Effect of leupeptin on the intracellular degradation of asialofetuin in isolated rat hepatocytes

The effect of the protease inhibitor leupeptin on the intracellular distribution of [14C]-sucrose-asialofetuin in isolated rat hepatocytes was investigated. Leupeptin had no effect on the uptake but reduced the degradation of asialofetuin. Fractionation of hepatocytes by isopycnic centrifugation in sucrose gradients indicated that prolonged treatment with leupeptin inhibited the uptake of asialofetuin into the lysosomes. Therefore, leupeptin inhibits degradation of asialofetuin both by inhibiting intralysosomal proteolysis and transport of endocytosed asialofetuin to the lysosomes.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes.

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of 125I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca2+ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca2+ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more than 30 min. When incubations were carried out for more than 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of ¹²⁵I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca²⁺ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca²⁺ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more that 30 min. When incubations were carried out for more that 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

The effect of leupeptin on intracellular digestion of asialofetuin in rat hepatocytes

The effect of leupeptin on the intracellular distribution of asialofetuin, endocytosed by isolated rat hepatocytes, was studied. By means of sucrose gradient centrifugation it was found that leupeptin led to accumulation of undegraded ¹²⁵I-labeled asialofetuin both in lysosomes and in an organelle of lower density (probably an endocytic vesicle). To decide whether the protease inhibitor interfered with the uptake of asialofetuin into lysosomes we studied its effect on the intracellular distribution of [¹⁴C]sucrose-asialofetuin. Acid-soluble radioactivity formed from [¹⁴C]sucrose-asialofetuin is trapped within the lysosomes and the rate of uptake of this ligand in the lysosomes can therefore be studied. Using [¹⁴C]sucrose-asialofetuin it was found that leupeptin, in addition to inhibiting proteolysis inside the lysosomes, retards the transport of asialofetuin into these organelles. Reduced uptake of asialofetuin into lysosomes was seen only after incubating the cells with leupeptin for more than about 30 min. The leupeptin effect on the transport of asialofetuin may therefore be secondary to accumulation of undegraded substrates inside the lysosomes.     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and degradation of 125I-labelled asialo-fetuin by isolated rat hepatocytes.

125I-Labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 - 10(-8) M asialo-fetuin. Non-parenchymal liver cells did not take up asialo-fetuin in vitro. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Asialo-fetuin consequently accumulated in the cells until the extracellular supply was exhausted. Asialo-fetuin degradation could be studied without concurrent uptake by incubating cells, previously exposed to asialo-fetuin, in asialo-fetuin-free medium. Degradation, as evidenced by increase in acid-soluble radioactivity, was inhibited by NH4Cl and chloroquine. The change with time in the intracellular distribution pattern of radioactivity in cells that had been exposed to 125I-labelled asialo-fetuin for 10 min was examined by means of differential centrifugation. Initially, the radioactivity was found mostly in the microsomal fraction. 60 min after the exposure to labelled protein, the distribution pattern of radioactivity resembled that of the lysosomal enzyme beta-acetylglucosaminidase. The possibility that asialo-fetuin digestion takes place in lysosomes is discussed.     

Sensitive measurement of polyethylene glycol-modified proteins

An IgM monoclonal antibody (AGP3) against polyethylene glycol (PEG) was used to assay PEG-modified proteins by ELISA. PEG-modified beta-glucuronidase could be measured at concentrations as low as 15 ng/mL, corresponding to 750 pg (1.8 fmol) of conjugate. This ELISA should be generally applicable to all PEG-modified proteins because AGP3 binds the backbone of the PEG chain independent of the linker used for PEG attachment.     

Uptake and degradation of desialylated luteinizing hormone by suspended hepatocytes

The uptake and degradation of desialylated human luteinizing hormone (asialo-LH) in suspended hepatocytes have been studied. Asialo-LH was taken up by the asialo-glycoprotein receptor at a rate which was somewhat lower than that of asialo-fetuin. The rate constants and equilibrium binding parameters were similar, but the rate of dissociation of the receptor-ligand complex was higher in the case of asialo-LH. The uptake was influenced by heterogeneity of the asialo-LH preparation. Degradation of endocytosed asialo-LH took place in the lysosomes. After fractionation of the cells by isopycnic centrifugation in a sucrose gradient, partially degraded asialo-LH (precipitable with trichloroacetic acid, but not with antibody) was found in the fractions containing endocytic vesicles, but not in the lysosomal fractions, indicating that the proteolysis of asialo-LH was initiated in the endocytic vesicles.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.     

Uptake and degradation of iodine-labelled chylomicron remnant particles by monolayers of rat hepatocytes.

1. Rat chylomicrons were labelled with 125I with 69--72% of the iodine in the protein moiety. Less than 1 nmol of iodine was incorporated per nmol of protein. Of the peptide radioactivity 44--56% was in apolipoprotein A-1, 30--40% in the C peptides and 11--15% in apolipoprotine B. The arginine-rich peptide, which accounted for about 14% of the chylomicron protein mass as determined by scanning of sodium dodecyl sulphate-polyacrylamide gels, contained very little radioactivity. 2. Chylomicron remnants generated with postheparin plasma from iodine-labelled chylomicrons showed a relative increase in the percentage of the arginine-rich peptide (76--90% of the apolipoprotein mass according to gel scanning). The major portion of the peptide iodine label was present in apolipoprotein A-1 (43--57%), B (22--32%) and C peptides (17--35%). 3. When iodine-labelled chylomicron remnants were added to rat hepatocytes in primary culture, labelled peptides were taken up and degraded by the hepatocytes by a saturable process. The Vmax. for the uptake was calculated to the 300ng of protein/h per mg of cell protein and the apparent Km as 7.7 microgram of protein/mg of cell protein. A larger proportion of the 125I-labelled lipids of the remnants (mainly polar lipids) was taken up. This suggest that these may also enter the cells by a mechanism that does not involve particulate uptake, such as phospholipid exchange. 4. The degradation of labelled peptides was inhibited by colchicine, concanavalin A, chloroquine and NH4Cl, which also inhibit degradation of the cholesteryl ester portion. All these drugs exerted their inhibition mainly after the uptake of labelled peptide. No degradation occurred at 4 degrees C, and also the uptake was markedly decreased. 5. The uptake of labelled chylomicron remnant peptide was 77 times as effective as that of labelled sucrose, which is likely to be taken up randomly by pinocytosis.     

