Lysosomal trafficking in rat cardiac myocytes.

By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. MPR was associated mostly with inner membranes, whereas lgp120 was predominantly bound to the outer limiting membrane. MPR, lgp120, and MEP were not detected in Golgi stacks, but some labeling was seen in the putative TGN. Our data suggest that the MPR structures are prelysosomes involved in lysosomal enzyme targeting in rat cardiac myocytes.

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.

Subpopulations of endosomes generated at sequential stages in the endocytic pathway of asialoganglioside-containing ferrite ligands in rat liver.

Subpopulations of endosomes generated at different stages of the endocytic pathway were isolated by a high-gradient magnetic separation followed by a Percoll density gradient centrifugation. Rat livers were perfused for 5 min with asialoganglioside (ASG)-containing ferrite particles and chased at 37 degrees C. At various times after the internalization, the endocytic vesicles containing ferrite particles were isolated by the magnetic separation. Isolated fractions contained endosomes until 15-min perfusion, after which most of the particles were transported to lysosomes. The endosomal fractions isolated after the 5- or 15-min perfusions were further analyzed by 30% Percoll density gradient centrifugation. The endosomes after 5-min perfusion showed peaks around the density of 1.05 g/ml (peak I) and 1.07 g/ml (peak Is), both of which contained asialoglycoprotein receptors. In the 15-min perfusion, another peak of endosomes (peak II) was observed at the higher density of 1.09 g/ml without the receptors, in addition to peak I. These endosomes had their own characteristic proteins. Some proteins were common in the subgroups of endosomes. These results suggest that the endosome I containing the ligands and the receptors was first produced after endocytosis and, through the endosome is, was scissioned into the endosome II containing the ligands. The endosome II was then fused with primary lysosomes for proteolytic cleavage of ligands.

Immunocytochemical localization of a calcium-binding phosphoprotein in hemocytes of heterodont bivalves.

Calcium-binding phosphoprotein particles are the most abundant extracellular proteins in the hemolymph of heterodont bivalves, and granular hemocytes are the most abundant cells in the same fluid. In this study, the hemocytes of Rangia cuneata were examined ultrastructurally and probed with anti-phosphoprotein IgG to demonstrate that the granulocytes are a probable source of the hemolymph phosphoprotein. The granulocyte cytoplasm is laden with large vesicles containing an amorphous homogenous matrix and variable numbers of electron-dense particles; the latter are ultrastructurally similar to the extracellular phosphoprotein. The vesicle particles and matrix are related forms of the hemolymph phosphoprotein as evidenced by heavy gold labeling when Lowicryl sections were sequentially treated with rabbit-anti-phosphoprotein IgG and colloidal gold-anti-rabbit IgG. The vesicles may be the loci for posttranslational phosphorylation and eventual secretion of the calcium-binding phosphoprotein, or alternatively the vesicles may be digestive structures which degrade internalized phosphoprotein.

Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol.

Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% (w/w) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and beta-D-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.

Crystals in an oligodendroglioma: an optical, histochemical, and ultrastructural study.

The report describes an oligodendroglioma that was examined in four biopsies and contained a large number of intracytoplasmic crystals. The crystals appeared in neoplastic cells with eosinophilic cytoplasms and eccentric nuclei. They were positive to periodic acid-Schiff stain and resistant to diastase. A lysosomal genesis of the crystals is proposed on the basis of a transition observed between lysosomal bodies with lipid droplets and those with crystalloid electron-dense structures. The morphologic and histochemical features of these crystals are compared to those in other tumors, lesions, and nonneoplastic cells.

Immunocytochemical localization of sphingolipid activator protein 2 (SAP-2) in normal and SAP-deficient fibroblasts.

