Electrophoretic assay of folylpolyglutamate synthetase activity

A method is described for the determination of the activity of folylpolyglutamate synthetase based upon incorporation of reaction products into a covalent, ternary complex with tritiated 5-fluoro-2′-deoxyuridylate and thymodylate synthetase followed by electrophoretic identification of the enzyme-bound polyglutamate species.     

A method for the determination of total,free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts

A radiochemical assay for thymidylate synthase (EC 2.1.1.45, dTMP synthase), which permits the accurate determination of total, free, and 5-fluoro-2′-deoxyuridylate (FdUMP)-bound enzyme in cells exposed to the 5-fluoropyrimidine anticancer agents, is described. The total intracellular concentrations of dTMP synthase (free plus FdUMP-bound enzyme) in extracts from CCRF-CEM leukemic cells incubated with 5-fluoro-2′-deoxyuridine were determined following dissociation of the covalent dTMP synthase-5,10-methylenetetrahydrofolate-FdUMP ternary complex in the presence of the substrate, 2′-deoxyuridine-5′-monophosphate. The addition of substrate prevented reformation of the ternary complex during the dissociation procedure, and allowed complete recovery of FdUMP binding sites in cells exposed to a high concentration of 5-fluoro-2′-deoxyuridine. After removal of the substrate by charcoal adsorption, the concentration of total FdUMP binding sites was determined by titration of the enzyme with a saturating concentration of [6-³H]FdUMP and 5,10-methylenetetrahydrofolate. The concentration of FdUMP-bound dTMP synthase was then calculated as the difference between the total and free (without prior ternary complex disruption) enzyme values. The high sensitivity of this assay coupled with its ability to accurately quantitate both free and FdUMP-bound dTMP synthase in cells exposed to a wide range of fluoropyrimidine concentrations should make it useful for a variety of experimental and clinical studies.     

5-Nitro-2'-deoxyuridylate: a mechanism-based inhibitor of thymidylate synthetase.

5-Nitro-2′-deoxyuridylate is a potent reversible inhibitor of thymidylate synthetase. Once the reversible binary complex is formed, a nucleophile of the enzyme covalently binds to the 6-position of this inhibitor. The interaction does not require the cofactor of the normal enzymic reaction, and spectrophotometric properties of the complex are ideal for detailed studies of the interaction.     

A single functional arginyl residue involved in the catalysis promoted by Lactobacillus casei thymidylate synthetase

The inactivation of Lactobacillus casei thymidylate synthetase by phenylglyoxal occurs by a pseudo-first-order, pH-dependent process which is 100-fold faster at pH 8.4 than at pH 7.4. The second-order rate constant for inactivation at pH 7.4 is 32 m^?1 min^?1. Although four or more arginyl residues of the 24 arginines per enzyme dimer can be modified, as determined by amino acid analysis or [2-¹⁴C]phenylglyoxal incorporation, only one arginine appears to be essential for activity. The association of this arginine with the catalytic process is supported by the finding that 2′-deoxyuridylate not only protects it from modification by phenylglyoxal, but in so doing prevents the enzyme from losing activity. Additional support is derived from the fact that the product of the reaction, 2′-deoxythymidylate, a competitive inhibitor of 2′-deoxyuridylate, also protects the enzyme, but 2′-deoxycytidylate and uridylate do not. Neither the enzyme's second substrate, 5,10-CH₂H₄folate nor the folylpolyglutamates protect the enzyme from inactivation by phenyglyoxal. These findings contrast with those recently reported by Cipollo and Dunlap (Biochemistry18, 5537, 1979), which indicate that the inactivation is associated with the modification of 4 arginines per mole of enzyme, 2 of which are protected by 2′-deoxyuridylate. The requirement for a single arginine in the catalytic process is consistent with the involvement of one essential cysteine (Noonan et al., Arch. Biochem. Biophys.184, 336, 1977, both amino acids apparently participating in the binding of 1 mol of 2′-deoxyuridylate per enzyme dimer. These findings suggest that the synthetase's two identical subunits function asymmetrically and that 2′-deoxyuridylate binds as a dianion.     

