Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte.

The phytohemagglutinin (PHA)-stimulated human lymphocytes demonstrated trace or no activity (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.     

Principal component regression analysis applied in structure-activity relationships. 1. selective dihydrofolic acid reductase inhibitors

The principal component regression analysis is used when the predicting variables correlate significantly against the regressor variables and, when a collinearity and multicollinearity exist among regressor variables. The example adapted from enzymology shows that no loss of information occurs with respect to the chemical constants in comparison with the reduced model of multivariate regression analysis.     

The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase

The nucleotide sequence of the human dihydrofolic acid reductase (DHFR) reading frame has been derived from the analysis of human DHFR cDNA. This sequence and the corresponding amino acid sequence have been compared with those available for the enzyme and its coding segment from other organisms. There is an 89% nucleotide sequence homology between the human DHFR reading frame and the mouse coding sequence. Furthermore, amino acid-sequence homologies of 74%, 81% and 89% has been found between human DHFR and chicken, bovine and mouse DHFR, respectively.     

Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.     

Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate.

The acidic dissociation constants in the range HO--1.5 to pH 7 of folic acid, dihydrofolic acid, methopterin (N(10)methylfolic acid), and methotrexate have been measured by potentiometric and spectrophotometric titrations. Assignment of these dissociations was made by comparison to model compounds, by proton magnetic resonance measurements, and by examination of associated ultraviolet absorbance changes. For folic acid, the dissociation constants are as follows: N(1), pK' 2.35; N(10), pK' 0.20; N(5), pK' greater than -1.5. For dihydrofolic acid: N(5), pK' 3.84; N(1), pK' 1.38; N(10), pK' 0.28. For methotrexate: N(1), pK' 5.71; gamma-carboxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5), boxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5) pK' greater than -1.5. For methopterin: acidic ionization of amide, pK' 7.68; gamma-carboxyl, pK' 4.62; N(1), pK' 2.40; N(10), pK; 0.36; N(5), pK' greater than -1.5. The pK' values were determined directly for the four compounds at 25 degrees near 0.1 ionic strength, or in 0.1 to 4 M HCl for pK ln 0.1 M NaCl.     

Intracellular location of nitrate reductase and nitrite reductase. II. Wheat roots

Approximately 15% of the total nitrite reductase of crude homogenates of wheat roots applied to sucrose gradients was separated with an organelle whose isopycnic density was about 1.22 g·cm⁻³. The activity recovered in the supernatant was thought to be particulate in origin, because similar ratios of activity of isoenzyme 1 and 2 of nitrite reductase were found in both particulate and supernatant fractions. The particle with nitrite reductase activity also contained glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triose phosphate isomerase and NADPH diaphorase. This root particle and whole chloroplasts from leaves had a similar isopycnic density as well as these enzymes, and thus the data suggest that the root particle may be a proplastid.

Nitrate reductase was found only in the supernatant and it was not associated with any of the root organelles.

Mitochondria from wheat roots had an equilibrium density of 1.18 g·cm₋₃ and contained both NAD and NADP glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triosephosphate isomerase and NADPH diaphorase but not nitrite reductase. Microbodies of wheat roots had an equilibrium density of about 1.20 g·cm⁻³ on the sucrose gradient and contained catalase and glycollate oxidase.     

NADH-cytochrome c reductase, succinate cytochrome c reductase and phospholipids.

Phospholipid peroxidation of isolated rat liver inner mitochondrial membranes induced by either ascorbate or cysteine was accompanied by a release of flavins and coenzyme Q. A straight correlation between this release and the alteration of molecular species of phosphatidylcholine and phosphatidylethanolamine containing one saturated and one unsaturated fatty acid has been found. Peroxidation induced on molecular species of phosphatidylcholine and phosphatidylethanolamine containing only unsaturated fatty acids were accompanied by losses in enzyme activities of NADH-cytochrome c reductase and succinate cytochrome c reductase.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

Reconstitution of succinate-Q reductase.

Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome $\text{b-c}{}_1$ complex. The reconstituted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome $\text{c}$ reductase or submitochondrial particles) does not show “low concentration ferricyanide reductase activity” as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms.