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F Maley

Publications and source records attributed to F Maley.

At least 145 records · Page 8Linked to original sources
PubMed1976

Factors affecting substrate binding in Lactobacillus casei thymidylate synthetase as studied by equilibrium dialysis.

The binding of deoxynucleoside 5'-monophosphates and various folate derivatives to Lactobacillus casei thymidylate synthetase was measured by equilibrium dialysis. The substrate, deoxyuridylate (dUMP), and the product, thymidylate (dTMP), were bound to the enzyme at a ratio of unity and appeared to compete for the same site. The binding of each was tighter in 50 mM Tris-HCl (pH 7.1) than in 50 mM potassium phosphate (pH 7.0). Folate derivatives increased the affinity of the enzyme for the substrate to a greater extent than for the product, although they themselves did not appear to be bound in the absence of substrate or substrate analogues. However, in the presence of enzyme, dUMP or 4-N-OH-dCMP, and either 7,8-dihydrofolate or methotrexate, a ternary complex was obtained, with the folate derivatives exhibiting single site binding. The binding of dUMP in the ternary complex was 25-fold greater than that of 7,8-dihydrofolate and 50-fold greater than that of methotrexate. Supporting evidence for the enhanced stability of the ternary complex was provided by heat inactivation studies. As in the case of deoxynucleotide binding to the synthetase, the ternary complex was more stable in Tris HCl than in potassium phosphate buffer. The binding characteristics of the substrate analogue 5-fluoro-2'-deoxyuridylate (FdUMP) could be clearly distinguished from that of dUMP by comparing their binding in phphate and Tris-HCl. While each deoxynucleotide exhibited only single site binding in phosphate, a second site was clearly demonstrated for FdUMP with Tris-HCl. The binding of FdUMP to each site appeared to be equal in the presence of methotrexate or (-)5,10-methylene tetrahydrofolate and was increased about 17-fold in Tris-HCl. Two sites were also obtained for FdUMP in the presence of 7,8-dihydrofolate, but Scatchard analyses revealed a biphasic curve, with the second site possessing a higher dissociation constant than the first. A second low affinity FdUMP binding site was also detected in phosphate buffer when 7,8-dihydrofolate or (-)5,10-methylene tetrahydrofolate was included in the binding assay. In the presence of (+)5,10-methylene tetrahydrofolate, however, 2 mol of FdUMP was bound stoichoimetrically to 1 mol of enzyme regardless of the buffer used. The significance of these results is discussed in relation to the presence of two apparently identical subunits in the native enzyme.

Binding Sites↗
PubMed1976

Amino acid sequence at the FdUMP binding site of thymidylate synthetase.

Cyanogen bromide treatment of thymidylate synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-14c] FdUMP and 5,10-methylene-tetrahydrofolate followed by S-carboxymethylation, yielded at least four visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo [I-14C] acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000= was labeled. Analysis of CN2 that had been labeled with [2-14C] FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-14C] acetate revealed that their amino-acid contents were almost identical except for the presence of two S-carboxymethyl (Cm)-cysteinyl residues in the latter peptide and only one in FdUMP-CN2. A nonapeptide was isolated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: Ala-Leu-Pro-Pro-[Cm-Cys]-His-Thr-Leu-Tyr. This peptide was found to be located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the fifth, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide.

Affinity Labels↗
PubMed1975

The isolation and structure of the core oligosaccharide sequences of IgM.

Methods are presented for separating the three IgM heavy chain sialoglycopeptides associated with asparagines 170, 332, and 395. The core glycopeptide units containing the disaccharide fucosyl-N-acetylglucosamine were obtained through the use of an endo-beta-N-acetylglucosamindase from Diplococcus pneumoniae, following exoglycosidase treatment of the sialoglycopeptides. In addition to the core glycopeptides, high yields of a tetrasaccharide, (Man)3GlcNAc, were obtained. The fucose in the core disaccharide is glycosidically linked to the 6-O position of the N-acetylglucosamine residue in Asn-GlcNAc. This core unit is resistant to glycosyl asparaginase, but becomes susceptible to hydrolysis on removal of the fucosyl residue by a purified hen oviduct alpha-L-fucosidase. The core sequence of the immunoglobulin M sialoglycopeptides appears to be similar to that of most other asparagine-linked oligosaccharides in consisting of a basic unit composed of beta-D-Man-(1 leads to 4)beta-D-GlcNAc(1 leads to 4)beta-D-GlcNAc(1 leads to 4), but with L fucose linked alpha-(1 leads to 6) to the proximal GlcNAc. The two nonreducing terminal ends of (Man)3GlcNAc are linked to beta-D-Man by alpha-(1 leads to 3) and alpha(1 leads to 6) bonds, respectively.

