Adenylate kinase: kinetic behavior in intact cells indicates it is integral to multiple cellular processes.

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by 18O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by 18O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K+ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2)ADP.

Suppression of adenylate kinase catalyzed phosphotransfer precedes and is associated with glucose-induced insulin secretion in intact HIT-T15 cells.

Adenine nucleotide metabolism was characterized in intact insulin secreting HIT-T15 cells during the transition from non-stimulated (i. e. 0.2 mM glucose) to the glucose-stimulated secretory state. Metabolic dynamics were monitored by assessing rates of appearance of 18O-labeled phosphoryls of endogenous nucleotides in cells incubated in medium enriched in [18O]water. Most prominent of the metabolic alterations associated with stimulated insulin secretion was the suppression in the rate of adenylate kinase (AK)-catalyzed phosphorylation of AMP by ATP. This was manifest as a graded decrease of up to 50% in the rate of appearance of beta-18O-labeled species of ADP and ATP and corresponded to the magnitude of the secretory response elicited over a range of stimulatory glucose concentrations. The only nucleotide exhibiting a significant concentration change associated with suppression of AK activity was AMP, which decreased by about 50%, irrespective of the glucose concentration. Leucine-stimulated secretion also decreased the rate of AK-catalyzed phosphotransfer. This secretory stimulus-related suppression of AK-catalyzed phosphotransfer occurs within 45 s of glucose addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium. The secretory stimulus-induced decrease in AK-catalyzed phosphotransfer, therefore occurs prior to or at the time of KATP+ channel closure but it is not associated with or a consequence of events occurring subsequent to KATP+ channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the KATP+ channel microenvironment; secretory stimuli-linked decreased rates of AK-catalyzed ADP generation from ATP (and AMP) would translate into an increased probability of ATP-liganded and, therefore, closed state of the channel.

Suppression of creatine kinase-catalyzed phosphotransfer results in increased phosphoryl transfer by adenylate kinase in intact skeletal muscle.

The kinetics of creatine kinase (CK) and adenylate kinase (AK) activities were monitored in intact diaphragm muscle by 18O phosphoryl oxygen exchange to assess whether these two phosphotransferases provide an interrelated function integral to high energy phosphoryl metabolism. This possibility was examined by quantitating the net rates of CK- and AK-catalyzed phosphoryl transfer in comparison to the total cellular ATP metabolic rate when CK activity in the intact diaphragm muscle was progressively inhibited by 2,4-dinitrofluorobenzene. In noncontracting muscle from untreated rats, net rates of CK- and AK-catalyzed phosphotransfer were equivalent to 88 and 7%, respectively, of the total ATP metabolic rate. These results were compared with reported 31P NMR analyses of total creatine phosphate flux to estimate that each creatine phosphate molecule produced undergoes about 50 unidirectional CK-catalyzed phosphotransfers in transit to an ATP consumption site in the intact muscles. Graded inhibition by 2,4-dinitrofluorobenzene of intracellular CK activity by up to 98% resulted in a progressive shift in phosphotransferase catalysis from the CK to the AK system; the sum of the net rates of phosphoryl transfer by combining the increasing AK and decreasing CK activities continued to approximate the total cellular ATP metabolic rate. These results indicate that in diaphragm muscle CK and AK operate as interrelated cellular high energy phosphoryl transfer systems through which the majority of newly generated ATP is processed prior to its utilization.

Adenylate kinase-catalyzed phosphoryl transfer couples ATP utilization with its generation by glycolysis in intact muscle.

