James W. Ogilvie

Reaction of tris with aldehydes: Effect of tris on reactions catalyzed by homoserine dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase

Tris buffer was observed to produce an apparent inhibition of the homoserine dehydrogenase (EC 1.1.1.3)-catalyzed reduction of aspartic beta-semialdehyde and an apparent inhibition of the glyceraldehyde phosphate dehydrogenase (EC 1.2.1.9)-catalyzed oxidation of glyceraldehyde 3-phosphate. In each case, the apparent inhibition was found to be due to a lowering of the substrate concentration as a result of a reversible reaction between the free base form of Tris and the substrate, an aldehyde. The product of the reaction was tentatively identified as an imine on the basis of its spectral properties. The inhibition of these two enzymatic reactions by Tris was employed to investigate the kinetics of the reaction of Tris with their substrates. Assuming that these aldehydes exist entirely as the free aldehyde in aqueous solution, equilibrium constants of 369 +/- 12M-1 and 68 +/- 1.5M-1 were determined at 25 degrees C for the reaction of the free base form of Tris with glyceraldehyde 3-phosphate and asparitic beta-semialdehyde, respectively. Correcting for the existence of the hydrated form of glyceraldehyde 3-phosphate in aqueous solution, an equilibrium constant of 1.1-10(4) M-1 was obtained for the reaction of this aldehyde with the free base form of Tris. Forward and reverse direction rate constants for the reaction of Tris with glyceraldehyde 3-phosphate were determined at pH 7.45 and pH 8.5, and both were found to be pH-dependent.

Active enzyme gel chromatography: I. experimental aspects

Transport properties of active enzyme species can be studied effectively by layering a small band of enzyme-containing sample on a gel chromatographic column previously saturated with substrate. The column is optically scanned at successive time intervals to yield profiles representing the appearance of chromophoric product or disappearnce of chromophoric substrate. These profiles permit determination of the specific activity and rate of transport of the active species. Initial studies on mechanic of the technique establish the feasibility of accurately determining transport properties of active enzyme species chromatographed on gel columns. Illustrative results are presented for L-glutamate dehydrogenase and for homoserine dehydrogenase studied in both forward and reverse reactions. It is shown that the partititon cross sections derived from the rates of motion of catalytic activity are the same as those determined by equilibrium saturation experiments which directly measure the degree of partitioning by the protein. These results establish the validity of the technique for a variety of future studies. Active enzyme gel chromatography appears comparable in precision to the active enzyme sedimentation technique at current stages of development.

Cleavage of phosphofructokinase at S-cyanylated cysteine residues

Rabbit muscle phosphofructokinase (E.C. 2.7.1.11; ATP: D-fructose-6-phosphate 1-phosphotransferase) consists of four protomers of 80 000 molecular weight, each of which contains 15--16 sulfhydryl groups. Specific cyanylation of the most reactive sulfhydryl group in each protomer with 2-nitro-5-thio-[14C]cyanobenzoic acid and cleavage of the S-cyanylated protomer yields two fragments--a 14C-labeled fragment of 72 500 molecular weight and an unlabeled fragment of 5400 molecular weight--indicating that the position of the cysteine residue bearing the most reactive sulfhydryl group is approx. 5400 daltons from the amino-terminal end of each protomer. The phosphofructokinase protomer also contains 5--6 sulfhydryl groups that are reactive in the denatured protomer but unreactive in the native protomer. The cleavage fragments, obtained from protomers specifically cyanylated at sulfhydryl groups reactive only in the denatured protomer, indicate that three of the cysteine residues bearing this class of sulfhydryl group occupy positions approx. 5000, 11 500, and 58 000 daltons from the carboxyl-terminal end of each protomer.