No abstract

Stephenson and Stickland (1931, a), set out the evidence for the presence in EschencJiia formica of au enzyme, hydrogenase, considered to catalyze the reaction, H2 = 2 H' 4-2e. Reversibly reducible dyes, nitrate, fumarate and molecular oxygen were shown to be the hydrogen acceptors. The reduction of sulphate to snlphide, and of carbon dioxide to methane in the presence of other bacteria was shown later to be catalyzed, probably by the same enzyme; Stephenson and iStiekland (1931, b;1933). Green, Stickland and Tarr (1934) demonstrated that the reaction was catalyzed in a completely reversible way by the hydrogenase system of E. coli. The E6-O-42 v is that of the most negative reversible oxidation-reduction system yet described in living cells. Woods (1938) studied the reduction of nitrate by Clostridmm welchn and E. coli ill the presence of molecular hydrogen. Using cells of Azoiobacter vinelandii and extracts from these cells ground with glass, Wilson and Wilson (1942) studied the effect of potassium cyanide, sodium azide, carbon monoxide and hydroxylamine on the Knall-gas reaction. Wilson^ Lee and Wyss (1941) had found that molecular hydrogen inhibited symbiotic nitrogen fixation. Kalnitsky and Werkman (1943) showed that hydrogenase was one of the enzymes in cell-free extracts of E. coli. Using suspensions of cells of Vroteus vvlgaris and B. lactis aerogevcf^ catalyzing the exchange of hydrogen hetween deuterium and water; Tloberman and Rittenberg (1943) obtained evidence that hydrogenase may be an iron protein, active only in the ferrons state.Evidence that hydrogenase may be an adaptive enzyme bas been obtained by Foster (1944) during a study of oxidations catalyzed by Rhodospirillum. The literature dealing with the role of hydrogen in the metabolism of tbe photosynthetic algae has been reviewed recently by Gaffron (1944).The present communication records observations on the utilization of molecular hydrogen by suspensions of E. eoli in the presence of (a) an artificial hydrogen acceptor and (b) molecular oxygen. METHODS.Organisms. A laboratory strain of E. coU was used; and maintained on nutrient agar. When required for suspensions, the organisms were grown on nutrient broth containing' 1 p.c. glu&ose, at 37° C. for 18 honrR. The pH of the medium was 7-4. The organisms were centrifuged, washed once with diatilled water and the final suspensions made up in distilled water. Only fresh suHpensions were used in these experiments. Dry weight determinations were made by drying to constant weight at 100° C.Hydrogenase activity. This was observed at 38° 0. uaing Warburg manometers with veseels carrying two side-arms. It was considered desirable to have sodiiim hydroxide and folded filter paper in the centre compartment during gas absorption except in those experiments where cyanide was present also. The most satisfactory hydrogen acceptor was methylene blue. The sample used waa that manufactured by Q. T. On'rr, London. The marked hydrogenase activity of these organisms necessitated the use of dilute anspensions....

CELL-free extracts of Bact. coli were found to contain a highly active alcohol enzyme requiring coenzyme I. This investigation deals with the properties of this enzyme. These are very similar to those of the alcohol enzyme isolated by Negelein & Wulff [1937] from yeast and would suggest the probable identity of the two enzymes. I. Preparation of the enzyme The organism used was Bact. coli commune Escherich (National Collection of Type Cultures No. 86). It was grown and maintained on agar to which was added tryptic digest of casein and inorganic salts. The cells from 30 hr. growth on 30 Roux bottles were washed off the medium with distilled water and centrifuged. The thick suspension of cells was ground for 2 hr. in the wet-crushing mill [Booth & Green, 1938]. The suspension of crushed cells, volume about 40 ml., was diluted with 60 ml. water and centrifuged for 40 min. at 3000 r.p.m. The supernatant was heated to 55'; cooled immediately and centrifuged for 45 min. at 12,000 r.p.m. The clear yellow supernatant fluid was then dialysed overnight against running water. Such preparations had a dry weight of 8-10 mg. per ml., and when kept on ice retained activity for several weeks. The activity of the enzyme was measured anaerobically by the Thunberg tube technique, and manometrically with Barcroft and Warburg manometers.

BlaCTERIUM COLI oxidizes pyruvate to CO2 and water [Cook & Stephenson, 1928]; and in the presence of dinitrophenol or NaN3 the oxidation is complete [Clifton & Logan, 1939]. Anaerobically, the main products of the breakdown of pyruvate are acetic, lactic, formic and succinic acids, ethyl alcohol, H2 and CO2 [Tikka, 1935]. The theories concerning the oxidation have been summarized' by Mazza & Cimmine [1934].. More recent work appeared in two papers by Kreks [1937, 1, 2). Cell-free extracts of Bact. coli prepared by crushing the cells in the Booth-Green mill were found to contain an enzyme system capable of oxidizing pyruvate. This paper reports the study of some of the properties of such extracts. I. Methods Pyruvic acid was estimated by the bisulphite fixation method of Clift & Cook [1932]. The cocarboxylase was prepared by Dr H. Weil-Malherbe, to whom the author is grateful. The activity of the enzyme system was followed by measuring the 02 uptake in Warburg manometers at-37°. Anaerobically, methylene blue acts as hydrogen acceptor for the reaction. II. Preparation of the extracts Bacterium coli-commune Escherich (National Collection of Type Cultures No. 86) was grown ol agar with tryptic digest of casein, lactate and yeast water. Yeast water was added to ensure an adequate supply of substances required for synthesis of cocarboxylase [Silverman & Werkman, 1939].' After 24-30 hr. growth the cells were washed off, centrifuged and crushed for 2 hr. in the wet-crushing mill [Booth & Green, 1938]. The crushed cells were diluted with an equal volume of distilled water and centrifuged at 12,000 r.p.m. for 40 mim. The clear yellow supernatant fluid was dialysed for 14 hr. against running water.

No abstract