Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii
'
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PETERJURTSHUK AND JOHNE. MCENTIRE Deprrrtmet~tof Biology, University of Horrston. Horrston, Texns 77004 Accepted June 2, 1975 JURTSHUK, P., and J. E. MCENTIRE.1975. Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotohncter vinelnndii. Can. J . Microbiol. 21: 1807-1814. The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azorohricter ~~inelrrndii concentrates in the membranous R, fraction that is directly associated with Azotohrrcter electron transport function. Sonically disrupted Azorobncrer cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R, membranous fraction which sedimentson ultracentrifugation at 144000 x g for2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activity occurred at pH 8.0; Mg2+ ion when added t o the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATPstoichiometry was 1:l on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R, fraction was stimulated 12-fold by treatment with trypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R, electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R, fraction had no effect on activity; overnight refrigeration at 4°C. however, resulted in a25% loss ofactivity a s compared with a 14% loss when the R, fraction was stored overnight at 25 "C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 "C), and subsequent sonicationfailed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R, electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation. JURTSHUK, P., et J. E . M C E N T I R E1975. . Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azorobrrcrer ~~inelrrndii.Can. J. Microbiol. 21: 1807-1814. est concentric L'activite de I'adenosine triphosphatase (ATPase) chez Azorobncter ~~inelrrndii dans la fraction membranaire R, qui est directement associee a la fonction de transport d'electrons chez Azorobrrcrer. Des cellules d'ilzotobrrcter brisees par sonification furent examinees pour detecter la distribution de I'activite ATPase. e t I'activite la plus specifique (et les unites d'activite) fut constamment trouvte dans la fraction membranaire morcelie R,, sedimentke par ultracentrifugation a 144 000 x g pendant 2 h. Lorsque I'intervalle de sonification fut augment&, I'activite ATPase liCe a la membrane n'a pu 2tre solubilisee ou liberee dans la fraction surnageante. L'activite ATPase optimale est survenue a pH 8.0; I'ion MgZ+ajoute a I'essai fut stimulateur. L'activite maximale est toujours survenue lorsque la stoechiomitrie Mg2+:ATP Ctait dans un rapport molaire 1: 1 a un niveau de concentration d e 5 mM. Les ions sodium et potassium n'ont pas eu d'effet stimulateur. Les reactions cinetiques furent lintaires durant les intervalles utilises (0-60 min). L'ATPase liCe la membrane dans la fraction R, fut stirnulee 12-fois plus par traitement a la trypsine et les etudes de fractionation ont demontre que les traitements la trypsine n'ont pas solubilisC I'activite ATPase de la fraction membranaire d e transport d'electrons R,. L'ATPase ne s'est pas aviree labile froid et la temperature au coursde la preparation d e la fraction R, n'a pas eu d'effet sur I'activite; cependant, la refrigeration a 4 "C, pour la nuit, a conduit a une perte d'activiti de 25% comparee a une perte de 14% lorsque la fraction R, Ctait entreposee a 25 "C pour la nuit. Une inactivation marquee (bien que variable et, habituellement, d'environ 60%) est resultee d'une congelation (-20 "C) durant une nuit; la sonification subsequente n'a pu retablir I'activite ATPase. Ceci est une indication que la reaggregation de la membrane (par congelation) n'est pas responsable de I'inactivation de 'Received February 5, 1975.
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I'ATPase. L'addition d'azide, ouabain, Z,CdinitrophCnol, ou d'oligomycine au systkme de I'essai n'a pas eu d'effet stimulateur ou inhibiteur sur I'activite ATPase. La proprikte de I'activation a la trypsine et que I'activite ATPase est la plus &leveedans la fraction de transport d'klectrons R, suggkrent que son r6le fonctionnel probable est le couplement du transport d'electrons a la phosphorylation oxidative. [Traduit par le journal]
Introduction Detailed studies on bacterial adenosinetriphosphatases (ATPase) (ATP phosphohydrolase, EC 3.6.1.3) have been performed on several genera of bacteria, i.e., Streptococcus (1, 2, 5, 27, 28), Micrococcus (3, 23), Bacillus (22), and Escherichia (8, 12). A substantial amount of information is now available, which suggests that ATPase activity in bacteria is bound to, or closely associated with, the cytoplasmic membrane. The specific function of the various bacterial ATPases is not completely understood, although it appears that this enzyme can serve as a marker enzyme for some energy-dependent processes like (a) ion transport (I), (b) couplingfactor activity in oxidative phosphorylation and photophosphorylation (4,7), (c) association with ATP-dependent reactions such as N A D + transhydrogenase (21), and (d) possibly other bioenergetic processes such as nitrogen fixation (6, 13). The multifunctional roles of ATPase have been derived primarily from the interpretation of dicyclohexylcarbodiimide (DCCD) inhibition data which assume that inhibition of a bioenergetically associated function is due directly to the inhibition of ATPase activity. Singh and Bragg (29) have shown more recently that DCCD inhibition of ATPase activity may not be a valid parameter on which to base functional significance, since DCCD appears able to react with sites other than the ATPase. Depending on the organism examined, the bacterial ATPase may have many features in common with either the mitochondria1 ATPasecoupling factor or the N a + + K+-activated ATPase of eucaryotic plasma membranes. Generally, the bacterial ATPases are ~ g ' +or Ca2+ stimulated enzymes which show little or no activation response to Na' and (or) K + ion. ATPases in bacteria may be cold labile (when they are removed from the membrane) in soluble form (8), and some are affected by treatment with proteases (9, 23,24). They are not generally sensitive to oligomycin, ouabin, or 2,4-dinitrophenol. Azotobacter vinelandii, a Gram-negative, aero-
bic, free-living nitrogen-fixing bacterium could theoretically contain an ATPase (or ATPases) associated with ion transport, coupling, transhydrogenation, nitrogen fixation, or with a variety of other energy-linked reactions that might, for example, be associated with the contractile properties of the 'microtubule' ultrastructure known to be present in isolated membrane fractions (25). Recent studies on oxidative phosphorylation in Azotobacter gave only fragmentary data concerning the major properties of the membrane-bound ATPase (9). Little is known about the Azotobacter ATPase. Bioenergetically this organism is of immense interest because of its unusually high respiratory capability which undoubtedly is related to its efficient free-living nitrogen-fixing potential. Since ATPases are now commonly considered to be valid marker enzymes for numerous bioenergetic processes, a study was undertaken to characterize the nature of the ATPase in membrane fractions and establish its functional relationship to the electron transport system of Azotobacter vinelandii strain 0.
Materials and Methods Bacterial Strain and Growth Conditions Azotobacter vinelandii strain 0 (ATCC2 12518) was grown on Burk's nitrogen-free medium with 1% acetate as sole carbon source. One-litre batch cultures, or seed inocula, were grown at 30 "C for 24-30 h and aerated by reciprocal shaking (using low-form cultureflasks). Large scale batch cultures were grown in glass carboys containing 14 litres of Burk's NF/acetate medium. After 30 h of growth at 30°C with forced-filtered aeration, the Azotobacter cells were harvested by a Sharples centrifuge; washed and standardized resting cell preparations were made in 25 mM Tris3-HC1 buffer, pH 8.0, as described elsewhere
(16). Preparation of Membrane Fractions Turbidimetrically standardized resting cell suspensions were disrupted by sonic oscillation using a Heat Systems Ultrasonics model M140 sonicator, power setting 7 (output 70). Most cell suspensions were sonically irradiated for 2-min intervals with adequate cooling for a total time of 10 min. Varying sonication times were used in one study. After sonication, the homogenate was differen-
'ATCC, American Type Culture Collection. 3Tris, tris(hydroxymethyl)aminomethane.
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tially centrifuged at the speeds and times previously described in other communications (14-19). Both a Beckman J-21B preparative centrifuge and a Beckman L3-50 ultracentrifuge were used to prepare cell-free extracts. All procedures were carried out routinely at 4"C, and where indicated, studies on cold lability were carried out at room temperature (25 "C), refrigerated overnight (4 "C, 16 h), or frozen overnight (--20 "C, 16 h). ATPase Assay The standard colorimetric assay system was used for all ATPase determinations. The enzyme reaction mixture contained 25 m M Tris-HCI, pH 8.0; 5 m M NaATP; 5 m M MgC1,; and 1-3 mg of protein in a total volume of 1.0 ml. After 10 min of incubation at 30 "C, the reaction was stopped by the addition of 0.5 ml of 15% (w/v) trichloroacetic acid (TCA). Inorganic phosphate released by ATP hydrolysis was determined calorimetrically (10). All specific activities for ATPase are expressed as pmol Pi (inorganic phosphate) liberatedlmin per milligram protein. In studying the effect of metal ions, Mg2+ ion was replaced in the standard assay system with other divalent or monovalent cations among which were C a 2 + , M n 2 + , C o 2 + ,Hg2+, Cu2+, Zn2+, N a + , K + , and both N a + and K + ions. Final concentration of all metal ions was 5 mM. Inhibitors of ATPase activity as well as electron transport activity were added t o the standard assay system at a final concentration of M , except for oligomycin of which only 5 pg was added. Trypsin Trearnzenr The Azorobacrer R3 electron transport fraction (protein concentration 10-30 mglml) was treated with trypsin (0.5 mg/mg R 3 protein) for 4 min at 25 "C. At the end of this time interval, an excess of soybean trypsin inhibitor (type 11) was added. Prorein Dererminarion Protein was determined by the Biuret method using bovine serum albumin as a standard (1 1).
Results The Relationship of ATPase Activity to the Membranous R , Electron Transport Fraction It has previously been shown that the Azotobacter R, fraction contains both the highest specific activity as well as the greatest amount of the membrane-bound enzymes associated with electron transport function (14, 15, 17-20). Initial ATPase fractionation studies revealed that the ATPase activity similarly concentrated in the R, fraction (see Table 1). A study was carried out to determine if increased sonication times would release the bound ATPase found in the R, fraction into the S, supernatant fraction. For this study, the sonication times were varied from 1 to 15 min and the activities found in the R, and S, fractions are shown in Table 2. The highest specific activity (0.059) for ATPase was found in the R, fraction prepared by sonicating whole
TABLE 1 Distribution of activity units for ATPase in fractions obtained by differential centrifugation from sonically disrupted resting cells of Azorobacrer vinelandii Activity Protein recovery, Fraction
%
Sp. act.'
Total unitsb
Recovery,
%
'Expressed as pmol Pi liberatedlrnin per milligram protein at 30DC. T o t a l activity units were calculated by multiplying total protein concentration by specific activity.
cells 9-10 min. This was the sonication time used routinely in preparing the Azotobacter R, electron transport fraction. Total activity units recovered in the R, fraction are also greatest for this 9- to 10-min sonication time interval. The specific activities for ATPase activity in the S, fraction ranged from 0.014-0.033 and never did become as high as the value found for the R, fraction. Increasing the time of sonication of whole cells past the 10-min time interval does not result in activity being released into the S, supernatant fraction although by increasing the sonication time interval, some inactivation occurred as evidenced by a 50% loss in specific activity in the R, fraction prepared from cells after a 15 rnin sonication time. This does not represent heat denaturation since precautions were taken to keep all temperature no higher than 10" during all exposures to sonication procedures. Therefore, all additional characterization studies for ATPase activity in the R, fraction were carried out using an R, electron transport fraction prepared after only a 10-min sonication interval. Kinetic Studies on the Azotobacter ATPase Activity EfSect of Reaction pH The effect of reaction pH on ATPase activity is shown in Fig. 1. Three buffer systems were used to obtain experimentally the desired pH range. Suitable assay controls were always run to insure that non-enzymatic ATP hydrolysis was negligible. Optimal ATPase activity occurs at pH 8.0 with considerable activity also occurring over the range pH 8.0-10.0.
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TABLE 2 Effect of time of sonication on distribution of protein and ATPase activity ATPase activity -
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Protein recovered, % of SZ fraction Time, min
R3
S3
R3
Activity recovered, % of Sz fraction
Sp. act." S3
R3
S3
I>
%Specific activity expressed as pmol Pi liberatedlmin per milligram protein at 30'C.
ATPase A C TlVlTY
-
4z0T0B4cTER
R3
Pr=38mg
1.6
-
Maximal ATPase activity occurs at that point where both ATP and M g 2 + ion were added in equimolar amounts at the 5-mM concentration g 2 + ion were pointequimolar is noted level. where The both other A T P and highM activity
-
5 I
0
7
1.2
o W l-
-
Trrs-IlC1
-
a LL W
m -1
.a
08
-
J
o
r x 0.4
-
C i t r a t e Buffer
-
I
0 4.0
6.0
I 8.0
,
P H
FIG.1 . Effect of reaction pH on ATP hydrolysis by the R3 fraction.
Azotobacter
Actioation by ~ g Ion~ + The effect ATP in the absence and presence of different concentrations of Mg2+ ion is shown in Fig. 2.
a t the 20-mM concentration level. The 1 :1 molar ratio (of Mg2+:ATP) probably indicated the optimal substrate to metal ion concentration needed t o form a Mg2+-ATP complex, the active substrate complex needed for enzyme turnover. The control, which contained no externally added Mg2+ ion, showed a decrease in activity with increasing ATP concentrations. The effect appears to be identical with those observed for the other curves where the A T P concentrations exceeds the M ~ ion ~ concen+ tration. The initial high activity for the control is probably due to residual ~ g ion~present + in the Azotobacter R 3 fraction. This endogenous ATPase activity was absent when the R 3 membrane fraction was pretreated by exposure t o 1 m M EDTA.4 Eflect of Other Cations As shown in Table 3, M n 2 + and Ca2+ ions could replace Mg2+ ion t o some extent in stimulating ATPase activity. All other cations tested were inhibitory. Titne-course of Reaction and Stability Figure 3 shows an ATPase kinetic study which defines the relationship of the amount of Pi released as a function of the reaction time. Both figures are essentially identical and ATPase "EDTA, ethylenediaminetetraaceticacid.