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.     

Lysosomal and non-lysosomal pathways of intracellular insulin degradation in isolated rat hepatocytes

The amount of 125I-insulin associated with freshly isolated hepatocytes was increased 50% in the presence of 0.2 mM chloroquine (CQ) after 2 h of incubation. The degradation of insulin by the hepatocytes incubated with CQ was significantly diminished as compared with control cells. Hepatocytes incubated with 125I-insulin in the presence of CQ showed a slower rate of ligand dissociation than control cells. More TCA-precipitable and less TCA-soluble material appeared in the dissociation buffer of CQ-treated cells. However, CQ inhibited only 25-35% of intracellular insulin degradation. Non-lysosomal intracellular insulin degradation appears to be responsible for the remaining portion of the ligand degradation by isolated hepatocytes.     

Uptake of thymidine into isolated rat hepatocytes. Evidence for two transport systems

Thymidine transport was studied in isolated rat hepatocytes. In these cells no phosphorylation of the substrate by thymidine kinase occurred subsequent to transport. Results from studies of the concentration-dependent uptake of thymidine indicated two transport systems with about 80-fold differences in their kinetic constants. These systems were denoted as high affinity [Km = 5.3 micron, V = 0.47 pmol/(10(6) cells X s)] and low affinity systems [Km = 480 micron, V = 37.6 pmol/(10(6) cells X s)]. From intracellular to extracellular distribution ratios of [3H]thymidine it could be concluded that the uptake by the high affinity system was a concentrative process while the transport by the low affinity system was non-concentrative. The uptake of [3H]-thymidine by the high affinity system could only be inhibited by unlabeled thymidine. In contrast, all other nucleosides tested (uridine, 2'-deoxycytidine, and 2'-deoxyguanosine) were equally effective in inhibiting the low affinity system competitively. The results would suggest that in hepatocytes lacking phosphorylation by thymidine kinase, thymidine is taken up by a high and a low affinity system working in tandem. The high affinity system seems to be an active transport process with narrow substrate specificity. Thymidine uptake by the low affinity system is a facilitated diffusion process. This system is considered to be a common transport route for nucleosides of different structures.     

Biosynthesis of cholesterol linoleate by polyethylene glycol-modified cholesterol esterase in organic solvents.

Cholesterol esterase modified with polyethylene glycol was able to dissolve in some highly hydrophobic solvents such as benzene and toluene, and catalyze the synthesis of cholesterol linoleate with time dependency in the reverse of the usual reaction in aqueous solvents. Enzymatic cholesterol linoleate synthesis followed Michaelis-Menten kinetics which depended on the concentration of cholesterol and linoleic acid. When more than 20 mM of both substrates was used, the specific activity in this esterification was 200-250 nmol/min/mg protein. The apparent Km value for cholesterol and linoleic acid was 3.7 and 7.6 mM, respectively. The possibility of using such a modified enzyme for the synthesis of less stable cholesterol esters is discussed.     

Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.     

Retinyl esters synthesis by polyethylene glycol-modified lipase in benzene

Summary Using polyethylene glycol-modified lipase we have succeeded in synthesizing retinyl palmitate through ester exchange reaction between retinyl acetate and palmitic acid in a transparent benzene solution. The product had much lower peroxide value than the one obtained by a conventional organic synthesis. As much as 85% of a substrate, retinyl acetate, was converted to the product in a day at 25°C. We have also succeeded in ester synthesis of retinyl oleate with very small peroxide value, although both substrates have double bonds and tend to be oxidized easily.     

Synthesis of eicosapentaenoyl phosphatidylcholines by polyethylene glycol-modified lipase in benzene

Summary Lipase fromCandida cylindracea was modified with activated polyethylene glycol, which was synthesized from -carboxymethyl--methoxypoly-(oxyethylene) and N-hydroxysuccinimide with dicyclohexylcarbodiimide. The modified lipase was soluble in organic solvents such as benzene and catalyzed the reaction of ester-exchange to synthesize eicosapentaenoyl phosphatidylcholines from dipalmitoyl phosphatidylcholine and eicosapentaenoic acid. Either one of palmitic acid at C-1 or C-2 position of the phosphatidylcholine was exchanged with eicosapentaenoic acid.     

Degradation of endogenous proteins and internalized asialofetuin in primary cultured hepatocytes of rats

1. The effects of various metabolic and enzyme inhibitors on the degradation of endogenous proteins and internalized asialofetuin were examined in the primary cultured hepatocytes of rats. 2. The results showed the important physiological role of bestatin-sensitive aminopeptidases in the degradation of endogenous and internalized proteins as well as in the degradation of both long- and short-lived proteins.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.