The intracellular localization of sphingolipid activator protein 2 (SAP-2) was determined immunocytochemically using an antiserum raised against a SAP-2 preparation from Gaucher spleen. The immunolabeling indicated that SAP-2 was largely localized in the lysosomes of fibroblasts from normal individuals. In some lysosomes the labeling was greatest around the perimeter of the matrix, suggesting an association between the activator and lysosomal membrane components. The labeling technique was also applied to fibroblasts from a patient with a unique sphingolipid storage disorder. Consistent with immunoblotting studies on electrophoretograms, both the patient and his affected fetal sibling were found to be deficient in immunoreactive SAP-2.

Ultrastructural changes in the pancreas of carbonyl iron-fed rats.

Diabetes mellitus is found with increased frequency in patients with both primary and secondary hemochromatosis. In these conditions, the pancreas shows fibrosis and iron overload of acini, interstitium, and islet B cells. Previous morphological studies have only described changes found in advanced stages of disease, while abnormalities of the initial stage of iron overload have, as yet, not been reported. Rats fed a carbonyl iron-supplemented diet for 4-15 months showed storage iron deposition (ferritin and hemosiderin) in many organs, in a pattern similar to primary human hemochromatosis. Electron microscopic examination of the pancreas showed ferritin particles segregated in lysosomes of acinar cells, as well as diffuse cytosiderosis of macrophages in the interstitial septa. In the islets, iron deposits were discrete and only in B cells. In the absence of electron-microscopic studies of incipient pancreatic cytosiderosis in human subjects, the present experimental animal study may contribute to a better understanding of the pathway leading to the extensive lesions found in the advanced stages of the human iron overloading diseases.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Sequestration and degradation of alpha 2u-globulin in rat kidney lysosomes.

alpha 2u-Globulin (M.W. = 18,709) is the major protein synthesized in the liver of adult male rats. alpha 2u-Globulin, secreted from the liver, is filtered by the kidneys where about a half of the protein is reabsorbed and the remainder is excreted in the urine. Highly purified lysosomes from rat kidneys were subjected to immunoblot analysis using antiserum against alpha 2u-globulin. A major immuno-reactive band was observed at the position about 1 k dalton smaller than that of the authentic alpha 2u-globulin. The immuno-reactive band in SDS-polyacrylamide gel was excised and the protein, eluted from the gel, was subjected to amino acid sequence analysis by micro Edman method. The N-terminal amino acid sequence of the intralysosomal alpha 2u-globulin was found to be leu-asp-val-ala-lys-leu-asn-gly-... which exactly corresponded to the sequence from the 10th leucine residue of the authentic urinary alpha 2u-globulin. This result suggests that alpha 2u-globulin, sequestered into the kidney lysosomes, is cleaved intralysosomally between the 9th asparagine and the 10th leucine, and the degradative intermediate, thus produced, is accumulated in the lysosomes. The immuno-blot assay gave the value of 210 +/- 70 mg alpha 2u-globulin/g kidney lysosomal proteins. The molecular basis for the resistance of the degradative intermediate of alpha 2u-globulin against further degradation in the kidney lysosomes was discussed in the light of the computer-predicted secondary structure of alpha 2u-globulin.

Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles.

To characterize the mechanism of internalization of beta-adrenergic catecholamine receptors on human epidermoid A431 carcinoma cells, their distribution was analyzed by immunocytochemistry using the monoclonal anti-receptor antibody BRK2. In preconfluent cultures, the receptors appeared to be randomly distributed on the cell surface. Exposure to the agonist isoproterenol induced an overall decrease in the number of cell surface receptors as determined by binding experiments and visualized by immunofluorescence. When cells were incubated at 4 degrees C with BRK2 and anti-mouse IgG-gold and then transferred at 37 degrees C, non-coated invaginations and vesicles were labeled. The addition of isoproterenol resulted in an increased rate of internalization of the receptor-BRK2-anti-IgG-gold complex. When incubation with the two antibody reagents was prolonged (with or without isoproterenol), non-coated vesicles fused in the endosomal compartment, and receptors were transferred to multivesicular bodies and lysosomes. At no stage in this process was there any indication that clathrin-coated pits or vesicles participated. Furthermore, we found that an intracellular potassium depletion treatment known to inhibit endocytosis, did not affect the normal pattern of desensitization of beta-adrenergic receptors.

