scholarly journals Isolation of pig thyroid lysosomes. Biochemical and morphological characterization

1985 ◽  
Vol 232 (2) ◽  
pp. 529-537 ◽  
Author(s):  
C Alquier ◽  
P Guenin ◽  
Y Munari-Silem ◽  
C Audebet ◽  
B Rousset
Keyword(s):  
Endoplasmic Reticulum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Average Diameter ◽  
Morphological Characterization ◽  
Thyroid Cell ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Thyroid Cells ◽  
Secondary Lysosomes ◽  
Isopycnic Centrifugation

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, β-galactosidase and β-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.

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

1988 ◽  
Vol 253 (2) ◽  
pp. 523-532 ◽  
Author(s):  
S Selmi ◽  
B Rousset
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Distribution Patterns ◽  
Acid Hydrolase ◽  
Percoll Gradient ◽  
Cell Organelles ◽  
Thyroid Cells ◽  
Percoll Gradients ◽  
Secondary Lysosomes ◽  
L1 And L2

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

scholarly journals THE EFFECT OF POLY-L-LYSINE ON THE UPTAKE OF REOVIRUS DOUBLE-STRANDED RNA IN MACROPHAGES IN VITRO

1973 ◽  
Vol 57 (2) ◽  
pp. 484-498 ◽  
Author(s):  
Rolf Seljelid ◽  
Samuel C. Silverstein ◽  
Zanvil A. Cohn
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Nucleic Acids ◽  
Acid Hydrolases ◽  
Cell Compartment ◽  
Double Stranded Rna ◽  
Secondary Lysosomes ◽  
Toxic Concentrations ◽  
Vacuolar System

The effect of polycations on cultured mouse peitoneal macrophages has been examined. Polycations, at concentrations greater than 5 µg/ml, are toxic for macrophages) as measured by failure of the cells to exclude vital dyes. At toxic concentrations polycations bind in large amounts to nuclei and endoplasmic reticulum, while at nontoxic levels polycations bind selectively to the cell surface. Nontoxic concentrations of polycations stimulate binding of reovirus double-stranded (ds) RNA to the macrophages by forming polycation-dsRNA complexes either in the medium or at the cell surface. These complexes enter the cell in endocytic vacuoles and are concentrated in secondary lysosomes. Despite exposure to the acid hydrolases within this cell compartment, the dsRNA and the polycation (poly-L-lysine) are conserved in a macromolecular form within the vacuolar system. The mechanism(s) by which the uptake of infectious nucleic acids and the induction of interferon by dsRNA are stimulated by polycations are discussed.

scholarly journals The expression of the sarco/endoplasmic reticulum Ca2+-ATPases in thyroid and its down-regulation following neoplastic transformation

2003 ◽  
Vol 30 (3) ◽  
pp. 399-409 ◽  
Author(s):  
F Pacifico ◽  
L Ulianich ◽  
S De Micheli ◽  
S Treglia ◽  
A Leonardi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Northern Blot ◽  
Blot Analysis ◽  
Thyroid Tissue ◽  
Neoplastic Transformation ◽  
Thyroid Cell ◽  
Rt Pcr ◽  
Down Regulation ◽  
Thyroid Cells ◽  
Rat Thyroid

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.

scholarly journals The sarcoplasmic–endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells

2006 ◽  
Vol 190 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Luca Ulianich ◽  
Maria Giovanna Elia ◽  
Antonella Sonia Treglia ◽  
Antonella Muscella ◽  
Bruno Di Jeso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Thyroid Cell ◽  
Myristate Acetate ◽  
Thyroid Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Thyroid Cell Line ◽  
Rat Thyroid ◽  
Stimulation Of

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.

