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The kinetics of creatine kinase (CK) and adenylate kinase (AK) activities were monitored in intact diaphragm muscle by 18 O 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 31 P 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.The view put forth by Bessman and co-workers (1, 2) that the metabolic function of creatine kinase (CK) 1 is to transfer energy-rich phosphoryls from intracellular generation to utilization sites is supported by kinetic studies in vitro and in vivo by the distinctive localization of isozymic forms of CK and their functional coupling to ATP-consuming and ATP-generating processes (for reviews see . In opposition to this view is the relatively intact physiological performance of muscle when CK activity is impaired following protracted ingestion of creatine analogs (6, 7) or deletion of the gene encoding the major cytosolic isoform of muscle CK (i.e., M-CK) (8). It is also contradicted by reports that the total unidirectional rate of CKcatalyzed transfer of phosphoryls assessed by 31 P NMR technology in intact skeletal muscle is workload-independent or even reduced with the onset of muscle contraction (9, 10).An importance in intracellular energy transfer similar to that of CK has been suggested for adenylate kinase (AK)-catalyzed phosphotransfer (4,11,12). From studies of AK kinetic behavior in intact diaphragm muscle, increases in net AK-catalyzed phosphotransfer were found to be directly proportional to the frequency of stimulated muscle contraction (12), and the stoichiometry between net AK-catalyzed phosphoryl transfer and anaerobic glycolytic ATP generation was nearly equivalent over a greater than 20-fold range of stimulated fluxes (13). The interpretation of these results wa...

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.

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.

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