Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway.

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n     

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines

Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. In conclusion: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid Preparation of a Distinct Lysosomal Population from Myelinating Mouse Brain Using Percoll Gradients

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.     

Age delays thyroglobulin progression towards dense lysosomes in the cream hamster thyroid

We have shown that large lysosomes appear in thyroids of aging male cream hamsters. To investigate the role of this lysosomal change in the age-dependent reduction in hormone secretion, thyroids of young (<4 months of age) and old (>22 months of age) male and female hamsters were labeled with ¹²⁵I at near isotopic equilibrium. Changes in thyroid morphology were analyzed by light- and electron-microscopic morphometry. Changes in thyroglobulin processing were analyzed by subcellular fractionation and identification of ¹²⁵I-compounds by sucrose gradients and reverse-phase high-pressure liquid chromatography (HPLC). Sexual dimorphism present in thyroids of young animals became more marked upon aging. The parallel increase in thyroid weight and thyroglobulin content was more conspicuous in old females than in old males. Two morphological observations were specific to old females: (1) large follicles with flat epithelium and evenly labeled colloid and (2) deposits of amyloid material (possibly immunoglobulin light chain-related) between follicles. Although lysosomes were enlarged in female and male aged thyroids, they did not accumulate iodine. However, after isopycnic centrifugation of crude lysosomal fractions in Percoll gradients, ¹²⁵I in old thyroids was not distributed mainly in the dense fraction L1 (lysosomes) as in young thyroids, but partly in particles of lower density (light L2 and buoyant fractions). ¹²⁵I in the lighter particles was mostly found in intact thyroglobulin and in large iodopeptides. This ¹²⁵I shift towards less dense particles was more marked in females than in males. These results indicate that age delays thyroglobulin progression towards dense lysosomes and suggest that the slower traffic of thyroglobulin in the endocytic pathway contributes to the reduction in thyroid hormone secretion in the aged cream hamster.     

Characterization of porcine adrenocortical lysosomes

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.     

Thyroid hormone residues are released from thyroglobulin with only limited alteration of the thyroglobulin structure

We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.     

Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria

A method for the preparation of lysosomes from rat liver is presented. The procedure requires only standard equipment and is completed within less than 3 h. Homogenization and differential centrifugation were performed at pH 7.4 in isotonic potassium phosphate-buffered sucrose medium. The addition of potassium phosphate, at the concentration used (10 mM), accelerated the sedimentation rate of mitochondria without altering that of lysosomes resulting in the decrease in the mitochondrial contamination of the final pellet. Further purification was achieved by isopycnic centrifugation in 45% isotonic Percoll performed in an angle rotor. Lysosomal fractions representing 51.5% of the original population were recovered over a density range of 1.09 to 1.15 g/ml. The most purified fraction (37-fold purified) contained 25.3% of lysosomal beta-N-acetylglucosaminidase, and only 0.9% of mitochondrial monoamine oxidase and 0.6% of peroxisomal urate oxidase original activities. It was practically devoid to endoplasmic reticulum contamination.     

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.     

Isolation of monkey aortic lysosomes using Percoll density gradient

A novel technique involving the Percoll density gradient and 0.01M phosphate buffer has been employed for the first time on aortic tissue for isolation of lysosomes. The purity of the lysosomes has been established by marker-enzymes, acid phosphatase and N-acetyl-beta-D-glucosaminidase and latent activities of lysosomal hydrolases. The heavier fraction (density 1.08) obtained after Percoll density gradient centrifugation showed high specific activities of lysosomal hydrolases and these enzymes were markedly latent. Moreover this heavier (lysosome rich) fraction has been noted to be free of other sub-cellular contaminants.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Evidence for the presence of a very high concentration of arylsulfatase A in the pig thyroid: identification of arylsulfatase A subunits as the two major glycoproteins in purified thyroid lysosomes

