Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.

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On the regulation of cyclic AMP level in bacteria II. In vitro regulation of adenylate cyclase activity. Solubilization and reconstitution of a functional membrane-bound adenylate cyclase system responsive to regulation by glucose

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(75)90357-8 ◽

1975 ◽

Vol 385 (2) ◽

pp. 294-304 ◽

Cited By ~ 9

Author(s):

M. Abou-Sabé ◽

S. Mento

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Adenylate Cyclase System ◽

Functional Membrane ◽

Membrane Bound

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Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

Biochemical Journal ◽

10.1042/bj1980631 ◽

1981 ◽

Vol 198 (3) ◽

pp. 631-638 ◽

Cited By ~ 1

Author(s):

Nicholas B. Lydon ◽

John L. Young ◽

David A. Stansfield

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Guanine Nucleotides ◽

Lag Phase ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Human Choriogonadotropin ◽

Activated State ◽

Cyclic Amp Synthesis

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ‘burst’ phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ‘lag’ phase with p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.

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Renal cyclic AMP accumulation and adenulate cyclase stimulation by erythropoietic agents

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1975.229.5.1387 ◽

1975 ◽

Vol 229 (5) ◽

pp. 1387-1392 ◽

Cited By ~ 2

Author(s):

GM Rodgers ◽

JW Fisher ◽

WJ George

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Regional Distribution ◽

Renal Medulla ◽

Iron Incorporation ◽

Erythropoietin Production ◽

Cyclic Amp Accumulation ◽

Stimulation Of

The regional distribution of cyclic AMP in the kidney was determined following erythropoietic stimulation with hypoxia and cobalt. Following these stimuli, increases in renal cyclic AMP concentrations were restricted to the cortex. The basis for this localization in the case of cobalt treatment was found to reside in the stimulation of renal cortical adenylate cyclase activity in vitro by concentrations of cobalt similar to those found in vivo. The level of cobalt in the cortex after cobalt treatment was found to approach 500 mumol/kg of tissue, whereas no detectable levels of cobalt were found in the renal medulla. Additionally, other agents such as parathyroid hormone and lactic acid, that are known to lack stimulatory effects on medullary adenylate cyclase, were found to stimulate the cortical enzyme. This stimulation of renal cortical adenylate cyclase correlates with enhanced erythropoiesis as demonstrated by increased radiolabeled iron incorporation into erythrocytes. These results support previous reports which suggest that renal cortical cyclic AMP mediates erythropoietin production in response to erythropoietically active agents.

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Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3873-3883.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3873-3883

Author(s):

M R Mitts ◽

D B Grant ◽

W Heideman

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Protein ◽

Adenylate Cyclase ◽

Adenylate Cyclase System ◽

Hydrodynamic Properties ◽

Yeast Saccharomyces Cerevisiae ◽

Peripheral Membrane Protein ◽

Mammalian Enzyme ◽

Regulatory Significance ◽

Stimulation Of

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.

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Antisecretory effects of berberine in rat ileum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1981.241.3.g253 ◽

1981 ◽

Vol 241 (3) ◽

pp. G253-G258 ◽

Cited By ~ 1

Author(s):

Y. H. Tai ◽

J. F. Feser ◽

W. G. Marnane ◽

J. F. Desjeux

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Short Circuit ◽

Short Circuit Current ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Rat Ileum ◽

Ion Secretion ◽

Stimulation Of

The in vitro antisecretory effects of the alkaloid berberine (1.0 mM) on intestinal ion secretion and mucosal adenylate cyclase and Na-K-ATPase activities were studied in the rat ileum. Mucosal berberine did not alter the individual basal net ion fluxes and basal adenylate cyclase activity but decreased short-circuit current (Isc) and increased the net absorption of chloride plus bicarbonate. In the cholera toxin-treated tissue, mucosal berberine stimulated absorption of Na and Cl and inhibited the increased adenylate cyclase activity but did not change the specific Na-K-ATPase activity, whereas serosal berberine stimulated Na secretion and decreased Isc. Mucosal berberine also decreased Isc, increased Cl permeability, and reversed the ion secretion induced by dibutyryl cyclic AMP, the heat-stable enterotoxin of Escherichia coli, and methylprednisolone administration. The antisecretory effects of mucosal berberine may be explained by stimulation of a Na-Cl-coupled absorptive transport process. The mechanism of action of serosal berberine remains to be elucidated. However, it is clear that mucosal berberine affects intestinal ion transport by mechanisms different from stimulation of the Na pump and probably at a step distal to the production or degradation of cyclic AMP or cyclic GMP.