1,25-Dihydroxyvitamin D3-mediated vesicular calcium transport in intestine: dose-response studies.

In order to further test the validity of the vesicular transport model of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated intestinal calcium absorption, dose-response studies were undertaken. Using previously established methodology for subcellular fractionation following 45Ca absorption from in situ ligated duodenal loops, radionuclide levels were found to increase gradually in endocytic vesicles prepared from 1,25(OH)2D3-treated (+D) chicks relative to controls (-D) achieving a plateau at greater than or equal to 260 pmol seco-steroid. By comparison, lysosomal 45Ca levels increased more readily, having +D/-D ratios of 1.88 +/- 0.35, 2.21 +/- 0.05, 2.17 +/- 0.88, 2.31 +/- 0.25, and 2.15 +/- 0.47 after 0.0104, 0.052, 0.26, 1.3, or 6.5 nmol of 1,25(OH)2D3, respectively. Net intestinal calcium absorption, as judged by appearance of 45Ca in the serum for the same range of doses, rose gradually to a plateau value at greater than or equal to 260 pmol. Since lysosomal 45Ca levels were maximally increased at 1,25(OH)2D3 doses lower than those required for fully stimulated transport, it was concluded that lysosomes are still candidates for cellular calcium carriers, but that other elements of the transport pathway are required. Analyses of gradient fractions for calbindin-D28K (the vitamin D-induced calcium binding protein), and potential 1,25(OH)2D3-mediated changes in vesicular ATPase (microtubule motive power for transcellular delivery of calcium) failed to identify the missing components.

Transferrin receptors and cation-independent mannose-6-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes.

The distribution of transferrin receptors (Tf-R) was determined in Clone 9 hepatocytes and compared to that of 215 kDa, cation-independent mannose-6-phosphate receptors (M6P-R) by double labeling. Cells were allowed to take up exogenous human transferrin (Tf) for 5 to 30 min, after which Tf, Tf-R, and M6P-R were localized by immunofluorescence using specific antibodies. All these proteins were found to be concentrated in the juxtanuclear or Golgi region. When Clone 9 cells were treated with NH4Cl to trap M6P-R in endosomes (Brown, W. J., J. Goodhouse, M. G. Farquhar: J. Cell Biol. 103, 1235-1247 (1986)), the distribution of the two receptors differed: Tf-R remained the same as in controls, but M6P-R were localized in large vacuolated endosomes. To carry out double labeling experiments at the electron microscope level, transferrin gold conjugates (Tf-Au) were prepared, and M6P-R were detected by immunoperoxidase labeling. Tf-Au binding to the cell surface was specific as it was reduced approximately 70 to 79% in the presence of excess native Tf. When Clone 9 cells were incubated with Tf-Au at 37 degrees C for 5 to 30 min, or binding of Tf-Au was carried out at 4 degrees C followed by warming to 37 degrees C, Tf-Au was found within a peripheral tubulovesicular network and within multivesicular endosomes that were not labeled with anti-M6P-R. Other multivesicular endosomes of similar size and morphology were heavily labeled for M6P-R but contained little or no Tf-Au. Tf-Au and M6P-R were also found in separate endosomes in cells treated with NH4Cl. Native Tf was localized in the same compartments as Tf-Au by immunoperoxidase labeling of both Clone 9 cells and mouse myeloma cells. We conclude that in Clone 9 hepatocytes, Tf/Tf-R internalized from the cell surface and M6P-R bearing newly synthesized lysosomal enzymes from the Golgi deliver their ligands to two different subpopulations of multivesicular endosomes. The endosomal subpopulation visited by Tf/Tf-R is known to correspond kinetically to early endosomes. The endosomal subpopulation heavily labeled for M6P-R presumably represent a later endosomal compartment which serves as the junction point where endocytosed ligands and newly synthesized lysosomal enzymes enroute to lysosomes meet.

