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Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase.

ABSTRACT: Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.

SUBMITTER: Snider DE

PROVIDER: S-EPMC1164630 | biostudies-other | 1977 Mar

REPOSITORIES: biostudies-other

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Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase.

Project description:Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.

| S-EPMC1164631 | biostudies-other

Characterization of the Intrinsic Phospholipase A1 Activity of <i>Bordetella pertussis</i> Adenylate Cyclase Toxin.

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Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

Project description:Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

| S-EPMC4642564 | biostudies-literature

The Rhizobium etli cyaC product: characterization of a novel adenylate cyclase class.

Project description:Adenylate cyclases (ACs) catalyze the formation of 3',5'-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis.

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| S-EPMC9416875 | biostudies-literature

Neurally mediated syncope diagnosis based on adenylate cyclase activity in Japanese patients.

Project description:The study aims to clarify the mechanism in patients with neurally mediated syncope (NMS), focusing on the adenylate cyclase (AC) activity level in lymphocytes. This study included 40 subjects: 22 healthy volunteers and 18 NMS patients. We investigated the changes in AC activity that occur during of syncope at rest and during the head-up tilt (HUT) test. We obtained 8 mL of blood at rest time and four times during the HUT test. Then, we measured the AC activity and the test reagent was added to the lymphocytes (10,000) and reacted for 30 min at room temperature. We were able to determine the standard value of AC activity when adrenaline (AD) and isoproterenol (IP) were added to lymphocytes. The results of our study showed one of the causes of NMS has a difference in AC activity level and classification of the patients into two different types of NMS was possible: either the vasodepressor type (VT) or mixed type (MT). At rest time, VT patients showed significantly higher AC activity (AD; 100 μM: p = 0.005, IP; 50 μM: p = 0.02) and MT patients showed significantly lower AC activity (AD; 10 μM: p = 0.02, IP; 50 μM: p = 0.004) than the average AC activity in healthy volunteers. Moreover, VT patients had significantly higher AC activity than healthy volunteers at the four points of the HUT test. MT patients had significantly lower AC activity (AD: p = 0.04 and IP: p = 0.04) than healthy volunteers at the rest time of HUT. Our study showed a significant difference in AC activities between NMS patients and healthy volunteers at rest. Therefore, a detailed NMS diagnosis can be made by examining AC activity levels in blood taken at rest time.

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Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain.

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| S-EPMC5565420 | biostudies-other

Expression data from human adipocytes after adenylate cyclase activation

Project description:Progenitors in human vasculature expanded in-vitro were differentiated with adipogenic cocktail for 12 days, following which they were stimulated with forskolin for 7 days Microarrays were used to detail the program of differentiation of thermogenic human adipose cells

2018-01-22 | GSE65190 | GEO

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