Comparative analysis of RBC membrane fatty acids, proteins and glycophorin in patients with heterozygous beta thalassemia and iron deficiency anemia.

Membrane lipid and protein composition was compared in erythrocytes from iron deficiency anemia (IDA) and heterozygous beta thalassemia patients. The study was planned to correlate the influence of iron deficiency with the intrinsic defect of the heterozygous condition on the membrane structural integrity as well as to investigate whether there are differences in membrane changes between the two conditions. Results indicate high levels of saturated fatty acids and low unsaturated fatty acids in both disorders although arachidonic acid and the unsaturation index were lower in heterozygous thalassemia than IDA. Nevertheless, neither of the conditions provoked any alterations in membrane protein or glycophorin suggesting alterations in the lipid moiety only. Present findings indicate that irrespective to the etiology, both, iron deficiency and the heterozygous condition show a common pattern of lipid derangement, which may in turn result in increased membrane rigidity and decreased cellular deformability.

Hydrolysis of capecitabine to 5'-deoxy-5-fluorocytidine by human carboxylesterases and inhibition by loperamide.

Capecitabine is an oral prodrug of 5-fluorouracil that is indicated for the treatment of breast and colorectal cancers. A three-step in vivo-targeted activation process requiring carboxylesterases, cytidine deaminase, and thymidine phosphorylase converts capecitabine to 5-fluorouracil. Carboxylesterases hydrolyze capecitabine's carbamate side chain to form 5'-deoxy-5-fluorocytidine (5'-DFCR). This study examines the steady-state kinetics of recombinant human carboxylesterase isozymes carboxylesterase (CES) 1A1, CES2, and CES3 for hydrolysis of capecitabine with a liquid chromatography/mass spectroscopy assay. Additionally, a spectrophotometric screening assay was utilized to identify drugs that may inhibit carboxylesterase activation of capecitabine. CES1A1 and CES2 hydrolyze capecitabine to a similar extent, with catalytic efficiencies of 14.7 and 12.9 min(-1) mM(-1), respectively. Little catalytic activity is detected for CES3 with capecitabine. Northern blot analysis indicates that relative expression in intestinal tissue is CES2 > CES1A1 > CES3. Hence, intestinal activation of capecitabine may contribute to its efficacy in colon cancer and toxic diarrhea associated with the agent. Loperamide is a strong inhibitor of CES2, with a K(i) of 1.5 muM, but it only weakly inhibits CES1A1 (IC(50) = 0.44 mM). Inhibition of CES2 in the gastrointestinal tract by loperamide may reduce local formation of 5'-DFCR. Both CES1A1 and CES2 are responsible for the activation of capecitabine, whereas CES3 plays little role in 5'-DFCR formation.

Comparative analysis of RBC membrane lipids in thalassemia, and iron deficiency anemia in relation to hypochromia and oxidant injury.

The effect of an intrinsic defect in the red cell and pronounced hypochromia on oxidative damage to RBC membrane lipids was compared in beta-thalassemia and iron deficiency anemia (IDA), which have a varied etiology but equivalent low hemogiobin content. The study was planned to correlate the etiology of the disorders to the severity of lipid imbalance and RBC hemolysis in membranes of both the conditions. Results indicated a fall of lysophosphatidylcholine(LPC), phosphatidylethanolamine(PE) and the unsaturated to saturated fatty acid ratio in both conditions, while phosphatidylcholine(PC) increased only in thalassemia. However, irrespective of the disease, sphingomyelin(SM), total cholesterol and phospholipid levels elevated and the hydrogen peroxide stress test indicated increased susceptibility of both pathologic RBCs to peroxidation. Present findings indicate that IDA and thalassemla, allow for considerable amounts of oxidative damage to membrane lipids, irrespective of their etiologles, and thus point hypochromia as an important contributor for inducing lipid imbalance and RBC hemolysis.

Identification of three key active site residues in the C-terminal domain of human recombinant folylpoly-gamma-glutamate synthetase by site-directed mutagenesis.

Three cysteines in human recombinant folylpoly-gamma-glutamate synthetase (FPGS) that were reactive with iodoacetamide were located in peptides that were highly conserved across species; the functions of two of these peptides, located in the C-terminal domain, were studied by site-directed mutagenesis. When cDNAs containing mutations in each conserved ionic residue on these peptides were transfected into AUXB1 cells, which lack endogenous FPGS activity, one mutant (D335A) did not complement the auxotrophy, and another (R377A) allowed only minimal growth. FPGS activity could not be detected in insect cells expressing abundant levels of these two mutant proteins from recombinant baculoviruses nor from a virus encoding an H338A mutant FPGS. Kinetic analysis of the purified proteins demonstrated that each of these three mutants was quite different from the others. The major kinetic change detected for the H338A mutation was a 600-fold increase in the K(m) for glutamic acid. For the D335A mutation, the binding of all three substrates (aminopterin, ATP, and glutamic acid) was affected. For R377A, the K(m) for glutamic acid was increased by 1500-fold, and there was an approximately 20-fold decrease in the k(cat) of the reaction. The binding of the K(+) ion, a known activator of FPGS, was affected by the D335A and H338A mutations. We conclude that these three amino acids participate in the alignment of glutamic acid in the active site and that Arg-377 is also involved in the mechanism of the reaction.

Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase.

The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations. rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximately 50-fold tighter than previously reported, with Kd and Ki values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor. The binding of the hexaglutamate of DDATHF to rmGARFT had Kd and Ki values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono- or hexaglutamate substrate did not yield different values for the Ki for the hexaglutamate form of DDATHF, in contradiction with previous reports. Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low Km values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.