JURTSHUK AND McENTIRE: ATPASE IN
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0 20
10
1811
VINELANDII
activity is plotted using two different time scales. In both studies the rate of release of Pi from ATP, for the various R, fractions, is essentially linear with respect to the time intervals examined. The R, fraction designated A was prepared at temperatures of 15-25 "C to examine whether the Azotobacter enzyme was cold labile; B represents an R, fraction prepared from the same batch of resting cells but all storage and preparatory temperatures were kept a t 4°C. Curves C and D represent the activity of the same Azotobacter R, fraction as in curve A, only after overnight storage a t room temperature and frozen, respectively. Curve E shows this same R, fraction after having been stored frozen (-20 "C) for 2 weeks. The similarity in activity patterns for both curves A and B shows that the Azotobacter membrane-bound ATPase is not cold labile in the classical sense, since preparation a t 4 ° C results in n o loss of activity, while overnight freezing results in a significant loss of as much as 60% in some samples. When the freezing time interval is extended, one markedly inactivates ATPase activity. The frozen R, fraction (activity pattern of curve E) was resonicated and it was not possible to recover the
ATPase A C T I V I T Y
0
A.
30
ATPose
ACTIVITY
ATP (mM)
FIG. 2. Effect of MgZ+ion concentration on ATP hydrolysis by the Azotobacter R3 fraction at varying ATP concentrations.
TABLE 3 Effect of various cations on ATPase activity of the Azotobacter R3 fraction Cationa
None Co2 Nat K Na HgZ CuZ ZnZ +
Sp. acLb
Activity, % '
+ K+
+
+
+
+
+
OFinal concentration of all metal cations was 5 rnM. bExpressed a s pmol Pi liberatedimin per milligram protein a t 30 " C .
FIG. 3. Typical time-course reactions for A T P hydrolysis by the Azotohacter R 3 fraction as plotted on two different time scales.
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original high ATPase activity. This suggests that reaggregation of membranes caused by prolonged freezing is not responsible for this inactivation. The temperature at which the Azotobacter R3 fraction is prepared has little effect on the ATPase activity, but the temperature and duration of storage are important factors in maintaining high activity in the R, fraction. Trypsin Activation Treatment by trypsin markedly increases the specific activity of ATPase in the Azotobacter R3 fraction, and this increase ranged from 12- to 14-fold after trypsinization for 4 min at 25 "C. Since some bacterial ATPases require a binding protein for attachment to the membrane (5), which might be affected by proteases, a fractionization study was carried out using the Azotobacter R3 fraction to determine whether trypsin treatment released the membranebound ATPase activity into the supernatant (S,) fraction. Table 4 shows the results of this study. An R3 fraction was trypsin-activated (and the reaction stopped by trypsin inhibitor) and ultracentrifuged at 144 000 x g for 2 h. The resulting residue (pellet) fractions were designated R,(heavy) which resembled the original R3 fraction, and R,(light) which was a membrane fluff layer which overlayed the R,(heavy) fraction. The supernatant fraction designated S, represented those proteins that might have been solubilized by the trypsin treatment. As shown, the bulk of the ATPase activity remained membrane-bound as evidenced by specific TABLE 4 Scheme outlining the fractionation of the trypsin-activated ATPase in the Azotobacter R3 fraction (A. uinelatidii) and distribution of protein and ATPase activity units among the soluble and particulate fractions
Sp. act.'
k?
0.06 Trypsin (0.5 mg/mg Pr) Tr-R3 0.74 000 x g, 120 Min0.85
Pr (Total) 150
1 50b 30.5
Units (Total) 9.0 110.9 25.9
(Heavy) 0.83
78
0.17
18.6b
64.7
(Light) 'Specific activity (pmol Pilmin per milligram at 30°C). bExcluding trypsin protein.
3.1
activities of 0.85 and 0.83 found in the R,(heavy) and R,(light) fractions as compared with the low activity of 0.17 found for the S, fraction. Furthermore, about 90 of the original 110 total activity units were accounted for in the two R, particulate fractions while only three units were recovered in the S, fraction. The activation caused by trypsin treatment shows that the membrane fraction is in some way altered to allow for high turnover rates, and this may involve some type of membrane conformational change. Inhibitor Studies No inhibition or stimulation resulted from addition of azide, 2,4-dinitrophenol, oligomycin, or ouabain to the standard reaction mixture.
