Abstract
A simple and rapid method is described for the isolation of the acidophilic and basophilic granules from the anterior pituitary gland of the rat. The method involves chromatography of pituitary particulates on columns of No. 545 Celite equilibrated and developed with 0.25 M sucrose. Mitochondria are retained quantitatively on the column. The granules and microsomes which are not retained on the Celite are further fractionated on a discontinuous density sucrose gradient and by differential centrifugation. Essentially homogeneous populations of acidophilic and basophilic granules were obtained as indicated by 1) extensive electron microscopic studies, 2) enzymatic determinations, and 3) fatty acid and RNA analyses on the granule pellets. Microfiltration studies indicated that the acidophilic granules were smaller than 450 mµ, but greater than 300 mµ in diameter. They were found, unlike the basophilic granules, to be partially stable to extraction with water, but were unstable on incubation in 1 mM EDTA at 37°. Magnesium ions were not detected in the granules. The acidophilic and basophilic granules contained, respectively, 5 and 8 per cent of the protein present in the whole homogenate. Extensive hormone studies showed that growth and lactogenic hormones were associated with the acidophilic granules, while thyroid-stimulating hormone and gonadotropin were associated with the basophilic granules. ACTH was not present in significant amounts in either of the granule fractions, but was localized in a particulate fraction which contained microsomes and small granules. The association of the pituitary hormones with specific granules and cell types is discussed.
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- CERIOTTI G. Determination of nucleic acids in animal tissues. J Biol Chem. 1955 May;214(1):59–70. [PubMed] [Google Scholar]
- CHERMS F. L., Jr, HERRICK R. B., McSHAN W. H., HYMER W. C. Prolactin content of the anterior pituitary gland of turkey hens in different reproductive stages. Endocrinology. 1962 Aug;71:288–292. doi: 10.1210/endo-71-2-288. [DOI] [PubMed] [Google Scholar]
- COOPERSTEIN S. J., LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed] [Google Scholar]
- DRYER R. L., TAMMES A. R., ROUTH J. I. The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem. 1957 Mar;225(1):177–183. [PubMed] [Google Scholar]
- GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
- GREENSPAN F. S., KRISS J. P., MOSES L. E., LEW W. An improved bioassay method for thyrotropic hormone using thyroid uptake of radiophosphorus. Endocrinology. 1956 Jun;58(6):767–776. doi: 10.1210/endo-58-6-767. [DOI] [PubMed] [Google Scholar]
- GREENSPAN F. S., LI C. H. Bioassay of hypophyseal growth hormone; the tibia test. Endocrinology. 1949 Nov;45(5):455-63, illust. doi: 10.1210/endo-45-5-455. [DOI] [PubMed] [Google Scholar]
- HARTLEY M. W., McSHAN W. H., RIS H. Isolation of cytoplasmic pituitary granules with gonadotropic activity. J Biophys Biochem Cytol. 1960 Apr;7:209–218. doi: 10.1083/jcb.7.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HERLANT M. Séparation des activités du lobe antérieur de l'hypophyse par la méthode des centrifugations différentielles. Ann Endocrinol (Paris) 1952;13(4):611–623. [PubMed] [Google Scholar]
- HYMER W. C., McSHAN W. H. Isolation of cytoplasmic pituitary granules by column chromatography. J Cell Biol. 1962 May;13:350–354. [PMC free article] [PubMed] [Google Scholar]
- MCSHAN W. H., ROZICH R., MEYER R. K. Biochemical properties of fractions obtained from rat anterior pituitary glands by differential centrifugation. Endocrinology. 1953 Feb;52(2):215–222. doi: 10.1210/endo-52-2-215. [DOI] [PubMed] [Google Scholar]
- PERDUE J. F., McSHAN W. H. Isolation and biochemical study of secretory granules from rat pituitary glands. J Cell Biol. 1962 Nov;15:159–172. doi: 10.1083/jcb.15.2.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riley V. T., Hesselbach M. L., Fiala S., Woods M. W., Burk D. Application of Chromatography to the Separation of Subcellular, Enzymatically Active Granules. Science. 1949 Apr 8;109(2832):361–364. doi: 10.1126/science.109.2832.361-a. [DOI] [PubMed] [Google Scholar]
- SCHACHTER D. The fluorometric estimation of magnesium in serum and in urine. J Lab Clin Med. 1959 Nov;54:763–768. [PubMed] [Google Scholar]
- SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. 6. Release of enzymes and ribonucleic acid from ribonucleoprotein particles. J Biophys Biochem Cytol. 1960 Jul;7:631–644. doi: 10.1083/jcb.7.4.631. [DOI] [PMC free article] [PubMed] [Google Scholar]