Amino Acids↗
PubMed1975

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.

Animals↗
PubMed1975

The effect of substrate analogs on the circular dichroic spectra of thymidylate synthetase from Lactobacillus casei.

Circular dichroism studies from 290 to 400 nm with the thymidylate synthetase from Lactobacillus casei revealed characteristic Cotton effects in the presence of various folate analogs plus 5-fluoro-2'-deoxyuridylate. Omission of either substrate analog prevented the appearance of the Cotton effects. When 5-fluoro-2'-deoxyuridylate and (+/-)-5,10-methylenetetrahydrofolate are mixed with the synthetase, a ternary complex results which yields distinctive minor negative ellipicity bands at 285 and 332 nm and a major negative ellipticity bands at 285 and 332 nm and a major positive band at 305 nm. Similar results were obtained with the ternary complex containing (+)-5,10-methylenetetrahydrofolate, but the enzymically inactive (-) diastereoisomer induced only the positive band at 305 nm. More intense Cotton effects were elicited by (+/-)-5,11-methylenetetrahydrohomofolate with a major positive ellipticity band at 308 nm and a minor negative band at 335 nm. A ternary complex was also formed with dihydrofolate, which provided a major circular dichroic band at 305 nm and a broad minor negative band in the region of 335 nm. Deoxyuridylate and thymidylate also formed ternary complexes with dihydrofolate, but their ellipicity bands were much less intense. Other folate analogs that formed ternary complexes with 5-fluoro-2'-deoxyuridylate to provide characteristic circular dichroic spectra were tetrahydrofolate, tetrahydrohomofolate, 10-methyltetrahydrofolate, and a 2-amino-4-hydroxyquinazoline derivative. By measuring the increment in ellipticity at 305 nm on addition of specific ligands to enzyme solutions, it was determined that the L. casei thymidylate synthetase contains two binding sites for 5-fluoro-2'-deoxyuridylate and for each of the diastereoisomers of 5,10-methylenetetrahydrofolate. An improved procedure is presented for the large-scale purification and crystallization of L. casei thymidylate synthetase.

Binding Sites↗
PubMed1975

Effect of methotrexate on thymidylate synthetase in cultured parenchymal cells isolated from regenerating rat liver.

The effect of methotrexate (MTX) on thymidylate synthetase activity during liver regeneration was examined with parenchymal cells isolated 22 and 44 hr after partial hepatectomy and cultured as a monolayer. The synthetase activity in these cells decreased with a half-life of 18 to 24 hr, but if MTX (1.5 X 10(-6) to 1.5 x 10(-5) M) was present in the culture media, this decline could be delayed for at least 48 hr. In contrast, thymidine kinase activity decreased at a rate which we unaffected by MTX. Dihydrofolate reductase was inhibited at all concentrations of MTX used to block the decrease in synthetase activity. Folic acid at 10(-4) M, although less effective than MTX, also delayed the decrease in synthetase activity. The addition of cycloheximide, puromycin, or antinomycin D to the culture media did not alter the response of the synthetase to MTX. The latter studies, coupled with those indicating that the rapid loss of synthetase activity in crude extracts could be prevented by MTX or, more effectively, by MTX plus deoxyuridine 5'-monophosphate, suggest that the primary effect of MTX on thymidylate synthetase in vivo is that of enzyme stabilization. Similar stabilizing effects were obtained in liver cell extracts with 10(-5) M deoxyuridine 5'-monophosphate in combination with 10(-4) M folate or 10(-4) M dihydrofolate.

Animals↗