Distribution of Hydrolases among Anterior Pituitary Cell Fractions (Anterior Pituitary Hydrolases)

The existence of intracellular particles with sedi-mentation characteristics intermediate between those of mitochondria and microsomes has been demonstrated in liver by studies of enzyme distribution among subcellular fractions (1-3), and the postulated intermediary particles were named "lysosomes" because of their manifold hydrolytic properties (1). The electron microscope shows that this intermediary fraction is composed primarily of small mitochondria and large microsomes, but Novikoff et aI. (4) have also encountered therein a few dense, polymorphic particles, less than 0.5 micron in diameter, which are assumed to be the lysosomes. The fraction under consideration is distinguished by the highest specific activity for such hydrolytic enzymes as acid phosphatase, ribonuclease, deoxyribonuclease, uricase, and cathepsin among all cell fractions. Bennett (5) has suggested that the lysosomes may be an essential component of the phagocytic cells of the liver. We have studied several hydrolases, as well as enzymes which in liver are localized in mito-chondria or microsomes, in the subcellular fractions of pig anterior pituitary glands and have observed their distinct intracellular distribution. Anterior lobes from fresh pig pituitaries were ho-mogenized in the cold in 0.25 ~ sucrose containing 0.02 per cent hepafin to make a 10 per cent (w/v) homogenate. Nuclei, unbroken cells, and debris were removed by spinning for 15 minutes in a model QV-110 International centrifuge at 1200 R.P.~. (approximately 700 g). The supernate was centrifuged at 7000 g for 20 minutes in a Spinco model L refrigerated centrifuge to give a milk white pellet, the aeidophilic granules. Centrifugation of the resultant supernate at 34,000 g for 15 minutes yielded a tan-colored mitochondrial fraction. This fraction consisted of a firm pellet with an overlying fluffy layer. In these experiments the fluffy material was kept separate, and the labels heavy and light mitochondria are used to denote the firm pellet and fluff, respectively. The microsomes were collected as a red, translucent gel by centrifuging the supernate from the previous centrifugation at 78,000 g for 3 hours. The final supernate was a perfectly clear, reddish solution. The granules identified as basophilic in electron micrographs of sectioned pituitaries could not be found in homogenates. In the preparation of each of the particulate fractions, each pellet was rehomogen-ized and respun two or three times at slightly increasing speeds. No washings were discarded. (The techniques of centrifugation and morphological examination will be presented in detail elsewhere.) Nitrogen was determined by Nesslerization (6). Succinic de-hydrogenase was assayed by …