Elimination of asialofetuin and asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic clearance mechanism.

The capacity of the liver to eliminate asialofetuin and asialoorosomucoid was investigated in intact rats. From plasma radioactivity curve measurements and assays on tissue homogenates the liver is shown to be able to dispose of an average of 19.8 microgram of asialofetuin/min per 100 g body weight. No other major route is identified for the disappearance of asialofetuin from the plasma, although trace amounts of the protein were detectable in the urine. From analyses of the plasma radioactivity curves the elimination process for asialoorosomucoid appears to be comparatively complex because of the existence of extrahepatic disposal routes. Quantification of labelled asialoorosomucoid in liver homogenates indicates, however, that the hepatic clearance rate for asialoorosomucoid is similar to that for asialofetuin. Urinary excretion significantly contributes to the disappearance of asialoorosomucoid from the plasma but the hepatic and renal routes do not account for all the protein lost from this compartment. At plasma concentrations above the maximal eliminative capacity of the liver, the hepatic clearance of asialofetuin obeys zero-order kinetics and is remarkably constant. Elimination of a quantity of asialoglycoprotein which exceeds the calculated total number of binding sites in the liver does not reduce the efficiency of the pathway, and studies of [3H]leucine incorporation indicate that the lectin, unlike the bound asialoglycoprotein, is not destroyed in the elimination process. Cytochalasin B (80 microgram/100 g body wt.) had no measureable effect on the hepatic clearance of asialofetuin. Administration of colchicine (10 mg/100 g body wt.) resulted in transitory accumulations of asialoorosomucoid in the liver, presumably due to interference with the intracellular transport of the endocytised protein.     

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α₁-acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.     

Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo.     

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.     

The effects of monensin on secretion of very-low-density lipoprotein and metabolism of asialofetuin by cultured rat hepatocytes.

Primary cultures of rat hepatocytes were used to study secretion of very-low-density lipoproteins and metabolism of asialofetuin. The ionophore monensin inhibited both secretion of very-low-density lipoproteins and binding and degradation of asialofetuin in a concentration-dependent manner. Secretion as well as receptor binding were markedly decreased after 15 min treatment with monensin. The inhibitory effect of the ionophore was fully reversible, and no effect on protein synthesis was observed at concentrations up to 50 microM. The secretion of apoproteins (B-small, B-large and E) and that of albumin were inhibited to the same extent as was triacylglycerol secretion. Secretion of very-low-density lipoproteins was more sensitive to low concentrations of monensin than was the metabolism of asialofetuin. Maximum inhibition of very-low-density-lipoprotein secretion was obtained at 5-10 microM-monensin, whereas 25 microM was required to obtain maximum inhibition of binding and degradation of asialofetuin. The number of surface receptors for asialofetuin decreased to about half when the cells were exposed to 25 microM-monensin. It is possible that monensin inhibits endo- and exo-cytosis via a similar mechanism, e.g. by disturbing proton gradients. Since secretion of very-low-density lipoproteins was more sensitive to low concentrations of monensin, it is likely that monensin independently inhibits endocytic and secretory functions in cultured hepatocytes.     

Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver

The uptake and degradation of a homologous rat serum asialoglycoprotein, 125I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. 125I-Asialoorosomucoid was rapidly taken up by the liver (t1/2 = 5.7 min) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20-30 min. These products accounted for 60-65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of 125I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of 125I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathepsin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat 125I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occurred in the native molecular weight of the starting glycoprotein.     

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 transport of asialoglycoproteins in rat hepatocytes : Evidence for two subpopulations of lysosomes

The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.     

125I-glycoconjugate labels for identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation

