Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

Sialidase in Cerebellar Granule Cells Differentiating in Culture

The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside

A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.     

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.     

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.     

Lysosomal sialidase deficiency in sialidosis with partial β-galactosidase deficiency

We analyzed the subcellular localization of sialidases in human lymphocytes from a patient with adult type sialidosis with partial β-galactosidase deficiency and normal controls. Sialidase activities were measured with α,2 → 3 NeuAc-lactitol, 4-methylumbelliferyl-NeuAc and G_M3 ganglioside as substrates. Sialidases in the lysosomes were sonication-labile and hydrolyzed mainly hydrophilic substrates such as NeuAc-lactitol and 4-methylumbelliferyl-NeuAc, but hydrolyzed subsidiarily G_M3 ganglioside. On the other hand, sialidases in the plasma membrane were sonication-stable and hydrolyzed both hydrophilic substrates and G_M3 ganglioside. In sialidosis with partial β-galactosidase deficiency, the sialidases of the lysosomes showed 3–5% activity toward hydrophilic substrates and 25% activity toward G_M3 ganglioside as compared with sialidase activities of the controls. However, there are no differences in the activities of the sialidases in the plasma membrane. These results demonstrate that the essential defect in this disease is the deficiency of a lysosomal sialidase.     

SUBCELLULAR FRACTIONATION OF GANGLIOSIDE SIALIDASE FROM HUMAN BRAIN

—A subcellular fractionation was performed on forebrain cortex from three human brains and the fractions obtained were assayed for ganglioside sialidase and four p-nitrophenyl glycohydrolases. Differences in the sedimentation patterns of the enzymes were observed. From 53 to 77 per cent of the recovered sialidase activity was found in the synaptosomal fraction, while the p-nitrophenyl glycosidases were mainly recovered in the lysosome-enriched fraction. Three possible interpretations of the sialidase sedimentation pattern are suggested: (1) The ganglioside sialidase is bound to the limiting membrane structure of the nerve ending. (2) The ganglioside sialidase is lysosomal, although bound to lysosomes of low density. (3) The enzyme occurs mainly in lysosomes primarily located in the nerve endings, being trapped under the formation of the synaptosomes.     

Characterization of the major sialidases of various types of rat blood cells: their comparison with rat liver sialidases

The substrate specificity and subcellular location of the major sialidases of three types of rat blood cells were characterized and compared with those of the known three types of rat liver sialidase, which have been designated intralysosomal, cytosolic, and plasma membrane-associated sialidases. Platelets and leucocytes contain mainly an acid sialidase, which is highly active towards oligosaccharides and 4MU-NeuAc, and erythrocytes possess a high level of a sialidase acting on gangliosides. A Percoll gradient centrifugation study showed that the former is located in lysosomes and the latter in plasma membrane. When the sialidase was solubilized and partially purified from erythrocyte ghosts, the enzyme was found to hydrolyze actively gangliosides but only poorly other substrates such as 4MU-NeuAc, oligosaccharides, and glycoproteins. The sialidase partially purified from rat liver membrane fraction exhibited the same substrate specificity. It is concluded that the major sialidase of platelets and leucocytes corresponds to hepatic intralysosomal sialidase while erythrocytes contain almost exclusively a ganglioside sialidase which corresponds to hepatic plasma membrane sialidase.     

Ganglioside sialidase distribution in mucolipidosis type IV cultured fibroblasts

The subcellular distribution of ganglioside sialidase in Mucolipidosis IV (ML IV) cells was characterized by a series of Percoll gradients. Similar to normal cells, the enzyme cosedimented with plasma membrane markers, although this activity was reduced and exhibited decreased solubility in ML IV cells. Only trace amounts of ganglioside sialidase (less than 5%) was found in the lysosomes of normal cells. This activity was apparently reduced in ML IV cells but its minute activity in controls excluded further characterization of these differences. Plasma membranes on 6.7 and 5.6% Percoll gradients were biomodally distributed. Ganglioside sialidase in normal cells was found to be in both the heavier and the lighter membrane fractions, whereas the enzyme in ML IV cells was associated mainly with the denser membrane fraction. These data indicate that the enzyme in ML IV cells is characteristically different from normal in that it exhibits reduced activity and solubility and a different plasma membrane distribution.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Lysosomal and plasma membrane ganglioside GM3 sialidases of cultured human fibroblasts. Differentiation by detergents and inhibitors

Cultured human fibroblasts contain two sialidases that degrade gangliosides such as GM3: a lysosomal activity that appears identical with the activity towards water-soluble substrates and that is deficient in the genetic lysosomal disorder sialidosis, and another enzyme that seems localized on the external surface of the plasma membrane. In this report we show that both enzymes can be differentiated in the presence of each other by choice of the detergent used for activation, and also by the inhibitory action of some polyanionic compounds such as sulphated glycosaminoglycans. The lysosomal ganglioside GM3 sialidase is greatly stimulated by sodium glycodeoxycholate and, to lesser degrees, by sodium glycocholate and sodium cholate. The ganglioside GM3 sialidase of the plasma membrane is not measurably active under the conditions of the lysosomal enzyme but is specifically activated by the non-ionic detergent Triton X-100. The glycodeoxycholate-stimulated, but not the Triton-activated, ganglioside GM3 sialidase activity was profoundly diminished in cell lines from patients with the lysosomal disorders sialidosis and galactosialidosis; however, both activities were normal in fibroblasts from patients with mucolipidosis IV, previously thought to be a ganglioside sialidase deficiency disorder. Both the lysosomal and the plasma membrane ganglioside GM3 sialidases were inhibited by sialic acids, suramin, dextran sulphate and sulphated glycosaminoglycans. Among the latter, heparin and heparan sulphate showed a much higher inhibitory potency towards the plasma membrane ganglioside GM3 sialidase than towards the lysosomal onw.(ABSTRACT TRUNCATED AT 250 WORDS)     

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.     

Desialylation of extracellular GD1a-neoganglioprotein suggests cell surface orientation of the plasma membrane-bound ganglioside sialidase activity in human neuroblastoma cells

The orientation of the catalytic site of a ganglioside-specific sialidase in the plasma membrane of SK-N-MC neuroblastoma cells was probed using water-soluble GD1a-neoganglioprotein substrate on intact cells and GM1-product detection by cholera toxin B. Desialylation of substrate was readily observed, whereas specific sialidase inhibitors prevented the reaction, and conditioned medium was inactive. Inhibitors of endocytosis and acidification had no effect on substrate degradation, and lowering temperature to 18 degrees C reduced activity but did not abolish it. We conclude that the ganglioside sialidase activity is cell surface-orientated and displays an in situ specificity that mirrors enzyme preparations in vitro.     

Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Sialidase Activity in Nuclear Membranes of Rat Brain

Abstract: A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N -acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N -acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane-bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain.     

The identification of lysosomal ganglioside sialidase in human cells

In this report we present evidence for the existence of a lysosomal ganglioside sialidase. The sialidase activity was solubilized by sonication and stimulated by cholate. The absence of ganglioside sialidase activity in sialidosis patients indicates that lysosomal sialidase is active towards gangliosides and glycoproteins. The plasma membranes were associated with two types of ganglioside sialidase activities, one was enhanced by cholate while the other was partially inhibited by this detergent.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.