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The Effect of Aging on β-Adrenoceptor-Stimulated Cyclic AMP Formation in Human Lymphocytes

Clinical Science ◽

10.1042/cs0600587 ◽

1981 ◽

Vol 60 (5) ◽

pp. 587-589 ◽

Cited By ~ 13

Author(s):

C. A. Kraft ◽

C. M. Castleden

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Binding Assay ◽

Human Lymphocytes ◽

Phosphodiesterase Inhibitor ◽

Adenylate Cyclase System ◽

Competitive Binding Assay ◽

Cyclic Monophosphate ◽

Maximal Stimulation ◽

Stimulation Of

1. Responsiveness of the β-adrenoceptor adenylate cyclase system was measured in lymphocytes from healthy young and old subjects by incubating the cells with isoprenaline in the presence of a phosphodiesterase inhibitor and by measuring production of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) with a competitive binding assay. 2. The two groups did not differ significantly in the levels of cyclic AMP produced or in the concentration of isoprenaline required to give half-maximal stimulation of the cells (ED50).

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The human eccrine sweat gland adenylate cyclase system: response to agonists

Clinical Science ◽

10.1042/cs0680433 ◽

1985 ◽

Vol 68 (4) ◽

pp. 433-439 ◽

Cited By ~ 2

Author(s):

A. K. Khullar ◽

V. Schwarz ◽

P. D. Wilson

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Cyclic Gmp ◽

Sweat Gland ◽

Sweat Glands ◽

System Response ◽

Renal Tubules ◽

Adenylate Cyclase System ◽

Cyclic Amp Accumulation

1. Cyclic AMP accumulation has been measured in whole human sweat glands. The mean rate in glands from 19 subjects was 0.519 ± 0.316 pmol of cyclic AMP formed 5 min−1 μg−1 of DNA, which is comparable with that reported for other tissues. 2. Cyclic AMP accumulation in the sweat gland is stimulated fourfold by prostaglandin (PG) E1 and fivefold by PGE2 (0.1 mmol/l), in accord with stimulation in renal tubules and medullary membranes. 3. Bradykinin (10 μg/ml) increases the rate threefold and this is substantially prevented by indomethacin (1.5 × 10−5 mol/l), as also is a five-fold stimulation by cyclic GMP (10−5 mol/l). 4. Mecholyl (10−2 mol/l) and isoprenaline (6 × 10−6 mol/l) increase the rate five- and four-fold respectively, and these agonist effects are largely abolished by atropine and propranolol. 5. The stimulation and inhibition pattern suggests a direct action of PGE, enhancement of prostaglandin synthetase by cyclic GMP and stimulation of guanylate cyclase by mecholyl and bradykinin. Isoprenaline presumably stimulates adenylate cyclase directly. 6. This complex chain of events, from cholinergic stimulation to an enhancement of adenylate cyclase, demonstrated in vitro, constitutes a potential for flexible and fine control of sweat gland function.

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Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3873 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3873-3883 ◽

Cited By ~ 22

Author(s):

M R Mitts ◽

D B Grant ◽

W Heideman

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Protein ◽

Adenylate Cyclase ◽

Adenylate Cyclase System ◽

Hydrodynamic Properties ◽

Yeast Saccharomyces Cerevisiae ◽

Peripheral Membrane Protein ◽

Mammalian Enzyme ◽

Regulatory Significance ◽

Stimulation Of

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.

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Stabilization and solubilization of bovine corpus-luteum adenylate cyclase. The effects of guanosine triphosphate, guanosine 5′-[β,γ-imido]triphosphate, sodium fluoride and Tris/hydrochloric acid concentration on enzyme activity

Biochemical Journal ◽

10.1042/bj1690133 ◽

1978 ◽

Vol 169 (1) ◽

pp. 133-142 ◽

Cited By ~ 5

Author(s):

John L. Young ◽

David A. Stansfield

Keyword(s):

Adenylate Cyclase ◽

Corpus Luteum ◽

Active State ◽

Adenylate Cyclase System ◽

Hydrochloric Acid Concentration ◽

Bovine Corpus Luteum ◽

Ionic Detergent ◽

Stimulation Of

1. Adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate is stimulated by p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate), the imido analogue of GTP, and to a lesser extent by GTP itself. Activation by p[NH]ppG is not reversed by extensive washing before assay, but can, however, be reversed by NaF. 2. Both p[NH]ppG and NaF stabilize the enzyme during incubation at 37°C. NaF also causes an irreversible activation, but only of part of the potentially NaF-activatable adenylate cyclase; there are possibly two components of the adenylate cyclase system, which can be distinguished by their response to NaF. 3. Solubilization of the adenylate cyclase activity in the 600g sediment, by using the non-ionic detergent Lubrol-PX, gave variable yields. A relationship between the magnitude of NaF stimulation of the 600g-sediment enzyme and the yield of soluble activity derived from the sediment was recognized. The results suggest that the pre-existing state of the enzyme complex in vivo is reflected by the response in vitro to NaF and may determine the success with which activity can be solubilized. 4. The absolute yields of soluble activity could be increased by p[NH]ppG preactivation of the 600g sediment. During the development of the maximally active state by preincubation with p[NH]ppG the enzyme passes through a stage in which Lubrol solubilization is increased, but the maximally active state is itself less amenable to solubilization. p[NH]ppG activation causes the appearance of NaF-inhibited states, which appear to be preferentially solubilized by Lubrol-PX.

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