Activity of some lysosomal enzymes and postnatal ontogenetic development of epileptic reactivity in rabbit brain.

The beta-glycerophosphatase (GP), p-nitrophenyl phosphatase (PNFP) and beta-glucuronidase (BG) activities in four neocortical rabbit areas have been determined in six successive electrocorticographic stages of postnatal ontogenetic development of epileptic reactivity. The lysosomal enzyme activity decreases gradually from rabbits aged 1 day to adult ones and is similar within the four neocortical areas. The activity of the three lysosomal enzymes is not parallel. Statistically significant decreases in beta-glycerophosphatase activity are present in rabbits at the age of 30 days versus adult age in all the areas investigated while beta-glucuronidase is higher at the same developmental stage only in beta retrosplenial (III) and motor (II) areas. Correlation of the electrophysiological and biochemical data cannot provide explanation for the particular phenomena of the epileptogenic reactivity in rabbit cerebral neocortex.

Morphological localization of a major lysosomal membrane glycoprotein in the endocytic membrane system.

We have raised specific polyclonal immunoglobulin G (IgG) against a major lysosomal membrane sialoglycoprotein (LGP107) taken from rat liver and have prepared a conjugate of its Fab' fragment with horseradish peroxidase (HRP-anti LGP107 Fab') as a probe for the subcellular antigen. Electron immunocytochemistry in primary cultured rat hepatocytes showed that LGP107 resided primarily within lysosomes and was associated with luminal amorphous materials as well as limiting membranes. In addition, LGP107 was shown to be substantially distributed throughout the endocytic vacuolar system. The glycoprotein was found clustered in coated pits at the cell surface and localized along the surrounding membranes in endocytic vesicles. When cultured cells were exposed to HRP-anti LGP107 Fab', the antibody which was bound to its antigen within the coated pits was internalized via a system of endocytic vesicles and transported to lysosomes. During 20 min of incubation at 37 degrees C, the HRP tracer appeared at an early stage in small vesicles and moved progressively to larger vesicles, including multivesicular bodies. After 1 h, the tracer could be clearly seen in lysosomes heterogeneous in shape and size. The existence of LGP107 in endocytic compartments and the uptake of anti LGP107 antibody by hepatocytes were not blocked by prior treatment of the cells with cycloheximide and excess amounts of anti LGP107 IgG. These data suggest that LGP107 circulates between the cell surface and lysosomes through the endocytic membrane traffic in hepatocytes.

Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys.

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.

Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways.

1. The distribution of the alpha- and beta-subunits of nucleotide-binding G-proteins among rat liver sinusoidal, lateral and canalicular plasma membranes, endosomes, Golgi membranes and lysosomes was investigated. 2. Pertussis-toxin-catalysed ADP-ribosylation identified a 41 kDa inhibitory alpha-subunit in all liver plasma-membrane functional domains as well as in endosomes. An antibody to a synthetic peptide corresponding to a C-terminal sequence of the inhibitory alpha-subunit also identified the 41 kDa polypeptide in all plasma-membrane domains, in 'early' and 'late' endosomes and in Golgi membranes; this polypeptide was not detected in lysosomes. The antibody-binding studies showed that bile-canalicular plasma membranes had the highest content of the inhibitory alpha-subunit. 3. Immunofluorescent microscopy confirmed the presence of the inhibitory alpha-subunit in all regions of the hepatocyte's cell surface. 4. An antibody recognizing the beta-subunit showed that a 36 kDa polypeptide was present in all plasma membranes and in 'early' and 'late' endosomes; it was not detected in lysosomes. The relative distribution among the fractions of this polypeptide was similar to the distribution of the inhibitory alpha-subunit. 5. The presence of high levels of the G-protein inhibitory alpha-subunit in bile-canalicular plasma membranes was confirmed by demonstration of its co-fractionation with marker enzymes in Nycodenz gradients and by free-flow electrophoresis. The significance of this location is discussed.

Polarized distribution of Na+,K+-ATPase in giant cells elicited in vivo and in vitro.

