Rat brain adenylate cyclase

Lynch, Thomas J.; Tallant, E. Ann; Cheung, Wai Yiu

doi:10.1016/0003-9861(77)90290-9

Cited by 83 publications

(11 citation statements)

References 29 publications

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“…Calmodulin was prepared from calf brains as described by Klee (20) with the following modifications: (i) the starting material was the first warm high-speed supernatant from microtubule preparations (see ref. 21 for details on procedures used for obtaining this supernatant), and (ii) the gel filtration step used a composite column (5 X 68 cm) composed, from top to bottom, of 31 cm of Sephacryl S-200 (superfine), 6 (10). Although the culture supernatant showed substantial, albeit decreased, activation by calmodulin (Table 1), the purified enzyme did not respond to activation by calmodulin plus Ca2+, and we attribute this to the loss of another factor needed by the system..…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Calmodulin activates prokaryotic adenylate cyclase.

Wolff¹,

Cook²,

Goldhammer³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

267

178

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The aden late cyclase of Bordetella pertussis is stimulated 100-to 1000I old in a dose-dependent manner by calf brain calmodulin. The system has the following properties. (i) The activation is prevented by ethylene glycol bis(faminoethyl ether) NNN :Ft enzyme * calmodulin * Ca2+(active).The relatively few adenylate cyclases so far shown to be regulated by calmodulin are from neural tissues (7-14), adrenal medulla (15), and pancreatic islets (16). -It seemed reasonable, therefore, to investigate the effect of this regulator protein on B. pertussis adenylate cyclase. The present study shows activation of this enzyme up to 100-to 1000-fold and is an example of such an effect of calmodulin in a prokaryote. METHODSGrowth of the Organism. B. pertussis (strain 114) organisms were grown from a 5% inoculum in 250 ml of modified Stainer-Scholte medium (in 500-ml flasks) on an oscillating shaker at 35.50C with room air as the gas phase as described (17); incubation was for 20-24 hr. The flasks were then stored at 4°C and the suspension was used for several weeks unless otherwise indicated. Under these conditions, basal adenylate cyclase activity increased with time but the maximal stimulation attainable did not change.Supernatant culture medium which contains 0.1-0.2 of the total cyclase activity (18) was obtained by centrifugation of the suspension at 5000 X g for 10 min. Washed cells were prepared from the cell pellet by resuspending in 0.154 M NaCl that had been passed through a Chelex-100 column to remove divalent cations. This was repeated four times. ,Ml containing 60mM N-tris(hydroxymethyl)methylglycine-HCI (pH 7.8), 10 mM MgCl2, 1 mM [a-32P]ATP (1-1.5 X 106 cpm), and 10 Al of the bacterial suspension. GTP has no effect on this enzyme and was not included. The order of addition of the components of the assay mixture proved to be of importance. In some experiments, bacteria and activator were preincubated for 10 min and then the reaction was started by addition of the prewarmed substrate and was allowed to run for 10 min as described (1, 2). In most assays, the bacteria were added lastto start the reaction as described in the legends.Preparation of Calmodulin. Calmodulin was prepared from calf brains as described by Klee (20) with the following modifications: (i) the starting material was the first warm high-speed supernatant from microtubule preparations (see ref. 21 for details on procedures used for obtaining this supernatant), and (ii) the gel filtration step used a composite column (5 X 68 cm)

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Section: Methodsmentioning

confidence: 99%

“…The relatively few adenylate cyclases so far shown to be regulated by calmodulin are from neural tissues (7)(8)(9)(10)(11)(12)(13)(14), adrenal medulla (15), and pancreatic islets (16). -It seemed reasonable, therefore, to investigate the effect of this regulator protein on B. pertussis adenylate cyclase.…”

mentioning

confidence: 99%

Calmodulin activates prokaryotic adenylate cyclase.

Wolff¹,

Cook²,

Goldhammer³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

267

178

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“…Although these regulatory proteins seem to be distinct from the Ca+2-dependent regulatory protein (11), their relationship to the putative hormone-sensitive GTPase (12) and to the Gpp(NH)p-sensitive retinal cyclic GMP phosphodiesterase (13) has not been elucidated.…”

