ISOLATION OF RAT PITUITARY GRANULES AND THE STUDY OF THEIR BIOCHEMICAL PROPERTIES AND HORMONAL ACTIVITIES

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.