David H. Ives

Rapid determination of nucleoside kinase and nucleotidase activities with tritium-labeled substrates☆

Abstract Methods suitable for the assay of ribonucleoside and deoxyribonucleoside kinases and of any enzyme that hydrolyzes nucleoside monophosphates have been described. Nucleotides are retained on disks of anion-exchange paper, while nucleosides are washed off. The technique of eluting the nucleotide from the disk within a liquid scintillation vial, followed by solution of the eluate in a suitable scintillation solvent, permits the use of tritium- as well as 14C-labeled substrates. It has been demonstrated that tritium-labeled nucleotides may be counted as reliably as 14C-labeled compounds if the samples are first eluted into the liquid phase. The abilities of diethylaminoethyl (DEAE) substituted paper and of Amberlite impregnated paper (SB-2) to retain nucleotides were compared as the salt concentration of the sample was increased. Under the conditions normally used for enzyme assays both papers retained 100% of the labeled nucleotide, while the percentage of nucleotide retained decreased as the salt concentration of the sample was increased. SB-2 paper was found to be substantially more retentive than DEAE paper, but both papers retained more than 50% of the labeled dCMP or dTMP from samples containing 1 M KCl.

Isolation and characterization of an aminopeptidase from lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.

Life on the Salvage Path: The Deoxynucleoside Kinases of Lactobacillus acidophilus R-26

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.

The stereochemical course of thiophosphoryl group transfer catalyzed by adenosine kinase

Adenosine kinase was partially purified form beef liver and used to catalyze the conversion of (γR)ATPγS,γ18O and adenosine to ADP and AMPαS,α18O. The configuration at phosphorus in AMPαS,α18O was established by subjecting it to stereospecific phosphorylation to (αS)ATPαS,α18O and showing that only the nonbridging oxygen bonded to the α-P was enriched with 18O. The configuration at α-P in AMPαS,α18O was therefore S, and the transfer of the [18O]thiophosphoryl group occurred with inversion of configuration.

The metabolism of deoxyribonucleosides in Lactobacillus acidophilus: Regulation of deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine kinase activities by nucleotides

Abstract 1. 1. The presence of deoxycytidine kinase, as well as of deoxyadenosine, deoxyguanosine and thymidine kinase activities, is reported in Lactobacillus acidophilus . 2. 2. Phosphorylation of each deoxynucleoside is inhibited most effectively by its homoogous deoxynucleoside triphosphate. Besides inhibiting deoxycytidine kinase, dCTP also inhibits deoxyguanosine kinase activity, but stimulates deoxyadenosine and deoxythymidine phosphorylation. 3. 3. The inhibition of deoxycytidine, deoxyguanosine or deoxyadenosine phosphorylation by the corresponding triphosphate is reversed by UTP and dUTP, but not by dTTP which augmented the inhibition in certain cases. The inhibition of thymidine kinase by dTTP was readily reversed by dCTP. 4. 4. Preliminary purification of the bacterial deoxycytidine kinase is described, and its properties are compared with those of the enzyme from calf thymus. The pH curve is biphasic, with optima at pH 7.6 and 10.5. The mol. wt., as estimated by gel filtration, is only about 35 000. The enzyme is slightly activated by mercurial reagents, apparently without loss of regulatory properties. Initial kinetic experiments yielded linear double-reciprocal plots when either deoxycytidine or ATP-Mg 2− was varied. The end-product inhibitor dCTP apparently is competitive; K i dCTP, 5.0·10 −5 M.

A new system for washing ion-exchange paper disks used in radiochemical enzyme assays

A new device has been developed for the removal of radioactive substrate from ion-exchange paper disks, leaving ionized product bound to the paper. It has a number of important advantages over older methods of washing the ion-exchange paper: (1) It is much more thorough and efficient; background absorption of unionized material is reduced to relatively low levels. (2) Fragile media such as DEAE-substituted paper (DE81) are not subjected to the erosive effects of stirrers or agitators. (3) The disks remain organized throughout the sample application and washing procedure, eliminating need for time-consuming sorting of wet radioactive disks after washing. A larger number of assays can therefore be processed conveniently at one time. (4) Radioactive nucleosides and other adsorbable substrates are collected in a cartridge, eliminating the handling and sink-disposal of large volumes or excessive quantities of radioactive materials.