In addition to their general function in cellular homeostasis, thyroid lysosomes play an essential role in the biosynthesis of thyroid hormones by cleaving the macromolecular prohormone, thyroglobulin. In the present work, we have attempted to determine whether the enzyme composition of thyroid lysosomes differs from that of lysosomes from other tissues. Lysosomal enzymes, cathepsin D, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase, and arylsulfatase A and B, were assayed in crude fractions from various pig tissues, heart, brain, liver, kidney, thyroid, adrenals, ovary, and spleen. It appeared that the specific activity of arylsulfatase A was at least 20 times higher in the thyroid than in most other tissues. Thyroid lysosomes purified by isopycnic centrifugation on Percoll gradients contained two major polypeptides with apparent molecular weights of 58,000 and 54,000 representing about 30% of the total protein. These polypeptides were glycosylated and were exclusively found in the intralysosomal soluble fraction obtained by osmotic pressure-dependent lysis. By fractionating intralysosomal soluble proteins by velocity sedimentation on sucrose gradients or gel permeation chromatography we identified a thyroid arylsulfatase A holoenzyme which corresponds to a 120,000 Mr species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of the gradient or column fractions showed that the 120-kDa protein peak with arylsulfatase A activity essentially contained the 58- and 54-kDa polypeptides in equivalent amounts. In conclusion, arylsulfatase A, a heterodimer of 120 kDa composed of two nonidentical subunits, is the major protein component of thyroid lysosomes. The superabundance of this protein in purified thyroid lysosomes is related to the very high specific activity of the enzyme in the thyroid as compared to other tissues.     

Coated vesicles from thyroid cells carry iodinated thyroglobulin molecules. First indication for an internalization of the thyroid prohormone via a mechanism of receptor-mediated endocytosis

We have tried to identify iodinated thyroglobulin molecules in purified thyroid-coated vesicles to determined whether the internalization of the thyroid prohormone could proceed via a mechanism of receptor-mediated endocytosis. Coated vesicles isolated from pig thyroids by differential centrifugation and centrifugation on 2H2O-sucrose cushion were characterized by transmission electron microscopy and analyses of the polypeptide composition by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot using anti-clathrin heavy chain and anti-thyroglobulin antibodies. Clathrin and thyroglobulin (Tg) appeared as the two major components of the purified thyroid coated vesicles (TCV). Purified TCV fraction was homogeneous when analyzed by isopycnic centrifugation on 30% Percoll gradient. TCV had an apparent buoyant density of 1.035 g/ml. The presence of Tg molecules inside TCV was ascertained by (a) immunogold labeling on cryosections of TCV pellet and (b) identification by gel electrophoresis and radio-immunoassay of a definite fraction of Tg (3-5% of total protein) in TCV treated by Triton X-100. The detergent-treated TCV also contained protein-bound iodine: 0.5-0.7 micrograms of iodine/mg protein. Pulse-chase experiments on in vitro reconstituted thyroid follicles have been used to further document the presence of iodinated Tg molecules in coated vesicles. TCV were isolated from reconstituted thyroid follicles previously labeled with [125I]iodide to radioiodinate Tg of the follicular lumen (the pre-endocytotic compartment) and incubated with or without thyrotropin or dibutyryl cyclic AMP to activate intraluminal 125I-Tg endocytosis. Autoradiographic analyses revealed the presence of 125I-Tg in purified TCV and Triton X-100-treated TCV. 125I-Tg present in TCV represented 1-2% of the total intracellular protein-bound radioactivity. Thyrotropin and dibutyryl cyclic AMP increased 2-3-fold the 125I-Tg content of TCV. Our results clearly show that iodinated Tg, the molecular form of the thyroid prohormone known to be internalized, is present into TCV. The data suggest that coated vesicles are involved in the uptake and transport of Tg from the follicular lumen to the lysosomal compartment and therefore, that the internalization of Tg could proceed, at least for a part, via a mechanism of receptor-mediated endocytosis.     

Density gradient separation of two populations of lysosomes from rat parotid acinar cells

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.     