We previously suggested that an importance of adenylate kinase (AdK) in skeletal muscle is to function as a high energy phosphoryl transfer system regulating ATP generation in correspondence with its consumption by specific cellular processes. The present experiments are intended to define the ATP-generating system coupled to and regulated by AdK-catalyzed phosphotransfer in skeletal muscle and also to examine the relationship between AdK- and creatine kinase (CK)-catalyzed phosphotransfer. Rates of phosphoryl transfer catalyzed by AdK were assessed in intact, isolated rat diaphragm by determining rates of AMP phosphorylation with endogenously generated [gamma-18O]ATP under conditions of altered anaerobic and aerobic ATP production. AdK-catalyzed phosphoryl transfer rates accelerated incrementally up to 12-fold in direct proportion to stimulated contractile frequency in parallel with equivalent increases in rates of ATP generation by lactate producing glycolysis. Stoichiometric equivalent increases of AdK-catalyzed phosphotransfer and anaerobic ATP production also occurred up to more than 20-fold when oxidative phosphorylation was impaired by either O2 deprivation or treatment with KCN or p-(trifluoromethoxy)-phenylhydrazone. These enhanced rates of AMP phosphorylation were balanced by virtually identically increased rates of AdK-catalyzed generation of AMP. This AMP was traced to arise from AdK-catalyzed phosphotransfer involving ADP generated by a muscle ATPase. Increased AdK-catalyzed phosphotransfer paired with the apparent compensatory increase in ATP generation by anaerobic glycolysis in oxygen-deprived muscle occurred coincident with diminished rates of CK-catalyzed phosphoryl transfer indicative of a pairing between oxidatively produced ATP and CK-catalyzed phosphotransfer. A metabolic model consistent with these results and conforming to the Mitchell general principle of vectorial ligand conduction is suggested.

Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls.

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.

Evidence for compartmentalized adenylate kinase catalysis serving a high energy phosphoryl transfer function in rat skeletal muscle.

The first characterization of the kinetics and subcellular compartmentation of adenylate kinase activity in intact muscle has been accomplished using rat diaphragm equilibrated with [18O]water. Rates of adenylate kinase-catalyzed phosphoryl transfer were measured by appearance of 18O-labeled beta-phosphoryls in ADP and ATP resulting from the transfer to AMP of newly synthesized 18O-labeled gamma-ATP. Unique features of adenylate kinase catalysis were uncovered in the intact cell not predictable from cell free analysis. This enzyme activity, which in non-contracting muscle is limited to 1/1000 of the estimated Vmax (cell free) apparently because of restricted ADP availability, is localized in subcellular compartments that increase in size and/or number with contractile frequency. Contraction also causes frequency-dependent increments in adenylate kinase velocity (22-fold at 4 Hz) as does oxygen deprivation (35-fold). These enhanced rates of adenylate kinase activity, equivalent to processing all the cellular ATP and ADP in approximately 1 min, occur when levels of ATP, ADP, and AMP are maintained very near their basal steady state. These characteristics of the dynamics of adenylate kinase catalysis in the intact cell demonstrate that rapid rates of AMP production from ADP are balanced by equally rapid rates of AMP phosphorylation with no net synthesis or accumulation of any adenine nucleotide. This rapid processing of nucleotide phosphoryls conforms to a proposed scheme whereby the adenylate kinase system provides the unique function of transferring, as beta-ADP, high energy phosphoryls generated by glycolytic metabolism to ATP-utilizing components in muscle.

Hepatic actinomycosis.

Primary hepatic actinomycosis is seldom seen, and almost all cases have been diagnosed by laparotomy. Treatment with high-dose parental penicillin for prolonged periods is recommended. In the past concomitant surgical drainage also has been recommended, but the nonoperative success reported here and by others suggests that surgical drainage is not essential to the treatment.

Non-identity of cGMP as the guanine nucleotide stimulated to bind to ROS by light and ATP.

Light, in the presence of ATP, has been reported to stimulate cGMP binding to a 58 kDa protein in ROS (rod outer segments, Fesenko and Krapivinsky, 1986b, Photobiochem. Photobiophys. 13 345-58). This apparent light-related redistribution of ROS cGMP has been suggested to eliminate any requirement for phosphodiesterase-promoted hydrolysis of cGMP in the mechanism subserving phototransduction. Using conditions identical to those previously reported, this effect of light and ATP was examined further by characterizing the metabolic products that arise and the nucleotides that become liganded. The increased binding of radiolabeled guanine nucleotide upon illumination of ROS in the presence of ATP was confirmed, but the species of guanine nucleotide that were stimulated to bind under these conditions were identified as [32P]GDP and [32P]GTP rather than [32P]cGMP. The precautions to prevent enzymic hydrolysis of cGMP, which included conducting the reactions at 0 degrees and the addition of 3-isobutyl-l-methylxanthine (250 microM) to the reaction mixture did not prevent about a 20-fold increase in the rate of phosphodiesterase-catalyzed hydrolysis of radiolabeled cGMP by light when ATP was also present. This stimulation of phosphodiesterase activity is undoubtedly related to transphosphorylation by exogenous ATP of endogenous GMP and GDP involving catalytic actions of guanylate kinase and nucleoside diphosphate kinase in isolated ROS. These enzymes can also serve to generate [32P]GDP and [32P]GTP, which subsequently bind to ROS components. Such a mechanism involving ATP as phosphoryl donor was supported by observing that an analog of ATP (beta,gamma-methyleneadenosine 5'-triphosphate), which cannot serve as a phosphoryl donor, did not increase radiolabeled guanine nucleotide binding. Although several ROS proteins can form filter-retainable complexes with GDP and GTP, the properties of the 58 kDa protein found to be photoaffinity labeled with radioactive guanine nucleotide are most characteristic of those attributable to tubulin. The previous report that illumination in the presence of ATP stimulates the binding of cGMP to ROS components finds no support from the data obtained in the present studies.

