412 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 22 (1956)

R E S P I R A T O R Y G R A N U L E S IN MICRO-ORGANISMS

P. M. NOSSAL, D. B. KEECH AND D. J. MORTON

Department o[ Biochemistry, University o] Adelaide (Australia)

In animal tissues, the enzymes for oxidative phosphorylation as well as those for respiration are located on mitochondria (sarcosomes). In the case of the enzymes catalysing the Krebs tricarboxylic acid cycle, it is envisaged 1 that there is a structurally organized complex for the terminal oxidation of many metabolites, and that this is confined to the mitochondrial fraction of homogenates. In carefully prepared animal mitochondria, respiration is accompanied by esterification of inorganic phosphate. Somewhat similar findings have also been reported for plant tissues 2, 3, 4.

The knowledge of respiratory and especially coupled phosphorylative processes in micro-organisms has lagged far behind. There have been few reportsS,e,7, s con- cerning oxidative phosphorylation in cell-free extracts of bacteria. One of the reasons may be the greater resistance of micro-organisms to mechanical disintegration. By virtue of their small size and the strength of their cell-membranes, bacteria require far more severe treatments for disruption than do animal tissues. Accordingly, it is not surprising that neither respiratory nor coupled phosphorylative processes have been studied as successfully in extracts of bacteria.

Using cytochemical techniques, the school of MUDDg, 1° has shown in several micro-organisms granules which stain with a number of dyes and which are electron- scattering. LINDEGREN ll, SARACHEK AND TOWNSEND TM a n d MUNDKUR 13 have referred to refractile granules in yeast. We have reported from this laboratory that freezing of yeast cells deranges the distribution of these granules around the nuclear vacuole 14. GEORGI et al. 1~ have prepared "red granules" from B. siearothermophilus which are rich in cytochromes, aldolase, ATPase, succinoxidase and malic dehydrogenase. HYNDMAN et al. 7 have reported that the hydrogenase of A. vinelandii is almost wholly confined to particles.

EPHRUSSI, SLONIMSKY AND HIRSCH~, ~7,~s have prepared a particulate fraction from cell-free yeast extracts. The fraction contains virtually all the cytochrome oxidase, succinic and a-glycerophosphate dehydrogenase and cytochrome c reductase activities of the whole extract, but seven other enzymes associated with respiration predominate in the supernatant fraction. We have shown TM that this failure to obtain high activities of the latter seven enzymes in the particulate fraction of yeast was due to the disintegration procedure employed, and that in the intact cell these enzymes are probably concentrated on cytoplasmic granules, just as in animal tissues. The high-speed shaker used in this laboratory ~° disintegrates micro-organisms far more rapidly than older machines 11, 33. Encouraged by the experiments with yeast, attempts were therefore made to study further the respiration and phosphorylation of yeast

References p. 420.

VOL. ~ 2 (1956) RESPIRATORY GRANULES IN MICRO-ORGANISMS 413

cytoplasmic granules. Also, to examine the question whether extracts of micro- organisms contain the equivalent of animal mitochondria.

An account of this work was given at the Third International Congress of Biochemistry 23. Since its completion, LINNANE AND STILL ~¢, using our shaker and slight modifications in the disintegrating media, have reported the preparation of yeast particles capable of oxidising pyruvate to completion.

METHODS

Organisms

Fresh baker ' s yea s t was suppl ied twice a week by the Eff ront Yeas t Co., Sou th Yarra , Vic. a n d was s tored in a h u m i d o r a t 2 °.

B o t h Proteus vulgaris O X 19 and Aerobacter aerogenes were g rown for 15 hours a t 380 on a m e d i u m cons is t ing of 13 g n u t r i e n t b ro th (Oxoid) and 4 g yea s t ex t r ac t (Oxoid) per IOOO ml. Aera t ion was ob ta ined by shak i ng in shal low cul ture . Cells were ha rve s t ed on a Sharples centr ifuge, washed once wi th disti l led wa te r and divided so t h a t each por t ion represen ted the yield f rom i25 ° ml med ium. Por t ions were suspended j u s t pr ior to d is in tegra t ion in o. i M PO 4 buffer p H 7.4 con ta in ing o.oo 3 M versene.