Discussion Many similarities exist among the various bacterial ATPases studied to date. They all have in common a requirement for divalent cations, either Ca2+ of Mg2+, and maximal activation occurs at a molar ratio of cation: ATP that ranges from 0.5 to 1 .O. They all appear to be intimately associated with cytoplasmic membranes but are usually solubilized by relatively mild procedures, like prolonged washing in cation-deficient buffers (22, 23, 27). Some ATPases may be activated by treatment with proteases (or heat) which apparently unmasks latent activity (9, 23), and in many cases allotopic effects are observed between bound and soluble states (23). Those ATPases purified thus far have molecular weights of about 350 000 and usually are made up of subunits (12, 22, 28). However, both latency and subunit structure may vary depending on the preparative procedures used (3, 26). Many of the ATPases, when solubilized, are inactivated by exposure to cold temperature (cold labile); the effect of inhibitors is variable. ATPases are believed to be functional in ion transport (I), nitrogen fixation (6, 13). ATP-dependent reactions (21), and as coupling factors in oxidative and photophosphorylation systems (4, 7). Many of the findings for the Azotobacter ATPase presented here are consistent with properties described for other bacterial ATPases. It should be emphasized that this study represents only a preliminary characterization of the membrane-bound ATPase of Azotobacter vinelandii, the results of which are a prerequisite for
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JURTSHUK AND McENTIRE: ATPASEIN A. VINELANDII
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terial membrane protein, nectin, essential for the atfurther studies on the isolation, purification, tachment of adenosine triphosphatase. J. Biol. Chem. and characterization of the membrane-bound 1542-1544. ATPase in this bioenergetically diverse organism. 6. 246: BULEN,W. A,, and J. R. LECOMTE.1966. T h e nitroAlthough some of the variables defined in this genase system from Azotobacter: Two-enzyme requirement for N, reduction, ATPdependent H, evolustudy were reported before (9), this study repretion, and A T P hydrolysis. Proc. Natl. Acad. Sci. U.S. sents the first attempt to determine conditions 979-986. for optimal hydrolysis of ATP by the membrane- 7. 56: B U T L I N ,J. D., G. B. COX, and F. GIBSON. 1971. bound ATPase and attempts to show its close Oxidative phosphorylation in Escherichia coli K-12. association with cytoplasmic membrane, especiMutations affecting magnesium ion- or calcium ionstimulated adenosine triphosphatase. Biochem. J. ally the Azotobacter R, fraction which is known 124: 75-81. to contain the highest concentration of electron P. L . , and P. D. BRAGG.1972. Properties of a transport enzymes. As shown herein, the Azoto- 8. DAVIES, soluble Ca+Z- and Mg+z-activated ATPase released bacter ATPase appears to be intimately associafrom Escherichiacoli membranes. Biochim. Biophys. Acta, 266: 273-284. ted with the membranous network that is very extensive in this organism. Preliminary indica- 9. E I L E R M A N NL,. J . M., H. G . PANDIT-HOVENKAMP, M. VAN DER MEER-VANB U R E N A. , H. J . K O L K ,and tions are that Azotobacter ATPase is tightly M. FEENSTRA.1971. Oxidative phosphorylation in bound to membranes since neither extended Azotobactrr vitlelatldii. Effect of inhibitors and unsonication nor trypsin treatment caused its couplers of P I 0 ratio, trypsin-induced A T P a s e and ADP-stimulatedrespiration. Biochim. Biophys. Acta, release into the soluble or supernatant fraction. 245: 305-3 12. This tight association with the membrane-bound 10. F I S K E ,C. H., and Y. SUBBAROW.1925. T h e colelectron transport system, its trypsin activation, orimetric determination of phosphorus. J. Biol. Chem. as well as its apparent ability to serve as a 66: 375-400. , G., C. J. B A R D A W I L L and , M. D A V I D . coupling factor in oxidative phosphorylation I I. G O R N A L LA. 1949. Determination of serum proteins by means ofthe (9) leaves little doubt that the ATPase of Azoreaction. J. Biol. Chem. 177: 751-766. robacter uinelandii, though possibly multifunc- 12. biuret HANSON,R. L., and E . P. K E N N E D Y1973. . Energytional, is at least involved in coupling and energy transducing adenosine triphosphatase from Eschericonservation. The multitude of energy-requiring cilia coli: Purification, properties, and inhibition by antibody. J . Bacteriol. 114: 772-781. processes in Azotobacter warrantspurification and further study of the ATPase to establish its 13. H A R D Y , R. W. F., and E . K N I G H T , J R . 1968. Reductant-dependent adenosine triphosphatase of relationship to structural, physiological, and nitrogen-fixing extracts of Azotobncter vinelandii. bioenergetic processes in this organism. Biochim. Biophys. Acta, 132: 520-531. 14. J U R T S H U KP., , A. J . B E D N A R ZP. , ZEY, and C . H. DENTON. 1969. L-Malate oxidation by t h e electron transport fraction of Azotobacter vinelandii. J . Bacteriol.. 98: 1 120-1 127. 15. JURTSHUK,P., and L. HARPER.1968. Oxidation o f D(-)-lactate by the electron transport fraction of Azotobacter vinelandii. J. Bacteriol. 96: 678-686. 16. JURTSHUK,P., S. M A N N I N Gand , C. R. BARRERA. 1968. Isolation and purification of the D(-)p-hydroxyABRAMS,A , , and J . B. S M I T H .1971. Increased membutric dehydrogenase o f A z o t o b a c t e r vit~elandii.Can. brane ATPase and K+ transport rates in StreptoJ . Microbiol. 14: 775-783. coccirs faecalis induced by K+ restriction during 17. JURTSHUK,P., A. K. M A Y , L. M. POPE, and P. R. growth. Biochem. Biophys. Res. Commun. 44: 14881495. ASTON. 1969. Comparative studies o n succinate and terminal oxidase activity in microbial and mammalian ABRAMS,A., J . B. S M I T H , and C. BARON. 1972. Carbodiimide-resistant membrane adenosine triphoselectron-transport systems. Can. J . Microbiol. 15: phatase in mutants of Streptococcirs faecalis . 1. 797-807. 18. JURTSHUK,P., and L. MCMANUS.1974. Non-pyridine Studiesofthe mechanismof resistance. J. Biol. Chem. nucleotide dependent, L-(+)-glutamate oxidoreduc247: 1484-1488. ANDREAU,J. M., J . A. ALBENDEA, and E . M u ~ o z . tase in Azotobacter vinelnndii. Biochim. Biophys. 1973. Membrane adenosine triphosphatase of MiActa, 368: 158-172. crococciis lysodeikticirs. Molecular properties of the 19. JURTSHUK,P., and L . OLD. 1968. Cytochrome c oxipurified enzyme unstimulated by trypsin. Eur. J . dation by the electron transport fraction of Azotobacter vinelandii. J. Bacteriol. 95: 1790-1797. Biochem. 37: 505-515. A , , H. GEST, and A. S. BACCARINE-MELANDRI, 20. JURTSHUK,P., and B. A. SCHLECH.1969. PhosPIETRO. 1970. A coupling factor in bacterial photopholipids of Azotobacter vinelandii. J. Bacteriol. 97: phosphorylation. J . Biol. Chem. 245: 1224-1226. 1507-1 508. 21. K A N N E R ,B. I., and D. L. GUTNICK.1972. Energy BARON,C., and A. ABRAMS.1971. Isolation of a bac-
Acknowledgments This study was supported by a grant awarded by the National Institute of General Medical Sciences, GM 17607-02 (USPHS). 1.
2.
3.
4. 5.
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22.
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23.
24. 25.
CAN. J. MICROBIOL. VOL. 21, 1975
linked NAD-transhydrogenase in a mutant of E. coli K12 lacking membrane Mg++-Ca++-activated ATPase. F E B S Lett. 22: 197-199. MIRSKY,R., and V. BARLOW.1973. Molecularweight, amino acid composition and other properties of membrane-bound ATPase from Bacillrrs rnegateriutn KM. Biochim. Biophys. Acta, 291: 480-488. M u ~ o z E, . , M. R. J. SALTON,M . H. NG, and M. T. SCHOR.1969. Membrane adenosine triphosphatase of Micrococclrs lysodeikticlrs. Purification, properties of the "soluble" enzyme and properties of the membrane-bound enzyme. Eur. J . Biochem. 7: 490-501. N E U J A H R H. , Y. 1970. T h e effect of proteases o n membrane ATPase from two lactic acid bacteria. Biochim. Biophys. Acta, 203: 261-270. POPE, L . , and P. JURTSHUK.1967. Microtubule in Aiotobncter vinelandii strain 0. J . Bacteriol. 94: 2062-2064.