Adenosine triphosphate utilization rates and metabolic pool sizes in intact cells measured by transfer of 18O from water.

The hydrolytic rates and metabolic pool sizes of ATP were determined in intact cells by monitoring the time courses of 18O incorporation from 18O-water into the gamma-phosphoryl of ATP and orthophosphate. To calculate the rate of ATP hydrolysis, a kinetic model is used to fit the time course of the 18O labeling. The size of the metabolic pool of ATP is calculated from the 18O distribution after isotopic equilibrium has been achieved. Metabolic pools have a binomial distribution of 18O whereas nonmetabolic pools exhibit negligible 18O labeling. The application and limitations of this approach are illustrated with data from isolated toad retinas and human platelets. At 22 degrees C, the time constant of ATP hydrolysis in the dark-adapted toad retina is about 30 s. Under these conditions, over 80% of the retinal ATP is involved in high-energy phosphate metabolism. It is calculated that when cGMP metabolic flux in the photoreceptors is maximally stimulated by light, it accounts for 10% of the ATP utilization by the entire retina. The time constant of ATP hydrolysis in human platelets at 37 degrees C is approximately 1 s, and 60% of the platelet ATP is involved in energy metabolism.

A Ca2+-linked increase in coupled cAMP synthesis and hydrolysis is an early event in cholinergic and beta-adrenergic stimulation of parotid secretion.

The dynamics and compartmental characteristics of cAMP metabolism were examined by 18O labeling of cellular adenine nucleotide alpha phosphoryls in rat parotid gland stimulated to secrete with beta-adrenergic and cholinergic agents. The secretory response occurred in association with a rapidly increased rate of cAMP hydrolysis apparently coordinated with an equivalent increase in the rate of cAMP synthesis, since the cellular concentration of cAMP remained unchanged. The magnitude of this metabolic response was equivalent to the metabolism of 10-75 times the cellular content of cAMP within the first minute of stimulation. This increased metabolic rate occurred only during the early (1-3 min) period of stimulation, in what appeared to be an exclusive cellular compartment distinguished by a unique distribution of 18O among adenine nucleotide alpha phosphoryls. This 18O distribution contrasted with that produced by forskolin, which increased cellular cAMP concentration and elicited only a delayed response missing the early secretory component. The early acceleration of cAMP metabolism appeared linked to a stimulus-induced increase in intracellular Ca2+ concentration, since the Ca2+ ionophore ionomycin produced the same metabolic response in association with secretion. These observations suggest that cAMP metabolism is involved in stimulus-secretion coupling by a Ca2+-linked mechanism different from that in which cAMP plays the role of a second messenger.

Voltage dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in peeled skeletal muscle fibers.

Excitation-contraction coupling in skeletal muscle is known to be under absolute control of plasmalemma voltage, but the steps from transverse (T)-tubule depolarization to Ca2+ release from the sarcoplasmic reticulum have not been elucidated. The effect of changing T-tubule membrane potential on inositol 1,4,5-trisphosphate (InsP3) stimulation of Ca2+ release from the sarcoplasmic reticulum was studied to explore a possible role for InsP3 as a chemical signal in excitation-contraction coupling. InsP3 was microinjected into peeled rabbit skeletal muscle fibers at a pipette concentration of 0.5 microM; Ca2+ release from the sarcoplasmic reticulum was monitored as an isometric tension transient. The response to 0.5 microM InsP3 was significantly larger when T-tubules were in a depolarized state than when they were in a polarized state, and this difference in response was independent of the ionic composition of the bathing solutions or the method for depolarizing the T-tubules. Thus, T-tubule depolarization may sensitize the sarcoplasmic reticulum to a preexisting low concentration of InsP3 and greatly reduce the need for InsP3 production. Plasmalemma voltage control of the stimulatory effects of InsP3 may have relevance for mechanisms in excitable nonmuscle cells.

