Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.     

In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2'-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl(2), polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.     

Polyphosphate kinase of Acinetobacter sp. strain ADP1: purification and characterization of the enzyme and its role during changes in extracellular phosphate levels.

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl(2). The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40 degrees C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per microg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P(i)) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P(i)-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P(i) conditions. This activity declined when phosphate was added.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge.

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1)

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.     

Allosteric activation of rat liver microsomal [hydroxymethylglutaryl-CoA reductase (NADPH)]kinase by nucleoside phosphates

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase activity is enhanced about 5 fold by 2 mM of either AMP or ADP. Activation constants, Ka, for AMP and ADP are 17 microM and 430 microM respectively, showing that AMP is a more potent activator than ADP. This property is expressed by increasing not only the rate of reductase inactivation but also the rate of reductase phosphorylation from [gamma-32P]ATP. GTP can replace ATP as substrate of reductase kinase but GMP and GDP cannot replace AMP as activators. Kinetic studies show that ATP can only act as a substrate. Nucleoside mono or diphosphates and nucleoside triphosphates, thus, appear to bind to different sites on microsomal HMG-CoA reductase kinase. Nucleoside mono or diphosphates act as allosteric activators of reductase kinase. The adenosyl moiety and the unaltered phosphate ester at the 5' position are two essential features of the activator molecule. Phosphorylation of reductase either by microsomal or cytosolic AMP-activated reductase kinase produces an 80% inactivation, with a concomitant incorporation of 0.8 mol of 32P per mol of reductase (Mr 55,000). In both cases exhaustive tryptic digestion of 32P-labeled HMG-CoA reductase, which had been denatured in 2M urea, yields two major phosphopeptides, the phosphoryl group being bound to serine residues.     

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.     

Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.     

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.     

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.     

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Activation and inhibition of ATP synthesis in cell envelope vesicles of Halobacterium halobium

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.     

Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction

The cytoplasmic protein-tyrosine kinase domain of the insulin receptor (residues 959-1355) has been expressed as a soluble protein in Sf9 insect cells via a Baculovirus expression vector (Ellis, L., Levitan, A., Cobb, M.H., and Ramos, P. (1988) J. Virol. 62, 1634-1639). The purified protein is a monomer as judged by its behavior in sucrose gradients and on gel filtration in the presence or absence of protamine. The initial rate of autophosphorylation using 3 mM MgCl2 is increased 20-30-fold by protamine. A maximum of 4-5 mol of phosphate are incorporated per mol of enzyme. The activity of the enzyme as a function of phosphorylation state was studied for three substrates: a synthetic dodecapeptide derived from the sequence of the major autophosphorylation site in the insulin receptor, poly(Glu, Tyr), 4:1, and histone 2B. Autophosphorylation of the protein to a stoichiometry of 4-5 mol of phosphate/mol increases its enzymatic activity as much as 200-fold; a 30-fold increase in activity occurs upon addition of 1 mol of phosphate/mol. The activities of unphosphorylated enzyme with the three substrates are 3.4, 2.3, and 0.44 nmol/min/mg, respectively. The activities of the autophosphorylated enzyme with the three substrates are 175, 274, and 45 nmol/min/mg, respectively. Exposure of the autophosphorylated enzyme to ADP results in a loss of phosphate from the enzyme which is associated with a decrease in enzymatic activity. Autophosphorylation of the kinase in the presence or absence of protamine displays a marked dependence on enzyme concentration. Furthermore, the rate of autophosphorylation decreases as the viscosity of the solution increases. Taken together, these data suggest that phosphorylation occurs via an intermolecular reaction.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.