Isolation of pig thyroid lysosomes. Biochemical and morphological characterization.
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, beta-galactosidase and beta-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.
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation; D003576:Electron Transport Complex IV / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure*; D008854:Microscopy, Electron; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000648:ultrastructure*
D011506:Proteins; D003576:Electron Transport Complex IV; D000135:Acid Phosphatase