Phosphorylation of myelin basic protein by an adenosine 3′:5′-cyclic monophosphate-dependent protein kinase (Short Communication)

Myelin basic protein was shown to be a substrate for protein kinase from rabbit muscle. One of the major sites of phosphorylation was the serine residue in the sequence Gly-Arg-Gly-Leu-Ser-Leu. The arginine residue in this sequence is known to be a substrate for a protein methylase.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3′:5′-cyclic monophosphate

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac(2)-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac(2)-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac(2)-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.     

The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Effects of insulin secretagogues on islet-cell protein kinase activity

1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.     

Modification and identification of glutamate residues at the arginine-recognition site in the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

It has been proposed that the active centre of cyclic AMP-dependent protein kinase contains an arginine-recognition site, which is considered to be essential for the function of the catalytic subunit of the kinase [Matsuo, Huang & Huang (1978) Biochem. J.173, 441-447]. The catalytic subunit can be inactivated by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide and glycine ethyl ester at pH6.5. The enzyme can be protected from inactivation by preincubation with histone, a protein substrate of the enzyme. On the other hand, ATP, which also serves as a protein kinase substrate, does not afford protection. Polyarginine, a competitive inhibitor of protein kinase, which is known from kinetic studies to interact specifically with the arginine-recognition site, partially protects the catalytic subunit from inactivation by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide. These results lead to the conclusion that the site of modification by carbodi-imide/glycine ethyl ester is most likely located at the arginine-recognition site of the active centre. A value of 1.7+/-0.2 (mean+/-s.d.) mol of carboxy groups per mol of catalytic subunit has been obtained for the number of essential carboxy groups for the function of protein kinase; a complete chemical modification of these essential carboxy groups results in total loss of catalytic activity. Finally, we have identified the essential carboxy group in the catalytic subunit of cyclic AMP-dependent protein kinase as being derived from glutamate residues. This is achieved by a three-step procedure involving an extensive proteolytic digestion of the [1-(14)C]glycine ethyl ester-modified enzyme and two successive high-voltage electrophoreses of the hydrolysate. It is concluded that 1.7mol of glutamyl carboxy groups per mol of catalytic subunit may be considered a component of the arginine-recognition site in the active centre of cyclic AMP-dependent protein kinase.     

Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3':5'-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N(6)-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP-Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50-100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N(6)-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP-Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release by isolated rat islets of Langerhans

1. Concentrations of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and rates of insulin release were measured in islets of Langerhans isolated from rat pancreas and incubated for various times in the presence of glucose, 3-isobutyl-1-methylxanthine, caffeine, theophylline, adrenaline and diazoxide. 2. Caffeine and theophylline produced small but significant increases in both cyclic AMP and release of insulin when they were incubated in the presence of 10mm-glucose. 3. 3-Isobutyl-1-methylxanthine produced a marked increase in the intracellular concentration of cyclic AMP in the presence of 5mm- and 10mm-glucose. However, insulin release was stimulated only in the presence of 10mm-glucose. 4. In response to rising concentrations of extracellular glucose (5-20mm) there was no detectable increase in the intracellular concentration of cyclic AMP even though there was a marked increase in the rate of insulin release. 5. In response to 10mm-glucose insulin release occurred in two phases and 3-isobutyl-1-methylxanthine potentiated the effect of glucose on both phases. The intracellular concentration of cyclic AMP remained constant with glucose and rose within 10min to its maximum value with 3-isobutyl-1-methylxanthine. 6. Adrenaline and diazoxide inhibited insulin release and lowered the intracellular concentration of cyclic AMP when islets were incubated with glucose or 3-isobutyl-1-methylxanthine. 7. It is suggested that glucose does not stimulate insulin release by increasing the concentration of cyclic AMP in islet cells. However, the concentration of cyclic AMP in islet cells may modulate the effect of glucose on the release process.     

Direct effects of adenosine 3′,5′-cyclic monophosphate and adenosine 5′-monophosphate upon tyrosinase activity

1. The direct actions of cAMP and 5′-AMP upon goldfish integumental and mushroom tyrosine activity were examined.2. cAMP and 5′-AMP produce similar alterations in goldfish enzyme activity, being stimulatory at low concentrations (5′-AMP being more effective than cAMP) and inhibitory at high concentrations.3. Theophylline stimulates goldfish tyrosinase activity.4. Mushroom tyrosinase activity is inhibited by cAMP and by theophylline.5. cAMP and 5′-AMP stimulation of goldfish tyrosinase activity may result from the direct action of these nucleotides on the enzyme in addition to cAMP stimulation of cAMP-dependent protein kinase.6. 5′-AMP stimulation of goldfish tyrosinase activity may indicate a key regulatory role of this nucleotide in cAMP-mediated events.     

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.     