26. SALTON,M. R. J., and M. T . SCHOR.1974. Release and purification ofMicrococclrs lysodeikticlrs A T P a s e from membranes extracted with N-butanol. Biochim. Biophys. Acta, 345: 74-82. 27. SCHNEBLI,H. P., and A. ABRAMS.1970. Membrane adenosine triphosphatase from Streptococclrs,faecnlis. Preparation and homogeneity. J. Biol. Chem. 245: 1115-1121. 28. S C H N E B L IH. , P., A. E. VATTER,and A. ABRAMS. 1970. Membrane adenosine triphosphatase f r o m Streptococclrs .fnecalis. Molecular weight, subunit structure, and amino acid composition. J. Biol. C h e m . 245: 1122-1 127. , . P., and P. D. BRAGG. 1974. Effect of dicy29. S I N G HA clohexylcarbodiimide o n growth and membranemediated processes in wild type and heptose-deficient mutants of Escliericl~iacoli K-12. J . Bacteriol. 119: 129-137.
'
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PETERJURTSHUK AND JOHNE. MCENTIRE Deprrrtmet~tof Biology, University of Horrston. Horrston, Texns 77004 Accepted June 2, 1975 JURTSHUK, P., and J. E. MCENTIRE.1975. Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotohncter vinelnndii. Can. J . Microbiol. 21: 1807-1814. The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azorohricter ~~inelrrndii concentrates in the membranous R, fraction that is directly associated with Azotohrrcter electron transport function. Sonically disrupted Azorobncrer cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R, membranous fraction which sedimentson ultracentrifugation at 144000 x g for2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activity occurred at pH 8.0; Mg2+ ion when added t o the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATPstoichiometry was 1:l on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R, fraction was stimulated 12-fold by treatment with trypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R, electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R, fraction had no effect on activity; overnight refrigeration at 4°C. however, resulted in a25% loss ofactivity a s compared with a 14% loss when the R, fraction was stored overnight at 25 "C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 "C), and subsequent sonicationfailed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R, electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation. JURTSHUK, P., et J. E . M C E N T I R E1975. . Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azorobrrcrer ~~inelrrndii.Can. J. Microbiol. 21: 1807-1814. est concentric L'activite de I'adenosine triphosphatase (ATPase) chez Azorobncter ~~inelrrndii dans la fraction membranaire R, qui est directement associee a la fonction de transport d'electrons chez Azorobrrcrer. Des cellules d'ilzotobrrcter brisees par sonification furent examinees pour detecter la distribution de I'activite ATPase. e t I'activite la plus specifique (et les unites d'activite) fut constamment trouvte dans la fraction membranaire morcelie R,, sedimentke par ultracentrifugation a 144 000 x g pendant 2 h. Lorsque I'intervalle de sonification fut augment&, I'activite ATPase liCe a la membrane n'a pu 2tre solubilisee ou liberee dans la fraction surnageante. L'activite ATPase optimale est survenue a pH 8.0; I'ion MgZ+ajoute a I'essai fut stimulateur. L'activite maximale est toujours survenue lorsque la stoechiomitrie Mg2+:ATP Ctait dans un rapport molaire 1: 1 a un niveau de concentration d e 5 mM. Les ions sodium et potassium n'ont pas eu d'effet stimulateur. Les reactions cinetiques furent lintaires durant les intervalles utilises (0-60 min). L'ATPase liCe la membrane dans la fraction R, fut stirnulee 12-fois plus par traitement a la trypsine et les etudes de fractionation ont demontre que les traitements la trypsine n'ont pas solubilisC I'activite ATPase de la fraction membranaire d e transport d'electrons R,. L'ATPase ne s'est pas aviree labile froid et la temperature au coursde la preparation d e la fraction R, n'a pas eu d'effet sur I'activite; cependant, la refrigeration a 4 "C, pour la nuit, a conduit a une perte d'activiti de 25% comparee a une perte de 14% lorsque la fraction R, Ctait entreposee a 25 "C pour la nuit. Une inactivation marquee (bien que variable et, habituellement, d'environ 60%) est resultee d'une congelation (-20 "C) durant une nuit; la sonification subsequente n'a pu retablir I'activite ATPase. Ceci est une indication que la reaggregation de la membrane (par congelation) n'est pas responsable de I'inactivation de 'Received February 5, 1975.
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I'ATPase. L'addition d'azide, ouabain, Z,CdinitrophCnol, ou d'oligomycine au systkme de I'essai n'a pas eu d'effet stimulateur ou inhibiteur sur I'activite ATPase. La proprikte de I'activation a la trypsine et que I'activite ATPase est la plus &leveedans la fraction de transport d'klectrons R, suggkrent que son r6le fonctionnel probable est le couplement du transport d'electrons a la phosphorylation oxidative. [Traduit par le journal]
Introduction Detailed studies on bacterial adenosinetriphosphatases (ATPase) (ATP phosphohydrolase, EC 3.6.1.3) have been performed on several genera of bacteria, i.e., Streptococcus (1, 2, 5, 27, 28), Micrococcus (3, 23), Bacillus (22), and Escherichia (8, 12). A substantial amount of information is now available, which suggests that ATPase activity in bacteria is bound to, or closely associated with, the cytoplasmic membrane. The specific function of the various bacterial ATPases is not completely understood, although it appears that this enzyme can serve as a marker enzyme for some energy-dependent processes like (a) ion transport (I), (b) couplingfactor activity in oxidative phosphorylation and photophosphorylation (4,7), (c) association with ATP-dependent reactions such as N A D + transhydrogenase (21), and (d) possibly other bioenergetic processes such as nitrogen fixation (6, 13). The multifunctional roles of ATPase have been derived primarily from the interpretation of dicyclohexylcarbodiimide (DCCD) inhibition data which assume that inhibition of a bioenergetically associated function is due directly to the inhibition of ATPase activity. Singh and Bragg (29) have shown more recently that DCCD inhibition of ATPase activity may not be a valid parameter on which to base functional significance, since DCCD appears able to react with sites other than the ATPase. Depending on the organism examined, the bacterial ATPase may have many features in common with either the mitochondria1 ATPasecoupling factor or the N a + + K+-activated ATPase of eucaryotic plasma membranes. Generally, the bacterial ATPases are ~ g ' +or Ca2+ stimulated enzymes which show little or no activation response to Na' and (or) K + ion. ATPases in bacteria may be cold labile (when they are removed from the membrane) in soluble form (8), and some are affected by treatment with proteases (9, 23,24). They are not generally sensitive to oligomycin, ouabin, or 2,4-dinitrophenol. Azotobacter vinelandii, a Gram-negative, aero-
bic, free-living nitrogen-fixing bacterium could theoretically contain an ATPase (or ATPases) associated with ion transport, coupling, transhydrogenation, nitrogen fixation, or with a variety of other energy-linked reactions that might, for example, be associated with the contractile properties of the 'microtubule' ultrastructure known to be present in isolated membrane fractions (25). Recent studies on oxidative phosphorylation in Azotobacter gave only fragmentary data concerning the major properties of the membrane-bound ATPase (9). Little is known about the Azotobacter ATPase. Bioenergetically this organism is of immense interest because of its unusually high respiratory capability which undoubtedly is related to its efficient free-living nitrogen-fixing potential. Since ATPases are now commonly considered to be valid marker enzymes for numerous bioenergetic processes, a study was undertaken to characterize the nature of the ATPase in membrane fractions and establish its functional relationship to the electron transport system of Azotobacter vinelandii strain 0.
