The effect of cyclic GMP on phosphofructokinase from rat tissues.

In view of the recently proposed hypothesis of biologic regulation through opposing influences of cyclic AMP and cyclic GMP, and since cyclic AMP is a well-known allosteric activator of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the effect of cyclic GMP on the activity of this enzyme from several rat tissues was investigated. It was found that cyclic GMP exerted an inhibitory effect on the activity of rat heart and skeletal muscle phosphofructokinase. This effect was most pronounced under conditions in which the enzyme was partially inhibited by ATP or by citrate. Cyclic GMP also antagonized the deinhibitory action of cyclic AMP and other allosteric activators, such as glucose 1,6-bisphosphate or AMP, on the ATP or citrate-inhibited heart or muscle phosphofructokinase. In contrast to the heart and skeletal muscle phosphofructokinase, the adipose-tissue enzyme was not affected by cyclic GMP to any significant degree. The antagonistic action of cyclic GMP to the activation of heart-phosphofructokinase, may suggest a mechanism by which the activity of phosphofructokinase is synchronized with the activity of glycogen phosphorylase, as a result of acetylcholine action in heart, to achieve a decrease in total glycogenolysis and glycolysis.     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3′:5′-cyclic monophosphate in androgen action

1. The ability of exogenously administered cyclic AMP (adenosine 3':5'-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as alpha-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2'-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16-24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized-castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a submaximal dose of testosterone (1.0mg/100g) on all those prostatic and seminal-vesicular enzymes that are increased by exogenous cyclic AMP. 11. The evidence indicates that cyclic AMP may be involved in triggering the known metabolic actions of androgens on secondary sexual tissues of the rat.     

Decrease of rat liver cysteine dioxygenase (cysteine oxidase) activity mediated by glucagon.

The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Some properties of phosphofructokinase from kidney cortex and their relation to glucose metabolism

1. Phosphofructokinase from rat kidney cortex has been partially purified by using a combination of isoelectric and ammonium sulphate precipitation. This preparation was free of enzymes which interfered with the measurement of either product of phosphofructokinase. 2. At concentrations greater than the optimum, ATP caused inhibition which was decreased by raising the fructose 6-phosphate concentration. This suggested that ATP reduced the affinity of phosphofructokinase for the other substrate. Citrate potentiated the ATP inhibition. 3. AMP and fructose 1,6-diphosphate relieved the inhibition by ATP or citrate by increasing the affinity of the enzyme for fructose 6-phosphate. 4. K(+) is shown to stimulate and Ca(2+) to inhibit phosphofructokinase. 5. The similarity between the complex properties of phosphofructokinase from kidney cortex and other tissues (e.g. cardiac and skeletal muscle, brain and liver) suggests that the enzyme in kidney cortex tissue is normally subject to metabolic control, similar to that in other tissues.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.     

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.     

Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments

Striking effects of F-actin and the reconstituted thin filament of muscle on the catalytic activity of rabbit muscle phosphofructokinase are demonstrated through direct measurements of enzymatic activity by using the pH stat. The addition of F-actin to solutions of phosphofructokinase at low ionic strength (10 mM KCl and 5 mM MgCl2) partially reverses the inhibition of the enzyme seen at high ATP concentrations and increases the apparent affinity of the enzyme for fructose 6-phosphate with slight effect on Vmax. F-Actin augments the activation of the enzyme obtained with AMP and partially counters the inhibition obtained with citrate. The maximum effect in the reversal of ATP inhibition is about the same for combinations of either F-actin or the thin filament with AMP as it is for AMP alone. In general, the effect of F-actin on the catalytic activity of phosphofructokinase is larger than that of the thin filament. The activation of phosphofructokinase by F-actin persists at physiological ionic strength.     