Disintegration and preparation o] cell-]ree extracts The h igh-speed shake r ha s been descr ibed elsewhere 2°. For th i s work, two pul leys were used, g iving s h a k i n g frequencies of 3ooo and 5600 s t rokes]ra in (high and low) speed respect ively. Af ter io to 3 ° sec d i s in tegra t ions ill a 2 o cold room, the t e m p e r a t u r e of the capsu la r con ten t s did no t exceed 15 °.

The p repa ra t ion and f rac t iona t ion of yea s t ex t r ac t s h a v e a l ready been descr ibed 1~, 3s. For the two bacter ia l species, t he m i n i m u m init ial cen t r i fuga t ion requi red to r emove all in tac t ceils was a b o u t 15 m i n a t 5000 g. Cent r i fuga t ion a t 35o0 g for up to 45 rain was also tried, b u t occasional ly ex t r ac t s t hus p repared con ta ined appreciable n u m b e r s of whole cells. Cen t r i fuga t ion a t 500o g yields ex t r ac t s which are v i r tua l ly cell-free. However , such cen t r i fuga t ion m a y also r emove some of t he more act ive par t i cu la te mate r ia l f rom t he ex t rac t . W h e n work ing wi th ex t r ac t s p repared a t 35o0 g, control e x p e r i m e n t s on the ra tes of O 3 u p t a k e wi th var ious s u b s t r a t e s were carr ied out , u s ing d i lu ted suspens ions of i n t ac t cells. The m e a s u r e d O 3 u p t a k e s of ex t r ac t s were far too g rea t to be ascr ibed to c o n t a m i n a t i o n by in t ac t cells.

Generally, t he cells f rom 125 ° ml m e d i u m were suspended in abou t 3 ° m l of a su i tab le m e d i u m and t he whole was d i s in tegra ted in 2 - 3 separa te operat ions . The yield of whole ex t r ac t (W) was abou t 18 ml. For fu r the r f rac t ionat ion, W was cent r i fuged for 3 ° m i n a t 25,000 g in a refr igerated Servall h igh-speed cent r i fuge a t - - 2 °. The s u p e r n a t a n t f ract ion (S) was poured off, a n d t he pa r t i cu la te f ract ion (R) r e suspended in o. i M p h o s p h a t e buffer p H 7.4. Yeas t R was occasional ly washed once wi th t he d is in tegra t ion med i um, s p u n down, and t h e n resuspended .

Measurement o] respiration Oxygen u p t a k e s were m e a s u r e d in s t a n d a r d W a r b u r g e q u i p m e n t a t 3 o°. 2.0 ml bacter ia l W were used in a to ta l v o l u m e of 3.0 ml. For f rac t iona ted ex t rac ts , final v o l u m e s were abou t 2 ml, of which up to 1.3 ml was t he s y s t e m to be t e s t e d . QO t ~ /A1 O 3 t aken u p / h r / m g d ry w t or pro te in as indicated.

Dry weights and protein estimation Dry weights were e s t i ma t ed by d ry ing to c o n s t a n t weight a t 12o °. Some ex t r ac t s or f rac t ions have a low d ry weight as c o m p a r e d wi th t he solids con t en t of t he m e d i u m in which t h e y were suspended . I n these, pro te in was e s t i ma t ed b y t h e m e t h o d of ROBINSON AND HOGDEN 36. R f rac t ions were f r equen t ly difficult to dissolve in N a O H alone a n d therefore 0.2 ml i ~ s o d i u m taurocho la te was added rou t ine ly to clear the t u rb id suspensions . Vigorous syr ing ing of t he m i x t u r e has also been found to disperse such f ract ions sa t is factor i ly .

