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D G Priest

Publications and source records attributed to D G Priest.

At least 55 records · Page 3Linked to original sources
PubMed1986

Micellar high-performance liquid chromatographic determination of folylpolyglutamate hydrolase activity.

A rapid and sensitive method for the quantitative determination of folylpolyglutamate hydrolase activity in crude tissue extracts was developed. The procedure is based on high-performance liquid chromatographic separation of folate analogue mono- and polyglutamates on a reversed-phase column using sodium dodecyl sulfate in water as the mobile phase. Interfering substances in tissue extracts were removed by gel filtration on centrifugally-eluted mini-columns of Sephadex G-25 prior to incubation of polyglutamate substrate with tissue extract hydrolase. Reactions were terminated by denaturation of the enzyme in sodium dodecyl sulfate, which subsequently served as the micellar solvent system for chromatographic separation of substrate from reaction products.

Animals↗
PubMed1986

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate.

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate is described based on the stable ternary complex formed from methylenetetrahydrofolate, tritiated fluorodeoxyuridylate and thymidylate synthase. Tissue extract 10-formyltetrahydrofolate is deacylated with 10-formyltetrahydrofolate deacylase and the tetrahydrofolate formed is converted to methylenetetrahydrofolate using formaldehyde. Mouse tissue 10-formyltetrahydrofolate levels and their stability to extraction procedures are described.

Animals↗
PubMed1986

Effects of methotrexate on folates in Krebs ascites and L1210 murine leukemia cells.

Changes in reduced folates upon exposure of Krebs ascites cells and L1210 murine leukemia cells to methotrexate (MTX) have been measured by stoichiometric entrapment of tissue methylenetetrahydrofolate into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxy-uridine-5'-monophosphate. Tetrahydrofolate and 5-methyltetrahydrofolate were determined after conversion to methylenetetrahydrofolate. In both tumor cell lines, treatment with methotrexate at levels which had little effect on methylenetetrahydrofolate and tetrahydrofolate concentrations resulted in nearly complete elimination of the methyltetrahydrofolate pool. Thus, an initial effect of methotrexate on folate metabolism appears to be on methyltetrahydrofolate.

Animals↗
PubMed1986

Biochemical basis for cisplatin and 5-fluorouracil synergism in human ovarian carcinoma cells.

The human ovarian cell line A2780 was exposed to either cisplatin (10 microM) or 5-fluorouracil (5FUra) (5 microM) for 1 hr. Cytotoxicity was less than 14% with either agent alone. Cisplatin (10 microM) and 5FUra (5 microM) in combination for 1 hr caused a 76% reduction in cell growth. Thymidine (dThd, 10 microM), if given concomitantly with the combination of cisplatin and 5FUra, completely protected the tumor cells. A 30-min exposure to cisplatin increased the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate 2.5-fold. The capacity of intact cells to form 5-fluorodeoxyuridylate (FdUMP)-thymidylate (dTMP) synthase complex when incubated with fluorodeoxyuridine (FdUrd) was enhanced 2.5-fold when the cells were pretreated with cisplatin. These experiments demonstrate that cisplatin can increase the availability of the reduced folate necessary for tight binding of FdUMP to dTMP synthase, thus enhancing the cytotoxicity of the cisplatin and 5FUra combination.

Carcinoma↗
PubMed1986

Effects of growth rate and methotrexate on folate polyglutamates and folylpolyglutamate hydrolase activity in Krebs ascites cells.

The activity of folylpolyglutamate hydrolase was measured throughout intraperitoneal growth of Krebs ascites cells in mice and after exposure to methotrexate. Hydrolase activity was lowest during the log phase of growth. Methotrexate administered intraperitoneally during log growth caused a dose- and time-dependent increase in hydrolase activity. Modest changes were observed in endogenous folate polyglutamate chain length distributions throughout growth and upon exposure to methotrexate, but these changes could not be correlated with hydrolase activity.

Animals↗
PubMed1986

Hydrolytic cleavage of methotrexate gamma-polyglutamates by folylpolyglutamyl hydrolase derived from various tumors and normal tissues of the mouse.

Folylpolyglutamate hydrolase (folyl hydrolase) activity derived from murine tumors and various normal tissues was measured by means of high performance liquid chromatography using methotrexate polyglutamates as substrate. Enzyme-mediated hydrolysis was considerably greater (10-20-fold) on a specific activity basis in extracts from all normal mouse tissues (kidney greater than bone marrow greater than small intestine approximately equal to liver) than from tumor cells (Sarcoma 180 greater than Ehrlich approximately equal to L1210 cells). Enzyme preparations from purified absorptive and crypt cell epithelium from mouse small intestine exhibited comparable levels of specific activity and were greater than that derived from the total organ. Folyl hydrolase from mouse kidney showed mixed endo- and exopeptidase activity while that derived from all other normal tissues and tumor cells was consistent with endopeptidase activity. Levels of cell-free folyl hydrolase activity derived from tumor cells varied substantially with the phase of growth in vivo. Also, levels were appreciably lower from the same cells grown in vitro. Hydrolysis by crude or partially purified enzyme preparations from mouse small intestine or tumor cells conformed to Michaelis-Menten kinetics (single saturable component). Rates of hydrolysis and Km values were proportional to gamma-glutamyl chain length in the case of L1210 cell-derived enzyme but not for enzyme derived from small intestine. Km values derived for 4-amino-10-methylpteroyldiglutamate were the same [Km = 80.4 +/- 9 (SE) microM] for small intestine and L1210 cells. However, with 4-amino-10-methylpteroyltetraglutamate Km values were 3-fold lower for tumor cell preparations and 8-fold lower for preparations derived from small intestine. Fourfold lower Km values for 4-amino-10-methylpteroyldiglutamate were obtained with enzyme derived from Sarcoma 180 cells as compared to the enzyme from L1210 or intestinal cells. Varying levels of folyl hydrolase activity for methotrexate polyglutamates in cell-free preparations from different tumor cells appeared to reflect differences in in situ hydrolytic activity shown for the same substrate when internalized. The relevance of these results to antifolate pharmacology and, specifically, to a role for polyglutamates of 4-aminofolate compounds in determining cytotoxicity and selective antitumor activity of these agents is discussed.

Animals↗
PubMed1985

Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms.

The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this cofactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodeoxyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methyltetrahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determined electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglutamate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates in extracts of normal mouse liver tissue.

Animals↗
PubMed1983

Response of methylenetetrahydrofolate levels to methotrexate in Krebs ascites cells.

Levels of methylenetetrahydrofolate in Krebs ascites cells subsequent to transplantation and the effects of methotrexate on these levels have been measured. To directly measure methylenetetrahydrofolate in tissue extracts, the cofactor was incorporated into a covalent ternary complex with thymidylate synthase and 3H-labelled fluorodeoxyuridine monophosphate. A 3-4-day lag preceded rapid growth of the tumour cells, and this same kinetic behaviour was observed for methylenetetrahydrofolate levels in the tumour cells. Liver and kidney tissue from the same animals also showed an increase in methylenetetrahydrofolate over the same time period. The impact of methotrexate on methylenetetrahydrofolate in the tumour cells depended upon concentration and the post-transplantation time at which treatment was initiated. Levels of methylenetetrahydrofolate in the tumour cells were most sensitive to the drug at the beginning of the rapid growth phase and were more sensitive to a given level of methotrexate in the presence of phospholipids. A slight but significant increase in methylenetetrahydrofolate occurred in some cases in response to the presence of methotrexate.

Animals↗
PubMed1983

Response of mouse hepatoma cell methylenetetrahydrofolate polyglutamates to folate deprivation.

The behavior of methylenetetrahydrofolate polyglutamate conjugates in cultured mouse hepatoma cells which were starved of folate has been investigated. Folate deprivation caused methylenetetrahydrofolate levels to decrease an order of magnitude. This diminished pool consisted essentially completely of the octaglutamate form. Replenishment of the media with folate caused a slow recovery to normal levels of methylenetetrahydrofolate with undetectable quantities of shorter chain length polyglutamates observable during recovery. Leucovorin, on the other hand, caused a much more rapid recovery to normal levels and gave rise to the early appearance of short chain length polyglutamate intermediates.

Animals↗
PubMed1983

Evaluation of folylpolyglutamates by electrophoretic separation of fluorodeoxyuridylate-thymidylate synthase-methylenetetrahydrofolate complexes.

The chain length of specific reduced folylpolyglutamates has been estimated by incorporation of the 5,10-methylenetetrahydrofolate form of the cofactor into a stable ternary complex with L. casei thymidylate synthase and tritiated fluorodeoxyuridylate followed by electrophoretic separation based on charge differences in complexed polyglutamates. The method is also applicable to tetrahydrofolate polyglutamates after conversion to the active cofactor form by introduction of formaldehyde. The method can be used to analyze less than one pmole of folylpolyglutamate and can be applied to evaluation of tissue polyglutamates or to monitor relevant enzyme catalyzed reactions.

Animals↗
PubMed1982

Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, muscle and brain.

The folylpolyglutamate hydrolase activities of mouse liver, kidney, muscle and brain were examined by incorporation of methylenetetrahydrofolate polyglutamate reaction products into a stable ternary complex with tritiated fluorodeoxyuridylate and L. casei thymidylate synthetase. Complexes were separated electrophoretically on the basis of charge associated with the polyglutamyl moieties to determine distribution of chain lengths throughout the time course of the reaction. Tissue folylpolyglutamate hydrolase activities were allowed to utilize endogenous folylpolyglutamate as substrates by incubating crude tissue extracts at pH 7.4 ang pH 4.5. Kidney and muscle contained relatively reactive hydrolases which were capable of generating intermediates of essentially all chain lengths from folylpentaglutamate, the predominant endogenous species. The relatively low activity in brain also gave rise to all possible intermediates. Liver contained a high concentration of methylenetetrahydrofolate but little hydrolase activity. The activity present in liver gave rise to essentially no intermediates but yielded only the monoglutamate form of the cofactor. When purified lysosomal preparations from liver and kidney were allowed to react with synthetic folylpolyglutamates, the same specificity with regard to reaction products was observed as with endogenous substrates.

Animals↗
PubMed1982

Cell cycle patterns of thymidylate synthetase and 5,10-methylenetetrahydrofolate polyglutamates in cultured mouse hepatoma cells.

The regulation of thymidylate synthetase activity was investigated throughout the first cell cycle after release from an isoleucine block in synchronous cultures of mouse hepatoma (Hepa) cells. Activity in cell extracts increased with the onset of S phase and the increased activity was attributed to a parallel increase in enzyme concentration as determined by titration with tritiated fluorodeoxyuridylate. The polyglutamate chain length of reduced folate cofactors, which could also influence thymidylate synthetase activity, was unchanged.

Animals↗