PRELIMINARY NOTES 247

5 D. W. WOOLLEY AND 1~. W. GOMMI, Proc. Natl. Acad. Sci. U.S., 53 (1965) 959. 6 0 . K. LANGLEY AND E, J. AMBROSE, Nature, 204 (1964) 53- 7 J, C. ROBINSON AND J. E. PIERCE, Nature, 204 (1964) 472. 8 S, BOGOCH, Biochem. J., 68 (1958) 319 . 9 s. ]3OGOCH, J..din. Chem. Soc., 79 (1957) 3286.

IO P. V. JOHNSTON AND B. I. ROOTS, Nature, 205 (1965) 778. i i D. W. WOOLLEY AND B. W. GOMMI, Nature, 202 (1964) lO74. 12 H. MclLWAIN, Biochem. J., 90 (1964) 442. 13 H. MclLwAIN, Chemical Exploration of the Brain, Elsevier, Amsterdam, 1963, p. 186. 14 R. WHITTAM, in J. F. HOFFMAN, The Cellular Functions of Membrane Transport, Prentice-Hall

Inc., Eaglewood Cliffs, N. J., 1964, p. 139. 15 J. D. JUDAH, K. AHMED AND A. E. M. McLEAN, Biochim. Biophys. Acta, 65 (1962) 472. 16 K. AHMED AND J. D. JUDAH, Biochim. Biophys. Acta, 93 (1964) 6o3. 17 t~. AHMED AND J. D. JUDAH, Biochim. Biophys. Acta, lO 4 (I965) 112. 18 R. L. POST AND A. K. SEN, J. Histochem. Cytochem,, 13 (1965) lO 5. 19 J. L. GLICK AND S. GITHENS III, Nature, 208 (1965) 88.

Received September 23rd, 1965 Biochim. Biophys. Aeta, 115 (1966) 244-247

PN 2 1 1 1 7

Nitrogen fixation in breis of soybean root nodules

Detached legume root nodules fix nitrogen 1 but fixation is reduced in sliced nodules and absent in crushed nodules 2. There have been many attempts to obtain nitrogen fixation in nodule extracts but only TURCHIN, BERSENEVA AND ZHIDKIKH 3 have claimed success. The recent work with cell-free extracts from nitrogen-fixing bacteria has stimulated us to repeat earlier unsuccessful attempts to obtain fixation with nodule preparations. The following is a preliminary report of experiments in which nitrogen fixation has been obtained in breis of soybean nodules prepared in a special press in a stream of argon and incubated for short periods in atmospheres containing high enrichments of 15N~.

The basic apparatus consisted of a gas-tight, ice-cooled stainless-steel press with two ports. To one of these was connected a source of pure argon and a means of drawing measured amounts of buffered medium into the press. The other port was connected to a specially constructed glass flask by means of a sidearm with a 4-mm bore stopcock. Each flask had two further stoppered sidearms for addition of sub- strates and was equipped with a gas-sampling attachment 4. The attachment was in turn connected to a glass manifold, vacuum pump, containers of the various gases and a mercury manometer.

Nodules aged 28-35 days were detached from glasshouse-grown soybean plants (var. Shelby, inoculated with strain CC7II of Rhizobium japonicum) and 7 g (fresh weight) was wrapped in two layers of washed organdie, placed in the press and the piston inserted. Argon was passed through the whole apparatus for IO min and then 3 ml buffered sucrose (o.I M KH2PO 4 (pH 7.o), 0.3 M sucrose, lO -3 M Mg 2+) which had previously been saturated with argon, was drawn into the press. The press was then tightened and slackened three times and the resulting brei was displaced into the flask by allowing some argon to flow through the system. The flask stopcock was then closed, disconnected from the press, and the flask flushed three times with pure argon before filling with gas mixture containing 15N 2 to a pressure of 700 mm Hg.

Bioehim. Biophys. Aeta, 115 (1966) 247-249

248 PRELIMINARY NOTES

A separate prepara t ion was made for each t r ea tmen t at intervals of 3o rain for each

exper iment . Flasks rout inely contained, in addi t ion to the brei, 0.8 ml buffered sucrose and

in the sidearms the const i tuents of an ATP-genera t ing system consisting of A T P

(i /~mole), py ruva t e kinase (0.5 rag), and phosphoenolpyruva te ( t r icyclohexylamine

salt ; 4/zmoles). The to ta l volume was 6 ml. Flasks were incubated with shaking at 23 ° in a Warburg bath. Gas samples for mass-spec t rometer analysis were taken at the

beginning and end of the incubations. Af ter incubat ion the breis were separated by

centr i fugat ion into the soluble, bacteroid and membrane fractions 5 and these were analyzed for 15N content .

Breis prepared and incubated in the manner described above consistent ly fixed

significant amounts of ni t rogen under certain conditions. Maximum fixation was ob-

ta ined at oxygen pressures of 46-48 toni Hg (about 6 % of I a t m ) as shown in Table I.

