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Stereospecificity of the allosteric NADH dehydrogenase from Mycobacterium tuberculosis
ARCHIVES
OF
BIOCHEMISTRY
Stereospecificity
AND
BIOPHYSICS
42&423
118,
of the Aliosteric Mycobacterium A. WORCEL
Tuberculosis
AND
(1967)
NADH
Dehydrogenase
tuberculosis’
DEXTER
S. GOLDMAN
Research Laboratory, Veterans Administration Hospital, and the Institute Research, Ciniversity of Wisconsin, Madison, Wisconsin 53706 Received
from
for Enzyme
June 17, 196G
The electron chain-linked, allosteric NADH dehydrogenase from Mycobacterium tuberculosis is a 4-B NADH specific enzyme, and does not catalyze NADH-II20 H-exchange. AMP activation of this dehydrogenase does not affect the stereospecificity of the H removal from NADH and has no effect on the lack of NADIIGHZO H-exchange. A soluble, AMP-sensitive NADH-menadione oxidoreductase can be extracted from the particulate NADH oxidase complex. This soluble diaphorase, as well as the soluble NADH-nitro-blue tetrazolium oxidoreductase present in cell-free extracts of this microorganism are also 4-B NADH specific enzymes and do not catalyze the NADH-H20 H-exchange reaction; this is taken as further proof that the soluble diaphorases from M. tuberculosis are derived from the membrane-bolrnd NADH oxidase complex.
Drysdale and Cohn (1) showed that the mitochondrial KADH oxidase system specifically removes the 4-B hydrogen (2) of NADH and also catalyzes an exchange reaction between the 4-B hydrogen of KADH and HzO. Ernster et al. (3, 4), in a confirmation and extension of these findings, reported that soluble KADH dehydrogenases derived from the mitochondria.1 electron chain-linked KADH dehydrogenase are also 4-B NADH specific and catalyze NADH-HZ0 H-exchange. The microsomal NADH-cytochrome b5 oxidoreductase, the DT diaphorase2 and the NADH-NADP transhydrogenase, on the other hand, were found to be 4-A NADH specific and devoid of the exchange reaction.
Previous work from this laboratory showed that the particulate NADH dehydrogenase of Mycobacteriuw tuberculosis is an allosteric enzyme activated by AMP (5). The AMP activat,ion, which causes a marked kinetic (6) and thermodynamic (7) change in the dehydrogenase is probably due to a conformational change in the enzyme (S). It was of interest to us to see if this allosteric activation had any effect on the stereospecificity of the enzyme. Studies on the stereospecificity of t#he particulate and soluble NADH dehydrogenases would also be of importance in the continuing investigations on the question of the derivation of the soluble NADH dehydrogenases from the membrane-bound ,\ADH dehydrogenases (9).
1 This research was supported, in part, by research grant, A102416.08 from the National Institute of Allergy and Infectious Diseases, USPHS. 2 Abbreviations: DT, diaphorase, NADH, NADPH: (acceptor) oxidoreductase, dicoumarol sensitive flavoenzyme; NBT, nitro-blue tetrazolium chloride, 2,2’-di-p-nitrophenyl-5,5’-diphenyl - 3,3’(3,3’ - dimethoxy - 4,4’ - biphenylene) dit,etraeolium chloride.