Measurement o/phosphorylation Inorganic p h o s p h a t e was m e a s u r e d by t he m e t h o d of FISKE AND SIg-BBAROW 37. The s y s t e m for t r a p p i n g t he A T P fo rmed cons is ted of t he following m i x t u r e : o.15 ml of a solut ion m a d e b y mix ing equal vo lumes of 0.5 M glucose, 0.4 M N a F and o.o 4 M ATP; o. i m l of hexok inase ~3 (Stage 3 A. specific ac t iv i ty 215, d i lu ted wi th two pa r t s of water) ; o. i m l o. i M MgC13.

Af ter t he exper iment , t he cup con t en t s were acidified wi th o. i ml i o N H3SO 4. i . o m l a l iquots were deprote in ised wi th 3 ml io % tr ichloroacet ic acid, a n d d i lu ted to Io ml wi th water , filtered t h r o u g h W h a t m a n No 42 f i l terpaper (or centr ifuged), and t he inorganic p h o s p h a t e de t e rmined on

Re]erences p. 420.

4 1 4 P . M . NOSSAL, D. B. KEECH, D. J. MORTON VOL. 2 2 (I956)

0. 5 to I.O ml fi l trate (or supe rna tan t ) . P /O ra t ios are expressed as u sua l in /zmoles inorganic p h o s p h a t e es ter i f ied/ / ta toms oxygen t a k e n up.

Substrates and coenzymes

Citric acid, May and Baker Ana ly t i ca l R eagen t ; formic acid, 95 ~o May and Baker ; a -ke toglu tar ic acid, L igh t ' s , found to con ta in 5-1o % succinic acid by t he succ inoxidase m e t h o d ; succinic and acetic acids, glucose, Br i t i sh D r u g Houses " A n a l a r " ; fumar ic , DL-malic, L-glutamic, L-aspart ic acids, l i t h ium lacta te , Br i t i sh D r u g Houses , L a b o r a t o r y Reagen t s ; 2 ,4-dini t rophenol (2,4-DNP), Br i t i sh D r u g H o u s e s spo t t e s t r eagen t ; sod i um p y r u v a t e (92%), Nut r i t iona l Biochemicals Corpora t ion ; L-malic acid, E a s t m a n K o d a k ; e thanol , absolu te ; dl- isoci tr ic acid lactone, syn the t i c p r epa ra t i on k ind ly dona ted by Dr. J. F. MORRISON; p h o s p h o r y l a t e d hexoses (HMP), Schwarz Labora tor ies ; adenos ine t r i p h o s p h a t e (ATP), P a b s t ; d iphosphopyr id ine nucleot ide (DPN), a 45 ~o l abora to ry p repa ra t ion or S i g m a " 6 5 " ; t r i phosphopyr id ine nucleot ide (TPN), a io % labora tory p repara t ion con ta in ing 9 % D P N ; t h i a m i n e p y r o p h o s p h a t e (TPP), a syn the t i c l abora to ry prepara- tion, no t assayed.

RESULTS

Yeast

As reported earlier 25, whole extracts of baker's yeast oxidise the acids of the Krebs cycle, ethanol and glutamate. On fractionation, differences were observed between fractions of extracts prepared by shaking for different periods (Table I); 6-sec and Io-sec particulate fractions oxidised ethanol, lactate and succinate rapidly, whereas the supernatants had little oxidative activity towards any substrate tested. 3o-sec high-speed extracts contained a particulate fraction which seemed mechanically damaged. The fraction did not pack down well even after prolonged centrifugation at 25,ooo g, and the supernatants were cloudy. On a dry weight basis, their particulate fraction oxidised ethanol and lactate much less actively than Io-sec particles, though succinate oxidation remained rapid. Further, 3o-sec supernatants showed appreciable 0 3 uptakes with various substrates. It is likely that the longer disintegration period caused partial mechanical destruction of the cytoplasmic granules of yeast, that the smaller fragments could not be centrifuged down at 25,ooo g, and that thus the supernatant contained small but still active fragments of the original respiratory granules TM.