Residualizing radioactive labels are designed to remain entrapped within cells following degradation of a carrier protein, and have been used for identification of the tissue and cellular sites of plasma protein catabolism. In this study we describe a convenient synthesis and purification of a series of 125I-labeled glycoconjugates, and an evaluation of their efficiency of retention in liver following degradation of a model carrier protein, asialofetuin. Glycoconjugates were prepared in 65-90% yield by reductive amination of reducing sugars with aromatic amines using NaBH3CN. The products were purified in a single ion-exchange chromatographic step, and then labeled with 125I. The derivatives prepared were mono-and disubstituted lactitol-,cellobiitol-and glucitol-[125I]tyramine and lactitol-[125I]tyrosine. 125I-Glycoconjugates were coupled to asialofetuin using either cyanuric chloride or, for lactose-containing labels, by treatment with galactose oxidase followed by reductive amination with NaBH3CN. Attachment of labels by either procedure did not affect the normal rapid clearance of asialofetuin from the rat circulation nor its uptake and degradation in liver lysosomes. Leakage of 125I-labeled degradation products from cells was measured by following the kinetics of loss of whole-body radioactivity. We observed that degradation products from larger, disubstituted glycoconjugates were retained more efficiently than those from smaller and monosubstituted derivatives, and that glycoconjugates coupled to protein via reductive amination were retained in the body more efficiently than those coupled by cyanuric chloride. Overall, dilactitol-[125I]tyramine coupled to protein by reductive amination was entrapped most efficiently in liver.     

Oxidant-increased proteolysis in rat liver slices: effect of bromotrichloromethane, antioxidants and effectors of proteolysis

Proteolysis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of the oxidant bromotrichloromethane and effectors of proteolysis. Proteolysis was evaluated by S-amino acids and lipid peroxidation by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. The increased release of S-amino acids by BrCl3C depended on incubation time and oxidant concentration. S-Amino acid release increased 30% over control value and TBARS increased from 22 to 124 nmol/g liver by incubation for 120 min with 1 mM BrCl3C. Release of S-amino acids and TBARS was decreased when liver slices were treated with nor-dihydroguaiaretic acid (NDG), butylated hydroxyanisole (BHA), Trolox C, or N,N'-diphenyl-1,4-phenylenediamine (DPPD) immediately prior to addition of oxidant, suggesting participation of lipid-soluble free radicals. Oxidant-induced release of S-amino acids but not of TBARS was decreased by mannitol, suggesting participation of hydroxyl radical or a species with similar reactivity; and by superoxide dismutase and catalase, suggesting participation of superoxide and hydrogen peroxide, respectively. The decrease of S-amino acid release by sodium fluoride, sodium arsenate, 2,4-dinitrophenol, chloroquine, leupeptin, phenylmethylsulfonyl fluoride, EDTA and o-phenanthroline was variable, suggesting the presence in liver of several proteases to remove oxidatively-modified proteins.     

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.     

Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat.

Plasma kinetics and liver metabolism of iodinated human corticosteroid-binding protein have been studied in ovariectomized female rats. 125I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected 125I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in 125I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. 125I labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma lebels. Asialofetuin also slowed the clearance of intact 125I-labeled human corticosteroid-binding globulin from the plasma (t1/2 = 90 min). Binding of 125I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. 125I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.     

Rapid subcellular fractionation of the rat liver endocytic compartments involved in transcytosis of polymeric immunoglobulin A and endocytosis of asialofetuin.

The distributions of two endocytosed radiolabelled ligands (polymeric immunoglobulin A and asialofetuin) in rat liver endocytic compartments were investigated by using rapid subcellular fractionation of post-mitochondrial supernatants on vertical density gradients of Ficoll or Nycodenz. Two endocytic compartments were identified, both ligands being initially associated with a light endocytic-vesicle fraction on Ficoll gradients, asialofetuin then accumulating in denser endosomes before transfer to lysosomes for degradation.     

Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver

The uptake and degradation of a homologous rat serum asialoglycoprotein, ¹²⁵I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. ¹²⁵I-Asialoorosomucoid was rapidly taken up by the liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5.7}\text{min}$) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20–30 min. These products accounted for 60–65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of ¹²⁵I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of ¹²⁵I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathespin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat ¹²⁵I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occured in the native molecular weight of the starting glycoprotein.     

