Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.     

Synthesis and applications of 8-azido photoaffinity analogs of P1,P3-bis(5'-adenosyl)triphosphate and P1,P4-bis(5'-adenosyl)tetraphosphate

32P-labeled photoaffinity analogs of bis(5'-adenosyl)-tetraphosphate and bis(5'-adenosyl)triphosphate which contain a single photoreactive 8-azidoadenosine group distal to the radiolabel have been synthesized from commercially available components using a combination of chemical and enzymatic procedures including a water-soluble carbodiimide. The method is simple, rapid, and produces yields of high specific activity products of around 60%. The analog of bis(5'-adenosyl)-tetraphosphate is very similar to the parent compound in its inhibition of rat liver adenosine kinase and its efficiency as a substrate for the bis(5'-nucleosidyl)tetraphosphate pyrophosphohydrolase from Artemia embryos. In the latter case, ATP and 8-azidoAMP are the preferred products. As would be expected, this analog is a much more effective photoprobe for both adenosine and adenylate kinases than the corresponding analog of bis(5'-adenosyl)triphosphate. Both compounds have been used to photoaffinity label crude extracts of Artemia, Vero cells, and Clostridium acetobutylicum and preferential specific labeling of different polypeptides by each analog has been shown. In extracts of C. acetobutylicum, the labeling of a polypeptide of Mr 48,500 by the bis(5'-adenosyl)tetraphosphate analog was totally dependent on the presence of Co2+ ions. These compounds should therefore prove valuable both for the active site labeling of purified binding proteins and for the detection and identification of new target proteins for these nucleotides.     

Inhibition of adenylate kinase by P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate and P1-(lin-benzo-5'-adenosyl)-P5-(5'-adenosyl pentaphosphate.

P1-(lin-Benzo-5'-adenosyl)-P5-(5'-adenosyl) penraphosphate and P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate have been synthesized from lin-benzoadenosine 5'-monophosphoromorpholidate and adenosine 5'-tetraphosphate and adenosine 5'-triphosphate. These mixed dinucleoside polyphosphates are potent inhibitors of porcine muscle adenylate kinase, with association constants of 2 x 10(5) M-1 for the pentaphosphate and 2 x 10(6) M-1 for the tetraphosphate, respectively, as determined by kinetics and fluorescence experiments. The increase in fluorescence intensities and fluorescence lifetimes of both inhibitors upon binding to adenylate kinase results from a breaking of the intramolecular stacking interaction observed when these ligands are free in solution and implicates their binding to the enzyme in an "open" or "extended" form. These results and the dimensional requirements of these inhibitors are discussed in relation to our current knowledge of the active site of adenylate kinase and to the known inhibitors of adenylate kinase, P1,P5-bis(5'-adenosyl) pentaphosphate and P1,P4-bis-(5'-adenosyl) tetraphosphate.     

Variation in intracellular P1P4-bis(5''-adenosyl) tetraphosphate (Ap4A) in virus-infected cells.

The effect of virus infection on the intracellular concentration of the proposed stress alarmone P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) has been examined in Vero cells. Compared with exposure to 0.8 mM-Cd2+, which causes a 30-fold increase in Ap4A, infection with simian virus 40 and poliovirus causes only a 2-fold increase, whereas herpes simplex virus type 1 results in a decrease in Ap4A during the course of the infection.     

Novel fluorescent labelled affinity probes for diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A)-binding studies

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

Interleukin-2 regulation of diadenosine 5',5'-p1 p4-tetraphosphate (Ap4A) levels and DNA synthesis in cloned murine T lymphocytes

The levels or diadenosine 5', 5'-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibition of TdT by P1, P5-bis(5'-adenosyl) pentaphosphate

