Shojiro Nakamura

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10−4, 22 × 10−4, and 13 × 10−4 m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.

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.

Specific reaction of concanavalin-A with sera of various animals.

Abstract 1. 1. Sera of various animals were electrophorezed in one dimension on a square of paper, then the extract of jack beans was applied on a line perpendicular to the zones of serum proteins and electrophorezed in the direction perpendicular to the first run. 2. 2. The line of concanavalin-A contained in the latter formed grooves at the crossings with the zones of serum globulins owing to their reaction, but not with that of serum albumin. The groovedline of concanavalin-A was named the crossing diagram of the serum against concanavalin-A, and was characteristic for each animal species. 3. 3. The reaction of concanavalin-A with a protein seemed to occur only when the latter was glycoprotein, as exemplified by the reaction of haptoglobin. The fact that the serum albumins of mammals and birds did not react with concanavalin-A was ascribed to the absence of carbohydrate moiety. 4. 4. The sera of tortoise, frogs and carp contained also a protein fraction, with resembled the serum albumin of mammals in that they did not react with concanavalin-A, migrated faster than the other fractions and bound bromophernol blue.

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.

Application of the two-dimensional technique to the "crossing" paper electrophoresis of immunological reactions.

Abstract The principle and procedure of two-dimensional crossing paper electrophoresis are described. In applying the technique to the antigen-antibody reaction, electrophoresis of the antiserum alone was carried out in one direction, then the antigen was applied on a line to encounter each fraction of the antiserum and electrophoresis was started in a direction at right angles to the first one. Peaks appeared on the line of antigen owing to the formation of complexes in the regions of antibody proteins contained in the antiserum, thus forming a “crossing diagram”. An anomaly was observed in several cases of the “crossing diagrams” of egg albumin-anti-egg albumin reaction; it was ascribed to mechanical disturbances of the paper surface. A “crossing diagram” of a diphtheria toxoid obtained by initial electrophoresis of the antiserum showed two peaks in the region of T-globulin and one in the region of γ-globulin of the antiserum. Reversing the order of electrophoresis (first toxoid, then antiserum) the “crossing diagram” showed two flat asymmetrical peaks in the γ-globulin zone. In general, the toxoid used must contain more than four antigens, of which two have their antibodies in T-globulin and the other two in γ-globulin. It is suggested that the antibodies have different equivalence zones for the same toxoid.

Distribution of trypsin inhibitors in the egg white proteins of various birds.

Abstract 1. 1. Egg white of bird (chicken, Japanese long-tail fowl, bantam, guinea hen, quil, turkey, peafowl, pheasant, golden pheasant, duck, goose, pigeon, zebra, parrakeet, kite, owl, penguin and ostrich) was separated into fractions by electrophoresis on a square sheet of filter paper. Then bovine trypsin was applied on a line perpendicular to the zones of the fractions and the second electrophoresis was carried out in the direction perpendicular to the first run. The trypsin line formed one or two peaks by the cross with the fractions of the egg white, and was named the cross diagram of the egg white against trypsin. The major peak was ascribed to the crossing of ovomucoid and the second minor one, to that of conalbumin 2. 2. Trypsin-inhibiting activity of each fraction isolated by sectioning the paper electropherogram was measured using benzoylarginine p -nitroanilide. Chymotrypsin-inhibiting activity was measured using benzoyltyrosine p -nitroanilide. 3. 3. The distribution curves of trypsin inhibitors measured enzymatically coincided with the cross diagrams of the egg whites against trypsin in all the cases of which the former were measured. 4. 4. The distribution curve of chymotrypsin was measured only with the egg] whites of the chicken, the quail and the golden pheasant. The curve of the golden pheasant egg white coincided with the cross diagram against trypsin as well as with that against chymotrypsin. But the inhibiting activity toward chymotrypsin was about six times higher than toward trypsin. In other cases, the peak of chymotrypsin inhibitor in the distribution curve corresponded to the minor second peak of the cross diagram against trypsin.