Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Fluorimetric determination of carbamoyl phosphate

A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP⁺, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

Studies on the specific activity of [γ-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

1. The specific activity of the γ-³²P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of ³²P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-³²P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [³²P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-³²P]ATP was 30–40% of the initial specific activity of the medium [³²P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [³²P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

Synthesis and Some Properties of UDP-(1)fructose

UDP-(1)fructose was synthesized essentially by the method of Michelson or Roseman et al. The product obtained was much more stable to acid than UDP-fructose isolated from Jerusalem artichoke tubers by Umemura et al.⁷⁾ and UDP-glucose. Hydrolysis time curves of UDP-(1)fructose and fructose-1-phosphate in 0.01N HGl and 0.1N HCl both at 100°C are presented. It was concluded from these curves that UDP-(1)fructose was first hydrolyzed into UMP and fructose-1-phosphate, and then fructose-1-phosphate was hydrolyzed more slowly into free fructose and inorganic phosphate.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

The influence of inorganic phosphate and ATP on the kinetics of bovine heart muscle pyruvate kinase

Summary The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/ mg (phosphate buffer, pH 7.6, 25 °C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.K_m(phosphoenolpyruvate) –0.04 mM, app.K_m(ADP) =0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the V_max without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in V_max was observed.The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app. K_m(P_i) = 20 mM and V_max = 167 units/ mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, P_i is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them.These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.Abbreviations PEP phosphoenolpyruvate - Pi inorganic phosphate - TEA triethanolamine - EDTA ethylenediaminetetraacetate     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

Measurement of nucleoside kinases in crude tissue extracts

The measurements of deoxyadenosine kinase, adenosine kinase, and deoxycytidine kinase were examined in human placental cytosol to achieve a valid and reliable assay linear with time and protein. Our studies confirm the need to inhibit deaminase enzymes, since deoxyadenosine and deoxycytidine undergo extensive deamination and phosphorolysis. The use of a uniformly labeled nucleoside substrate introduced an artifact because the chromatographic behavior of the deoxyribose-1-phosphate, formed during the assay, was difficult to distinguish from the deoxynucleoside phosphate product. Accurate product identification was also essential. Finally, the substitution of GTP in place of ATP as the phosphate donor, the addition of a sulfhydryl reducing agent and a monovalent cation need to be considered when an assay is optimized. The use of these methods have lead to valid assays in placental cytosol that are linear with time and protein. Consideration of these important principles are necessary when establishing a valid and reliable nucleoside kinase assay in a crude tissue preparation.     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach

³¹P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g⁻¹ wet wt.), ATP (3.6), inorganic phosphate (P_i, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of P_i. ¹H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. ³¹P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T₁ value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g⁻¹ wet wt.·s⁻¹. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

The effect of potassium depletion on the initial kinetics of glycolysis in ascites tumor cells

Ehrlich ascites carcinoma cells depleted of K⁺ and provided with 5.5 mM K⁺ in isosmotic 50 mM tris(hydroxymethyl)methylglycine buffer at pH 7.4 and 38 °C take up K⁺ from the medium at a rate of 6 μmoles/ml intracellular fluid per min. Depleted cells exposed to K⁺ for 2 min prior to glucose addition exhibit a higher initial rate of glycolysis, a lower glycose-6-P accumulation, and a higher fructose-1,6-P₂ accumulation than depleted cells incubated in a K⁺-free medium. Both the K⁺ transport and the effect of K⁺ on glycolysis are blocked by 2 mM oubain.Calculation of thein vitro velocities of glycolytic enzymes from the rates of accumulation of lactate and glycolytic intermediates shows that the presence of K⁺ accelerates the velocities of fructose-6-phosphate kinase and lactate dehydrogenase about 2-fold and the velocity of hexokinase about 1.5-fold during the first 15 s. In either the presence or absence of K⁺, the hexokinase velocity is highest immediately after glucose addition and declines sharply with time; this decline is greater than would be predicted by product inhibition by the accumulated glucose-6-P. The maximal stimulation of fructose-6-phosphate kinase attibutable to the increasing intarcellular K⁺ concentration is only 1.25-fold. These observations indicate that the initial acceleration in glycolysis is not simply mediated through a direct K⁺ activation of fructose-6-phosphate kinase.The calculated theoretical rate of ATP generation by glycolysis shows that glycolysis is an ATP-utilizing system for the first 5–10 s both in the presence and in the absence of K⁺. Hence, the initial stimulation of glycolysis by K⁺ is not a consequence of an increased rate of ATP hydrolysis associated with K⁺ transport, although this mechanism may be responsible for the stimulation of steady-state glycolysis.The initial rate of phosphate ester (hexose and triose phosphates) accumulation corresponds to be rate of ATP generation by the “tail-end” of glycolysis, or twice the rate of lactate accumulation, in either the absence or presence of K⁺, but both the rate and the maximal level of ester accumulated are higher in the presence of K⁺. This implies that the oxidatively generated pool of ATP which is diverted from endogenous reactions to hexokinase and fructose-6-phosphate kinase on the introduction of glucose is larger in the presence of K⁺.Valinomycin (0.27 μM) under certain conditions can produce effects on the glycolysis of non-depleted cells which superficially resemble the effects of K⁺ on depleted cells. However, unlike K⁺, valinomycin stimulates the initial rate of glycolytic ATP generation, and abolishes the initial correspondence between the ATP generation by the “tail-end” of glycolysis and phosphate ester accumulation. These observations are interpreted to mean that valinomycin introduces an ATPase activity effective on glycolytically generated ATP.Comparison of the theoretical ATP generation in the presence and absence of K⁺ indicates that approximately one ATP is hydrolyzed for each K⁺ transported.     

The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity.

The purpose of this study was to examine the effects of lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpio L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10 degrees C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca(2+)-activated ATPase activity was assessed by an ATP regenerating assay that coupled the myofibrillar ATPase to pyruvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85%. Lactate had no effect on myofibrillar ATPase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not to the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many ³²P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-³²P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-³²P] phosphate from [γ-³²P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]₀ e^?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [³²P] phosphate from [γ-³²P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [³²P]phosphorylated peptide, unreacted [γ-³²P]ATP, and ³²P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.