Regulation of cyclic GMP metabolism in toad photoreceptors. Definition of the metabolic events subserving photoexcited and attenuated states.

Photoreceptor metabolism of cGMP and its regulation were characterized in isolated toad retinas by determining the intensity and time dependence of light-induced changes in the following metabolic parameters: cGMP hydrolytic flux determined by the rate of 18O incorporation from 18O-water into retinal guanine nucleotide alpha-phosphoryls; changes in the total (protein-bound and unbound) concentrations of the guanine nucleotide metabolic intermediates; and changes in the concentration of metabolic (unbound) GDP calculated from the fraction of the alpha-GDP that undergoes labeling with 18O. The latter is interpreted to reflect the state of the equilibrium between GDP- and GTP-complexed forms of G-protein. With narrow band 500 nm light that preferentially stimulates red rod photoreceptors, a range of intensities covering approximately 5 log units produced increases of over 10-fold in cGMP metabolic flux. However, the characteristics of the cGMP metabolic response over the first 2.5 log units of intensity are readily distinguishable from those at higher intensities which exhibit progressive attenuation by an intensity- and time-dependent process. Over the range of low intensities (0.6-3 log photons.micron-2.s-1) the metabolic response is characterized by 1) increases in cGMP hydrolytic flux of up to 8-fold as a logarithmic function of intensity of photic stimulation that are sustained for at least 200 s; 2) small increases or no change in the concentration of total cGMP; 3) large increases of up to 10-fold in the concentration of metabolically active GDP as a linear function of intensity with no significant change in the tissue concentrations of total GDP or GTP; and 4) amplification of the photosignal by the metabolism of approximately 10,000 molecules of cGMP per photoisomerization with the major site of amplification at the level of the interaction of bleached rhodopsin with G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP opposes IP3-induced calcium release from permeabilized human platelets.

Platelets permeabilized by means of a high voltage electric field demonstrated time- and ATP-dependent uptake of 45Ca++. Submicromolar concentrations of inositol-1,4,5-trisphosphate (IP3) caused a rapid release of 45Ca++ which was followed by a slower reuptake. Adenosine 3':5'-cyclic monophosphate (cAMP) did not affect 45Ca++ uptake but did reduce IP3-mediated calcium release in a concentration-dependent manner over the range of 1-100 microM. Because cAMP concentrations in this range occur following exposure of platelets to prostacyclin and other agents which interfere with platelet function, it is proposed that cAMP-mediated inhibition of the action of IP3 may play a role in the antithrombotic activity of compounds believed to elevate levels of this cyclic nucleotide.

The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide alpha-phosphoryls.

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining rates of 18O incorporation from 18O-water into the alpha-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide alpha-phosphoryl labeling ranged from 180 to 244 pmol X mg protein-1 X min-1. Sodium nitroprusside (SNP) caused a sustained 3.4-fold increase in this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol X mg protein-1 X min-1) corresponded with the sum of the low (1.7 microM) and high (34 microM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol X mg protein-1 X min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of 18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of 18O labeling of alpha-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.

Inositol trisphosphate stimulates calcium release from peeled skeletal muscle fibers.

The effects of inositol phosphates (tris (InsP3), bis (InsP2), mono (InsP)) on rabbit adductor magnus and soleus muscles were determined using mechanically peeled fibers (sarcolemma removed). Isometric force generation of each fiber was continuously monitored and was used along with 45Ca to detect calcium release from internal fiber stores. All experiments were conducted at a physiological Mg2+ concentration (10(-3) M) of the bathing solutions. The inositol phosphates did not directly activate the contractile apparatus. At bath concentrations of 100-300 microM, only InsP3 was capable of stimulating Ca2+ release. In contrast, 1 microM InsP3 maximally and selectively stimulated Ca2+ release when microinjected into the myofilament lattice. Calcium releasing effects of InsP2 and InsP were manifested at 10 microM when they were microinjected. The end-to-end internal Ca2+ release and subsequent fiber force generation stimulated by the locally applied microinjected InsP3 suggests that the InsP3-induced Ca2+ release mechanism may involve propagation, but not via the Ca2+-induced Ca2+ release, since procaine did not inhibit this response. These findings support the possibility that InsP3 plays a role in skeletal muscle excitation-contraction coupling.

Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors.

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.