The regulation of growth hormone secretion from the isolated rat anterior pituitary in vitro. The role of adenosine 3′:5′-cyclic monophosphate

1. The release of growth hormone from isolated fragments of rat anterior pituitary tissue incubated in vitro was studied by employing a double-antibody radioimmunoassay. 2. In the absence of added stimuli, two phases of hormone release could be distinguished, an early phase of 2h duration and a subsequent late phase. In the early phase, hormone release was rapid but could be significantly decreased by calcium depletion and by 2,4-dinitrophenol whereas the rate of release in the late phase was uninfluenced by these incubation conditions. These results have been interpreted as indicating the existence of a secretory component in the early phase of release. 3. In subsequent experiments, the effects of various agents on the rate of hormone output during the late phase of incubation were investigated. Hormone release was increased by theophylline and by dibutyryl cyclic AMP (N(6)-2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate), the response to both of these agents being related to the concentration of the stimulant employed. 4. The stimulation of growth hormone output by theophylline was significantly decreased by calcium deprivation and by 2,4-dinitrophenol. The response to dibutyryl cyclic AMP was diminished by 2,4-dinitrophenol, iodoacetate and 2-deoxyglucose but not by malonate or colchicine. 5. Arginine, beta-hydroxybutyrate, albumin-bound palmitate and variation in the glucose concentration of the incubation medium over a wide range were without any statistically significant effect on the rate of hormone release from either control pituitary fragments or those subject to secretory stimulation by dibutyryl cyclic AMP. 6. It is suggested that the regulation of growth hormone secretion is mediated by cyclic AMP (adenosine 3':5'-cyclic monophosphate). The secretion observed in response to cyclic AMP requires the presence of ionized calcium and a source of metabolic energy but is independent of pituitary protein synthesis de novo. The integrity of the glycolytic pathway of glucose metabolism appears to be essential for cyclic AMP-stimulated growth hormone secretion to occur.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

Adenosine 3′:5′-cyclic monophosphate-binding protein from the cellular slime mould Dictyostelium discoideum (Short Communication)

During the differentiation of myxamoebae of Dictyostelium discoideum strain Ax-2 grown in axenic medium there is a seven- to ten-fold increase in the specific activity of cyclic AMP-binding protein(s). Evidence is presented for the belief that cyclic AMP phosphodiesterase and cyclic AMP-binding protein are distinct molecular species.     

Protein kinase activity stimulated by adenosine 3′:5′-cyclic monophosphate in synaptic-membrane fragments from ox brain. Inhibition of intrinsic activity by free and membrane-bound calcium ions

1. Cyclic AMP-stimulated protein kinase activity phosphorylating intrinsic substrates in preparations of synaptic-membrane fragments from ox cerebral cortex was examined in relation to (a) the content of membrane-bound Ca(2+) in the preparations and (b) added Ca(2+) in the assay medium. 2. Centrifugal washing of synaptic-membrane fragments with buffered ethane dioxybis(ethylamine)tetra-acetate solutions decreased bound Ca(2+) from 2.8+/-0.4 (s.d.) to 0.9+/-0.3nmol/mg of protein. In washed preparations basal protein kinase activity was increased by about 40% and the cyclic AMP-stimulated activity by about 15%. Addition of Ca(2+) in the concentration range 5-50mum to the assay medium progressively inhibited the kinase activity of the washed preparations; in this range of Ca(2+) concentration the basal activity was inhibited more than the stimulated activity. 3. In unwashed preparations concentrations of Ca(2+) above 100mum inhibited the cyclic AMP-stimulated activity more than the basal activity. 4. The inhibitory effect of several concentrations of Ca(2+) was examined in relation to cyclic AMP concentration; no evidence for competition between Ca(2+) and cyclic AMP for a site on the enzyme was observed.     

Adenosine cyclic 3′,5′-monophosphate-dependent protein kinase from human platelets

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit.The apparent K_m for ATP in the presence of 10 μM Mg²⁺ was 4 μM (plus cyclic AMP) and 4.3 μM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 μM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 μM (presence of MgATP) were exhibited by the “protein kinase-cyclic AMP complex”. The enzyme required Mg²⁺ for maximum activity and showed a pH optimum of 6.2 with histone as substrate.In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28 000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.     

Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.     

A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate     

The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Preparation and properties of islet-cell protein phosphokinase

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3':5'-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15x10(-8)m, which was similar to the K(m) for cyclic AMP (1.11x10(-8)m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a K(m) for ATP of 1.1x10(-5)m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.     

Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in a cyclic 3′:5′ adenosine monophosphate-dependent protein kinase mutant of Neurospora crassa

The possibility that plant growth regulators may relate to a cyclic 3:5 adenosine monophosphate (cAMP)-dependent protein kinase through the control of cAMP level in the conidial germination process of Neurospora crassa was examined using a cAPM-dependent protein kinase mutant (cpk mutant) which is thought to be cAMP-independent because of defect in the regulatory subunit of cAMP-dependent protein kinase. IAA, 2,4-D and GA₃ promoted conidial germination and elongation of young hyphae in the mutant as well as in the wild-type. The result suggests that the effects of auxin and gibberellin on germination and hyphal elongation are not mediated by cAMP.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

Involvement of adenosine 3′:5′-cyclic monophosphate in lipid mobilization in Locusta migratoria

The rôle of cyclic AMP in hormone-induced lipid mobilization in Locusta migratoria was investigated. Injection of a corpus cardiacum extract into adult female locusts resulted in an increased level of cyclic AMP in the fat body. The cAMP concentration is maximal at about 5 min of incubation and returns to the resting level after about 10 min. The dose-response curve is linear up to about 0.01 corpus cardiacum pair equivalents.Dibutyryl-cyclic AMP mimics the lipid mobilizing effect of corpus cardiacum extract. After flight the cyclic AMP concentration in fat body increased. Injection of corpus cardiacum extract had no effect on flight muscle cyclic AMP concentration.