Saburo Funahashi

Phytase (myoinositolhexaphosphate phosphohydrolase) from Wheat Bran

Pure inositol tetra- and hexaphosphate were isolated as the main components of rice phytin by Dowex 1 column chromatography. Phytase of wheat bran was purified more than 1,500 folds. This phytase preparation showed 1) broad substrate specificity, 2) pH optimum at about 5.0, 3) no activation by magnesium ion, and 4) inhibition by fluoride ion for all the substrates tested; and was concluded as nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The effects of metalic ions, thiol reagents, chelating agents, arsenate, alkylarsenate and hydrolysis products for this enzyme were studied. Michaelis constants and activation energy by this enzvme to various substrates were also calculated.

Phytase from Wheat Bran

myo-Inositol mono-, di-, tri-, tetra-, and pentaphosphate were prepared by enzymic hydrolysis of myo-inositol hexaphosphate with a 1,500-fold purified phytase preparation from wheat bran and the subsequent Dowex 1 column chromatography. Relative initial rates of hydrolysis of these inositol phosphates by phytase were nearly the same each other and the activation energy of hydrolysis was about 11,000 cal. per mole for all these substrates. Km values did not vary widely with the substrates. The hydrolysis of inositol phosphates proceeded in a complicated way, except inositol monophosphate, where the reaction was of the first order. The enzyme hydrolyzed the substrates in the manner that removed phosphate group of them one by one. When mixed substrate was used the enzyme showed a preferential attack on the highest member of the phosphates present. From the mixed substrate test, it was concluded that wheat bran phytase is a single enzyme.

Allantoinase from Mung Bean Seedlings

Mung bean allantoinase was purified sixty folds by calcium phosphate gel treatment, ammonium sulfate fractionation and acetone precipitation. The purified allantoinase hydro-lyzed allantoin to allantoic acid almost completely and the reaction had a broad pH optimum between 7.5 and 8.3. The accumulation of allantoic acid during the germination of mung bean was also noted. The allantoic acid content of seedlings was higher in hypocotyl than in leaf and root.

Pantothenic Acid and It's Derivatives

A new and improved method is described for synthesis of cystamine hydrochloride. β-Bromoethylamine hydrobromide [II]. was treated with equimolecular amount of thiourea in ethanol to give β-aminoethylisothiouronium bromide [III] quantitatively. The bromide was subjected to alkaline hydrolysis, the reaction mixture was neutralized with sulfuric acid, oxidised with iodine, and the cystamine hydroiodide was converted to hydrochloride thus cystamine hydrochloride was obtained as white crystals. Overall yield was more than 60 per cent from monoethanolamine [I].

The Metabolism of Germinating Plants

1. A CoA preparation of 2.2 units per mg. was isolated from soybean seedlings. 2 The preparation played a part in vitro both in the acetylation of sulfanilamide of p-aminoazobenzene and in the citrate sysnthesis from oxalacetate plus acetate, with pigeon liver enzyme as a catalist. 3. The preparation was inactivated in the presence of pigeon liver enzyme plus intestinal phosphatase in a similar manner as reported in the case of CoA sample from animal sources. 4. The occurrence of CoA in soybean seedlings may be taken as one of the evidences that TCA cycle was a major metabolic pathway of this plant tissue.