A new method for the isolation of eukaryotic nuclear proteins

Abstract A new two-step fractionation procedure has been developed which rapidly but quantitatively and qualitatively separates HeLa cell nuclear proteins from nuclear DNA and RNA. Purified HeLa cell nuclei, chromatin (prepared by two different procedures), or isolated heterogeneous ribonucleoprotein particles were first dissociated with sodium dodecyl sulfate detergent, and nuclear proteins and nuclear nucleic acid were partitioned after the addition of sodium chloride and chloroform:isoamyl alcohol. When isolated nuclei of HeLa cells (pulse labeled with [ 3 H]leucine) were subjected to this fractionation procedure, over 95% of the radiolabeled nuclear protein was found in the combined interphase and organic phase of the extraction mixture. In parallel experiments, over 90% of the 3 H-uridine labeled nuclear RNA and 95% of the [ 3 H]thymidine labeled nuclear DNA were consistently partitioned into the aqueous phase of the centrifuged suspensions. Acid soluble nuclear proteins (mostly histone), acid insoluble nuclear proteins (nonhistone), or total nuclear proteins were prepared utilizing this procedure and were analyzed by sodium dodecyl sulfate polyacryalmide gel electrophoresis.

cis-Active Ras G2-like Sequence Implicated in the Heterotropic Activation of the Deoxyadenosine Kinase of Lactobacillus acidophilus R-26

Deoxyadenosine kinase (dAK) forms a heterodimer with either deoxyguanosine kinase (dGK) or deoxycytidine kinase (dCK), and is heterotropically activated 3-5 times by dGuo or dCyd. Expressed alone, dAK is inactive and exhibits no response to dGuo or dCyd; activity and heterotropic response are fully restored upon reassociation with dGK or dCK. However, turnover of independently expressed dGK or dCK is nearly maximal, being further activated only 50-100% upon reassociation with dAK. In neither case is the heterotropic activation due to ligand-induced heterodimer formation. A proline/alanine substitution within a dAK segment homologous to loop G2 of Ras proteins yielded a heterodimer with dAK permanently cis-activated 2-fold, with a corresponding reduction in heterotropic activation by dGuo. A chimeric dAK, with 25% of its C terminus substituted by the homologous sequence from dGK, was inactive alone, and its characteristics were unchanged in the reconstituted heterodimer. Superimposing the Pro/Ala substitution on this chimera also reduced heterotropic activation by half. Cross-linking the dimer by 1,5-difluoro-2,4-dinitrobenzene was inhibited by ATP, dATP, dGTP, and dAdo, suggesting the proximity of the active site(s) to the interface. These data suggest that dAK depends on dGK or dCK in a manner resembling the reliance of Ras upon GTPase activating protein.

Evaluation of Carboranyl 2′-Deoxyuridine Derivatives as Substrates for Human Thymidine Kinases 1 and 2

Cellular nucleoside kinases play a pivotal role in the use of nucleosides for cancer and antiviral therapy.1 For BNCT, the cytosolic thymidine kinase (TK1) may be a particularly important target enzyme. TK1 activity is present in proliferating cells but is virtually absent from all quiescent cells.2 Boron-containing thymidine derivatives, that are good substrates for TK1, may be entrapped in proliferating neoplastic cells after conversion to the monophosphate. Therefore, such agents may be excellent vehicles for the selective delivery of boron-10 to those compartments of a tumor consisting of viable cells. A substantial number of nucleosides modified with various boron moieties at different positions have been synthesized and evaluated biologically for use in BNCT.3 Some thymidine derivatives were found to be phosphorylated in vitro 4 and in phos-phoryl transfer assays with purified human thymidine kinases.5 In these experiments, the observed rates of phosphorylation were generally low compared to natural nucleosides and did not distinguish between phosphorylation by TK1 and mitochondr-ial thymidine kinase (TK2) which appears to be equally active in proliferating and non-proliferating cells, thus providing no basis for selective uptake of boronated nucleosides in tumor cells.6

Synthesis and Biochemical Evaluation of 5-Tethered Boron-Containing Pyrimidine Nucleosides for BNCT

In order for BNCT to be successful, a major need is the development of boron compounds, which penetrate the blood-brain barrier (BBB), target tumor cells selectively, and maintain a relatively high concentration of boron-10 in the tumor, ca. 30 ug/g of tumor tissue, vis-a-vis surrounding normal tissues. Boron-containing nucleosides that use a carrier-mediated transport process for penetrating the CNS and have the potential for becoming incorporated into proliferating cells may be useful agents for BNCT.1 This fact led to the attempts to synthesize carborane-containing nucleoside derivatives. Yamamoto2 and Schinazi3 have attached the carborane moiety to the 5-position on the pyrimidine nucleosides, either directly or through a bulky substituent. Unfortunately, these compounds are very poor substrates for nucleoside kinases, and are unlikely to demontrate the requisite biological activity. This may be due to the fact that having a bulky substituent, like the carborane moiety, attached directly on the 5-position may inhibit enzymatic conversion of the 5-carboranyl-2′-deoxyuridine to their corresponding nucleotides. The formation of the latter is a necessary precondition for the incorporation of such nucleotides into DNA.