Adenylate cyclase catalytic subunit

The catalytic component of the adenylate cyclase complex, i.e., the subunit which catalyzes the conversion of ATP to cAMP, has proved more difficult to purify than the other components. It has recently been purified from myocardium and brain [50-52] using affinity chromatography on forskolin linked to agarose and other chromatographic procedures. Forskolin is a diterpene which can activate the catalytic moiety directly [53]. The heart enzyme was purified approximately 60000-fold and exhibited two major peptides of MT 150000 and 42000 on NaDodS04 polyacrylamide gel electrophoresis [50]. The lower A/r peptide was probably 05, whereas the higher Mt peptide was probably the catalytic subunit. Crosslinking of the partially purified catalytic subunit with a Gs preparation, after [32P]ADP-ribosylation 

There is abundant evidence that glucagon elevates cAMP levels in isolated liver parenchymal cells, in perfused liver and in the liver in vivo [58,59], As illustrated in Fig. 2, this occurs rapidly and with concentrations of the hormone [59] within the range found in portal venous blood in vivo i.e., 0.2-2 x 10-10 M. When sufficiently sensitive and accurate methods are employed to measure cAMP, an increase in the nucleotide is consistently observed in situations where the hormone induces metabolic responses [58,59]. However, an increase of only 2- to 3-fold is capable of inducing full stimulation of some major hepatic responses, e.g., phos-phorylase activation (Fig. 2) and gluconeogenesis [58,59]. Since higher concentrations of the hormone can elevate cAMP 10-fold or more [59] it appears that there is considerable receptor reserve for these responses. 

The changes in cAMP induced by glucagon in isolated hepatocytes are well correlated with the changes in the activation state of the protein kinase [58,59]. This is illustrated in Fig. 2. Careful examination of the correlations between the increases in cAMP and cAMP-dependent protein kinase activity induced by very low concentrations of glucagon illustrates some cooperativity in the effect of the nucleotide on the kinase [59] consistent with the synergistic interaction between the 

The catalytic subunit of the cAMP-dependent protein kinase has been purified to homogeneity from several tissues including liver [69]. It has an Mr of 39000-42000 (depending on the method used) and appears to be similar in all tissues in terms of its chemical, physical, catalytic and immunological properties [60]. It also has a similar substrate specificity and can interact with either the Type I or Type II regulatory subunit. 

It has a broad substrate specificity [60], but the preferred amino acid sequence around the phosphorylated serine or threonine includes a pair of basic amino acids on the amino terminus side [60], Two arginine residues removed by one residue from the phosphorylated amino acid or a Lys-Arg sequence removed by two residues are preferred. 

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The receptors (R) interact with a complex (GE) of the adenylate cyclase catalytic subunit (E) and the guanyl-nucleotide binding regulatory protein (G) with a 1 1 stoichiometry and (t/) complexes of GE are activated when an equal number (y) of receptors are occupied. 

Gsa -the a subunit of a guanine nucleotide-binding protein that stimulates the adenylate cyclase catalytic unit... 

Figure 6.3. Mechanism of action of heterotrimeric G-proteins. Upon receptor occupancy, the Ga-subunit binds GTP in exchange for GDP, and then moves in the membrane until it encounters its target enzyme, shown here as adenylate cyclase (alternatively, a phospholipase). The activated target enzyme then becomes functional. Inherent GTPase activity within the a-subunit then hydrolyses bound GTP to GDP, and the a-subunit dissociates from its target enzyme (which becomes inactive) and rebinds the / - and ysubunits. Upon continued receptor occupancy, further catalytic cycles of GTP exchange and target enzyme activation may occur. The scheme shown is for a stimulatory G-protein (Got,), but similar sequences of events occur with inhibitory G-proteins (Gcx,) except that the interaction of the a-subunit with adenylate cyclase will result in its inhibition. The sites of action of pertussis and cholera toxins are shown.

Figure 14-2. Regulation of cyclic AMP-dependent protein kinase A (PKA) by cyclic AMP. Activation of adenylate cyclase by binding of G( -GTP amplifies the signal by synthesis of many molecules of cyclic AMP. Cyclic AMP binding to PKA causes dissociation of the regulatory subunits from the catalytic subunits, which carry on the signal. Phosphodiesterase regulates the concentration of cyclic AMP by catalyzing its hydrolysis to AMP, which shuts off the signal.

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

Guanylate cyclases, which form cyclic GMP, occur in particulate and soluble forms.908 The latter have been of great interest because they are activated by nitric oxide (NO). The soluble guanylate cyclases are a(3 heterodimers. The C-terminal regions of both a and (3 subunits are homologous to the catalytic domain of adenylate cyclase. The N-terminal domain of the a subunits contains heme whose Fe atom is coordinated... 

Fig. (9). A) Schematical representation of the activation of intact adenylate cyclase (AC) b forskolin and Gsa (GTP- bound stimulatory G protein a subunit). Mammalian ACs consists o 12 transmembrane helices and two cytoplasmic catalytic domains (referred to as Q and C2 represented as lightly shaded and black respectively), (a) Hypothetical basal state, (b) Th suggested forskolin-activated state, (c) Forskolin and GSa-activated state.

Activation of G-proteins by occupied receptors results usually in the dissociation of the G-protein complex, releasing an activated a subunit. In the case of Gs, the activated a subunit interacts with andfactivates the catalytic unit of adenylate cyclase by increasing the affinity of the enzyme for Mg2+, whereas the activated a subunit of transducin stimulates cyclic GMP phosphodiesterase (PDE) activity by causing the dissociation of a y subunit from the PDE, hence relieving an inhibitory effect. G appears to exert its inhibitory effect on adenylate cyclase in two distinct... 

G protein subunits into a and [iy subunits. Both the a and [fy subunits may elicit secondary events such as the activation or inhibition of the catalytic unit of adenylate cyclase. 

Some ligand-activated membrane receptors transmit their signal by stimulating adenylate cyclase activity in the cell to produce cAMP. This activation pathway is mediated by a receptor-associated G protein called GS (Chapter 16). In mammals, the most common mechanism by which cAMP serves as a second messenger involves cAMP binding to the regulatory subunit of cAMP-dependent protein kinase A (PKA). Dissociation of the regulatory subunit allows the catalytic sub-... 

At the time this model was proposed, no precise information was available on the mechanism by which the cAMP-receptor complex activates adenylate cyclase. Therefore a simple hypothesis was retained, namely that the receptor and adenylate cyclase form the regulatory (R) and catalytic (C) subunits of an allosteric complex, which bind extracellular cAMP and intracellular ATP, respectively moreover, it is assumed that this complex possesses a tetrameric structure of the type RjQ. The input of the substrate ATP - but not the level of this metabolite - was considered to be constant in the course of time. 

Forskolin (29), a natural diterpene, has become an important tool in cell physiology due to its unique mechanism of action. 112-114 it exerts its activity either by direct action on the catalytic subunit of adenylate cyclase or by indirect action via a previously unrecognized... 

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