Transcellular iodide transport and iodination on the apical plasma membrane by monolayer porcine thyroid cells cultured on collagen-coated filters

1990 ◽  
Vol 126 (2) ◽  
pp. 275-281 ◽  
Author(s):  
Y. Nakamura ◽  
T. Kotani ◽  
S. Ohtaki
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Basal Medium ◽  
Experimental System ◽  
Total Radioactivity ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Iodide Transport ◽  
Cell Functions ◽  
Residual Radioactivity

ABSTRACT Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate 125I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0·1–0·3 mU/ml. Addition of ClO4− to the basal medium inhibited transcellular transport, whilst addition of ClO4− to the apical medium was much less effective. Constituents labelled with 125I in the apical medium were analysed. The amount of protein-bound 125I measured by acid precipitation was 3–8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate–polyacrylamide gel electrophoresis revealed that most of the 125I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium. The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential. Journal of Endocrinology (1990) 126, 275–281

A possible role for calmodulin in human thyroid cell metabolism

1983 ◽  
Vol 99 (2) ◽  
pp. 251-260 ◽  
Author(s):  
C. A. Ollis ◽  
S. MacNeil ◽  
S. W. Walker ◽  
B. L. Brown ◽  
R. M. Sharrard ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Thyroid Cells ◽  
Cell Extracts

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine–Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

scholarly journals FURTHER BIOCHEMICAL AND MORPHOLOGICAL STUDIES OF GRANULE FRACTIONS FROM RABBIT HETEROPHIL LEUKOCYTES

1970 ◽  
Vol 45 (3) ◽  
pp. 586-597 ◽  
Author(s):  
Marco Baggiolini ◽  
James G. Hirsch ◽  
Christian de Duve
Keyword(s):  
Alkaline Phosphatase ◽  
Low Density ◽  
Acid Hydrolase ◽  
Membrane Structures ◽  
Acid Hydrolases ◽  
Lysosomal Acid ◽  
Morphological Studies ◽  
Vesicular Membrane ◽  
Primary Granules ◽  
Isopycnic Centrifugation

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5–0.8 µm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25–0.40 µm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.

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

1973 ◽  
Vol 132 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Alfred Goldstone ◽  
Harold Koenig ◽  
Rajinder Nayyar ◽  
Charles Hughes ◽  
Chung Y. Lu
Keyword(s):  
Gel Electrophoresis ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Isolation And Characterization ◽  
The Matrix

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM2) 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 RM2 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 (RM1) 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 RM2 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.

scholarly journals Normal rabbit alveolar macrophages. II. Their primary and secondary lysosomes as revealed by electron microscopy and cytochemistry.

1976 ◽  
Vol 144 (4) ◽  
pp. 920-932 ◽  
Author(s):  
B A Nichols
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Rough Endoplasmic Reticulum ◽  
Alveolar Macrophages ◽  
Digestive Enzymes ◽  
Acid Hydrolases ◽  
Secondary Lysosomes ◽  
Enzymatic Machinery ◽  
Cytochemical Markers

In this investigation, vacuoles containing tubular myelin proved to be digestive compartments with cytochemical reactivity for acid phosphatase and arylsulfatase. These cytochemical markers identify the secondary lysosomes, known to contain enzymes capable of hydrolyzing phospholipids like surfactant. Therefore, it appears that alveolar macrophages possess the enzymatic machinery for the degradation of the tubular myelin found in their digestive vacuoles. Although it thus appears evident that alveolar macrophages participate in the turnover of surfactant, the quantitative significance of this route of disposal is undetermined. This investigation has also established that acid hydrolases, so prominently displayed in the secondary lysosomes, are also localized in the rough endoplasmic reticulum and in Golgi-endoplasmic reticulum-lysosomes (GERL). Moreover, small vesicles which are produced from GERL serve as primary lysosomes in transporting digestive enzymes to the vacuoles.

scholarly journals CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN GRANULE FRACTIONS FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

1972 ◽  
Vol 54 (1) ◽  
pp. 141-156 ◽  
Author(s):  
Marilyn G. Farquhar ◽  
Dorothy F. Bainton ◽  
Marco Baggiolini ◽  
Christian de Duve
Keyword(s):  
Acid Phosphatase ◽  
Polymorphonuclear Leukocytes ◽  
Mononuclear Cells ◽  
Acid Hydrolases ◽  
Cytochemical Localization ◽  
Dense Bodies ◽  
Pmn Leukocytes ◽  
Secondary Lysosomes ◽  
Isopycnic Centrifugation

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes

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