Nelson D. Goldberg

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.(Mol Cell Biochem 184:: 169–182, 1998)

Effects of insulin treatment on muscle 3′,5′-cyclic adenylate levels in vivo and in vitro

The role of 3′,5′-cyclic adenylate in the insulin-promoted conversion of glycogen transferase (UDP glucose: α-I,4 glucan α-4-glucosyltransferase, EC 2.4.1.11) from the glucose 6-phosphate dependent (D) to the independent (I) form was investigated. Insulin administration stimulates a rapid increase (45–65%) in vivo of muscle glycogen transferase-I activity. This activation is associated with an increase (36–78%) in tissue 3′,5′-cyclic adenylate levels rather than a decrease which would be more predictable from the presently understood effects of the cyclophosphate on the conversion mechanism. There is also no lowering of 3′,5′-cyclic adenylate levels in rat diaphragms incubated with insulin nor any effect of insulin to diminish the epinephrine action of raising tissue cyclophosphate levels in incubations of rat diaphragms in vitro.

Increased cyclic GMP and decreased cyclic AMP levels in the hyperplastic, abnormally differentiated epidermis of psoriasis

Summary The genetic skin disease psoriasis has been examined as a model system that may provide an understanding of the control of normal epidermal specialization (differentiation) and the perturbed regulatory processes in proliferatioe diseases . The excessive glycogen accwnulation, increased proliferation and decreased tissue specialization characteristic of psoriasis involve cellular pro- cesses that have been shown to be regulated by cyclic AMP in other cells and tissues . It has also been suggested that cyclic GMP is a cellular ef- fector that may be involved in promoting cell pro- liferation and other events that oppose those be- lieved to be mediated by cyclic AMP . It was postulated, therefore, that the epidermis of the psoriasis lesion might exhibit an imbalance in the cellular concentrations of these two cyclic nucleo- tides . In this study the levels of cyclic AMP were measured in the involved epidermis (IE) and unin- volved epidermis (UE) from 25 psoriasis patients . The concentrations of cyclic AMP were found as reported previously using a different analytical procedure, to be significantly lower in IE based on protein and DNA . A comparison of the levels of cyclic .GMP in IE versus UE of 12 other psoriasis patients showed the levels of this cyclic nucleo- tide to be significantly increased in IE based on protein, DNA and wet weight . We suggest that this imbalance in the ratio of these two cyclic nucleo- tides may have pathophysiological relevance to the initiation and/or the maintenance of the psoriasis lesion .

Enzymic analysis of cyclic 3′, 5′-AMP in mammalian tissues and urine☆

The details are presented for the analysis of 3′,5′ cyclic adenosine monophosphate (3′5′CAMP) in milligram amounts of mammalian tissues (muscles, liver, brain, and kidney) and in microliter samples of urine. An examination of the sources of difficulty and how they are effectively handled is also included. In the determination of tissue 3′5′CAMP the cyclic nucleotide is first separated from 5′-nucleoside mono-, di-, and triphosphates by cellulose thin-layer chromatography following Ba(OH)2-ZnSO4 precipitation of extracts. After quantitative recovery 3′,5′CAMP is converted to 5′ AMP and subsequently to ATP by the actions of phosphodiesterase, myokinase, and pyruvate kinase. Enzymic cycling with the hexokinase-pyruvate kinase system is then used to produce a proportional concentration of G-6-P equivalent to several thousand fold the ATP concentration and the G-6-P measured fluorometrically. Cyclic adenylate in urine samples is determined directly without prior separation from any urinary components. Examples are presented of the analytical procedures applied to the measurement of 3′5′CAMP levels in tissues and urine after various experimental treatments. These include the effects of epinephrine in skeletal muscle in vitro and in vivo, of adrenalectomy and hydrocortisone in liver, of ischemia in brain, and of hypertonic infusion on urinary excretion of 3′5′CAMP.

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 β-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 K+ATP channel closure but it is not associated with or a consequence of events occurring subsequent to K+ATP channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the K+ATP 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.

ATP (Mg2+) induced inhibition of cyclic AMP reactivity with a skeletal muscle protein kinase

Abstract Exposure of a skeletal muscle protein kinase (peak I DEAE fraction) to μmolar ATP in the presence of Mg2+ before initiating the binding reaction or the cAMP activable phosphotransferase reaction with cAMP increases the requirement for this cyclic nucleotide in both events about 10 fold. The binding of ATP (Mg2+) to a protein in the kinase preparation was shown by Millipore and Sephadex filtration to parallel the inhibitory effect of the triphosphate. The value of Ks for ATP was in the 10−7 M range and 50% inhibition of cAMP (10−8 M) binding occurred at approximately 2 × 10−7 M ATP. ATP binding activity was associated with protein peaks exhibiting both cAMP binding and phosphotransferase activity after subjecting the kinase (holoenzyme) to sedimentation in sucrose density gradients or electrophoresis by electrofocusing.

Cyclic Nucleotides and Cell Function

The “second messenger” function of the cyclic nucleotides, CA MP and CG MP, through which external stimuli arriving at the cell membrane are made internally effective, is thought to depend on their ability to activate a class of enzymes known as protein kinases. These then set in motion a cascade of biochemical processes that modulate and enormously amplify the metabolic activities carried on within the cell.

Hormonal and non-hormonal control of glycogen synthesis-control of transferase phosphatase and transferase I kinase.

Abstract Transferase, the enzyme that catalyzes the synthesis of the α-1,4 linkages of glycogen, is subject to control by several mechanisms including hormonal as well as non-hormonal. These controls are observed to occur rapidly in a number of tissues which are sensitive to the actions of the hormones. An important biochemical mechanism consists of interconverting two forms of the enzyme with second stage interconverting enzymes. These catalyze the phosphorylation and dephosphorylation of the two forms of transferase. The site of the non-hormonal control by glycogen is identified as the phosphatase, while the site of the hormonal control by insulin and epinephrine, the kinase. Insulin acts at the kinase site to bring about a greater dependence on cyclic AMP and thus inactivate the kinase, with no decrease in cyclic adenylate tissue concentrations. Epinephrine acts to increase tissue levels of cyclic adenylate, and thus promote kinase action.

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.

Separation of 5′-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography☆

Abstract The isocratic separation of six naturally occurring 5′-ribonucleoside monophosphates by reverse-phase high-performance liquid chromatography has been achieved using solvents containing tetramethylammonium, tetraethylammonium, or tetrabutylammonium ions. Their effectiveness in resolving the nucleoside monophosphates increases with increasing akyl chain length (butyl > ethyl > methyl). In an acidic aqueous solvent containing methanol, these ion-pair reagents effect discrete separations of all the nucleoside monophosphates. The systems described also provide for virtually quantitative recovery of the separated nucleotides and utilize phosphate-free solvents comprised of volatile components easily removed by evaporation.