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1.
Diverse treatments, which have been shown by Slayman, C. L. (1977) in Water Relations in Membrane Transport in Plants and Animals (Jungreis, A., Hodges, T. K., Kleinzeller, A., and Schultz, S. G., eds) pp. 69-86, Academic Press, New York, to depolarize the plasma membrane of Neurospora, increase levels of adenosine 3':5'-monophosphate (cyclic AMP) in the organism. The treatments include those producing large transport fluxes of metabolizable or nonmetabolizable compounds, rapid temperature drops, and addition of agents which uncouple oxidative phosphorylation. Severe mechanical stress, which may also act to depolarize the plasma membrane, leads to increases in cyclic AMP. The maximal depolarization appears to precede the maximal cyclic AMP levels. It is proposed that the membrane depolarization produces the increased cyclic AMP levels by stimulating the plasma membrane-bound adenylate cyclase and that cyclic AMP may be important to the maintenance of membrane integrity.  相似文献   

2.
It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.  相似文献   

3.
Cyclic AMP and cyclic GMP were released into the growth medium of mycelia of Neurospora crassa wild-type strains St.L.74A and Em5297a and by white collar-1 and white collar-2 mutant strains. After growth for 6 days at 18°C, there were 2.19 (St.L.74A), 5.83 (Em5297a), 1.38 (white collar-1), and 1.10 (white collar-2) nanomoles of cyclic AMP per gram dry weight of mycelia in the growth medium. These values corresponded to concentrations of cyclic AMP of between approximately 10 and 50 nanomolar. The corresponding values for extracellular cyclic GMP were typically less than 6% of the values for cyclic AMP. Following transfer to fresh medium, cyclic AMP efflux was demonstrated for each of the strains, and the amount of cyclic AMP exported into the fresh medium was greater at 25°C than 6°C. Intracellular cyclic AMP and cyclic GMP were also measured in each of the strains. The values for cyclic AMP were in the same range as those in the literature (approximately 0.5 to 1.5 nanomoles per gram dry weight of mycelia). However, the corresponding intracellular cyclic GMP values were less than 1% of the cyclic AMP values, i.e. more than 50 times lower than the value previously reported for the St.L.74A wild-type. Transfer of mycelia after 6 days at 18°C to fresh media and incubation for 2 hours at 25°C or 6°C did not consistently affect the intracellular level of cyclic AMP or cyclic GMP in the strains examined. We could detect no change in intracellular cyclic AMP when mycelia of the St.L.74A wild-type strain were irradiated with blue light for periods of up to 3.0 hours at 18°C, or in cyclic AMP and cyclic GMP for irradiation times of up to 1 minute at 6°C. We propose that the plasma membrane of Neurospora crassa is permeable to cyclic nucleotides, and the export of cyclic nucleotides into the growth medium may be a means of regulating intracellular levels. We conclude that three factors that affect carotenogenesis in Neurospora crassa (blue light, temperature, and the white collar mutations) have no appreciable effect on the total measurable intracellular cyclic nucleotides in this organism. There was no extracellular or intracellular cyclic AMP or cyclic GMP in the crisp-1 mutant strain, which suggested either that adenylate cyclase (which is absent in crisp-1) catalyzes the synthesis of both cyclic AMP and cyclic GMP or that the crisp-1 mutation somehow results in a deficiency of two enzymes (adenylate and guanylate cyclase).  相似文献   

4.
Several saturated, monosaturated, and polyunsaturated fatty acids produce rapid increases in cyclic AMP levels in the fungusNeurospora crassa when added to the growth medium at 10–50 M. The time courses of cyclic AMP increase resembled those previously shown to be induced by other agents, reaching peak cyclic AMP levels at about 2 min after fatty acid addition. These fatty acids had little or no influence on adenylate cyclase fromNeurospora crassa in vitro. On the basis of previous evidence that uncouplers of oxidative phosphorylation increase cyclic AMP levels and that fatty acids can act as uncouplers, we suggest that the fatty acids in vivo may act to increase cyclic AMP levels by acting as uncouplers of oxidative phosphorylation. In agreement with this suggestion, two fatty acids were shown to produce decreased ATP-ADP ratios inNeurospora at concentrations producing cyclic AMP increases.  相似文献   

5.
Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.  相似文献   

6.
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.  相似文献   

7.
Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.  相似文献   

8.
Wild-type Salmonella typhimurium could not grow with exogenous cyclic adenosine 3',5'-monophosphate (AMP) as the sole source of phosphate, but mutants capable of cyclic AMP utilization could be isolated provided the parental strain contained a functional cyclic AMP phosphodiesterase.All cyclic AMP-utilizing mutants had the growth and fermentation properties of cyclic AMP receptor protein (crp) mutants, and some lacked cyclic AMP binding activity in vitro. The genetic defect in each such mutant was due to a single point mutation, which was co-transducible with cysG. crp mutants isolated by alternative procedures also exhibited the capacity to utilize cyclic AMP. crp mutants synthesized cyclic AMP at increased rates and contained enhanced cellular cyclic AMP levels relative to the parental strains, regardless of whether or not cyclic AMP phosphodiesterase was active. Moreover, adenylate cyclase activity in vivo was less sensitive to regulation by glucose, possibly because the enzyme II complexes of the phosphotransferase system, responsible for glucose transport and phosphorylation, could not be induced to maximal levels. This possibility was strengthened by the observation that enzyme II activity (measured both in vitro by sugar phosphorylation and in vivo by sugar transport and chemotaxis) was inducible in the parental strain but not in crp mutants. The results suggest that the cyclic AMP receptor protein regulates cyclic AMP metabolism as well as catabolic enzyme synthesis.  相似文献   

