PRELIMINARY NOTES 517

appeared to be homogeneous giving a single band moving rapidly to the anode (cf. Fig. 2). Free-boundary electrophoresis at pH 4.95 revealed the presence of two minor fractions in addition to the main fraction. Analysis of the amino acid compo- sition of the flavoprotein shows the presence of most of the amino acids and a par- ticularly high content of dicarboxylic amino acids and serine.

The flavoprotein always contains small amounts of firmly bound vitamin Bx, which can be only split off by autoclaving at high temperature. The content of vitamin B12 determined microbiologically with Euglena gracilis "z ''8 was 21. 5/,g/g protein and 16/zg/g protein with Ochromonas malhamensis 9. It is difficult to decide at present whether both vitamins are bound by the same protein or whether the complex with B12 is a contamination of the flavoprotein. Further studies are in progress which will elucidate the question.

Some of the estimations (ultracentrifugal analysis, free-boundary electrophoresis, spectrofluorometric titration) were carried out by one of us (W.O.) at the Medicinska Nobel-institutet in Stockholm; the authors wish to express their heartfelt gratitude to Professor H. THEORELL and his coworkers for hospitality and encouragement extended to us.

Department of Physiological Chemistry, Medical Academy, Krakow, Kopernika (Poland)

WLODZIMIERZ OSTROWSKI

BOLESLAW SKARZYI~SKI

ZDZISLAW ZAK

1 R. KOHN AND H. RUDY, Ber., 69B (1936) 2557. H. THEORELL AND A. P. NYGAARD, Acta Chem. Scand., 8 (1954) 1649.

s IV[. B. RHODES, N. BENNETT AND R. E. FEENEY, J. Biol. Chem., 234 (1959) 2o54. 4 K. YAGI, J. Biochem. (Tokyo), 38 (1951) 161. 5 R. J. BLOCK, E. L. DURRUM AND G. ZWEIG, in Paper Chromatography and Paper Electrophoresis,

Academic Press, Inc. , New York, 1955, p. 296. e L. G. WHITBY, Biochem. J., 54 (1953) 437. ? E. G. ROBERTS AND E. E. SNELL. J. Biol. Chem., 163 (1946) 499. 8 S. M. HUTNER, M. K. BACH AND G. I. M. ROSS, J. Protozool., 3 (1956) lO4. 9 j . E. FORD, Brit. J. Nutrition, 7 (1953) 299.

Received March I9th, 1962 Biochim. Biophys. Acta, 59 (1962) 515-517

Discontinuity of amidase formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa grows readily in a medium containing acetamide as sole source of carbon and nitrogen. Extracts of the organism thus grown are rich in amidase (EC 3.5.1.4) activity, as measured by the hydrolysis of acetamide or propionamide. Only traces of amidase activity are found in extracts of the organism grown on suc- cinate as carbon source unless non-substrate acetamide analogues, such as N-methyl- acetamide, are also added to the growth medium 1.

I t is the main purpose of this communication to show that the formation of amidase by P aeruginosa 86o2/A during growth on acetamide proceeds in two distinct phases, and to suggest that either the nature of the amidase formed, or the mode of

Biochim. Biophys. Acta, 59 (1962) 517-519

518 PRELIMINARY NOTES

its formation, is different when elicited by non-substrate inducers than when elicited by acetamide.

When succinate-grown P. aeruginosa 86o2/A were placed in fresh medium con- taining 50 mM acetamide as sole source of carbon and nitrogen, the onset of growth was preceded by the formation of amidase, which thereafter continued, over two generations, at a rate proportional to the increase in cell density (Fig. IA). When the concentration of acetamide in the medium, which fell rapidly as a consequence of amidase action (Fig. IB), had decreased to less than one half of that initially present,

° A

B O F-

2 2 .~ u L

C ~

¢ < 0.1 0.2 0.3 0.4 0.5 0 6 0.7

Cell density (m9 dr), wt./rnt

Fig. I. Formation of amidase by P. aeruginosa 86o21A during growth on acetamide. Succinate- grown cells were placed in medium containing 5o m M acetamide (O) or 5o m M acetamide + 5 mM cyanoacetamide (O) and the amidase formed during growth was measured as the rate of hydrolysis of propionamide in sonic extracts of cells taken during the course of growth. Total amidase is expressed as the product of the specific activity of amidase (#moles of propionamide hydrolysed/mg of soluble protein/min) and the cell density (mg dry wt./ml). The acetamide content of the medium

(&) was measured as acetohydroxamate 1.

amidase formation ceased despite continued logarithmic growth of the organism. However, a second phase of rapid amidase formation began when acetamide could no longer be detected in the medium and continued throughout the subsequent growth of the cells.

These two phases of amidase formation could be observed independently of one another. When succinate-grown P. aeruginosa 86o2/A were placed in the 50 mM acetamide medium to which 5 mM cyanoacetamide was also added, the subsequent growth of the organism, the utilization of acetamide and the first phase of amidase formation were identical with those observed in the absence of cyanoacetam;de. However, the presence of cyanoacetamide, which has been shown I to abolish the induction of amidase by cells growing on succinate in the presence of non-substrate acetamide analogues, also abolished the second phase of amidase formation (Fig. IC).

Similarly, the second but not the first phase of amidase formation was observed when succinate-grown P. aeruginosa continued to grow in a medium containing 50 mM acetate as sole carbon source (Fig. 2A). Addition of 5 mM cyanoacetamide to this medium, initially or during the course of growth, abolished (Fig. 2F) or arrested (Fig. 2B-E) the formation of amidase without affecting the rate of growth.

Bioc~im. Biophys. Acta, 59 (1962) 517-519

PRELIMINARY NOTES 519

These results indicate that the process of amidase formation during growth on acetamide, which is unaffected by cyanoacetamide, differs from that observed during the later stages of growth on acetate or during growth on succinate in the presence of non-substrate acetamide analogues. Since abolition of amidase production does not affect growth of the organism on acetate, and since no amidase is produced during growth on butyrate, malonate or other substrates known to yield acetyl-coenzyme A (ref. 2), it is difficult to envisage a physiological role for the amidase formed during

~3

E

~2

0.1

growth on acetate.

A/•o

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Cell density (rn 9 dry w t . /m l )

Fig. 2. Effect of cyanoacetamide on amidase format ion during growth on acetate. Succinate-grown .cells were placed in medium containing 5 ° m M acetate (A) or 5 ° m M acetate + 5 m M cyano- acetamide (F). At points marked with arrows, samples of the culture growing on acetate were t ransferred to flasks containing cyanoacetamide, to final concentrat ion 5 mM, and continued to grow ( B - E ) . The amidase formed during growth was determined as described under Fig. I.

We thank Miss E. SEAMAN for technical assistance, and gratefully acknowledge support fiom the Medical Research Council, under a Grant for Scientific Assistance, and from the Air Force Office of Scientific Research, OAR, through the European Office, Aerospace Research, U.S. Air Force, under grant AF EOAR 61-12.

Department of Biochemistry, University of Leicester, Leicester (Great Britain)

M. KELLY H. L. KORNBERG

i M. KELLY AND P. H. CLARKE, J. Gen, Microbiol., 27 (1962) 305. 2 H. L. KORNBERG AND S. R. ELSDEN, Advances in Enzymol., 23 (1961) 4oi.

Received April 2nd, 1962 Biochim. Biophys. Acta, 59 (1962) 517-519