Affinity purification of cardiac adenylate cyclase: dependence on prior hydrophobic resolution.

Abstract

The interaction of cardiac adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.11 with a variety of nucleotide affinity resins was systematically investigated. None of these resins effectively bound the native, detergentsolubilized enzyme. However, after hydrophobic resolution on an uncharged resin consisting of long-chain alkyl groups linked to agarose via ether bonds, 40% of the adenylate cyclase activity biospecifically adsorbed to an ATP affinity resin. Gel filtration without detergent after hydrophobic chromatography demonstrated that the enzyme eluted in the identical position as the native enzyme chromatographed in the presence of detergent. This preparation almost completely biospecifically adsorbed to the same ATP-resin and was not eluted with 5 mM cyclic AMP, pyrophosphate, or GTP. If the GTP-washed immobilized enzyme was subsequently desorbed with ATP, then expected Gpp(NH)p (5'-guanylyliminodiphosphonate) sensitivity persisted. A preliminary purification scheme that resulted in an approximate 5000-fold increase in specific activity is presented. These observations indicate that a membrane-bound enzyme may appear to be intrinsically hydrophobic only by virtue of aggregation with other hydrophobic constituents and that prior separation of hydrophobic chromatography may permit such proteins to be fractionated subsequently by methods conventionally applied to hydrophilic proteins. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] is a membrane-bound enzyme present in almost all cell types which catalyzes the conversion of ATP to cyclic AMP. A number of polypeptide hormones as well as catecholamines, histamine, and prostaglandins exert all or part of their action through stimulation of this enzyme (1, 2). Cyclic AMP also may exert direct effects on a variety of cellular regulatory mechanisms, including microtubule assembly (3, 4), mobility, and chemotaxis (5), secretion (6, 7), and neoplastic transformation '(8, 9). Despite the critical role of this enzyme, minimal progress has been made in its purification. This difficulty arises for several reasons. First, the enzyme probably constitutes less than 0.005% of the total membrane protein (10). Second, the enzyme is notoriously labile, with a half-life at room temperature of only minutes (11). Third, nonionic detergents are required to solubilize the enzyme. Following such a procedure, several investigators have achieved only minimal success using classical procedures including gel filtration and ion-exchange chromatography (12, 13). We have recently investigated the interaction of the solubilized enzyme with a variety of long-chain alkyl resins linked to uncharged matrices via ether bonds. These gels have been shown to demonstrate all of the properties expected for hydrophobic interaction chromatography (14). Such resins effect an apparent separation of cyclase from more hydrophobic proteins, as demonstrated by the behavior of the enzyme on subsequent gel filtration and ion-exchange chromatography.These results suggest...

7‐O‐hemisuccinyl‐deacetyl forskolin—sepharose: a novel affinity support for purification of adenylate cyclase

“…This may in part be explained by its extreme instability and by the lack of suitable ligands for construction of affinity supports which otherwise proved to be extremely useful for isolation of minute quantities of total cellular protein [9,10]. The most notable success in purification of adenylate cyclase has been achieved with the canine myocardial enzyme by successive hydrophobic and affinity chromatography on ATP-agarose [11]. However, the procedure in [11] failed to resolve C and G since the purified material was still activated by Gpp(NH)p. The first successful separation of the catalyst and the regulatory G-protein has been achieved by *To whom correspondence should be addressed affinity chromatography of solubilized pigeonerythrocyte membrane adenylate cyclase on GTPSepharose: While the G-protein was selectively retained by the resin, the catalytic part was exclusively found in the column pass-through [2].…”

“…The most notable success in purification of adenylate cyclase has been achieved with the canine myocardial enzyme by successive hydrophobic and affinity chromatography on ATP-agarose [11]. However, the procedure in [11] failed to resolve C and G since the purified material was still activated by Gpp(NH)p. The first successful separation of the catalyst and the regulatory G-protein has been achieved by *To whom correspondence should be addressed affinity chromatography of solubilized pigeonerythrocyte membrane adenylate cyclase on GTPSepharose: While the G-protein was selectively retained by the resin, the catalytic part was exclusively found in the column pass-through [2]. Resolution of G and C by means of gel-filtration in the presence of cholate and high concentrations of ammonium sulfate has been reported independently by two groups [12,13].…”

Solubilization and Physical Characterization of the Adenylate Cyclase from Rat-Liver Plasma Membranes

“…[53] for 5'-nucleotidase ( M , 140000-150000); [51] for leucine aminopeptidase ( M , 320000) and [55] for y-glutamyltransferase ( M , It has been stressed [28] recently that hydrophobic membrane proteins such as adenylate cyclase are capable of interacting strongly with other membrane proteins thus leading to an apparent increase in their molecular weight. Hydrophobic chromatography has been proposed to promote separation of adenylate cyclase from other proteins of varying hydrophobicity [28].…”

“…The non-ionic detergents (Triton X-100 or Lubrol PX) which have been used have a very similar Hydrophilic Lipophilic Balance number (13.5 and 13.8). In a few cases, it has been established that cyclase activity was preserved in the absence of detergent [3, 27,28], but at the expense of an aggregation of the enzyme molecule, incompatible with further biophysical studies. Even when the solubilization process was performed under optimal conditions, all the cyclase systems studies have been shown to differ markedly with respect to their yield of solubilization and specific activity, stability, sensitivity to hormonal and nonhormonal effectors, and size.…”

Molecular cloning and amplification of the adenylate cyclase gene.

“…1]-has been only partially purified from mammalian tissues (15,16) or E. coli (17,18). The instability of the enzyme activity and the low abundance of the protein have accounted for the difficulty in its purification.…”