PubMed Health⌕ Search

Biomedical subjects

G F Maley

Publications and source records attributed to G F Maley.

At least 73 records · Page 4Linked to original sources
PubMed1979

The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.

Amino Acid Sequence↗
PubMed1979

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.

Amino Acid Sequence↗
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 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↗
PubMed1972

Relationship between Escherichia coli B titer and the level of deoxycytidylate deaminase activity induced on bacteriophage T2r + infection.

The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.

Aminohydrolases↗