Uptake of tyramine cellobiose by rat liver.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Association of purified thyroid lysosomes to reconstituted microtubules

We report the characteristics of the interaction between reconstituted microtubules and purified thyroid lysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 125I by conjugation with the Bolton-Hunter reagent. Thyroid lysosomes were purified from pig thyroid by isopycnic centrifugation on Percoll gradients. The formation of microtubule-lysosome complexes has been studied by electron microscopy, using negative staining, and by differential centrifugation. The association of lysosomes to microtubules is time- and temperature-dependent (between 25 degrees C and 37 degrees C). The rate of microtubule-lysosome complex formation is related to the concentration of lysosomes. The higher the lysosome concentration is, the higher also is the rate of the interaction. Changes in microtubule concentration merely alter the amount of complex formed; there is a linear relationship between the amount of complexes and the microtubule concentration. However, lysosomes seem to possess a limited number of 'microtubule-binding sites', since a saturation of the complex formation can be obtained at high microtubule concentration. Two main types of complex have been observed by electron microscopy on negatively stained samples; simple complexes composed of a lysosome in close contact with a microtubule and complexes formed by a lysosome surrounded by several microtubules. The formation of microtubule-lysosome complexes was totally inhibited in the presence of 100 microM N-ethylmaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 microM). The interaction we describe here in an acellular system might be relevant to the association of lysosomes to microtubules observed in intact cells (Collot, M., Louvard D. and Singer S.J. (1984) Proc. Natl. Acad. Sci. USA 81, 788-792) and will constitute a useful model to study the regulation mechanisms of microtubule-vesicle interaction.     

Isolation and characterization of a rough microsomal fraction from rat kidney that is enriched in lysosomal enzymes

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM(2)) from the bulk of rough microsomal material in rat kidney because of its greater density. 2. The specific activities of five acid hydrolases in the RM(2) fraction were approximately one-half those of a purified lysosomal (L) fraction and 10- to 30-fold greater than those of an ordinary rough microsomal (RM(1)) fraction. 3. These special rough microsomes have a distinctive ultrastructure and electron-cytochemical properties. Their cisternal content resembles the matrix of lysosomes in that it is electron-dense, osmiophilic and plumbophilic and gives a positive reaction for acid phosphatase activity. 4. Polyacrylamide-gel electrophoresis of soluble proteins from the L fraction resolved nine anionic glycoproteins, most of which exhibit acid hydrolase activities (Goldstone & Koenig, 1970, 1973; Goldstone et al., 1971a). The most anionic glycoprotein is the acidic lipoglycoprotein of the lysosomal matrix (Goldstone et al., 1970). 5. Polyacrylamide-gel electrophoresis of soluble proteins from the RM(2) fraction resolved two cationic glycoproteins with acid hydrolase activities (Goldstone & Koenig, 1973) and an anionic glycoprotein with the same electrophoretic mobility as the lysosomal lipoglycoprotein, but without its lipid constituents or capacity to bind the basic fluorochrome Acridine Orange. These constituents are considered to be the precursors of the lysosomal glycoproteins.     

Purification of gibberellic acid-induced lysosomes from wheat aleurone cells.

Using isopycnic density gradient centrifugation, lysosomes were concentrated in a single region of a sucrose-Ficoll gradient (p = 1-10 g cm-3), well separated from most other cell organelles. Gibberellic acid-induced lysosomes were found to be rich in alpha-amylase and protease but not ribonuclease. The lysosomal band also contained a majority of the NADH2-cytochrome c reductase, a marker enzyme for endoplasmic reticulum, found in the gradient. Examination of electron micrographs revealed that a purified band of lyosomes contained at least 3 vesicle types, ranging in size from 0-1 to 0-5 mum. The significance of these findings to proposed mechanisms of action of gibberellic acid is discussed.     

A simple procedure for the isolation of highly purified lysosomes from normal rat liver

A procedure for the isolation of highly purified lysosomes from normal rat liver is described. The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 1 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation. The lysosomal fraction obtained by our method was enriched more than 120-fold in terms of the marker enzymes with a yield of 25%. The electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.     

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.