Fasciola hepatica: the presence of particle-associated and soluble nonspecific acid phosphatases.

The kinetic and physical properties of acid phosphatases in the lysosomal and microsomal fractions of F. hepatica were found to be similar, indicating that they are one and the same enzyme. In contrast, the biochemical properties of the soluble acid phosphatase (EC 3.1.3.2) were quite different from those of the lysosomal and microsomal fractions. This indicated the presence of two distinct forms of the enzyme one particle associated and the other soluble. Electrophoretic heterogeneity of these two types of acid phosphomonoesterase was seen. Two bands of activity were observed in both lysosomal and microsomal fractions and three bands in the soluble fraction.     

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in (Na+ + K+)-ATPase has been isolated from the outer medulla of pig kidney. (Mg2+ + K+)-activated ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K+-activated phosphatase, a partial reaction of the (Na+ + K+)-ATPase.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.     

Intracellular transport of mouse kidney β-glucuronidase induced by gonadotrophin

1. The response of renal beta-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in beta-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of beta-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal beta-glucuronidases were compared.     

Effect of potassium deficiency on mouse kidney lysosomal enzymes.

Mice of inbred strains A/J, C57BL/6J and C57BL/6J beige were kept on a K+-deficient diet for up to 40 days to determine the magnitude and mechanism of changes in tissue lysosomal enzymes. From days 10 to 40 glucuronidase activity increased 3-fold in kidney of K+-deficient mice, but there was little effect on beta-galactosidase or acid phosphatase activity. Similar increases in kidney glucuronidase activity occurred in inbred strains known to have genetically altered control of the synthesis (A/J) and secretion (C57BL/6J beige) of glucuronidase in kidney proximal-tubule cells. Deprivation of K+ did not affect glucuronidase activity in liver, spleen, lung and brain, but there was a 2-3-FOld increase in glucuronidase activity in heart in the C57BL/6J and C57BL/6J beige strains. As shown by specific antibody titration, increased glucuronidase activity in kidney of K+-deficient mice was accompanied by accumulation of enzyme molecules. Likewise in kidney of deficient mice there was an increased rate of synthesis of glucuronidase as measured by incorporation of labelled leucine into immunoprecipitable glucuronidase. In kidney of K+-deficient mice the elevated glucuronidase activity was found in both collecting-tubule and interstitial cells of the medulla. It is probable therefore that a significant fraction of the increased kidney lysosomal synthesis and enzyme activity is due to infiltrating cells.     

The effect of TEPC-183 plasmacytoma growth on beta-glucuronidase activity in serum and tissues of tumor-bearing mice

beta-Glucuronidase activity increased in the serum of BALB/c mice during the growth of the IgM-secreting plasmacytoma, TEPC-183. The increase appeared to correlate with tumor burden. The beta-glucuronidase activity in tissue homogenates of spleen, liver, and kidney from tumor-bearing mice also increased significantly compared to the levels found in corresponding tissues from normal control mice. Assays of lysosomal and microsomal fractions from livers of TEPC-bearing mice indicated that approximately 70% of the enzyme activity was associated with the lysosomal fraction and the remainder with the microsomal fraction. A similar distribution was found in homogenates prepared from the plasmacytoma itself. In contrast to this the beta-glucuronidase activity in livers from normal BALB/c mice is nearly equally distributed between lysosomal and microsomal fractions.     

Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113-0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.     

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.     

ATP activation of protein degradation by extracts of crude and purified lysosomal preparations

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.     

An isoelectric focusing study of acid phosphatase heterogeneity in rat liver

The different forms of acid phosphatase (EC 3.1.3.2) in rat liver homogenates, lysosomal, mitochondrial, microsomal fractions and cytosol were studied with isoelectric focusing. Evidence is presented that isoelectric focusing of acid phosphatase in subcellular fractions shows individual changes and time related patterns. Mild autolysis shifted all enzyme activity peaks of isoelectric focusing patterns to the one at pH 7.04.     

Is Na + K ATPase a Myelin-Associated Enzyme?

The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter-enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. This discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29 degrees C) than for the microsomal fraction (21 degrees C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions, Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10-3 M-strophanthidin. The evidence thus indicated that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggest that this is not a significant factor and that Na + K ATPase may be a myelin-associated enzyme.     

Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.     

Isolation and properties of lysosomes from dark-grown potato shoots

Summary A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and -glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 and density of 1.10 g cm^-3 containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 and density of 1.07 g cm^-3 with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

Antigenic Makeup of Subcellular Fractions of Trypanosoma cruzi

Trypanosoma cruzi epimastigotes from cultures were separated into nuclear, mitochondrial, lysosomal, microsomal, and cell-sap fractions. Enzymic and ultrastructutal controls served to determine the cleanness of separation. The bulk of the DNA was in the nuclear (78%) and mitochondrial (12%) fractions. RNA was found in microsomal (74%) and cell-sap (14%) fractions. Marker enzyme distribution (succinic dehydrogenase, acid phosphatase, and glucose-6-phosphatase) was compared with their distribution in mammalian tissues. Subcellular localization of antigens by Ouchterlony tests revealed 2 specific precipitin lines in cell sap and 1 in the microsomal fraction. These antigens may prove diagnostically and immunoprotectively valuable.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r