T A B L E I

E F F E C T O F D I S I N T E G R A T I O N P E R I O D ON T H E R E S P I R A T I O N O F F R A C T I O N A T E D Y E A S T E X T R A C T S

Dis in tegra t ion in o.i M p h o s p h a t e buffer p H 7.6; cups con ta in io / ,moles MnClz, IOO / ,moles subs t r a t e ; cofactors (DPN and T P N 5o #g, T P P IOO pg) as follows: Succ ina te and lactate , none; e thanol , D P N ; blank, ci trate, i soc i t ra te and a -ke tog lu ta ra te , TPN, DPN, T P P . Qo, va lues

ca lcula ted on a d ry wt basis .

Expt. Disintegration Fraaion No. period (sec)

Max. Q oz values with the/olloming substvates :

None Succinate Ethanol Citrate a-Ketogluta- Lactate rate

I 6 R (unwashed) 4 50 41 33 18 - - R (washed) 2 39 24 - - 13 - - S 1.2 i . i 1.2 0.8 - - - -

I I ~o R (unwashed) - - 49 35 - - - - 22 30 R (unwashed) - - 32 14 - - - - 8 I O S - - 1. 7 5 . 0 - - - - 1 . 3

30 S - - 7.5 15 - - - - 4 .2 i soc i t ra te

I I I IO R (washed) I 25 15 15 14 9

Re/erences p . 420.

VOL. 22 (1956) RESPIRATORY GRANULES IN MICRO-ORGANISMS 415

Various oxidations by unwashed Io-sec granules and cofactor effects are shown in Table II. Such granules have previously been found to contain bound DPN and their dehydrogenase activities were not decreased by washing TM. The present work showed ethanol and isocitric oxidases to be comparatively inactive without added cofactors. Further, a single washing with disintegration medium reduced the observed O 3 uptakes with these substrates to about half those of unwashed granules. Succinate oxidation was much less affected by washing. Lactate oxidation was variable and not affected by DPN. Occasionally, very good O 3 uptakes were obtained, at other times hardly any oxidation was found. This irregularity may be connected with the complex nature of yeast lactic dehydrogenase (APPLEBY AND MORTON29). 09. uptakes with succinate were decreased by DPN, but increased (and maintained over a longer period) by the boiled supernatant fraction, S. No work has been done on the mechanism of this activation.

TABLE II

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

Each flask contained 4 °/,moles substrate, io/*moles MnCI$, IOO/*moles phosphate buffer pH 7.6, cofactors as shown; total vol. 2.0 ml.

01 uptake Substrate C°fact°rs~ (Itl/3o min]io r~g dry wt)

None 3 4 Succinate None 1 8 3

Succinate I 208 Ethanol None 12 Ethanol 2 135 Malate 2 33 Citrate None 17 Citrate 3 61 isoCitrate None 17 isoCitrate 3 7 2

* Cofactor solutions as follows: I. Boiled S fraction, I.O ml. 2.0.2 /,mole DPN. 3.0.2 /,moles each of DPN and TPN, i.o/,mole TPP.

The most notable defect of the granules was the low rate of malate oxidation. However, even in the absence of added DPN, there is appreciable dehydrogenation of malate in dialysed W 1~, although ethanol dehydrogenation is nil in the same system without added DPN. This may mean that there is a DPN-independent malic enzyme apart from the usual malic dehydrogenase. Alternatively, DPN may be more firmly bound to this malic dehydrogenase. There are some indications from isotope studies that the malic and succinic dehydrogenases in our extracts can be coupledS°; since we have no evidence of any DPN requirement in succinate oxidation, the coupling could be interpreted in favour of a cytochrome or flavoprotein-like malic system.

Electron-transport phosphorylation is readily demonstrated with IO sec yeast granules during succinate, isocitrate, lactate, ethanol and a-ketoglutarate oxidation. P /0 ratios of up to I.O were obtained, and the phosphorylation was wholly uncoupled by 2,4-DNP. 3o-sec granules gave very low P /0 ratios. Details of these studies will be published elsewhere (UTTER, KEECH AND NOSSAL31).