mentioning

confidence: 99%

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

Hebdon¹,

Vine²,

Sahyoun³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process that produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.The molecular mechanisms of activation of adenylate cyclase by NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p], and the relevance of such mechanisms to the hormonal modulation of this enzyme, have been difficult to investigate due to the lack of appropriate techniques for the resolution and reconstitution of the various molecular components of the adenylate cyclase system. We have reported (1) the development of such a technique which involves the preparation of a Gpp(NH)p-and NaF-insensitive brain particulate adenylate cyclase by differential detergent extraction. The enzyme regained its characteristic Gpp(NH)p and NaF responses upon the addition of one or more proteins solubilized from membrane preparations of various tissues (1). These regulatory proteins can be partially separated from solubilized adenylate cyclase by gel filtration on Ultrogel AcA 34 columns. We report here the utilization of this reconstitution technique to help elucidate the mode of interaction of the various components of this multifactorial system. Thermal and trypsin inactivation of the Gpp(NH)p-and NaF-reconstituting activities suggests that the two activities are functionally separable. Gpp(NH)p and NaF appear to interact reversibly with the corresponding regulatory proteins in a divalent cation-independent step, followed by a divalent cation-dependent step that leads to the persistent state of enzyme activation. Techniques that separate regulatory from catalytic components can also reverse the enzyme activation.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3693 The Gpp(NH)p-and NaF-insensitive adenylate cyclase was prepared by a modification of a published procedure (1, ...

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“…(mg protein)-' at pH 8 and 30°C. Protein determinations were performed according to Lowry [12], using bovine serum albumin as the standard.…”

Section: Purification Of Adenylate Cyclasementioning

confidence: 99%

“…Activity was measured at 30 "C. The standard incubation mixture contained 100 mM Tris-HC1 pH 8, 8 mM MgSO, (in excess of the added nucleotides) and 10 -30 pg protein/ml. In routine assays this mixture was supplemented with 10 mM sodium pyruvate.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Steady‐State Kinetic Studies on Adenylate Cyclase from Brevibacterium liquefaciens

Özer

Scheit

1978

European Journal of Biochemistry

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Kinetic data of basal adenylate cyclase activity were found to be in agreement with a negatively cooperative model for this enzyme. The model assumes a dimeric structure for the enzyme with two binding sites per dimer. The binding of the first substrate molecule increases the dissociation constant for substrate molecules increases the dissociation constant for substrate molecules at the second site. However, the doubly occupied enzyme has a greater catalytic efficiency per site than the singly occupied species. The effect of various nucleoside 5'-triphosphates on the basal enzymatic activity was investigated. At low concentrations of substrate and analogue relative to K, and K,, compounds closely related to ATP, like 3'-deoxy-adenosine 5'-triphosphate, 2-amino-adenosine 5'-triphosphate and 3'-deoxy-3'-amino-adenosine 5'-triphosphate activate adenylate cyclase. An increase in the concentration of either substrate or analogue reverses the activation effect. GTP, UTP, CTP and 2-aminopurineriboside 5'-triphosphate are purely competitive inhibitors of basal adenylate cyclase activity at low substrate concentrations. This behaviour can be explained by the assumption that coexistence of substrate and inhibitor fails to induce the increase in catalytic efficiency. Kinetic analysis of the activation of adenylate cyclase by pyruvate suggested that the primary effect of the activator is to abolish the negative cooperativity between subunits, at the same time inducing a better fit between the enzyme and its substrate. A tentative model, which is in reasonable agreement with the kinetic data is presented. Mixed inhibition of pyruvate-stimulated adenylate cyclase was observed with GTP. Kinetic analysis revealed that this was the result of normal competitive inhibition of ATP binding, on which an increase of the apparent affinity of the enzyme for pyruvate was superimposed.The adenylate cyclase of Brevihacterium liquefuciens is the first species of its kind to have been purified to crystalline homogeneity [l]. A dimer of molecular weight 92400, it catalyzes the reversible conversion of ATP to cAMP and inorganic pyrophosphate [2,3]. The activity extends to dATP and AdoP [NH]PP, but the enzyme is not active on the other naturally occurring nucleoside triphosphates. A characteristic feature of the enzyme is its stimulation in vitro, as well as in vivo, by 2-0x0-monocarboxylic acids, especially by pyruvate [1,4-81. The present report deals with the kinetic details of pyruvate activation of adenylate cyclase from Brevibacterium liquefuciens. We furthermore investigated the response of pyruvate-stimulated as well as basal adenylate cyclase activities to purine and pyrimidine nucleoside triphosphates as effectors.

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Rat brain adenylate cyclase

Cited by 83 publications

References 29 publications

Calmodulin activates prokaryotic adenylate cyclase.

Calmodulin activates prokaryotic adenylate cyclase.

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

Steady‐State Kinetic Studies on Adenylate Cyclase from Brevibacterium liquefaciens

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