Giant cell formation was analyzed to determine whether it results in the high level of Na+,K+-ATPase expression that characterizes multinucleated cells such as osteoclasts. Giant cells and fusing alveolar macrophages were subjected to morphological, immunological, and biochemical studies. Both subunits of the Na+,K+-ATPase were found to be present on the plasma membrane of giant cells. Their localization was restricted to the non-adherent domain of the cell surface. Dynamic studies of giant cell differentiation demonstrated that on culture and/or multinucleation, an increase in sodium pump alpha-subunit synthesis occurred and led to a high level of expression of Na pumps. Conversely, the adherent plasma membrane of giant cells was enriched in a lysosomal membrane antigen. This study demonstrates that culture and/or multinucleation induces a significant increase in the expression of sodium pumps. The polarized distribution of these pumps and of a lysosomal component suggests that fusing macrophages undergo biochemical and morphological alterations which prepare them for a new and specialized function in chronic inflammatory reactions. Giant cells may offer a suitable model system to study the differentiation of other related multinucleated cells, such as osteoclasts.

Intracellular distribution of copper in the liver of copper-loaded sheep--a subcellular fractionation study.

Eighteen ewes in two groups were dosed orally with CuSO4 to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of acid AP activity in the serum in group 1 sheep and on the first day of haemolysis in group 2 sheep. Liver samples were obtained 1 week prior to the start of Cu dosing, at the first rise of acid phosphatase (AP) activity in serum and on the first day of haemolysis. These liver samples were homogenized and were separated into nuclear (N), heavy mitochondrial (MH), light mitochondrial (ML), microsomal (MI) and cytosolic (CY) fractions by centrifugation. The Cu concentration and specific activities of AP were determined in the liver, LH and subcellular fractions. The composition of the fractions was studied by light and electron microscopy. In the predosing biopsies, the concentration and percentage of Cu and the total specific activity of AP were highest in the ML fractions. With increasing Cu loading, the concentration of Cu in all fractions increased; the percentage of Cu increased in the N and MH fractions, decreased in the ML and MI fractions and was maintained at a constant level in the CY fractions. The total specific activities of AP in LH, N, MH, MI and CY fractions were increased and the activity was highest in the MH fraction. The results indicate that the increase in the concentration of Cu in liver cells was predominantly in lysosomes and cytosol. Furthermore, it is suggested that the necrosis of isolated hepatocytes observed in chronic Cu-poisoned sheep may be due to a saturation of the uptake of Cu into the lysosomal system of the cell, leading to the accumulation of toxic levels of Cu in the cytosol.

Intravenously administered tetra-thiomolybdate and the removal of copper from the liver of copper-loaded sheep.

Eighteen ewes divided into two groups were dosed orally with CuSO4 in order to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of serum acid phosphatase activity in sheep of group 1 and on the first day of haemolysis in sheep of group 2. Tetra-thiomolybdate was administered intravenously to five group 1 sheep (group 1B) and to group 2 from the cessation of Cu dosing. Following thiomolybdate administration, in groups 1B and 2, there was a reduction in the concentration of Cu in the liver and liver fractions, the number and size of electron-dense lysosomes in particulate liver fractions, the volume density and the mean volume of electron-dense lysosomes in hepatocytes and the number of necrotic cells in the liver. Thiomolybdate appeared to remove Cu from the lysosomes and the cytosol of Cu-loaded liver cells. However, neither the total specific activity of acid phosphatase in liver homogenate and liver fractions nor the numerical density of electron-dense lysosomes in hepatocytes decreased significantly. This may be due to the production of new lysosomes in the liver cells. Furthermore, following thiomolybdate administration, Mo concentration in the liver and liver fractions increased indicating that Mo of thiomolybdate was entering liver cells. The percentage distribution of Cu and Mo in the liver fractions was similar. This may suggest that Mo is bound to Cu and that they remain together with each fraction. The decrease in Cu concentration may indicate that the liver retains its ability to excrete copper via bile.