preliminary degradation period. Prior inhibition of degradation with chloroquine was additive to the 30% inhibition caused by simultaneous infusion of 50-200 nM-isoprenaline. This additivity suggests that the adrenergic-controlled process is independent of the lysosomal degradative pathway. After discontinuation of drug infusions, the isoprenaline-inhibited degradation rate returned to the previous baseline; however, the chloroquine-inhibited degradation rate transiently exceeded the previous baseline. NaN3 (0.3 mM) caused a decrease of left-ventricular myocardial ATP content of approx. 60% at 14 min and extreme impairment of contractile function; however, the total lysosomal and non-lysosomal protein degradation was not changed at this time. Conversely, left-ventricular tissue ATP content was not changed during proteolytic inhibition by 10 nM-isoprenaline or 10 microM-chloroquine at 14 min. The results indicate that depletion of myocardial energy stores in this preparation is neither necessary nor sufficient to cause inhibition of the total of lysosomal and non-lysosomal protein degradation.     

Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain

The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.     

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

The effect of analogues of chymostatin on lysosomal and non-lysosomal components of protein degradation in isolated hepatocytes

Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Beta-glucosidase 1 (GBA1) is a second bile acid β-glucosidase in addition to β-glucosidase 2 (GBA2). Study in β-glucosidase deficient mice and humans

Beta-glucosidase 1 (GBA1; lysosomal glucocerebrosidase) and β-glucosidase 2 (GBA2, non-lysosomal glucocerebrosidase) both have glucosylceramide as a main natural substrate. The enzyme-deficient conditions with glucosylceramide accumulation are Gaucher disease (GBA-/- in humans), modelled by the Gba-/- mouse, and the syndrome with male infertility in the Gba2-/- mouse, respectively. Before the leading role of glucosylceramide was recognised for both deficient conditions, bile acid-3-O-β-glucoside (BG), another natural substrate, was viewed as the main substrate of GBA2. Given that GBA2 hydrolyses both BG and glucosylceramide, it was asked whether vice versa GBA1 hydrolyses both glucosylceramide and BG. Here we show that GBA1 also hydrolyses BG. We compared the residual BG hydrolysing activities in the GBA1-/-, Gba1-/- conditions (where GBA2 is the almost only active β-glucosidase) and those in the Gba2-/- condition (GBA1 active), with wild-type activities, but we used also the GBA1 inhibitor isofagomine. GBA1 and GBA2 activities had characteristic differences between the studied fibroblast, liver and brain samples. Independently, the hydrolysis of BG by pure recombinant GBA1 was shown. The fact that both GBA1 and GBA2 are glucocerebrosidases as well as bile acid β-glucosidases raises the question, why lysosomal accumulation of glucosylceramide in GBA1 deficiency, and extra-lysosomal accumulation in GBA2 deficiency, are not associated with an accumulation of BG in either condition.     

Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Role of Ca2+ for protein turnover in isolated rat hepatocytes.

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.     

人直肠癌β-葡糖苷酶同工酶的动力学研究

 本文对人直肠癌及癌旁组织(对照)的β-葡糖苷酶同工酶的分离、底物动力学和某些物质的影响做了初步探索。结果表明:在这两种组织中分别存在三种同工酶。一种是胞液酶,Km值为1.18mmol/L(对照为1.13mmol/L),受NaCl和抗癌药WB非竞争性抑制;第二种是溶酶休可溶性酶,Km值为2.38mmol/L(对照为2.94mmol/L),不受NaCl影响,受WB的混合型抑制,直肠癌此酶受顺铂的竞争性抑制;第三种是溶酶体膜结合酶,受NaCl激活,使底物动力学由负协同性酶变为米氏酶。肿瘤组织中三种同工酶活性均高于癌旁组织中相应的酶活性。     

Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain.

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic beta-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic beta-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl beta-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity beta-glucosidase compared with the placental glucocerebrosidase; for example, with octyl beta-D-glucoside the Ki values were 10 microM and 1490 microM for the cytosolic and lysosomal beta-glucosidases respectively. Furthermore the higher affinity of the cytosolic beta-glucosidase than glucocerebrosidase for the amphipathic alkyl beta-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [delta delta G0 (K,)/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic beta-glucosidase are discussed with regard to its potential physiological substrates.     

Efficient routing of glucocerebrosidase to lysosomes requires complex oligosaccharide chain formation

The biosynthesis and intracellular transport of the membrane-associated lysosomal enzyme glucocerebrosidase was studied in the monoblast cell line U937. Addition to the cultures of the oligosaccharide trimming inhibitors swainsonine or deoxymannojirimycin led to an increased intracellular activity of glucocerebrosidase. This was due to prevention of the lysosomal degradation of the enzyme. When homogenates of control cells were fractionated on Percoll gradients glucocerebrosidase, like beta-hexosaminidase, was distributed in two peaks, one at low density and one at high density. When homogenates of cells cultured in the presence of oligosaccharide trimming inhibitors were fractionated beta-hexosaminidase was still distributed in two peaks but glucocerebrosidase was found mainly in low density fractions also containing galactosyltransferase activity. It is concluded that complex type oligosaccharide chain formation is required for efficient routing of glucocerebrosidase to the lysosomes in U937 cells.     

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.     

Long-term ingestion of ammonium inhibits lysosomal proteolysis in rat liver

A standard diet was supplemented with ammonium acetate (20%, w/w). The effect on liver protein degradation of oral administration of the ammonium diet to rats for 6 weeks has been studied. It is shown that lysosomal proteolysis is markedly decreased (by 62%) while non-lysosomal proteolysis is inhibited by 11%. This is the first report showing that ammonium ingestion inhibits liver proteolysis.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Inhibition by cyanate of the processing of lysosomal enzymes

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.     

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes

The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets.These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells.