Distribution and characterization of angiotensin-converting enzyme-like activity in tissues of the blue crab Callinectes sapidus.

Angiotensin-converting enzyme-like activity (ACELA) was determined in tissue homogenates of the blue crab, Callinectes sapidus, using the synthetic substrate hippuryl-histidyl-leucine (Hip-His-Leu) and the specific inhibitor, captopril. ACELA was highest in gill homogenates followed by the hepatopancreas and hemolymph with specific activities of 1.69, 0.37 and 0.10 nmol/min/mg protein, respectively. Gill enzyme activity was membrane-associated and no difference in activity was noted between anterior and posterior gills. The enzyme preparation from gill membranes was activated by chloride and had a Km of 4.1 +/- 0.4 mmol and a Vmax of 39.5 +/- 2.0 nmol/min/mg protein. The enzyme was strongly inhibited by captopril and lisinopril with IC50 s of 3.8 x 10(-8) and 2.6 x 10(-8) M. The enzyme was less strongly inhibited by angiotensin II and SQ-20881 with IC50 s of 5.1 x 10(-5) and 2.5 x 10(-6) M, respectively.

Direct injection assay of angiotensin-converting enzyme by high-performance liquid chromatography using a shielded hydrophobic phase column.

A rapid and sensitive method for the determination of angiotensin-converting enzyme activity in serum and tissue extracts is described. The procedure is based on the high-performance liquid chromatographic separation of the synthetic substrate hippuryl-L-histidyl-L-leucine from the hydrolysis product hippuric acid. The separation is accomplished by direct injection of biological assay mixtures onto a shielded hydrophobic phase column with isocratic elution.

Role of methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured L1210 cells.

The effect of low methotrexate levels on methylenetetrahydrofolate and four other reduced folate pools in cultured L1210 cells has been examined over a 48-h period. Media folate levels and methotrexate were used to alter intracellular levels of reduced folates, and the distribution among individual reduced folates, so that they could be evaluated in terms of their effects on thymidylate synthesis and cell proliferation. Over the media folate concentration range of 0.25-50 microM, growth rate and thymidylate synthesis remained essentially unchanged while total intracellular reduced folates, determined from the summation of the five individual pools measured, increased approximately 25-fold. The 5-methyltetrahydrofolate and 10-formyltetrahydrofolate pools accounted for over 90% of the total reduced folate at the highest media folate level, while low media folate resulted in a much more equal distribution among the five reduced folates examined. Methotrexate, over the concentration range of 0.25-30 nM, caused extensive growth and intracellular thymidylate synthesis inhibition at media folate levels used in RPMI 1640 media (2.5 microM) and lower. However, growth inhibition was much less at the highest media folate level used, and thymidylate synthesis was not inhibited to a statistically significant extent. Intracellular reduced folates also responded differently to methotrexate depending upon the level of media folate. Depletion of the thymidylate synthase substrate, methylenetetrahydrofolate, could not account for diminished growth or thymidylate synthesis inhibition, since at 0.25 and 2.5 microM media folate no depletion occurred in response to methotrexate and only slight depletion was observed at 50 microM media folate. Dihydrofolate showed a tendency to increase at each of the media folate levels used with the least increase at the highest folate level. However, the ratio of dihydrofolate to total reduced folates was quantitatively most consistent with thymidylate synthesis and growth inhibition results.

Enhancement of polyacrylamide gel slice dissolution in hydrogen peroxide by cupric sulfate.

An improved method is described for quantitation of radio-labelled protein by scintillation counting after polyacrylamide gel electrophoresis. The method is based upon copper catalyzed dissolution of gel slices in hydrogen peroxide under ambient conditions. Complete dissolution of gel sections was accomplished by incubation at 25 degrees C in 30% H2O2 that contained 0.9 mM CuSO4. Recovery of tritiated protein was greater than 90% under these conditions while in the absence of CuSO4 recovery was less than 50%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Electrophoretic identification of poly-gamma-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes.

A method has been developed to characterize the poly-gamma-glutamates of 5,10-methylenetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[3H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-gamma-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.