MATERIALS
AND
METHODS
TzO was purchased from New England Nuclear Corp., Boston, Massachusetts. (T)NAD+, prepared by the method of San Pietro (10) as modified by Krakow et al. (II), was 70yo pure when assayed spectrophotometrically by NADH formation wit,h glllcose and glucose dehydrogenase, and had a 420
NADH
DEHYI)ROGENASE TABLE
STEREOSPECIFICITY OF T REMOV.IL
FI~OM NADT
421
STEREOSPECIFICITY I BY THE ALLOSTERIC NADH
OXID:SE
FROM
.%I. TUBERCULOSIP Substrate Experimental
-AMP
Expt. 1 AA&mitt cpm in Hz0 To T removed Expt. 2 cpm in Hz0 yc T removed
0.219 599 24 262 9
A-NADT +4MP
0.291 479 19 241 8
--Enzyme
0 358 14 254 9
B-NADT +AMP
-AMP
0.219 2,156 8(i 2,638 93
--Enzyme
0.291 2,450 97 2,778 97
0 622 25 108 4
(1The incubation mixtttre contained 5 rmoles of NADT (total cpm in Expt. 1 was 1.0 X IO5 aud in Expt. 2 was 1.13 X 105), 40 @moles of phosphate buffer, pH 7.5, 1.2 mg of oxidase protein aud water to a final volume of 4.0 ml. AMP, when added, was at a final concentration of 1 mM. The reaction proceeded for 5 minutes at 30”; the reaction was stopped by immersing the tubes in a boiling wat,er bath for 2 minutes. Radioactivity was det,ermined on 0.1.ml aliquots as described above. specific activity of 2 X lo5 counts per minttte per pmole at 20% counting efficiency. 4-A NADT [with the tritium on the A-side of the nicotinamide plane (2)] was prepared by reducing the (T)NAD+ with the B-specific glucose dehydrogenase purified from beef liver (12) and 4-B NADT was prepared by reducing (T)NAD+ with the A-specific yeast alcohol dehydrogenase (Sigma Chemical Co., St. Lotus, Missouri) essentially as described by Krakow et al. (11). The preparation of the particttlate NADH oxidase and the soluble NAI)H-nitroblue tetrazolium oxidoredttctase from extracts of hf. tttberculosis has been previortsly described [(l(i), (13)l. The soluble NADH-menadione oxidorednctase of JI. fubercdosis was obtained from t,he particulate N.4DH oxidase. The particles were ext,racted twice at -2O”, each time with 20 volrtmes of acet,one. The suspension was filtered under suction, washed twice with ethyl ether, air dried, and ground to a fine powder. The dry powder was extracted with 0.1 M Tris buffer (pH 7.5) equal in volume to the original suspension of particles. The homogenized srtspension was centrifttged at, 105,OtMg for A0 minutes. The restdting yellow supernatant solution contained the NADHmenadione oxidoredttctase activity. NAD+ reduct,ion and NAI)H oxidation were followed spectrophotometrically (6). The reaction was run in two steps; in the first, (T)NAl>+ was incttbated wit)h the appropriate enzyme and sttbstrate until completely redriced, the enzyme was denatured by heating the reaction mixture in boiling water for 2 minutes, and after cooling, the NADT was reoxidized in t.he secotid step by addition of the NAI)H dehydrogenase or oxidase and fitrther incttbation (11). After heatiiig for 2 millrites to stop the oxidation reaction, II20 was re-
covered from the reaction mixture by sublimation (4). Tritium in the H& was measured with a Packard Instrument Co. scintillation counter. RESULTS
When t#heparticulate NADH oxidase from M. tuberculosis is incubated with t,he 4-R
Y
20 Minutes
J 40
FIG. 1. Time course of B-NADT oxidation and T removal by particulate NADII oxidase. Conditions as in Table I, except t,hat 0.4 mg NADH oxidase protein was ttsed. Incubation temperatrtre was 20”. At times indicat,ed, aliquot samples were removed and analyzed for t,ritiat.ed water. Radioactivity has been corrected for a zero-time control. (a---@) counts/mm. in water; (X-X) ratio betweeii the rates of removal of tritium and the oxidation of stthstrate (isotope etl’ect).
422
WORCEL
AND
TABLE II ABSENCE OF NADT-Hz0 T-EXCHANGE DURING B-NAI)T OXIDATION BY THE NADH OXID~ISE OF M. TUBERCULOSIS~ Incubation Conditions Aerobic Anaerobic
cpm in Hz0 y. T removed
-AMP
+AMP
3224 100
3208 100
;;$
-AMP
193 6
692 22
+AMP 843 26
a Conditions as in Table I except that incubation was carried out for 30 minutes at 30”, the final volume was 3.0 ml, and the total cpm was 0.96 X 105. Anaerobiosis was obtained by flushing 02-free Nz through Thunberg-type reaction tubes for 10 minutes prior to enzyme addition.
