Ligand-induced conformational changes of thymidylate synthase detected by limited proteolysis.

Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP.

Interaction of pyridoxal phosphate with thymidylate synthase: spectral and equilibrium dialysis studies.

1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents. 2.

1-Phenyl-3-trimethylaminopropyl carbodiimide: a new inhibitor of thymidylate synthase.

Thymidylate synthase (EC 2.1.1.

Inhibition of thymidylate synthase by glyceraldehyde 3-phosphate.

1. A number of common metabolites which had carbonyl and/or phosphate groups were tested for their ability to alter the activity of thymidylate synthase from Lactobacillus casei. Glyceraldehyde 3-phosphate was found to be an effective inhibitor of thymidylate synthase.

Inhibition of thymidylate synthase by pyridoxal phosphate.

1. Pyridoxal phosphate (PLP) reversibly inhibited thymidylate synthase from Lactobacillus casei with a KI of 0.6-0.

Mitogenic activity in ovine uterine fluids: characterization of a growth factor activity which specifically stimulates myoblast proliferation.

Fluids produced by the uterus of pregnant sheep (OUF-ovine uterine fluids) were assayed for mitogenic activity in a thymidine incorporation assay. A dose-dependent mitogenic activity was observed in OUF which exceeded that of adult ovine plasma or fetal bovine serum. Uterine fluids were capable of stimulating thymidine incorporation in mouse 3T3 fibroblasts, rat L6 myoblasts, ovine trophoblast-derived cells, HeLa S3 cells, and bovine aortic endothelial cells.

Purification and properties of alkaline phosphatase from boar seminal plasma.

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity.

Reaction of methylated urates with 1,1-diphenyl-2-picrylhydrazyl.

The kinetics of the reaction between the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and methylated urates was studied. Urates that had methyl groups on the 1,3,9, or on the 1 and 3 or 1 and 9 nitrogens reacted with DPPH 15 to 77% faster than uric acid. Urates substituted with methyl groups on the 7 nitrogen or on both the 3 and 9 nitrogens reacted with DPPH at rates that were less than 0.

Microcomputer simulation of steady-state enzyme kinetics for educational purposes.

A BASIC program to assist the instruction of steady-state enzyme kinetics has been developed for the IBM PC microcomputer. Its purpose is to simulate laboratory experiments in order to minimize the time required to obtain kinetic data from which students deduce kinetic mechanisms and determine kinetic constants of enzyme-catalyzed reactions. The program randomly selects a kinetic scheme from various sequential, ping pong, and iso reaction sequences as well as values for the kinetic constants.

Purification and properties of beta-N-acetyl-D-hexosaminidase from boar seminal plasma.

beta-N-Acetyl-D-hexosaminidase has been purified ca. 190-fold to homogeneity from boar seminal plasma. It catalyzed the hydrolysis of the p-nitrophenyl-N-acetyl derivatives of both beta-D-glucosaminide and beta-D-galactosaminide but was inactive with the o- or p-nitrophenyl glycosides of other monosaccharides.

The inactivation of thymidylate synthase by periodate.

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of IO4- at 0 degree C. Nearly complete inhibition resulted when the IO4- concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO4- was in excess.

Oxidation of thymidylate synthase by inorganic compounds.

Thymidylate synthase from methotrexate-resistant Lactobacillus casei was rapidly and completely inactivated by low concentrations of permanganate, periodate, or potassium triiodide at 0 degree C. The enzyme was not inactivated to any appreciable extent by iodate, iodide, ferricyanate, iodosobenzoate, or hydrogen peroxide. The inactivation by permanganate was retarded by the substrate 2'-deoxyuridylate and, to a lesser extent, by phosphate.

Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate.

The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al.

The inactivation of thymidylate synthase by diethyl pyrocarbonate.

Thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.

Kinetics of thymidylate synthase inhibition by disulfides.

The inactivation of thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed.

Nutritional alteration of the fatty acid composition of a thermophilic Bacillus species.

The fatty acid composition of a thermophilic Bacillus sp. was altered by the addition of isobutyrate, isovalerate, alpha-methylbutyrate, leucine, and isoleucine to the growth medium. With isobutyrate, 81% of the fatty acids had 16 carbon atoms and 79% were iso-fatty acids with an even number of carbon atoms.

Fatty acid composition of lipid extracts of a thermophilic Bacillus species.

Fatty acids having 16 or 17 carbon atoms accounted for over 80% of the fatty acids produced by a thermophilic Bacillus species. Under most conditions, branched-chain fatty acids were more abundant than normal fatty acids. The proportion of unsaturated fatty acids varied inversely with the growth temperature and was never greater than 14%.

Occurrence of isocitrate lyase in a thermophilic bacillus species.

A thermophilic, sporeforming bacterium has been isolated from soil on a medium containing acetate as a carbon source. This organism is similar to Bacillus stearothermophilus in most respects but differs in its inability to hydrolyze starch. Isocitrate lyase is present in cell-free extracts of organisms grown in a medium with acetate as a carbon source.