Materials and Methods Bacterial Strain and Growth Conditions Azotobacter vinelandii strain 0 (ATCC2 12518) was grown on Burk's nitrogen-free medium with 1% acetate as sole carbon source. One-litre batch cultures, or seed inocula, were grown at 30 "C for 24-30 h and aerated by reciprocal shaking (using low-form cultureflasks). Large scale batch cultures were grown in glass carboys containing 14 litres of Burk's NF/acetate medium. After 30 h of growth at 30°C with forced-filtered aeration, the Azotobacter cells were harvested by a Sharples centrifuge; washed and standardized resting cell preparations were made in 25 mM Tris3-HC1 buffer, pH 8.0, as described elsewhere
(16). Preparation of Membrane Fractions Turbidimetrically standardized resting cell suspensions were disrupted by sonic oscillation using a Heat Systems Ultrasonics model M140 sonicator, power setting 7 (output 70). Most cell suspensions were sonically irradiated for 2-min intervals with adequate cooling for a total time of 10 min. Varying sonication times were used in one study. After sonication, the homogenate was differen-
'ATCC, American Type Culture Collection. 3Tris, tris(hydroxymethyl)aminomethane.
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tially centrifuged at the speeds and times previously described in other communications (14-19). Both a Beckman J-21B preparative centrifuge and a Beckman L3-50 ultracentrifuge were used to prepare cell-free extracts. All procedures were carried out routinely at 4"C, and where indicated, studies on cold lability were carried out at room temperature (25 "C), refrigerated overnight (4 "C, 16 h), or frozen overnight (--20 "C, 16 h). ATPase Assay The standard colorimetric assay system was used for all ATPase determinations. The enzyme reaction mixture contained 25 m M Tris-HCI, pH 8.0; 5 m M NaATP; 5 m M MgC1,; and 1-3 mg of protein in a total volume of 1.0 ml. After 10 min of incubation at 30 "C, the reaction was stopped by the addition of 0.5 ml of 15% (w/v) trichloroacetic acid (TCA). Inorganic phosphate released by ATP hydrolysis was determined calorimetrically (10). All specific activities for ATPase are expressed as pmol Pi (inorganic phosphate) liberatedlmin per milligram protein. In studying the effect of metal ions, Mg2+ ion was replaced in the standard assay system with other divalent or monovalent cations among which were C a 2 + , M n 2 + , C o 2 + ,Hg2+, Cu2+, Zn2+, N a + , K + , and both N a + and K + ions. Final concentration of all metal ions was 5 mM. Inhibitors of ATPase activity as well as electron transport activity were added t o the standard assay system at a final concentration of M , except for oligomycin of which only 5 pg was added. Trypsin Trearnzenr The Azorobacrer R3 electron transport fraction (protein concentration 10-30 mglml) was treated with trypsin (0.5 mg/mg R 3 protein) for 4 min at 25 "C. At the end of this time interval, an excess of soybean trypsin inhibitor (type 11) was added. Prorein Dererminarion Protein was determined by the Biuret method using bovine serum albumin as a standard (1 1).
Results The Relationship of ATPase Activity to the Membranous R , Electron Transport Fraction It has previously been shown that the Azotobacter R, fraction contains both the highest specific activity as well as the greatest amount of the membrane-bound enzymes associated with electron transport function (14, 15, 17-20). Initial ATPase fractionation studies revealed that the ATPase activity similarly concentrated in the R, fraction (see Table 1). A study was carried out to determine if increased sonication times would release the bound ATPase found in the R, fraction into the S, supernatant fraction. For this study, the sonication times were varied from 1 to 15 min and the activities found in the R, and S, fractions are shown in Table 2. The highest specific activity (0.059) for ATPase was found in the R, fraction prepared by sonicating whole
TABLE 1 Distribution of activity units for ATPase in fractions obtained by differential centrifugation from sonically disrupted resting cells of Azorobacrer vinelandii Activity Protein recovery, Fraction
%
Sp. act.'
Total unitsb
Recovery,
%
'Expressed as pmol Pi liberatedlrnin per milligram protein at 30DC. T o t a l activity units were calculated by multiplying total protein concentration by specific activity.
cells 9-10 min. This was the sonication time used routinely in preparing the Azotobacter R, electron transport fraction. Total activity units recovered in the R, fraction are also greatest for this 9- to 10-min sonication time interval. The specific activities for ATPase activity in the S, fraction ranged from 0.014-0.033 and never did become as high as the value found for the R, fraction. Increasing the time of sonication of whole cells past the 10-min time interval does not result in activity being released into the S, supernatant fraction although by increasing the sonication time interval, some inactivation occurred as evidenced by a 50% loss in specific activity in the R, fraction prepared from cells after a 15 rnin sonication time. This does not represent heat denaturation since precautions were taken to keep all temperature no higher than 10" during all exposures to sonication procedures. Therefore, all additional characterization studies for ATPase activity in the R, fraction were carried out using an R, electron transport fraction prepared after only a 10-min sonication interval. Kinetic Studies on the Azotobacter ATPase Activity EfSect of Reaction pH The effect of reaction pH on ATPase activity is shown in Fig. 1. Three buffer systems were used to obtain experimentally the desired pH range. Suitable assay controls were always run to insure that non-enzymatic ATP hydrolysis was negligible. Optimal ATPase activity occurs at pH 8.0 with considerable activity also occurring over the range pH 8.0-10.0.
CAN. J . MICROBIOL. VOL.
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TABLE 2 Effect of time of sonication on distribution of protein and ATPase activity ATPase activity -
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Protein recovered, % of SZ fraction Time, min
R3
S3
R3
Activity recovered, % of Sz fraction
Sp. act." S3
R3
S3
I>
%Specific activity expressed as pmol Pi liberatedlmin per milligram protein at 30'C.
