Ryosuke Suzuno

Crystallization of concanavalins A and B and canavalin from Japanese jack beans

Abstract A purification and crystallization method for concanavalins A and B and canavalin from Japanese jack beans is described. Concanavalin A was identified with a specific protein of jack beans, which had been named provisionally “Protein J,” by comparing electrophoretic mobility and reactions with serum proteins. Canavalin was identified with “Protein A,” which migrated toward the cathode and did not react with serum proteins.

Separation of monoclonal antibodies from antihapten antisera by two-dimensional affinity electrophoresis

A high-resolution two-dimensional affinity electrophoresis (2D-AEP) method was developed, using capillary polyacrylamide gel (PAG) isoelectric focusing in the first and slab PAG affinity electrophoresis in the second direction. Using this method, anti-hapten antibodies were separated into a number of monoclonal antibody [immunoglobulin G (IgG)] families, each of which is composed of several IgG spots having an identical affinity to the hapten but different isoelectric points. 2D-AEP may offer a powerful tool for solving fundamental problems in immunochemistry such as antibody heterogeneity, its hapten binding specificity and antigen-dependent somatic mutation.

Trypsin inhibitors and chymotrypsin inhibitors in the sera of some animals.

Abstract 1. 1. The sera of human, cattle, sheep, pig, horse, rabbit, rat, guinea pig, dog, cat, tortoise, frog, bull frog, carp, and eel were separated on paper electrophoresis. The localization of trypsin and chymotrypsin inhibitors were observed with the serum protein fractions, using anilide substrates. 2. 2. The localization curves were compared with the cross diagrams of the sera against trypsin and chymotrypsin, except tortoise. 3. 3. Bovine α2-globulin fraction was found to activate the hydrolysis of benzyol - spl - tryosine -p- nitroanilide by chymotrypsin.

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.