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F Maley

Publications and source records attributed to F Maley.

At least 55 records · Page 3Linked to original sources
PubMed1994

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Base Sequence↗
PubMed1993

Catalytically active cross-species heterodimers of thymidylate synthase.

Thymidylate synthase (TS) is a highly conserved homodimeric enzyme with two active sites, each of which contains amino acid residues from both subunits. We show that the conservation at the subunit interface between Escherichia coli TS and Lactobacillus casei TS is sufficient to permit the formation of a cross-species heterodimer between subunits of E. coli TS and L. casei TS. Heterodimer formation was monitored by the generation of catalytic activity when combinations of inactive E. coli homodimers and inactive L. casei homodimers were mixed under conditions of reversible unfolding and dissociation. The inactive L. casei mutant enzymes (Lc)C198A, (Lc)C198L, and (Lc)V316Am were tested as Arg donors to the active sites of the inactive E. coli mutant enzymes (Ec)R126Q and (Ec)R126E, while the inactive E. coli mutant enzymes (Ec)K48Q, (Ec)C146S, (Ec)R166Q, and (Ec)I264Am were tested as Arg donors to the active site of inactive (Lc)R178F. Except for (Lc)V316Am, all of the mutant enzymes tested were able to form catalytically active cross-species heterodimers. (Lc)C198A and (Ec)R126Q were cotransformed on compatible plasmids into a thymine-requiring E. coli host, and this combination was able to form sufficient active TS in vivo to support growth. Titration of (Ec)R126Q with (Lc)C198A showed that the cross-species heterodimer formed with the same probability as the intraspecies homodimers in the refolding mixture. The single active site formed by this pair has kcat and Km values similar to those of an intraspecies heterodimer.

Binding Sites↗
PubMed1993

Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli.

The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.

Amino Acid Sequence↗
PubMed1993

Properties of an affinity-column-purified human deoxycytidylate deaminase.

Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.

Antibodies↗
PubMed1993

T4-phage deoxycytidylate deaminase is a metalloprotein containing two zinc atoms per subunit.

Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per subunit by atomic absorption spectroscopy. One zinc appears to be involved in catalysis, as described for adenosine deaminase (Sharaff, A. J., Wilson, D. K., Chang, Z., and Quiocho, F. A. (1992) J. Mol. Biol. 226, 917-921) and cytidine deaminase (Yang, C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). This thesis is supported by the finding that the enzyme loses about 80% of its activity in the presence of o-phenanthroline. It has also been found that zinc is released when the enzyme is denatured in the presence of the metallochromic indicator, 4-(2-pyridylazo)resorcinol. Renaturation of the deaminase to an active form occurred in the presence but not in the absence of zinc. The second atom of zinc is proposed to be located in a region of T4-dCMP deaminase that resembles a zinc finger. This region, which has the sequence His-X3-Cys-X14-His-X3-His, would represent a zinc-binding motif that has not been described previously.

Amino Acid Sequence↗
PubMed1993

Identification of an RNA binding site for human thymidylate synthase.

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of approximately 36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.

Base Sequence↗
PubMed1993

Overcoming inclusion body formation in a high-level expression system.

Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex. However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism's cellular protein. Insoluble inclusion bodies were obtained with less rich media. This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies. The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.

Bacteriophage T4↗
PubMed1991

Purification and substrate specificity of a T4 phage intron-encoded endonuclease.

The T4 phage td intron-encoded endonuclease (I-Tev I) cleaves the intron-deleted td gene (td delta I) 23 nucleotides upstream of the intron insertion site on the noncoding strand and 25 nucleotides upstream of this site on the coding strand, to generate a 2-base hydroxyl overhang in the 3' end of each DNA strand. I-Tev I-157, a truncated form in which slightly more than one third (88 residues) of the endonuclease is deleted, was purified to homogeneity and shown to possess endonuclease activity similar to that of I-TEV I, the full-length enzyme (245 residues). The minimal length of the td delta I gene that was cleaved by I-Tev I and I-Tev I-157 has been determined to be exactly 39 basepairs, from -27 (upstream in exon1) to +12 (downstream in exon2) relative to the intron insertion site. Similar to the full-length endonuclease, I-Tev I-157 cuts the intronless thymidylate synthase genes from such diverse organisms as Escherichia coli, Lactobacillus casei and the human. The position and nature of the in vitro endonucleolytic cut in these genes are homologous to those in td delta I. Point mutational analysis of the td delta I substrate based on the deduced consensus nucleotide sequence has revealed a very low degree of specificity on either side of the cleavage site, for both the full-length and truncated I-TEV I.