Hormonal regulation of collagen degradation in the uterus: Inhibition of collagenase expression by progesterone and cyclic AMP

Dibutyryl cyclic AMP markedly increases the ability of progesterone to prevent the expression of collagenase activity in cultures of post-partum rat uterus. Dibutyryl cyclic AMP itself and, to a lesser extent, native cyclic AMP, are capable of producing a partial decrease in enzyme activity, but complete abolition is not observed at high cyclic nucleotide concentrations (5 mM) in the culture medium. Theophylline, when added to cultures, mimics the effect of dibutyryl cyclic AMP. Other cyclic nucleotides were without effect on levels of collagenase activity in the uterine cultures.When non-inhibitory concentrations of either dibutyryl cyclic AMP (1 · 10^?4 M) or theophylline (1 · 10^?4 M) are added to cultures together with a non-inhibitory concentration of either progesterone (5 · 10^?6 M) or the potent progesterone analogue Provera (1 · 10^?8 M) the ability of the tissue to produce collagenase is decreased by 40–70%. Collagenase activity is consistently diminished more than additively by combinations of steroid and cyclic nucleotide. Theophylline mimics the effect of dibutyryl cyclic AMP on steroid activity in culture. In the presence of dibutyryl cyclic AMP, diminution of collagenase activity can be observed at concentrations of steroid more than two orders of magnitude lower than the normal minimally inhibitory dose. Reduction of collagenase activity is reflected in all experiments by a concomitant decrease in the normal proteolytic degradation of collagen in the tissue ex-plants. The possibility that progesterone acts in the uterus to raise cyclic AMP levels is suggested by the fact that uterine tissue, when cultured in the presence of progesterone, contains reduced levels of cyclic nucleotide phosphodiesterase.These data suggest that, in some way a cyclic AMP-mediated system is critically involved in the control of collagenase activity by progesterone in the rat uterus.     

Attenuation of epinephrine-induced increase in liver cyclic AMP by endogeneous insulin in vivo.

1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin-deficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscule cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhaces epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cylase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.     

Phosphofructokinase from mollusc muscle is activated by phosphorylation.

Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.     

Ribose 1,5-bisphosphate regulates rat kidney cortex phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.     

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U     

Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis

1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Synthesis of adenosine 3',5'-monophosphate by guanylate cyclase, a new pathway for its formation.

The 105 000 X g gupernatant fractions from homogenates of various rat tissues catalyzed the formation of both cyclic GMP and cyclic AMP from GTP and ATP, respectively. Generally cyclic AMP formation with crude or purified preparations of soluble guanylate cyclase was only observed when enzyme activity was increased with sodium azide, sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine, sodium nitrite, nitric oxide gas, hydroxyl radical and sodium arachidonate. Sodium fluoride did not alter the formation of either cyclic nucleotide. After chromatography of supernatant preparations on Sephadex G-200 columns or polyacrylamide gel electrophoresis, the formation of cyclic AMP and cyclic GMP was catalyzed by similar fractions. These studies indicate that the properties of guanylate cyclase are altered with activation. Since the synthesis of cyclic AMP and cyclic GMP reported in this study appears to be catalyzed by the same protein, one of the properties of activated guanylate cyclase is its ability to catalyze the formation of cyclic AMP from ATP. The properties of this newly described pathway for cyclic AMP formation are quite different from those previously described for adenylate cyclase preparations. The physiological significance of this pathway for cyclic AMP formation is not known. However, these studies suggest that the effects of some agents and processes to increase cyclic AMP accumulation in tissue could result from the activation of either adenylate cyclase or guanylate cyclase.     

Effect of epinephrine and insulin on adenosine 3'5'-cyclic monophosphate--dependent protein kinase in human skeletal muscle in vivo.

Cyclic AMP dependent protein kinase has beeen identified in human skeletal muscle tissue. In crude muscle extracts the enzyme was 3--5 fold activated by cyclic AMP. The cyclic AMP-dependent activity (corresponding to the inactive holoenzyme) was completely inhibited by the heat stable inhibitor of protein kinase. Reciprocal changes of the cyclic AMP-dependent activity in skeletal muscle were observed after administration of epinephrine and insulin in vivo. Infusion of epinephrine in healthy volunteers increased the level of cyclic AMP and decreased the activity of the cyclic AMP-depenent form (i.e. the inactive form) of protein kinase. These changes were reversible after cessation of epinephrine administration. The results are consistent with an activation of protein kinase in vivo due to an epinephrine mediated increase of the concentration of cyclic AMP. I.v. injection of insulin had the opposite effect on the enzyme in skeletal muscle, leading to increased activity of the cyclic AMP-dependent form of protein kinase. Insulin had no effect on the level of cyclic AMP, but promoted a transient increase of cyclic GMP 1 min. after insulin injection. The effect by insulin on protein kinase cannot be related to the level of cyclic AMP or cyclic GMP.