ATPase A C TlVlTY
-
4z0T0B4cTER
R3
Pr=38mg
1.6
-
Maximal ATPase activity occurs at that point where both ATP and M g 2 + ion were added in equimolar amounts at the 5-mM concentration g 2 + ion were pointequimolar is noted level. where The both other A T P and highM activity
-
5 I
0
7
1.2
o W l-
-
Trrs-IlC1
-
a LL W
m -1
.a
08
-
J
o
r x 0.4
-
C i t r a t e Buffer
-
I
0 4.0
6.0
I 8.0
,
P H
FIG.1 . Effect of reaction pH on ATP hydrolysis by the R3 fraction.
Azotobacter
Actioation by ~ g Ion~ + The effect ATP in the absence and presence of different concentrations of Mg2+ ion is shown in Fig. 2.
a t the 20-mM concentration level. The 1 :1 molar ratio (of Mg2+:ATP) probably indicated the optimal substrate to metal ion concentration needed t o form a Mg2+-ATP complex, the active substrate complex needed for enzyme turnover. The control, which contained no externally added Mg2+ ion, showed a decrease in activity with increasing ATP concentrations. The effect appears to be identical with those observed for the other curves where the A T P concentrations exceeds the M ~ ion ~ concen+ tration. The initial high activity for the control is probably due to residual ~ g ion~present + in the Azotobacter R 3 fraction. This endogenous ATPase activity was absent when the R 3 membrane fraction was pretreated by exposure t o 1 m M EDTA.4 Eflect of Other Cations As shown in Table 3, M n 2 + and Ca2+ ions could replace Mg2+ ion t o some extent in stimulating ATPase activity. All other cations tested were inhibitory. Titne-course of Reaction and Stability Figure 3 shows an ATPase kinetic study which defines the relationship of the amount of Pi released as a function of the reaction time. Both figures are essentially identical and ATPase "EDTA, ethylenediaminetetraaceticacid.
JURTSHUK AND McENTIRE: ATPASE IN
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0 20
10
1811
VINELANDII
activity is plotted using two different time scales. In both studies the rate of release of Pi from ATP, for the various R, fractions, is essentially linear with respect to the time intervals examined. The R, fraction designated A was prepared at temperatures of 15-25 "C to examine whether the Azotobacter enzyme was cold labile; B represents an R, fraction prepared from the same batch of resting cells but all storage and preparatory temperatures were kept a t 4°C. Curves C and D represent the activity of the same Azotobacter R, fraction as in curve A, only after overnight storage a t room temperature and frozen, respectively. Curve E shows this same R, fraction after having been stored frozen (-20 "C) for 2 weeks. The similarity in activity patterns for both curves A and B shows that the Azotobacter membrane-bound ATPase is not cold labile in the classical sense, since preparation a t 4 ° C results in n o loss of activity, while overnight freezing results in a significant loss of as much as 60% in some samples. When the freezing time interval is extended, one markedly inactivates ATPase activity. The frozen R, fraction (activity pattern of curve E) was resonicated and it was not possible to recover the
ATPase A C T I V I T Y
0
A.
30
ATPose
ACTIVITY
ATP (mM)
FIG. 2. Effect of MgZ+ion concentration on ATP hydrolysis by the Azotobacter R3 fraction at varying ATP concentrations.
TABLE 3 Effect of various cations on ATPase activity of the Azotobacter R3 fraction Cationa
None Co2 Nat K Na HgZ CuZ ZnZ +
Sp. acLb
Activity, % '
+ K+
+
+
+
+
+
OFinal concentration of all metal cations was 5 rnM. bExpressed a s pmol Pi liberatedimin per milligram protein a t 30 " C .
FIG. 3. Typical time-course reactions for A T P hydrolysis by the Azotohacter R 3 fraction as plotted on two different time scales.
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CAN. J. MICROBIOL. VOL. 21, 1975
original high ATPase activity. This suggests that reaggregation of membranes caused by prolonged freezing is not responsible for this inactivation. The temperature at which the Azotobacter R3 fraction is prepared has little effect on the ATPase activity, but the temperature and duration of storage are important factors in maintaining high activity in the R, fraction. Trypsin Activation Treatment by trypsin markedly increases the specific activity of ATPase in the Azotobacter R3 fraction, and this increase ranged from 12- to 14-fold after trypsinization for 4 min at 25 "C. Since some bacterial ATPases require a binding protein for attachment to the membrane (5), which might be affected by proteases, a fractionization study was carried out using the Azotobacter R3 fraction to determine whether trypsin treatment released the membranebound ATPase activity into the supernatant (S,) fraction. Table 4 shows the results of this study. An R3 fraction was trypsin-activated (and the reaction stopped by trypsin inhibitor) and ultracentrifuged at 144 000 x g for 2 h. The resulting residue (pellet) fractions were designated R,(heavy) which resembled the original R3 fraction, and R,(light) which was a membrane fluff layer which overlayed the R,(heavy) fraction. The supernatant fraction designated S, represented those proteins that might have been solubilized by the trypsin treatment. As shown, the bulk of the ATPase activity remained membrane-bound as evidenced by specific TABLE 4 Scheme outlining the fractionation of the trypsin-activated ATPase in the Azotobacter R3 fraction (A. uinelatidii) and distribution of protein and ATPase activity units among the soluble and particulate fractions
Sp. act.'
k?
0.06 Trypsin (0.5 mg/mg Pr) Tr-R3 0.74 000 x g, 120 Min0.85
Pr (Total) 150
1 50b 30.5
Units (Total) 9.0 110.9 25.9
(Heavy) 0.83
78
0.17
18.6b
64.7
(Light) 'Specific activity (pmol Pilmin per milligram at 30°C). bExcluding trypsin protein.
3.1
activities of 0.85 and 0.83 found in the R,(heavy) and R,(light) fractions as compared with the low activity of 0.17 found for the S, fraction. Furthermore, about 90 of the original 110 total activity units were accounted for in the two R, particulate fractions while only three units were recovered in the S, fraction. The activation caused by trypsin treatment shows that the membrane fraction is in some way altered to allow for high turnover rates, and this may involve some type of membrane conformational change. Inhibitor Studies No inhibition or stimulation resulted from addition of azide, 2,4-dinitrophenol, oligomycin, or ouabain to the standard reaction mixture.
