Folates binding activity

There is very little urinary loss of folate, some 5 to 10 nmol of microbiologically active material per day. Not only is most folate in plasma bound to proteins (either folate binding protein for unsubstituted folate or albumin for methyl-tetrahydrofolate), and thus protected from glomerular filtration, but also the renal brush border has a high concentration of folate binding protein that acts to reabsorb any that is filtered. 

Folate binding protein has been prepared from goat s milk by adsorption to a methotrexate-Sepharose column [94]. The folate binding protein was then coupled to cyanogen bromide activated Sepharose and used in the purification of folates and for the removal of endogenous folates from reaction mixtures. Cortisol hemisuccinate coupled to an amino Sepharose has been used to purify glucocorticoid binding protein(s) from rat liver... 

Receptor-binding activity of the protein-folate conjugate can be determined by competitive displacement of a known quantity of [3H]-labeled folic acid (available from NEN Life Science Products, Boston, MA) from receptor-expressing cells. Alternatively, if the protein can be labeled with a fluorescent probe (e.g., fluorescein isothiocyanate), its uptake by folate receptor-bearing cells can be monitored by fluorescence microscopy (8). 

Tetrahydrofolic acid, THF, folate-H coenzyme F 5,6,7,8-tetrahydropteroylglutamic acid, the coenzyme responsible for the binding, activation and transfer of all active one carbon units, with the exception of carbon dioxide (the F in coenzyme F stands for formylation). M, 445.4 (after chemical preparation involving acetic add, it retains two molecules CH3-COOH and has M, 565.4), 298 nm in 0.01 M... 

Figure 3. Three of the modules comprising methionine synthase. At the top center is the Bi2- binding fragment [651-896], a structure with two domains, one a four-helix bundle that serves to cap the cofactor, and the other an a/p fold that interacts with the lower face of the corrin macrocycle and binds the nucleotide tail of cobalamin. Measurements of the rates of photolysis of the C0-CH3 bond indicate that the cap domain covers the upper face of the corrin in the substrate-free form of the intact enzyme (7). On the lower right is the activation domain [897-1227] with bound AdoMet. This helmet-shaped single domain is an unusual fold with no resemblance to other well-characterized AdoMet-binding domains (8). On the lower left is the structure of the methyltransferase AcsE from Moorella thermoaceticum, which we take as a surrogate for the folate-binding domain of MetH.

A plot of the binding activity of each folate derivative on the lAC column with respect to that of folic acid is shown in Figure 23.12. This demonstrates that our lAC method for the determination of folic acid presents a high specificity toward its detection and thus very low cross-reactivity was observed (Ho et al. 2009b). 

Figure 23.12 Plot of the binding activity of each folate derivative on the lAC column with respect to that of folic acid.

J. Holm, S. I. Hansen, and J. Lyngbye. High affinity folate finding in chronic myelogenous leukaemia serum, relationship of binding characteristics to activity of disease and binder concentration. Scand. J. Haematol. 26 153-155 (1981). 

The carbon-donating cofactor for this reaction is N, N methylenetetrahydrofolate, which is converted to dihydrofolate. The reduced folate cofactor occupies an allosteric site on thymidylate synthetase, which allows for the covalent binding of 5-FdUMP to the active site of the enzyme. 

Many drugs interact with folate to affect its absorption, antagonize its biochemical activity, or increase its loss from the body. These drugs include ethanol, phenytoin, and oral contraceptives. Salicylates can compete with foUc acid for plasma protein binding. Methotrexate, a cytotoxic agent, is a folate antagonist that inhibits the biosynthesis of this coenzyme. 

---