The Cellular Location of Adenyl Cyclase in the Pigeon Erythrocyte

Abstract

It has been reported by Sutherland, Rail, and Menon (1) that particulate preparations from a number of tissues, including pigeon erythrocytes, are able to synthesize adenosine 3’) 5’-phosphate (cyclic 3’,5’-AMP) when they are incubated in the presence of adenosine triphosphate and Mgff. The name adenyl cyclase was proposed for the enzyme system catalyzing the synthesis. The adenyl cychse activity of rat liver homogenate sedimented at 1000 X g in the so-called “nuclear” fraction, and it was not associated with preparations of either mitochondria or microsomes. This “nuclear” fraction may have contained both nuclei and cell membranes (2, 3), and it was concluded that these two cell components were the principal candidates for the location of the adenyl cyclase (4). The pigeon erythrocyte was chosen for the present investigation because of its relatively simple intracellular structure and because it provided a uniformity of cell type not found in more organized tissues such as liver, brain, and muscle, where blood vessels, connective tissue, and a multiplicity of cell types occur. The work of Rajam and Jackson (2) and of Neville (3) shows that, under certain conditions of homogenization, both the nuclei and the cell membranes can be sedimented by centrifugation at low speed. The centrifugal behavior of a particle is determined by its inherent properties such as size, shape, and density; however, this behavior may be modified by the mechanical or other association of the particle with other cell components during centrifugation. It is probable that the sedimentation behavior of the membranes of pigeon erythrocytes, which were prepared by exposure of cells to hypotonic conditions, is due at least in part to the envelope of the cell collapsing around the heavy nucleus. Whether the simultaneous sedimentation of membranes and nuclei is due to their mechanical association or to the size of the membrane, it has been found that these two components can be separated effectively by utilizing a dispersion procedure which extensively fragments the membranes and yet does little damage to the nuclei. Disruption of cells by the use of a pestle homogenizer or of units such as the Waring Blendor has the basic disadvantage that it is difficult to control the disruptive force applied to the cells, and, furthermore, the contents of the broken cells are subjected to further disruption. For the present investigation a device was con-