9.
Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.  相似文献   

10.
Effects of morphine administration were studied on cyclic AMP metabolism in several regions of rat brain. In the cortex, cerebellum and thalamus-hypothalamus, morphine dependence did not alter the activity of either adenylate cyclase or phosphodiesterase. However, during withdrawal from the opiate treatment, adenylate cyclase activity declined in all three regions studied. In contrast, the striatal cyclic AMP metabolism was enhanced during morphine treatment as reflected by elevated endogenous cyclic AMP and increased adenylate cyclase. Furthermore, narcotic dependence produced significant increases in acetylcholinesterase activity of rat striatum. Whereas morphine withdrawal reversed the changes in striatal acetylcholine levels and acetylcholinesterase activity, the enhanced striatal dopamine remained unaltered. Although the activity of striatal adenylate cyclase was significantly reduced when compared to the morphine-dependent rats, the drop in cyclic AMP levels was not significant. Methadone replacement did not affect the changes in striatal dopamine seen in morphine-withdrawn rats. Whereas dopamine stimulated equally well the striatal adenylate cyclase from control or morphine-dependent animals, it failed to stimulate the striatal enzyme from rats undergoing withdrawal. The crude synaptosomal fraction of the whole brain from morphine-dependent rats exhibited an increase in cyclic AMP which was accompanied by elevated adenylate cyclase and protein kinase activity. Naloxone administration suppressed this rise in cyclic AMP and reversed the morphine-stimulated increases in the activities of adenylate cyclase and protein kinase. Following the withdrawal of morphine treatment, alterations in cyclic AMP metabolism were similar to those noted in morphine-naloxone group. Furthermore, substitution of morphine with methadone antagonized the observed alterations in cyclic nucleotide metabolism during withdrawal.  相似文献   

11.
In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis.  相似文献   

12.
The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.  相似文献   

13.
Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-biphosphate at levels of 2t nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-biphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-biphosphate. Thus, the above-described regulation of fructose 2,6-biphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-biphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-biphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-biphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-biphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.  相似文献   

14.
The phosphorylation of fructose-1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP-dependent protein kinase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Possible mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby lead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that neither sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoacridine fluorescence after addition of fermentable sugars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cyanide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-1,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately 2-fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent Km for ATP with no change in Vmax. These studies suggest that activation of adenylate cyclase by decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose-1,6-bisphosphatase.  相似文献   

15.
Cyclic AMP is thought to have a general role in stimulating the breakdown of carbohydrate reserves and subsequent glycolytic activity. This would be expected to increase the availability of reducing equivalents in the form of cytoplasmic NADH. The current study examines another potential reaction controlling cytoplasmic NADH in the fungus Neurospora crassa, that of lactate dehydrogenase, to determine whether it is also regulated by cyclic AMP. The cr-1, adenylate cyclase and cyclic AMP-deficient mutant, grown with and without exogenous cyclic AMP was compared with an isogenic wild type. The results show that cyclic AMP raises pyruvic acid pools and lowers both lactic acid pools and lactate/pyruvate ratios. It does that, in part or in whole, by lowering lactate dehydrogenase activity. The possibility that cytoplasmic NAD+/NADH is a major target of cyclic AMP control is discussed. The high performance liquid chromatography procedures used in these studies are applicable to the measurement of intracellular pools of tricarboxylic acid cycle and other organic acids.  相似文献   

16.
The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.  相似文献   

17.
Regulation of adenosine 3':5'-monophosphate efflux from animal cells.   总被引:9,自引:0,他引:9  
Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.  相似文献   

18.
Mutants of Vibrio fischeri MJ-1 (wild type) apparently deficient in adenylate cyclase (cya-like) or cyclic AMP receptor protein (crp-like) were isolated and characterized. Compared with MJ-1, the mutants produced low levels of luminescence and luciferase. Addition of cyclic AMP restored wild-type levels of luminescence and luciferase in the cya-like mutant but not in the crp-like mutant. The results are consistent with the hypothesis that in V. fischeri cyclic AMP and cyclic AMP receptor protein are required for induction of the luminescence system.  相似文献   

19.
UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 mumol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30-37 degrees C than those grown at 25 degrees C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.  相似文献   

20.
Yeast cells with a nonsense adenylate cyclase mutation, cyr1-3, required cyclic AMP for growth. This phenotype was suppressed by the byc1 mutation; however, cyr1-3 bcy1 cells produced no detectable level of adenylate cyclase or cyclic AMP. On induction, the bcy1 and cyr1-3 bcy1 mutant cells produced the same levels of galactokinase and alpha-D-glucosidase as did the wild-type cells and fourfold-higher levels of invertase. Since galactokinase synthesis was severely repressed by glucose in the constitutive GAL81 mutants, irrespective of the cyr1-3 bcy1 genotype, cyclic AMP may not be involved in catabolite repression.  相似文献   

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