Re]erences p. 42o.

416 P. M. NOSSAL, D. B. K E E C H , D. J . MORTON VOL. 2 2 (I956)

T A B L E I I I

OXIDATION IN WHOLE AND FRACTIONATED EXTRACTS OF Proteus vulgaris

Cond i t i ons as in T a b l e I I . Q v a l u e s w i t h r e f e r e n c e to p r o t e i n , a n d f r o m 02 u p t a k e s

Expt. Disint. period No.

Centri/ugal ]orce at which it Qo2 with the Buffer pH System was prepared None Formate Succinate

I 15 sec (h igh-speed) 7.5 2 3 ° sec (h igh-speed) 7.5

3 30 sec (h igh-speed) 7.0

4 15 sec (h igh-speed) u n b u f f e r e d

W 5o00 g for 15 m i n 2 85 58 W 5ooo g for i o r a in o 62 62

R W b y 5ooo g for 15 r a in I 3o5 215 IR a n d S f r o m W b y

S 25,000 g for 25 m i n o 15 17 R W b y 5000 g for IO m i n o - - - -

R a n d S f r o m W b y S 25,000 g for 3 ° m i n 5 - - - -

Proteus vulgaris

Intact cells of Proteus oxidised a variety of substrates: Qo, values are given in parenthesis: L-aspartate (218), fumarate (2o8), succinate (2o5), formate (2oo), L- glutamate (i8o), L-malate (I47), DL-lactate (I37), glucose (I2O), citrate (96), pyruvate (95), a-ketoglutarate (84), sucrose (55), acetylphosphate (5o), ethanol (5o), DL-alanine (4o), ribose (35), acetate (2o), glycollate (8), none (3.2). Cell-free extracts showed much less activity and failed to oxidise many of the substrates. Fractionation of the extracts yielded a particulate fraction which oxidised only succinate, lactate and formate (Table III), the latter particularly rapidly. In order to obtain a particulate fraction capable of oxidising a greater variety of substrates, variations in technique were ex- amined. Neither milder distintegrations, addition of versene, nor changes in pH from 5.9 to 7.6 produced the desired result. Similarly, longer centrifugation periods and higher centrifugal forces during fractionation of the extracts also failed to increase the number of substrates oxidised. We therefore conclude that under our methods of disintegration the only oxidative activities associated with Proteus particles are succinic, lactic and formic oxidases.

T A B L E I V

£79roteus pI-IOSPHORYLATION

I s - s e c h i g h - s p e e d e x t r a c t in 0. 9 % KCl - - o . 0 o 3 M v e r s e n e . W p r e p a r e d b y 15 r a in c e n t r i f u g a t i o n a t 25,ooo g. E x p t . p e r i o d : 3 ° ra in .

Amount 2,4-DNP O, uptake Inorganic plwsphage System (rag protein) Substrate (0. 4 ,umoles) (l,atoms) uptake (l~moles) P/O

W 6. 5 B l a n k - - 1.3 o.8 o.62 W 6. 5 L a c t a t e - - 9.8 4. i o.42 W 6.5 L a c t a t e + 8.o 2.o o.25 S 5-5 B l a n k - - 1.8 - - 1 . 2 ( o u t p u t ) - - S 5.5 L a c t a t e - - 3 . I 0. 4 o . 5 2 " S 5.5 L a c t a t e + 2.6 - - 2 . o (ou tpu t ) - - IR 4.3 B l a n k - - o o o R 4.3 L a c t a t e - - 8.6 o o R 4.3 L a c t a t e + 8. 3 o o

* C a l c u l a t e d a f t e r c o r r e c t i n g for increase in p h o s p h a t e in t h e b l a n k .

Re#rences~ p.' ,42o.

VOL. 22 (1956) RESPIRATORY GRANULES IN MICRO-ORGANISMS 417

o v e r 3 ° m i n .

