[51] Urease

[51] Urease

378 ENZYMES OF PROTEIN METABOLISM [51] Activators and Inhibitors. Dialysis of cell-free extracts or of acetonedried cells results in preparations w...

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378

ENZYMES OF PROTEIN METABOLISM

[51]

Activators and Inhibitors. Dialysis of cell-free extracts or of acetonedried cells results in preparations which are completely inactive unless supplemented as above with 5-AMP or ADP, Mg ++, and orthophosphate. The Mg ++ requirement can be replaced by Mn ++, Co ++, or Zn ++. When the phosphate requirement is replaced by equivalent amounts of arsenate, neither Mg ++ nor 5-AMP (or ADP) is then required. The enzyme is inhibited by fluoride, which produces 50% inhibition of 10 mg. of S. faecalis acetone-dried cells at 1 × 10-3 M. This inhibitor is effective in the presence or absence of added Mg ++, i.e., on phosphorolysis or arsenolysis. The citrullinase reaction is inhibited by excess ornithine and runs to only 75 to 85% completion unless this product is removed (as by ornithine decarboxylase16). Hg ++ and organic mercurials are potent inhibitors at 1 X 10-4 M or higher; this inhibition is relieved by BAL or GSH. Arsenite, IAA, and DNP at 1.7 X 10-8 M are without effect. Phosphate inhibits the reaction run in the presence of arsenate. Effect of pH. The pH optimum of the cell-free enzyme extract, with either the phosphate or arsenate system, is 6.2 to 6.3. Distribution. The enzyme has been reported to be present in S. faecalis, 11.12.14S. lactis, 13 Pseudomonas,16 and C. perfringens.4

[51] U r e a s e

By JAMES B. SUMNER Jack B e a n M e a l

It is usually possible to purchase satisfactory jack bean meal from American supply houses. The author has purchased jack beans high in urease from Charles Martin, Waldron, Arkansas, or from Ernest Nelson, Route 1, Waldron, Arkansas. The beans can be ground in a Mikro Sample Mill, sold by the Pulverizing Machinery Company of Summit, New Jersey. The beans must be "bone d r y " when ground. Preparation of Crystalline U r e a s e 1

Place 100 g. of jack bean meal, finely powdered and rich in urease, in a 1000-ml. beaker. Add 500 ml. of 32 % c.p. acetone at 28 °. At once stir for 3 or 4 minutes, using preferably a wooden stick. Pour on 32-cm. Whatman No. 1 pleated filter paper. When 100 to 150 ml. of extract has filtered through, place the filtering material in the ice chest at 0 to 5 °, and allow to stay overnight. The next day centrifuge the urease crystals 1j. B. Sumner, J. Biol. Chem. 69, 435 (1926).

[51]

UREASE

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in the filtrate, using a refrigerated centrifuge. Decant the supernatant liquid, and dissolve the urease crystals in water. The water employed for this preparation should be distilled from pyrex glass.

RecrystaUization of Urease The method of Dounce ~ is as follows: Dissolve the crystals of crude urease by adding 3 ml. of water for every 100 g. of jack bean meal. Centrifuge the urease solution practically clear in a refrigerated centrifuge, or else filter and refilter it until it is practically clear in the ice chest. Then for every 20 ml. of urease solution add 1 ml. of 0.5 M citrate buffer, pH 6.0. Next add with stirring 0.2 vol. of pure acetone. Place the preparation in the ice chest. Crystallization of the urease will be complete in about 30 minutes. The crop of urease crystals can be increased by adding acetone, a few drops at a time, until the acetone concentration is about 25 %. Urease Paper Centrifuge down crystals of urease and dissolve in water, using about 10 ml. for the crystals from 100 g. of jack bean meal. Add about 100 mg. of neutral cysteine and 10 mi. of 0.5 M citrate buffer, pH 6.2, and mix. Moisten strips of filter paper with this solution, and dry. Place the dry strips in a brown glass bottle, and stopper. Acting on urea, one square centimeter of urease paper should turn 1 ml. of 0.5 M citrate buffer, pH 6.0, alkaline to phenol red in 6 minutes at 25 °. This urease paper will remain serviceable for several years. About 0.3 sq. cm. will be sufficient for each analysis of blood urea. Estimation of Urease Activity 3 Place 1 ml. of properly diluted crystalline urease in a test tube that is free from heavy metals, immerse this tube in a thermostat bath at 20 °, and allow it to come to this temperature. Then from a 1-ml. pipet blow into the urease solution 1 ml. of 3 % urea, 9.6 % phosphate, pH 7.0, which is at 20 °, starting a stop watch at this time. Mix, and allow to digest for 5 minutes. At this time add 1 ml. of N hydrochloric acid, and mix. Transfer the solution to a 200-ml. volumetric flask, dilute to about 150 ml., and add 10 ml. of Nessler solution. Dilute to mark, mix, and read in a photoelectric colorimeter, using a green glass filter. The milligrams of ammonia nitrogen present represent urease units. The amount present should be 0.2 to 0.5 mg. If crude urease is being analyzed, it will not be possible to nesslerize directly, but the ammonia will first have to be aerated into acid. 2A. L. Dounce, J. Biol. Chem. 140, 307 (1941). J. B. Sumner and D. B. Hand, J. Biol. Chem. 76, 1t9 (1928).