Akira Kuwahara

Study of the interaction between phosphorylase and hydrophobic groups by means of affinity electrophoresis

A homologous series of water-soluble alkyl-dextrans varying in the length of their hydrocarbon side-chain [-NH-(CH2)n-CH3; n = 1-5] were synthesized. When alkyl-dextrans were entrapped in polyacrylamide gel, the electrophoretic mobility of phosphorylase was retarded by hydrophobic interaction between phosphorylase and the immobilized alkyl groups. The dissociation constants of rabbit brain phosphorylase, rabbit skeletal muscle phosphorylase a and b and potato glycogen and starch phosphorylases were calculated from the extent of the retardation of mobility as a function of the concentration of the alkyl groups. As the length of the hydrocarbon side-chains of alkyl groups increased, the affinity of the phosphorylases for the alkyl groups increased. The introduction of a hydroxyl or an amino group at the terminal position of the hydrocarbon side-chain diminished the affinity.

An exploration of the binding site of aldolase using alkanediol monoglycolate bisphosphoric esters

Alkanediol monoglycolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The binding constants estimated from difference spectra correlate well with Ki values for the substrate analogues. Propanediol monoglycolate bisphosphoric ester protected aldolase from inactivation by 1,2-cyclohexanedione, which preferentially attacks arginine-55. However, propanol phosphate had little protective effect. The synthesized phosphate compounds protected the enzyme against inactivation by trypsin, and also against spontaneous denaturation. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site, which tends to keep the distance constant between the two phosphate-binding sites for the open-chain form of fructose 1,6-bisphosphate, and stabilize the natural conformation of the enzyme. Both arginine-55 and lysine-146 are shown to participate in the phosphate-binding site for the C-1-phosphate of fructose 1,6-bisphosphate.

Two-Dimensional Affinity Electrophoresis: Its Application to Separation of Individual Immunoglobulins from Heterogeneous Antibodies

Abstract We have developed a two-dimensional affinity electrophoresis carrying in the first direction by isoelectric focusing (IEF) and in the second direction by affinity electrophoresis (AEP). Using Dnp- and Tnp-polyacrylamide conjugates as affinity ligands for AEP, rabbit anti-Dnp antibodies were separated. They consisted of several groups of monoclonal IgG families. Dissociation constants of the individual IgG spots to the Dnp- and Tnp-haptens calculated from the affinity patterns ranged from 2.0 × 10 -6 M to 1.1 × 10 -4 M and from 1.4 × 10 -5 M to 1.3 × 10 -4 M, respectively.