]ollowing substrates

Lactate Citrate L-Malate L-Aspartate

- - 2o II II

13 6 13 - -

_ _ _ _ 41 - -

- - - - 19 - -

44 - - - - - -

12 - - - - - -

Proteus extracts were examined for phosphoryla- tion coupled with the oxidation of lactate, succinate, and formate. In the presence of lactate, inorganic phosphate disappeared in W and S, but not in R (Table IV). In both W and S, esterification of phos- phate was reduced by 2,4-DNP. As far as we know, this is the first instance of 2,4-DNP uncoupling of phosphorylation in an apparently non-particulate system. Succinate and formate caused an increased lib2ration of inorganic phosphate. The slight blank respiration was accompanied by esterification of phos- phate, which may be largely or wholly (in neutralised extracts) offset by phosphatase activity; but in the presence of the more readily oxidised succinate and formate, this blank respiration may be supressed, and

thus phosphatase activity more apparent.

Aerobacter aerogenes

Suspensions of intact cells of Aerobacter grown in the medium used for Proteus oxidised a variety of substrates. Their oxidative activity was much higher than that of cells grown on a synthetic medium. Qo, values are given in parenthesis: lactate (3oo), glucose (263), glucose-i-PQ (253), succinate (25I), fumarate (I34), acetate (I3O), L-malate (I3O), L-aspartate (99), L-glutamate (65), citrate (60), pyruvate (I8), ethanol (8), none (9).

Cell-free extracts of the organism oxidised, at decreasing rates, lactate, glucose-l- phosphate, glucose, citrate, succinate, L-aspartate, L-malate and fumarate; acetate was oxidised very slowly if at all. Freezing reduced all the rates of oxidation (Table V), suggesting that the particulate fraction is involved 14. On fractionation, R oxidised only lactate and succinate appreciably, whereas S oxidised, even if slowly, all the substrates tested. It seemed as though S might contain unusually small particles

T A B L E V

OXIDATIONS IN" WHOLE AND FRACTIONATED EXTRACTS OF A erobacter aerogenes

I 5 - s e c h i g h - s p e e d d i s i n t e g r a t i o n , p H 7.5, o .o2 M s u b s t r a t e s : lO -3 M M g S O i , c o e n z y m e s : f o r c i t r a t e a n d b l a n k s : i o o / ~ g each D P N , T P N , T P P ; a l s o 4 / , m o l e s A - 5 - P . F o r g l u c o s e a n d g l u c o s e -

I - P O 4 : 2 0 0 / , g D P N a n d 4 / z m o l e s A T P . F o r l a c t a t e , L - a s p a r t a t e a n d f u m a r a t e : 2 o 0 / ~ g D P N .

Q o~ with the ]ollowing substrates : Expt. System No. None Lactate Glucose- 1-POt Glucose Citrate Succinate L-Aspartate Fumarate

I W 4 lO5 63 53 47 39 36 29 I I W 1. 5 68 34 3 ° 29 3 ° 2o 22 I I I R o i 4 7 i o 8 i o 36 - - IO

* S 2 61 36 38 41 25 - - 30 I V R o 195 o o I 181 o 4 ** S 2 16 i o IO 18 I I 16 9

* W f r a c t i o n a t e d a t 2 5 , o o o g. ** W f r a c t i o n a t e d a t 14o, o o o g.

References p. 420.

418 P.M. NOSSAL, D. B. KEECH, D. J. MORTON VOL. 22 (1956)

responsible for these var ious oxidat ions . Accordingly, ano ther ex t rac t was f rac t iona ted on a p repa ra t ive Spinco ul t ra-centr i fuge at I40,000 g ins tead of the usual 25,00o g. Al though the resu l tan t R showed be t t e r O 3 up takes wi th succinate and lac ta te , no ox ida t ion of glucose-6-phosphate, glucose, c i t ra te , ma la te , a spa r t a t e or g lu t ama te could be obta ined. Fur the r , S st i l l oxidised all these subs t ra tes , a l though more slowly. Our findings suppor t those of TISSIERES 3~ and suggest t ha t e i ther the ex t rac t s conta in some wholly "soluble" oxidase systems, or t h a t the "pa r t i c l e s " are so small t h a t t hey cannot be sed imented b y shor t - te rm cent r i fuga t ion at ve ry high speeds.