The catalysis of DNA synthesis by calf thymus terminal deoxynucleotidyltransferase (TdT) is strongly inhibited in the presence of Ap5A, while replicative DNA polymerases from mammalian, bacterial, and oncornaviral sources are totally insensitive to Ap5A addition. The Ap5A-mediated inhibition of TdT seems to occur via its interaction at both the substrate binding and primer binding domains as judged by classical competitive inhibition plots with respect to both substrate deoxynucleoside triphosphate (dNTP) and DNA primer and inhibition of ultraviolet light mediated cross-linking of substrate dNTP and oligomeric DNA primer to their respective binding sites. Further kinetic analyses of Ap5A inhibition revealed that the dissociation constant of the Ap5A-enzyme complex, with either substrate binding or primer binding domain participating in the complex formation, is approximately 6 times higher (Ki = 1.5 microM) compared to the dissociation constant (Ki = 0.25 microM) of the Ap5A-TdT complex when both domains are available for binding. In order to study the binding stoichiometry of Ap5A to TdT, an oxidized derivative of Ap5A, which exhibited identical inhibitory properties as its parent compound, was employed. The oxidation product of Ap5A, presumably a tetraaldehyde derivative, binds irreversibly to TdT when the inhibitor-enzyme complex is subjected to borohydride reduction. The presence of aldehyde groups in the oxidized Ap5A appeared essential for inhibitory activity since its reduction to alcohol via borohydride reduction or its linkage to free amino acids prior to use as an inhibitor rendered it completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation control of cardiac acetyl-CoA carboxylase by cAMP-dependent protein kinase and 5'-AMP activated protein kinase.

Acetyl-CoA carboxylase (ACC) is regarded in liver and adipose tissue to be the rate-limiting enzyme for fatty acid biosynthesis; however, in heart tissue it functions as a regulator of fatty acid oxidation. Because the control of fatty acid oxidation is important to the functioning myocardium, the regulation of ACC is a key issue. Two cardiac isoforms of ACC exist, with molecular masses of 265 kDa and 280 kDa (ACC265 and ACC280). In this study, these proteins were purified from rat heart and used in subsequent phosphorylation and immunoprecipitation experiments. Our results demonstrate that 5' AMP-activated protein kinase (AMPK) is able to phosphorylate both ACC265 and ACC280, resulting in an almost complete loss of ACC activity. Although cAMP-dependent protein kinase phosphorylated only ACC280, a dramatic loss of ACC activity was still observed, suggesting that ACC280 contributes most, if not all, of the total heart ACC activity. ACC280 and ACC265 copurified under all experimental conditions, and purification of heart ACC also resulted in the specific copurification of the alpha2 isoform of the catalytic subunit of AMPK. Although both catalytic subunits of AMPK were expressed in crude heart homogenates, our results suggest that alpha2, and not alpha1, is the dominant isoform of AMPK catalytic subunit regulating ACC in the heart. Immunoprecipitation studies demonstrated that specific antibodies for both ACC265 and ACC280 were able to coimmunoprecipitate the alternate isoform along with the alpha2 isoform of AMPK. Taken together, the immunoprecipitation and the purification studies suggest that the two isoforms of ACC in the heart exist in a heterodimeric structure, and that this structure is tightly associated with the alpha2 subunit of AMPK.     

Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis.

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) in sea urchin embryos

Sea urchin embryos were labeled with [3H]adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP4A fraction. The present result suggests that AP4A is actively synthesized in the sea urchin embryos.     

Ecto-diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A)-hydrolase is expressed as an ectoenzyme in a variety of mammalian and human cells and adds new aspects to the turnover of Ap4A

Ap4A and other dinucleotides participate in the regulation of hemostasis and blood pressure control. With the exception of two previously reported surface anchored ectoAp4A-hydrolases on bovine aortic endothelial and chromaffine cells, all Ap4A-hydrolases reported are intracellular or freely soluble. We demonstrated that ectoAp4A-hydrolases are present on a broad variety of cell types of different species: rat mesangial, bovine corneal epithelial, human Hep-G2 and peridontal cells. Ectoenzyme properties were evaluated on rat mesangium cells. Chromatography of purified plasma membranes on Sephacel 300 resulted in enrichment of ectoAp4A-hydrolase and in separation from ectoATPase. In contrast to ATPase, Ap4A-hydrolase was stable at room temperature. EctoAp4A-hydrolase also recognized ATP as substrate, and therefore is not highly specific. The molecular weight was 180 kD. Unlike ectoAMPase ectoAp4A-hydrolase was not attached via a glycosyl-phosphatidylinositol (GPI)-moiety. Concentrations of PI-PLC 10-100-fold higher than effective for ectoAMPase cleavage (10-100 mU/ml) plus extensively extended incubation times up to eight hours did not result in cleavage of ectoAp4A-hydrolase. The enzyme ectoAp4A-hydrolase might presage a direction for pharmaceutical manipulation in the control of blood pressure and hemostasis.