Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase

Sodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base.     

Stereoselectivity of sodium borohydride reduction of saturated steroidal ketones utilizing conditions of Luche reduction

A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.     

Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis.

Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.     

Preparation of N-nosyl-3,4-epimines derived from levoglucosan by sodium borohydride reduction

Starting from 1,6-anhydro-beta-d-glucopyranose 1 (levoglucosan), N-o-nitrobenzenesulfonyl (nosyl) 3,4-epimino derivatives with d-allo, d-galacto, and d-talo configurations have been prepared via NaBH(4) reduction of suitably substituted azido tosylates. The benefits and limitations of this method over the classical LiAlH(4) reduction method are discussed.     

The kinetics of suppression of proliferation of oxidized murine lymphoid cells by sodium borohydride reduction.

Murine splenic lymphoid cells are stimulated to proliferate following mild oxidation with sodium periodate. To assess the class of cells responding, we used periodate treatment alone or in association with concanavalin A, a T-cell mitogen, or lipopolysaccharide (LPS), primarily a B-cell mitogen. Brief periodate treatment followed by culturing with concanavalin A gave no additive proliferative response to that seen using concanavalin A alone, while culturing periodate-treated cells with LPS gave approximately an additive response. Furthermore, periodate failed to stimulate spleen cells from neonatally thymectomized mice while LPS produced significant stimulation of proliferation, suggesting that periodate is stimulating a class of T lymphoid cells or a subpopulation of T cells. Studies were performed to determine an optimal concentration of borohydride which would suppress proliferation in lymphoid cells initially oxidized with periodate. It was observed that 2 mM borohydride would suppress proliferation of oxidized cells yet permit a normal response of these cells to another T-cell mitogen, concanavalin A. Higher concentrations of borohydride, from 3 to 5 mM, would also suppress proliferation of oxidized cells but would interfere with the ability of these cells to respond to concanavalin A, perhaps due to cell damage. Studies were performed to determine when it was possible to suppress periodate-induced mitogenesis by reducing with borohydride at various times after the initial oxidation. It was observed that 2 mM borohydride treatment could suppress stimulation through 8 hr after the original periodate oxidation and that from 12 hr through 20 hr after the initial periodate oxidation, borohydride was incapable of inhibiting proliferation. Additional studies demonstrate that optimal mitogenesis induced by periodate or concanavalin A is contingent upon a serum factor.     

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.     

The alkaline cleavage and borohydride reduction of cartilage proteoglycan

A method for the rapid isolation and purification of proteoglycan by using neutral solutions of LiBr for extraction and density-gradient centrifugation is described. The effect of 0.5m-KOH on isolated proteoglycan has been studied by using NaB(3)H(4) to reduce and label the chondroitin sulphate chains released. This study has established: (a) that at least 95% of the chondroitin sulphate chains are attached to the proteoglycan by alkali-labile bonds between xylose and serine; (b) that random degradation of the chondroitin sulphate chains does not occur to any significant extent; (c) that the method is convenient for the determination of polysaccharide number-average molecular weights.