Various subs t ra tes showed coenzyme requirements , a l though pa r t i a l a c t i v i t y was ob ta ined in W wi thout a d d e d coenzymes (Table VI). The most s t r ik ing coenzyme dependence was the DPN- l inked lac t ic oxidase system, which was a lmost wholly inac t ive wi thout added DPN. This enzyme therefore differs from the yeas t lact ic oxidase (cytochrome b,) which is a f lavocytochrome, independen t of D P N ~9.

TABLE VI

COENZYME DEPENDENCE OF SOME OXIDATIONS IN" Aerobacter EXTRACTS

i5-sec high-speed W, lO -3 M MgSO~. Coenzymes for individual substrates as in Table V. Ca. 5 mg protein/cup.

02 uptakes (1~113o rain) :

Substrate With Without

coenzymes coenzymes

Glucose 78 25 Glucose-i-PO 4 95 25 Citrate 77 5 i L-Aspartate 106 56 Lactate 183 6 None 5 o

A e r o b a c t e r W showed a phosphory la t ion in the presence of the t r a p p i n g sys tem wi thou t fur ther addi t ions. This process was insensi t ive to 2,4-DNP. None of the oxidisable subs t ra tes t es ted gave apprec iab le increases in phospha te up take , over the b lank conta in ing only the t r a p p i n g sys tem except l ac ta te , which consis tent ly caused a small increase. However , since l ac t a t e increased the 0 2 u p t a k e great ly , the P/O ra t io was much lower t h a n t ha t of the b l ank (Table VII). W i t h c i t ra te , there was a consis tent i n c r e a s e in the inorganic phospha te level dur ing the exper imenta l period. The reason for th is c i t r a te effect is not known.

TABLE VII

Aerobacter PHOSPHORYLATION

Preparation of W and fractionation (at 25,ooo g) as in Table V except that the crude extract was neutralised with 0.2 N NaOH.

Amount 02 uptake Inorganic phosphate P]O System (rag protein) Substrate (#atoms) uptake (#moles)

W 4.7 Blank 6.8 4,7 0.69 , W 4.7 Lactate 16.6 5.5 0.33 S 4.3 Blank 3.4 4.3 1.27 S 4.3 Lactate 8. 4 5. i o.6i R 1.4 Blank 0.2 o o R 1.4 Lactate 8.o 1.3 o.I6

References p. 420.

VOL. 22 (1956) RESPIRATORY GRANULES IN MICRO-ORGANISMS 419

Fractionation of the extracts by high-speed centrifugation into particulate and supernatant showed (Table vii) that the enzymes for the oxidation of lactate were more active in the R fraction but the phosphorylating mechanism and the "blank" oxidation were found in S.

DISCUSSION

The present work adds to the evidence for a certain similarity in the intracellular distribution of enzymes in micro-organisms, animals and higher plants. We have prepared from three micro-organisms, particulate, microscopically visible fractions which contain the enzymes necessary for the aerobic oxidation of one or more of the following substrates: isocitrate, succinate, a-ketoglutarate, lactate, ethanol, formate. We have reported TM that increasing periods of distintegration (from IO to 9 ° sec) cause a progressive shift of several enzymes from the particulate to the supernatant fraction. It is therefore probable that even io-sec mechanical shaking on our machines has a deleterious effect on the respiratory activities of the particulate fraction. Any treat- ment which is designed to disrupt small cells with strong membranes will almost certainly also affect cytoplasmic granules. Therefore our failure to prepare bacterial granules with a variety of oxidising activities may be due to the detachment of certain dehydrogenases particularly susceptible to the experimental procedure. Alternatively, osmotic conditons widely different from those optimal for animal mitochondria may be necessary to maintain bacterial granules in vitro. Thus far, no extended investigation of osmotic conditions has been attempted.

