Phosphate Acceptor Research Articles

Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization.

Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.

In vitro and in vivo regulation of hepatic mitogen-activated protein kinases in fetal rats.

We have studied the role of mitogen-activated protein (MAP) kinases in fetal hepatocyte growth in vitro and in vivo. With myelin basic protein (MBP) as the phosphate acceptor, kinase activity in cultured fetal hepatocyte lysates increased fourfold after exposure to transforming growth factor-alpha (TGF-alpha) for 10 min. This TGF-alpha-responsive MBP kinase activity was accounted for by five distinct MAP kinase isoforms detected by Western immunoblotting. All had negligible activity in cultured fetal hepatocytes under basal conditions. Treatment of fetal hepatocytes with hepatocyte growth factor led to activation of the predominant isoforms, relative molecular weight (M(r)) = 42,000 and 44,000 in a manner indistinguishable from TGF-alpha, whereas insulin had no effect. All five of the immunoreactive MAP kinases were present in both fetal and adult liver homogenates. The M(r) = 42,000 and 44,000 isoforms were only minimally activated in vivo. We conclude that the mitogen-independent growth exhibited by fetal hepatocytes in primary culture is not associated with tonic activation of the MAP kinase system. Our data support the possibility that fetal hepatic growth may be, in part, independent of the action of growth factors as mediated via the MAP kinase system.

Structural determinants of substrate selection by the human insulin-receptor protein-tyrosine kinase.

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Activation of PKN, a Novel 120-kDa Protein Kinase with Leucine Zipper-like Sequences, by Unsaturated Fatty Acids and by Limited Proteolysis

PKN, a novel protein kinase with catalytic domain homologous to PKC family and unique amino terminal leucine zipper-like sequences, was purified partially from COS7 cells transfected with the cDNA construct encoding human PKN for enzymatic characterization of the enzyme. Using serine containing synthetic peptides based on PKC pseudosubstrate sites as the phosphate acceptors, kinase activities estimated from partially purified PKN were not stimulated by Ca2+/phosphatidylserine/diolein but were activated several-fold to several tens-fold by 40 μM unsaturated fatty acids, such as arachidonic acid, linoleic acid, and oleic acid. Autophosphorylation of the immunoprecipitates using anti-PKN antiserum was also stimulated by various unsaturated fatty acids. Limited proteolysis of PKN with trypsin induced an enhancement of the peptide kinase activity that was almost independent of arachidonic acid.

Asp129 of low molecular weight protein tyrosine phosphatase is involved in leaving group protonation

Site-directed mutagenesis was used to explore the functions of a number of acidic residues of bovine low molecular weight protein tyrosine phosphatase. Residues Asp-129, Asp-56, and Asp-92 were mutated to Ala or Asn. The mutant enzymes D56A, D56N, and D92A showed no significant changes in Vmax values, although they did exhibit significantly altered Km values. In contrast, the D129A mutant enzyme exhibited a greater than 2000-fold reduction in Vmax, using p-nitrophenyl phosphate as a substrate. The Vmax values of D129A also exhibited a leaving group dependence, an altered solvent isotope effect of VmaxH/VmaxD of 0.78, and a lack of dependence on the presence of alternative phosphate acceptor alcohols, all properties that distinguish this mutant from wild type enzyme. The differences are due to a change of the rate-limiting step of the catalytic reaction. Asp-129 is concluded to be the proton donor to the leaving group in the phosphorylation step, and its mutation to alanine results in a reduced Vmax value and a change in the rate-limiting step of the catalysis from dephosphorylation to phosphorylation. Mechanistic considerations suggest that other phosphotyrosyl phosphatases having cysteine at the active site may be expected to have a similar requirement for a proton donor.

Enzymatic phosphorylation and pyrophosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key metabolite in the pathway for activation of the anti-HIV (human immunodeficiency virus) agent 2',3'-dideoxyinosine

2',3'-Dideoxyadenosine-5'-monophosphate (ddAMP) is a key intermediate in the metabolic pathway involved in the activation of the anti-retroviral agent 2',3'-dideoxyinosine (ddI) to 2',3'-dideoxyadenosine-5'-triphosphate (ddATP). The potential phosphorylation of ddAMP by adenylate kinase (myokinase) and pyrophosphorylation by the reverse reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase were investigated. Using ATP as phosphate donor, ddAMP was phosphorylated by adenylate kinase with an efficiency of 8.8% of that for AMP, as estimated from the V max/ K m ratios. In the presence of PRPP, Escherichia coli and rat PRPP synthetases catalysed the pyrophosphorylation of ddAMP with efficiencies of 52 and 35% of that determined for AMP, respectively. Two carbocyclic phosphonate analogues of ddAMP were not substrates of adenylate kinase. Yet, they were pyrophosphorylated by both PRPP synthetases, albeit less efficiently than ddAMP. In vivo, the usual function of PRPP synthetase is to synthesize PRPP from ribose-5-phosphate and ATP. In the forward reaction ddATP proved to be a substrate as efficient as ATP for rat PRPP synthetase. ddATP was also studied as a potential phosphate donor in the reaction catalysed by adenylate kinase with AMP as phosphate acceptor and found to be as efficient as ATP. The relevance of these in vitro results to the in viuo situation is discussed.

Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus.

The capsid protein of duck hepatitis B virus (DHBV) is phosphorylated at multiple sites during viral infection. A cluster of sites is located near the C terminus of the 262-amino-acid protein. We have used site-directed mutagenesis to show that three serines and one threonine serve as phosphate acceptor amino acids in the C terminus. An additional six potential phosphate acceptor sites in this region were apparently not utilized. Each serine or threonine that served as a phosphate acceptor was adjacent to a downstream proline, while all six serines that were not acceptors for phosphate residues lacked adjacent downstream prolines. Mutation of the downstream proline to glycine at each site had the same effect as mutating the serine itself, suggesting an SP or TP motif as an essential feature for capsid protein phosphorylation. Phosphorylation at these four sites resulted in complex shifts in electrophoretic mobility in sodium dodecyl sulfate gels of the capsid protein or of a C-terminal peptide containing the phosphorylated sites, suggesting that specific conformations of the C terminus are associated with different combinations of phosphorylated serines. We speculate that distinct functions of the C terminus may be associated with different phosphorylated domains on the intact capsid.

Structural basis by which a recessive mutation in the alpha-subunit of the insulin receptor affects insulin binding.

Recently, a mutation substituting Leu for Ser323 in the alpha-subunit of the human insulin receptor has been identified in an insulin-resistant patient. The Leu323 mutation leads to a severe impairment in insulin binding without significantly altering the processing or cell surface expression of the receptor. In order to study how alpha beta half-receptors interact to form the insulin-binding site, we cotransfected NIH-3T3 cells with two insulin receptor cDNA constructs: a truncated insulin receptor lacking the C-terminal 43 amino acids (delta 43) and the full-length Leu323 mutant receptor. A clonal cell line from cotransfected cells expresses a hybrid receptor consisting of a Leu323 half-receptor and a delta 43 half-receptor. We demonstrate that the Leu323-delta 43 hybrid receptor binds insulin with high affinity. Furthermore, by cross-linking 125I-insulin to immobilized hybrid receptors, we show that only the alpha beta delta half of the hybrid receptor binds insulin. Since the isolated half-insulin receptor has low affinity for insulin, this suggests that the addition of even a non-binding alpha-subunit can result in high affinity binding to the holoreceptor (alpha alpha mut beta delta beta). Both beta and beta delta-subunits of the Leu323-delta 43 hybrid receptor are phosphorylated in vivo and in vitro in an insulin-dependent manner, suggesting an intramolecular transphosphorylation mechanism and that the presence of the Leu323 mutant receptor that lacks an intrinsic high affinity binding site does not prevent the associated beta-subunit from functioning either as a tyrosine kinase or as a phosphate acceptor in the hybrid insulin receptor molecule (alpha alpha mut beta delta beta). Furthermore, we show that the hybrid receptor can phosphorylate insulin receptor substrate-1 (IRS-1) in response to insulin and can be coimmunoprecipitated together with IRS-1 by anti-IRS-1 antibody.

Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.

In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor.

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.

Purification and Characterization of 100-kDa Glycyrrhizin (GL)-Binding Protein (gp100) as an Effective Phosphate Acceptor for CK-II and the Effect of GL on the Phosphorylation of gp100 by CK-II in Vitro

By means of successive GL-affinity and Mono S column chromatographies (HPLC), a 100 kDa GL-binding protein (gp100) was purified from the partially purified CK-II fraction of EAT cells as an effective phosphate acceptor for CK-II. It was found that (i) gp100 (pI 9.0) is copurified with CK-II, Hsp-90 and p34 or p70; (ii) gp100 cross-reacts with anti-human GR; (iii) phosphorylation of gp100 by CK-II is significantly stimulated by 1 μM GL or 0.3 μM oGA; and (iv) GL as well as DEX inhibit it at doses above 3 μM. Data are provided to suggest that the GL-induced selective inhibition of the CK-II catalyzed phosphorylation of gp100 may be involved in the anti-inflammatory effects of GL.

Identification of a Latent Ca2+/Calmodulin Dependent Protein Kinase II Phosphorylation Site in Vascular Calpain II1

The Ca(2+)-dependent protease, calpain II, isolated from vascular smooth muscle was found to be a substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in vitro. Phosphorylation was dependent upon prior autolysis of the regulatory subunit of calpain II. The stoichiometry of phosphorylation of native, unautolyzed calpain II was 0.02 +/- 0.01 mol PO4/mol enzyme while for autolyzed calpain, the stoichiometry was 1.04 +/- 0.15 mol PO4/mol enzyme. All phosphate was incorporated into the 76 kDa catalytic subunit of calpain II. A single serine residue in domain III of the catalytic subunit was identified as the phosphate acceptor: RGS*TAGGCR. Phosphorylation doubled enzyme activity measured both as proteolysis of an exogenous substrate (alpha-casein) as well as by intermolecular catalytic subunit autolysis. The effects of phosphorylation could be reversed by dephosphorylation using a type IIA phosphoprotein phosphatase. These results demonstrate that calpain II possesses a latent CaM kinase II phosphorylation site that is unmasked by autolysis.

Biochemical characterization of a 34 kda ribonucleoprotein (p34) purified from the spinach chloroplast fraction as an effective phosphate acceptor for casein kinase II

A 34 kDa ribonucleoprotein (p34) was purified to homogeneity from a 1.0 M KCl extract of spinach chloroplasts and characterized as an effective phosphate acceptor for casein kinase II (CK-II). The N-terminal 21 residues (W-V-A-Q-T-S-E-E-E-Q-E-G-S-T-N-A-V-L-E-G-E) of p34 were 95% identical with the sequence reported for 28RNP (plastid mRNA 3' end processing factor in chloroplast). Moreover, the findings that DNAs as well as RNAs significantly stimulate the CK-II catalyzed phosphorylation of p34 in vitro and induce its conformational change, suggest that the physiological activity of p34-bound RNA or DNA in chloroplast post-transcriptional regulation is controlled by specific p34 phosphorylation by CK-II.

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: a single assay for a bifunctional enzyme

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase has been purified from calf thymus. Since the same protein is able to catalyze both the hydrolysis and the interconversion of several nucleoside monophosphates, it is necessary to study the effect of different metabolites and assay conditions on both activities in order to elucidate their physiological roles. We describe herein a method which allowed us to follow both activities contemporaneously in the same assay mixture. The method takes advantage of the observation that deoxyGMP is both a good substrate for hydrolysis and a good phosphate donor for the phosphotransferase reaction, but its dephosphorylated product, deoxyguanosine, is not a phosphate acceptor. As a consequence, it is possible to follow both the deoxyguanosine production and the transfer of phosphate from deoxyGMP to the best phosphate acceptor, inosine, during the reaction, applying a method for the chromatographic separation on HPLC of both substrates (inosine and deoxyGMP) and both products (IMP and deoxyguanosine). The method was applied to the determination of the KM for inosine.

Varicella-Zoster virus thymidine kinase: Characterization and substrate specificity

The varicella-zoster virus (VZV) thymidine kinase (TK) (EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent K m values for MgATP of 0.10 and 900 μM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2′-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2′,3′-dideoxyribonucleosides, and the acyclic moiety of acyclovir.

Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta.

Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced by self-assembly in vitro, the bicistronically expressed beta subunit was autophosphorylated during formation of the holoenzyme in bacteria. Electrospray ionisation mass spectrometry (ESI) revealed Ser2 (second amino acid, first serine) as the only phosphate acceptor site. Kinetic data obtained with either the phosphorylated or the unphosphorylated form of CK-2 did not differ significantly, suggesting that the autophosphorylation had no influence on the kinetic parameters Km and Vmax. In parallel, native rat liver CK-2 beta subunit was shown to incorporate 0.1 mol phosphate/mol holoenzyme, which suggests that the enzyme is already heavily phosphorylated. ESI analysis also revealed Ser2 as the only phosphorylated residue at the amino terminus. In the case of both recombinant human CK-2 and native rat liver CK-2, treatment with alkaline phosphatase readily reversed the phosphorylated form of the beta subunit to the faster migrating dephosphorylated polypeptide.

Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.

Purification and Biochemical Characterization of the Protein Kinase Encoded by the US3 Gene of Herpes Simplex Virus Type 2

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to succesive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 μM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.

Membrane-bound 5′-nucleotidase/nucleoside phosphotransferase from Bacillus cereus

1. 1. A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5′-nucleotidase. (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2′,3′-dideoxyinosine. (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor. 2. 2. Bacterial and vertebrate 5′-nucleotidases with phosphotransferase activity diner for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.