Isolation and Biochemical Study of Secretory Granules From Rat Pituitary Glands

Abstract

A B S T R A C TSecretory granules from anterior pituitary glands of young adult male castrate rats were isolated by differential centrifugation, microfiltration, and discontinuous density gradient centrifugation. The granules wcre obtained as pellets, sectioned, and studicd with the electron microscope. A major part of the gonadotropin and a substantial amount of the TSH were associated with the isolated granules. Negligiblc amounts of growth hormone and prolactin were present as contaminants. Succinic dehydrogenasc, glucosc-6-phosphatase, acid protease, and acid and alkaline phosphatascs were not found in the granules. Alkaline proteasc was the only enzyme found to be associated with the granules, and it is suggested, in the light of these results, that the alkaline protease may be involved in the release of the hormones. I N T R O D U C T I O NElectron microscope studies of anterior pituitary glands from rats by Farquhar and co-workers (1-5) have shown that the two representative cell types, acidophils and basophils, contain secretory granules of a specific maximum diameter. Certain of the acidophilic cells contain 350-m# and others 600-m# secretory granules reported to be associated, respectively, with growth and lactogenic hormones, while the two basophilic cell types have granules 140 m# (thyrotropin) and 200 m/.t (gonadotropins) in diameter (6).The separation of pituitary cytoplasmic particulates into various fractions and the determination of the hormones associated with them have been the objective of several workers employing differential centrifugation procedures similar to those used by Hogeboom et al. (7) for the preparation of liver particulates. These pituitary studies (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) were done with glands from different species, using different homogenizing media, and different times and speeds of centrifugation. The results of these studies are of interest in that they provided information concerning the hormonal activities of the fractions. The degree of purity of these fractions was not established and, as a consequence, generalizations cannot be made as to the specific particulates with which the hormonal activities are associated. In a recent study, however, Hartley et al. (6,21), using differential centrifugation, microfiltration, and isopycnic gradient centrifugation succeeded in isolating highly purified granules, 200 m# in diameter, from anterior pituitary glands of castrate rats. A substantial amount of the original homogenate's gonadotropic hormone activity was associated with these granules.The present report is concerned with the extension of this work, in that a shorter procedure was developed for obtaining the granules, young 159 on May 12, 2018 jcb.rupress.org Downloaded from

Subcellular Localization of Proinsulin to Insulin Conversion in Isolated Rat Islets

“…There are strong arguments for the enzyme being a trypsin-like enzyme and probably it is an alkaline proteinase (9). In view of this, it is interesting that alkaline proteinase has been demonstrated in association with isolated pituitary granules (46).…”

The Fine Structure of the Human Pituitary Chromophobe Adenoma with Special Reference to the Classification of this Tumor

“…16) (CARDELL and KNIGHTON, 1966;FUKUMITSU, 1964;GUSEK, 1962;LUSE, 1962;SCHELIN, 1962;WECHSLER and HOSSMANN, 1965 RINEHART, 1954 a, b, 1955;HYMER, MCSHAN and CHRISTIANSEN, 1961;LUNDIN and SCHELIN, 1962;rat, BARNSE, 1962;SANG, 1962;UCHIDA, 1967;YAMADA and SANO, 1960;mouse, SALAZAR, 1963;YOUNG, FOSTER andCAMERON, 1965, 1966;rabbit, CARDELL, 1964 a, b;salamander, OZTAN, 1966;fish MCSHAN and RIS, 1960;HYMER and MCSHAN, 1963;MCSHAN, 1964;PERDUE and MCSHAN, 1962).…”

Protease and ribonuclease activities in bovine pituitary lobes

“…'Alkaline proteinase' activity has been demonstrated in purified anterior-pituitary-lobe granule fractions (Perdue & McShan, 1962;Tesar et al 1969) and in other endocrine tissues, the adrenal gland and the thyroid gland (Todd & Trikojus, 1960). It seemed possible that alkaline ribonuclease activity would be localized in the granule fraction from homogenates of posterior pituitary lobes, especially as Philippu & Schumann (1964) have shown that adrenal-medullary granules contain ribonuclease activity.…”

“…The demonstration of 'alkaline protease' activity in purified anterior-pituitarylobe granule fractions (Perdue & McShan, 1962;Tesar, Koenig & Hughes, 1969) led us to wonder whether alkaline ribonuclease is localized in the granule fraction of homogenates of posterior pituitary lobes.…”

“…DISCUSSION Acid and alkaline proteinase activities were first described in the anterior and the posterior lobes of fresh pig and ox pituitary glands by Adams & Smith (1951). These authors used an assay in which the trichloroacetic acid-soluble digestion products of denatured haemoglobin were estimated by measuring the E280, a modification (Kunitz, 1947) Adams & Smith (1951) and in the subsequent reports that have confirmed pituitary-gland alkaline proteinase activity by measuring E280 (Meyer & Clifton, 1956;LaBella & Brown, 1959;Reichert, 1961Reichert, , 1962Perdue & McShan, 1962;Tesar et al 1969) did not contain the enzyme preparation alone. The need for adequate controls in protease assays because of nuclease activity has been emphasized by Marrink & Gruber (1966) and Umania (1968).…”