Under conditions where oxidative phosphorylation by yeast granules occurs, Proteus granules fail to show inorganic phosphate uptake, despite their ability to oxidise succinate and other substrates. It is possible that thegranules have a sufficiently high phosphatase activity to break down any ATP or hexose phosphate as it is formed. On the other hand, there was no liberation of inorganic phosphate from ATP or HMP in the granule "blank" during the experimental period. Alternatively, biological uncoupling substances may be present which could make it appear as though no phosphorylation was occurring (NossAL, UTTER AND KEECH31). Lastly, the phosphorylation systems may be soluble. There has been hitherto no evidence for "natural ly" soluble phosphorylation systems at anything other than the substrate level. In this connection, the recent results of BRODIE AND GRAY s are of much interest : Cell-free sonic extracts of M. phlei show oxidative phosphorylation with succinate, fl-hydroxybutyrate, pyruvate, malate, fumarate and a-ketoglutarate, with P/O ratios greater than I. Yet neither the particulate nor the supernatant fractions obtained by high-speed centrifugation could individually oxidise succinate or support phosphorylation.

During the preparation of this manuscript there appeared new results on enzyme distribution in bacteria: LINNANE AND STILL 33, using our shaker 2° and essentially the same fractionation procedure, found that in Serratia marcescens there are also granules rich in dehydrogenases and oxidases. The authors further report the same effects of prolonged disintegration (detachment of some enzymes from the granules) as we described earlier for yeast TM.

ACKNOWLEDGEMENTS

We are grateful to the Rockefeller Foundation and the National Heal th and Medical

Re/erences p. 420.

4 2 0 v . M . NOSSAL, D. B. KEECH, D. J. MORTON VOL. 2 2 (1956)

R e s e a r c h C o u n c i l o f A u s t r a l i a f o r s u p p o r t i n g t h i s w o r k , t o M i s s N . ATKINSOI~ fo r t h e

u s e o f a l a r g e t h e r m o s t a t i c a l l y c o n t r o l l e d s h a k e r f o r g r o w i n g t h e b a c t e r i a a n d t o t h e

E f f r o n t Y e a s t Co. , S o u t h Y a r r a , V i c . f o r a r e g u l a r s u p p l y o f y e a s t .

S U M M A R Y

I. A h igh-speed shake r has been used for t he p repa ra t ion of ac t ive ly respir ing ex t r ac t s of baker ' s yeast , A erobaeter aerogenes and Proteus vulgaris O X 19. Dis in tegra t ion per iods r anged f rom io to 4 ° seconds.

2. io -second y e a s t ex t r ac t s con ta in granules which oxidise isocitrate, a-ke tog lu ta ra te , succinate , l ac ta te and e thanol , a n d which accoun t for a large p ropor t ion of t he resp i ra t ion of t he whole ext rac t .

3. I5-second ex t r ac t s of Proteus oxidise m a n y subs t ra t e s , and the isolated granules oxidise succinate , l ac ta te a n d fo rmate . W i t h l ac ta te as subs t ra te , whole ex t r ac t s and, to a lesser ex ten t , h igh-speed supe rna t a n t s , show a phospho ry l a t i on sens i t ive to 2 ,4-dini t rophenol ; t he g ranu les alone show no phosphory la t ion .

4. IS-Second ex t r ac t s of Aerobacter also oxidise a va r i e ty of subs t ra tes , b u t the isolated granules oxidise succ ina te and lac ta te only. Whole ex t r ac t s and h igh-speed s u p e r n a t a n t f ract ions, b u t no t the granules , show a phospho ry l a t i on insens i t ive to 2 ,4-dini t rophenol wi th glucose as t he subs t ra te . The g ranu les h a v e s l ight p h o s p h o r y l a t i n g ac t iv i ty wi th l ac ta te as subs t r a t e .

5- The relat ion of the granules of mic ro -o rgan i sms to an ima l mi tochondr i a is discussed.

R E F E R E N C E S

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