The effect of Raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex.

Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Evidence from chemical degradation studies for a covalent bond from 5-fluoro-2'-deoxyuridylate to N-5 of tetrahydrofolate in the ternary complex of thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate

In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.     

19F NMR studies of the binding of 5-fluoro-2′-deoxyuridylate to thymidylate synthase

Summary In the presence of thymidylate synthase, the ¹⁹F signal of 5-fluoro-2-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands at these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a ¹⁹F signal shifted 12.5 ppm upfield and broadened to 120 Hz.Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex.     

A Phosphoramidite-Based Synthesis of Phosphoramidate Amino Acid Diesters of Antiviral Nucleosides

Abstract

A general synthetic procedure is presented for the preparation of 5′-amino acid phosphoramidates of zidovudine (AZT), 3′-deoxy-2′,3′-didehydrothymidine (D4T), and 3′-fluoro-3′-deoxythymidine (FLT) from their corresponding phosphoramidites. These water soluble amino acid phosphoramidates are more non-polar than the parent nucleoside and exhibit high stability in aqueous media.     

Repair of protein-linked DNA double strand breaks: Using the adenovirus genome as a model substrate in cell-based assays

The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5′ termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5′ terminus of its dsDNA genome, for studying the repair of 5′ protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5′ termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5′ termini of DSBs.     

Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides

Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end. They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter. Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of [alpha-32P]UTP were elongated, so that highly selective affinity labelling occurred. With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place. Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom). In both cases, the amino acid residues labelled were histidines.     

Thymidylate synthetase catalyzed dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate

Thymidylate synthetase catalyzes a facile dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate in the presence of dithiothreitol. The chloro and fluoro nucleotides are not dehalogenated under similar conditions. A mechanism for this reaction is proposed which is in complete accord with previously ascertained features of the catalytic mechanism as well as model chemical reactions. This reaction is likely an important pathway in the biological dehalogenation of 5-halogenated uracil derivatives.     

SYNTHESIS OF 5-ETHYNYL-2′-DEOXYURIDINE-5′-BORANOMONO PHOSPHATE AS A POTENTIAL THYMIDYLATE SYNTHASE INHIBITOR

The 5-ethynyl-2′-deoxyuridine nucleoside and the 5′-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2′-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2′-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.     

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.     

Solid Phase Synthesis of 2′, 5′-Oligoadenylates Containing 3′-Fluorinated Ribose

Abstract

2′, 5′-phosphodiester bond-linked oligoadenylate trimers with 3′-fluoro-3′-deoxyadenosine residues incorporated at specific positions of the nucleotide sequence were synthesized by the solid phase phosphite triester (phosphoramidite) method. The syntheses were in the 2′ to 5′ direction and were performed manually using commercially available microcolumns. The oligonucleotides were 5′-end phosphorylated on the support before deprotection.     

Molecular cloning of the murine branched chain α-ketoacid dehydrogenase E2 subunit: presence of 3′ B1 repeat elements

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain α-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5′ sequence encoding the amino-terminal preprotein sequence and 3′ untranslated region are less well conserved. The 3′-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3′-noncoding region of human E2 cDNA.     

Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.

B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

Chemical synthesis of 2''-deoxyoligonucleotides containing 5-fluoro-2''-deoxycytidine.

2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific positions of the nucleotide sequence are tools of great potential in the study of the catalytic mechanism by which DNA cytosine methyltransferases methylate the 5-position of DNA cytosine residues in specific sequence contexts. Chemical synthesis of such oligonucleotides is described. Two alternative approaches have been developed, one of which proceeds via a fully protected phosphoramidite of 5-fluoro-4-methylmercapto-2'-deoxyuridine 2, the other via a fully protected phosphoramidite of 5-fluoro-2'-deoxycytidine 3. Either building block can be used in automated oligonucleotide synthesis applying standard elongation cycles and deprotection procedures exclusively. The methylmercapto function of 2 is replaced by an amino group in the final ammonia treatment used for cleavage from support and base deprotection.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.