In vivo quantification of receptor-mediated uptake of asialoglycoproteins by rat liver

The in vivo kinetics of hepatic clearance of 125I-asialo-orosomucoid and 125I-asialofetuin was determined with a portal vein injection technique in barbiturate-anesthetized rats. Nonlinear regression analyses of saturation data gave the following parameters for asialo-orosomucoid, Km = 0.26 +/- 0.06 mg/ml, Vmax = 320 +/- 70 micrograms/min/g, and for asialofetuin, Km = 0.32 +/- 0.07 mg/ml, Vmax = 240 +/- 40 micrograms/min/g. Unlabeled asialofetuin inhibited the clearance of 125I-asialo-orosomucoid with a Ki = 0.25 +/- 0.04 mg/ml. Based on a model assuming that in vivo receptor concentration much greater than receptor KD, then the maximal binding capacity of the external surface of liver cells in vivo for asialo-orosomucoid is 2Km or 520 micrograms/ml or 52 micrograms/g of liver, assuming the liver interstitial space is 0.1 ml/g. Our estimate of in vivo binding capacity approximates in vitro estimates of total hepatic binding capacity, but is 10-fold greater than in vitro estimates of binding capacity on the external surface of liver cells. These results suggest the large majority of asialoglycoprotein receptors are located on the external surface of liver cells. The saturability of 125I-asialo-orosomucoid clearance was also demonstrated with a portal vein double bolus technique, wherein the portal injection of 20-1000 micrograms of unlabeled asialo-orosomucoid was followed 30 s later by the portal injection of tracer. Maximal inhibition of uptake was obtained with a portal vein injection of greater than or equal to 500 micrograms of asialo-orosomucoid. The specific extraction of the 125I-asialo-orosomucoid, which was near zero shortly after a 400-micrograms loading dose, gradually increased toward normal levels with a t1/2 of 21 min. This t1/2 may represent the in vivo rate of receptor recycling, since the gradual increase in unoccupied receptor sites is consistent with the model of receptor binding, internalization, and recycling.     

Quantitative aspects of the uptake and degradation of lysozyme in the rat kidney in vivo

Uptake and degradation of lysozyme in the rat kidney were studied in vivo. The protein was labeled with 125I by way of a moiety (tyramine-cellobiose or 'TC') which remained trapped inside the cells even after proteolysis of the peptide chain (in contrast, the label from conventionally labeled proteins escapes after degradation). Following the injection of 'trapped-label' lysozyme, the radioactivity in the kidneys represented the total amount of lysozyme that was taken up during the experiment. Proteolysis could be followed by determining the amount of acid-soluble degradation products. By adding the radioactivity in the urine to that in the kidneys, a measure of the total filtered load was obtained. When only a trace dose of 125I-labeled TC lysozyme was injected into rats, the amount of radioactivity in the kidneys increased on average by 0.09% per min, after the concentration in the blood had become nearly stable. After 100 min, 30% of the injected dose was recovered in the kidneys. The labeled protein was degraded to acid-soluble molecules of Mr less than 1000. There was apparently a 'lag period' between the endocytosis in the kidneys and the start of degradation. 40 min after the injection of a trace dose, about 0.6% of the 'trapped-label' lysozyme in the kidneys was degraded per min.; subsequently, there was a decline in the fraction which was degraded per min. The amount of lysozyme in the urine increased after the injection of increasing amounts of lysozyme, showing that the capacity of the uptake mechanism was being exceeded, but truly saturating levels of lysozyme could not be reached in vivo.     

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.     

Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells. Overall, these experiments indicate that 125I-InTn should provide greater sensitivity and more accurate quantitative information on the sites of catabolism of long-lived circulating proteins in vivo.     

Endocytosis of bovine lactoperoxidase by two carbohydrate-specific receptors in rat liver

A perfused rat liver took up bovine lactoperoxidase (LPO) by a Ca2+-dependent, saturable process. This endocytosis was accomplished by both hepatocytes and Kupffer or other nonparenchymal cells (NPCs). The mediating receptors were the Gal/GalNAc lectin of hepatocytes and the Man/GlcNAc lectin of NPCs. Blocking either one of these receptors caused a large shift in distribution of accumulated LPO into the cell type whose receptor was left unblocked, but the extent of uptake was unaffected and the rate was only moderately reduced. Effective inhibition of overall uptake into the perfused organ required the presence of competitors for both receptors. Conversely, LPO was an effective competitor of other ligands (asialoorosomucoid or mannan) for either of the two receptors. The major clearance capacity for LPO was associated with hepatocytes which in suspension took it up by a process completely inhibitable by asialofetuin (ASF) and at a rate more than three times greater than for ASF. A faster cycling time for Gal/GalNAc receptors when bound to LPO is suggested. The glycoprotein selectively lost its affinity for Man/GlcNAc receptors when digested by endoglycosidase H (endo H), suggesting that LPO contains mannose-rich oligosaccharides.