TABLE I

T H E E F F E C T O F O X Y G E N U P O N N I T R O G E N F I X A T I O N B Y B R E I S F R O M S O Y B E A N N O D U L E S

Flasks contained 6 ml extract and 20 % (14o mm Hg), 15N 2 (95 atom %) and 02 as shown. Incu- bation: i h at 23 °. 15N excess measured in the non-protein N of the soluble fraction.

Partial pressure of 0 2 (mm Hg)

o.I 7.4 11,9 46.2 48.3 58.I

o / I~N excess (atom /o) o.ooo O.Ol 4 0.026 0 .082 0 . 0 8 8 0.077 15N excess (#g) o 0.8 1. 3 4.0 4.5 3-7

TABLE II

T H E D I S T R I B U T I O N O F 1 5 N E X C E S S I N C E N T R I F U G E D F R A C T I O N S O F N O D U L E B R E I S

Flasks contained 6 ml extract, 20% (14o mm Hg) 15N 2 (98 atom %) and 2.5 % (17.5 mm Hg) O, z. Incubated i h at 23 °. #g 15N excess values are given for the amounts of the fractions recovered from the breis.

15N excess 15N excess (atom %) (/*g)

Soluble fraction, non-protein N Bacteroid fraction, non-protein N Menlbrane fraction, total N

o.123 5.16 0.o34 o.38 o.0o7 o.o3

Ear ly exper iments lasted for I h but more recent ly it has been shown tha t ni t rogen f ixat ion proceeded in a l inear fashion with t ime for 20-4o rain. In some preparat ions there was a lag of 5-1o rain before the m a x i m u m fixation rate was at ta ined. In all cases the ~SN enr ichment at I h was a reasonable measure of the

ac t iv i ty of the preparat ion. Ni t rogen fixation by intact , de tached nodules is sustained in a linear fashion with t ime for at least I h (ref. 6). This suggests tha t the breis are

inac t iva ted or tha t an essential substra te is exhaus ted more rapidly than is the case in the in tac t system.

The ~SN excess was found most ly in the soluble fraction only small enr ichments occurring in the bacteroid and membrane fractions (TaMe II).

Biochim. Biophys. Acta, 115 (I966) 247-249

PRELIMINARY NOTES 249

The ATP-generating system was included routinely because ATP has been found to be necessary for nitrogen fixation in cell-free extracts from Clostridium and Azoto- bacter 7-9. Omission of the ATP generator decreased fixation only slightly, suggesting tha t endogenous ATP production was relatively high or alternatively that the pyru- vate kinase system was not very active in the breis. Pyruvate kinase is inhibited by Na + and Ca 2+ and no particular precautions were taken to control the level of these ions.

Examination with the hand spectroscope showed that the leghaemoglobin in the breis was completely in the oxidised state after 20 rain in flasks containing pres- sures of oxygen higher than 0.I mm Hg and it is possible that this was a factor in limiting the time for which nitrogen fixation proceeded. Omission of the argon flushing of the nodules before crushing in the press resulted in inactive preparations. This obser- vation, coupled with the results described, suggests that although the experimental system needed small concentrations of oxygen for nitrogen fixation, oxygen could also limit the nitrogen fixing activity by inactivating some component of the system during the preparation of the breis and also by shortening the period for which the breis fixed nitrogen during incubation.

Microscopic examination of the breis showed large numbers of intact bacteroids in suspension, but in addition quite large numbers of what appeared to be groups of bacteroids enclosed in their membrane envelopes 1° were also present and these may have contributed to the nitrogen-fixing activity of the preparations.

Because of the nature of the methods, progress of the work has been slow but now that reproducible nitrogen fixation can be obtained experiments are in progress which will be the subject of a later, more detailed report.

Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra (Australia)

F. J . .BERGERSEN

i M. H. APRISON AND 1~. H. BURRIS, Science, 115 (1952) 264. 2 M. H. APRISON, W. E. M~AGEE AND R. H. BURRIS, J. Biol. Chem., 208 (1954) 29. 3 F. V. TURCHIN, Z. N. BERSENEVA AND G. G. ZHIDKIKH, Dokl. Akad. Nauh S.S.S.R., 149 (x963)

731 . 4 F. J. BERGERSEN, Australian J. Biol. Sci., 16 (1963) 669. 5 F. J. BERGERSEN, J. Gen. Microbiol., 22 (196o) 671. 6 F. J. BERGERSEN, Australian J. Biol. Sei., 18 (1965) I. 7 R. W. F. HARDY AND A. J. D'EUSTACHIO, Biochem. Biophys. Res. Commun., 15 (1964) 314 . 8 A. J. D'EUSTACHIO AND 1~. W, F. HARDY, Biochem. Biophys. Res. Commun., 15 (1964) 319 . 9 W. A. BULEN, P~, C. BURNS AND J. I~. LECOMTE, Proc. Natl. Acad. Sci. U.S., 53 (1965) 532.

io F. J. BERGERSEN AND M. J. BRIGGS, J. Gen. Microbiol., I9 (1958) 482.

Received September 27th, 1965

Biochim. Biophys. Acta, 115 (1965) 247-249