Base Sequence↗
PubMed1991

Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris.

Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.

Carbohydrate Sequence↗
PubMed1990

Properties of bacteriophage T4 thymidylate synthase following mutagenic changes in the active site and folate binding region.

Amino acid replacements have been introduced in specific sites of bacteriophage T4 thymidylate synthase (T4-TS) to assess the role that these changes have on enzyme activity. Each of the conserved amino acids in the active-site region of T4-TS was modified, and the effects that these changes had on the kinetic and physical properties of this enzyme were measured. The mutations introduced were Pro-155-Ala (P155A), Cys-156-Ser (C156S), and His-157-Val (H157V) with the resulting synthases possessing kcat's of 10.3, 0.008, and 2.70 s-1, respectively, relative to that of the wild-type enzyme of 11.8 s-1. Equilibrium dialysis was performed on the wild-type and mutant enzymes to determine the binding constants for 2'-deoxyuridylate and 5-fluoro-2'-deoxyuridylate, and while in most cases the extent of binding of these nucleotides to the mutant proteins was reduced when compared with wild-type TS, the number of binding sites involved remained about 1 or less for the binary complex and almost 2 for the ternary complex. Heat and urea stability studies revealed that the mutant with the highest enzyme activity, P155A, was the most unstable, while spectrofluorometric analyses revealed that the structures of P155A and H157V were perturbed relative to the C156S and wild-type TSs. These studies are in agreement with others implicating the phylogenetically conserved active-site cysteine as playing an essential mechanistic role in the catalytic process promoted by TS. The proximal amino acids on either side of this cysteine, although also highly conserved, do not appear to affect the catalytic mechanism directly, but may do so indirectly through their influence on the conformation at the active site as well as other regions of the enzyme. Amino acids replacements were introduced also into the folate and deoxynucleotide 5'-phosphate binding sites of the T4-phage TS to ascertain the potential role that these amino acids play in the catalytic process. These positions were selected on the basis of previous chemical modification and X-ray crystallographic studies on Lactobacillus casei TS. Amino acid residues 48 and 49, which are in the putative folate binding site, were converted from lysines to arginines; in the former case, the mutated enzyme had less than 7% of the wild-type activity while in the latter, the mutated enzyme still retained about 60% of its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate.

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.

Binding Sites↗
PubMed1990

Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis.

Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent. The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for Asp-23 in the catalytic process.

Acetylglucosaminidase↗
PubMed1990

Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.

Carcinoma, Hepatocellular↗
PubMed1990

Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum.

A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.

Amidohydrolases↗
PubMed1990

Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase.

External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme [Chu, F.K., Watorek, W., & Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E. (1987) J. Biol. Chem. 262, 4395-4401]. To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions. In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied. Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status. However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases. The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction. Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase. By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers. These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.

Chlorides↗
PubMed1990

Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene.

The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced. The deduced amino acid sequence was found to be 193 residues long compared with 188 for the corresponding enzyme from bacteriophage T2. There were nine amino acid differences between the two enzymes in addition to a 5-residue insert near the carboxyl terminus of the T4 deaminase which was not present in the T2 deaminase. The cd-containing fragment also contained all of gene 31 (Nivinskas, R., and Black, L. W. (1988) Gene (Amst.) 73, 251-257) and thus precisely locates the two genes relative to one another within the T4 phage genomic map. Attempts to place the cd gene within a high expression vector have not been successful so far due to possible toxic effects of the gene product. However, placement of the gene within pUC18 resulted in a degree of expression which is about 10-20 times that found in T4-infected Escherichia coli. The enzyme was purified to homogeneity and found to possess properties similar to T2 phage deoxycytidylate deaminase.

Amino Acid Sequence↗