Solubilization, stabilization, and partial purification of brain adenylate cyclase from rat

Abstract

Membrane-bound adenylate cyclase from rat brain has been solubilized with Lubrol-PX. The soluble enzyme is nonsedimentable at 165,000g for 2 hr, is filterable through 0.22 /x Millipore filters and is responsive to F_. The solubilized adenylate cyclase has been stabilized for at least 7 days by the addition of 5 mM NaF and 5 mM MgS04. The enzyme has an estimated molecular weight of 800,000 which was obtained on the basis of a calibrated Bio-Gel A-15 m (agarose) column. The enzyme has been purified 18-fold representing the removal of over 99% of the starting protein. Addition of fractions removed during purification did not increase enzyme activity. The partially purified adenylate cyclase has ATPase and adenosine 3',5'-cyclic monophosphate phospho-A L Vdenylate cyclase catalyzes the conversion of adenosine triphosphate to cAMP,* 1 and plays a pivotal role in mediating the effects of a wide variety of hormones (Sutherland et cil., 1965). The physiological importance of adenylate cyclase has long been recognized and its properties as a particulate, hormone-responsive membrane-associated enzyme have been extensively described and are the subject of investigation (

Cyclic 3′,5′-Adenosine Monophosphate in Isolated Bone Cells. II. Responses to Adenosine and Parathyroid Hormone1

“…Theophylline and its analogues would then block the interaction between adenosine and this putative superficial phos-* phodiesterase, whereas dipyridamole analogues would not. Indeed, a partially purified preparation of adenylate cyclase from rat brain has been shown to contain phosphodiesterase activity (37). Adenosine failed to increase bone cell ATP levels significantly, but we cannot exclude the possibility that adenosine expands a small, membrane-located ATP pool that is preferentially utilized for cyclic 3',5'-AMP formation.…”

Second Messenger System in Neoplasia

“…It has been isolated and studied (within or from membrane fragments) from the renal medulla [45,206], renal cortex [17,45], adrenal glands [233,265], lungs [341], lymphocytes [295], erythrocytes [19,246], skeletal muscle [274], cardiac muscle [296], adipose cells [11,25,42,117,118], Islets of Langerhans [160], brain [74,307], thyroid [38,134,188,351,352,358], prostate [173], bone [46], liver [23,24,123,211,226,237,248,308], and Ehrlich tumor cells [350], Several models of adenylate cyclase action have been proposed from these studies. The adenyl cyclase system must have at least two components: a receptor component and a catalytic component.…”

Preparation of a stable, highly‐active solubilized adenylate cyclase from rat cerebellum

“…Levey [3] described the use of the non-ionic detergent Lubrol-PX to solubilize adenylate cyclase from mammalian tissues. Attempts to purify this detergent solubilized enzyme have been hindered by the low specific activity of the enzyme in some tissue [4,5] and its inherent instability in the absence of detergent [5][6][7].…”

“…Levey [3] described the use of the non-ionic detergent Lubrol-PX to solubilize adenylate cyclase from mammalian tissues. Attempts to purify this detergent solubilized enzyme have been hindered by the low specific activity of the enzyme in some tissue [4,5] and its inherent instability in the absence of detergent [5][6][7]. The central nervous system in mammals has the highest activity of adenylate cyclase [6,8] and therefore provides an attractive source material for the purification of the enzyme.…”