Discussion Many similarities exist among the various bacterial ATPases studied to date. They all have in common a requirement for divalent cations, either Ca2+ of Mg2+, and maximal activation occurs at a molar ratio of cation: ATP that ranges from 0.5 to 1 .O. They all appear to be intimately associated with cytoplasmic membranes but are usually solubilized by relatively mild procedures, like prolonged washing in cation-deficient buffers (22, 23, 27). Some ATPases may be activated by treatment with proteases (or heat) which apparently unmasks latent activity (9, 23), and in many cases allotopic effects are observed between bound and soluble states (23). Those ATPases purified thus far have molecular weights of about 350 000 and usually are made up of subunits (12, 22, 28). However, both latency and subunit structure may vary depending on the preparative procedures used (3, 26). Many of the ATPases, when solubilized, are inactivated by exposure to cold temperature (cold labile); the effect of inhibitors is variable. ATPases are believed to be functional in ion transport (I), nitrogen fixation (6, 13). ATP-dependent reactions (21), and as coupling factors in oxidative and photophosphorylation systems (4, 7). Many of the findings for the Azotobacter ATPase presented here are consistent with properties described for other bacterial ATPases. It should be emphasized that this study represents only a preliminary characterization of the membrane-bound ATPase of Azotobacter vinelandii, the results of which are a prerequisite for
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JURTSHUK AND McENTIRE: ATPASEIN A. VINELANDII
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terial membrane protein, nectin, essential for the atfurther studies on the isolation, purification, tachment of adenosine triphosphatase. J. Biol. Chem. and characterization of the membrane-bound 1542-1544. ATPase in this bioenergetically diverse organism. 6. 246: BULEN,W. A,, and J. R. LECOMTE.1966. T h e nitroAlthough some of the variables defined in this genase system from Azotobacter: Two-enzyme requirement for N, reduction, ATPdependent H, evolustudy were reported before (9), this study repretion, and A T P hydrolysis. Proc. Natl. Acad. Sci. U.S. sents the first attempt to determine conditions 979-986. for optimal hydrolysis of ATP by the membrane- 7. 56: B U T L I N ,J. D., G. B. COX, and F. GIBSON. 1971. bound ATPase and attempts to show its close Oxidative phosphorylation in Escherichia coli K-12. association with cytoplasmic membrane, especiMutations affecting magnesium ion- or calcium ionstimulated adenosine triphosphatase. Biochem. J. ally the Azotobacter R, fraction which is known 124: 75-81. to contain the highest concentration of electron P. L . , and P. D. BRAGG.1972. Properties of a transport enzymes. As shown herein, the Azoto- 8. DAVIES, soluble Ca+Z- and Mg+z-activated ATPase released bacter ATPase appears to be intimately associafrom Escherichiacoli membranes. Biochim. Biophys. Acta, 266: 273-284. ted with the membranous network that is very extensive in this organism. Preliminary indica- 9. E I L E R M A N NL,. J . M., H. G . PANDIT-HOVENKAMP, M. VAN DER MEER-VANB U R E N A. , H. J . K O L K ,and tions are that Azotobacter ATPase is tightly M. FEENSTRA.1971. Oxidative phosphorylation in bound to membranes since neither extended Azotobactrr vitlelatldii. Effect of inhibitors and unsonication nor trypsin treatment caused its couplers of P I 0 ratio, trypsin-induced A T P a s e and ADP-stimulatedrespiration. Biochim. Biophys. Acta, release into the soluble or supernatant fraction. 245: 305-3 12. This tight association with the membrane-bound 10. F I S K E ,C. H., and Y. SUBBAROW.1925. T h e colelectron transport system, its trypsin activation, orimetric determination of phosphorus. J. Biol. Chem. as well as its apparent ability to serve as a 66: 375-400. , G., C. J. B A R D A W I L L and , M. D A V I D . coupling factor in oxidative phosphorylation I I. G O R N A L LA. 1949. Determination of serum proteins by means ofthe (9) leaves little doubt that the ATPase of Azoreaction. J. Biol. Chem. 177: 751-766. robacter uinelandii, though possibly multifunc- 12. biuret HANSON,R. L., and E . P. K E N N E D Y1973. . Energytional, is at least involved in coupling and energy transducing adenosine triphosphatase from Eschericonservation. The multitude of energy-requiring cilia coli: Purification, properties, and inhibition by antibody. J . Bacteriol. 114: 772-781. processes in Azotobacter warrantspurification and further study of the ATPase to establish its 13. H A R D Y , R. W. F., and E . K N I G H T , J R . 1968. Reductant-dependent adenosine triphosphatase of relationship to structural, physiological, and nitrogen-fixing extracts of Azotobncter vinelandii. bioenergetic processes in this organism. Biochim. Biophys. Acta, 132: 520-531. 14. J U R T S H U KP., , A. J . B E D N A R ZP. , ZEY, and C . H. DENTON. 1969. L-Malate oxidation by t h e electron transport fraction of Azotobacter vinelandii. J . Bacteriol.. 98: 1 120-1 127. 15. JURTSHUK,P., and L. HARPER.1968. Oxidation o f D(-)-lactate by the electron transport fraction of Azotobacter vinelandii. J. Bacteriol. 96: 678-686. 16. JURTSHUK,P., S. M A N N I N Gand , C. R. BARRERA. 1968. Isolation and purification of the D(-)p-hydroxyABRAMS,A , , and J . B. S M I T H .1971. Increased membutric dehydrogenase o f A z o t o b a c t e r vit~elandii.Can. brane ATPase and K+ transport rates in StreptoJ . Microbiol. 14: 775-783. coccirs faecalis induced by K+ restriction during 17. JURTSHUK,P., A. K. M A Y , L. M. POPE, and P. R. growth. Biochem. Biophys. Res. Commun. 44: 14881495. ASTON. 1969. Comparative studies o n succinate and terminal oxidase activity in microbial and mammalian ABRAMS,A., J . B. S M I T H , and C. BARON. 1972. Carbodiimide-resistant membrane adenosine triphoselectron-transport systems. Can. J . Microbiol. 15: phatase in mutants of Streptococcirs faecalis . 1. 797-807. 18. JURTSHUK,P., and L. MCMANUS.1974. Non-pyridine Studiesofthe mechanismof resistance. J. Biol. Chem. nucleotide dependent, L-(+)-glutamate oxidoreduc247: 1484-1488. ANDREAU,J. M., J . A. ALBENDEA, and E . M u ~ o z . tase in Azotobacter vinelnndii. Biochim. Biophys. 1973. Membrane adenosine triphosphatase of MiActa, 368: 158-172. crococciis lysodeikticirs. Molecular properties of the 19. JURTSHUK,P., and L . OLD. 1968. Cytochrome c oxipurified enzyme unstimulated by trypsin. Eur. J . dation by the electron transport fraction of Azotobacter vinelandii. J. Bacteriol. 95: 1790-1797. Biochem. 37: 505-515. A , , H. GEST, and A. S. BACCARINE-MELANDRI, 20. JURTSHUK,P., and B. A. SCHLECH.1969. PhosPIETRO. 1970. A coupling factor in bacterial photopholipids of Azotobacter vinelandii. J. Bacteriol. 97: phosphorylation. J . Biol. Chem. 245: 1224-1226. 1507-1 508. 21. K A N N E R ,B. I., and D. L. GUTNICK.1972. Energy BARON,C., and A. ABRAMS.1971. Isolation of a bac-
Acknowledgments This study was supported by a grant awarded by the National Institute of General Medical Sciences, GM 17607-02 (USPHS). 1.
2.
3.
4. 5.
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