NADT, more than 90 % of the T is recovered in the Hz0 (Table I). On the other hand, no T removal occurs when the enzyme oxidizes 4-A NADT. There is no change in the stereospecificity of the T removal when the oxidase is activated by AMP. The time course of T removal from the 4-B NADT is shown in Fig. 1. The rate of detritiation measured by the T recovered in the Hz0 is slower than the rate of NADH oxidation followed spectrophotometrically at TABLE B-NADT
OXIDATION
III
AMP
<0.005 420 15
AA340 mr/min cpm in Hz0 y0 T removed
340 rnp, which results in a time-dependent increase in the specific activity of both the residual NADT as well as the TzO formed. The same pattern was observed in the presence of AMP. The loss of T from NADT could be due either to the oxidation of NADT or to a T exchange reaction between NADT and Hz0 catalyzed by the NADH dehydrogenase. The exchange reaction was ruled out by incubating the enzyme with 4-B NADT under anaerobic conditions. As seen in Table II after 30 minutes at 30” only 20 % of the T could be recovered in the HzO. Aerobically, oxidation and T removal were completed in 5 minutes. The small anaerobic T removal was due to oxidation of NADT during the heating and handling of the mixture. The soluble NADH-menadione oxidoreductase extracted from the NADH oxidase particles a,lso shows 4-B specificity. T removal and NADH oxidation occur only in the presence of menadione (Table III). These data also show that the dehydrogenase does not catalyze T exchange between NADT and H,O. This soluble NADH dehydrogenase is activated by AMP; as
BY NADH-MENBDIONE
NOW2
Additions to enzyme
GOLDMAN
REDCCTASE~
Menadione
<0.005 451 16
0.180 2335 83
Menadione AMP
-Enzyme
0.230 2482 87
0 376 13
a Conditions as in Table I except that 200 pg of soluble NADH-menadione oxidoreductase and 30 pg of menadione used in 1.0 ml final volume. Total cpm was 2.84 X 104. TABLE NADT
OXIDATION
IV
BY SOLUBLE NADH-NBT
L>I.~PHOR.GE~ B-NADT
A-NADT -NBT AASO mM/min cpm in Hz0 y0 T removed
0 268 8
protein
+NBT
-Enzyme
0.340 370 11
0.080” 244 8
-NBT 0 260 8
+NBT 0.260 3,064 96
a Conditions as in Table I, except that 200 gg of soluble NADH-NBT oxidoreductase protein and 0.2 pmole NBT were used in 1.0 ml final volume. The pH of t,he reaction mixture was adjusted to 10.0 with N KOH. Total cpm was 3.18 X 104. b Reduction of NBT due to the glucose remaining from the prior reaction forming A-NADT.
NADH
DEHYDROGENASE
with the particulate enzyme, AZUP activation changes neither the stereospecificity nor the lack of the T exchange reaction. Finally, the NADH-NBT oxidoreductase of M. tubenxlosis (13) is also 4-B specific and lacks the NADT-Hz0 T-exchange reaction (Table IV). DISCUSSION
STEREOSPECIFICITY
423
proof that t’he soluble NADH diaphornses from ext’racts of M. tuberculosis are derived from the membrane-bound i\‘ADH dchydrogenase and are probably released by the mechanical disruption of the bacillus. ACKNOWLEDGMENTS Radioactivity counting ey\lipment was generously provided by I>rs. John W. Porter and A. ,4. MacKinney, Jr. of this hospital. Meat byprodrlcts were generously furnished by Oscar Mayer & Co., Madison, Wisconsin.
Ernster et al. (3) reported that the 4-B specific mitochondrial electron chain-linked NADH dehydrogenase, as well as the various REFERENCES soluble NADH dehydrogenases derived from it, catalyze H exchange between 1. I)KYSUALE, Cr. It., .IKD COHN, M., Biochinz. Biophys. Acta 21, 397 (1956). NADH and HzO, while the 4-A specific 2. CORNFORTH,J. W., RYBACK, G., P~P.J.IK, G., NADH dehydrogenases (NADH-NADP I~ONNINGER, C., .\ND SCHROEPFER,G., Riotranshydrogenase, DT diaphorase, microthem. Biophys. Res. Commun. 9, 371 (1962). somal NADH-cytochrome bg reductase) do 3. EILNSTER, L., HoBERM.~N, 11. I)., Ho\x\m), not catalyze H exchange. They suggested R. L., KING, T. E., LEE, C. I'., MXKLER, that this may reflect a general difference in B., .',ND sOTTOC.\SA, c:., ;VUtUJX 207, 940 the mechanism of action between A and B (IQ&i). specific dehydrogenases. That this is not so 4. LEE, C. P., SIX~AI~D-I)IIQUESNE,N., EIINSTEII, can be seen from the results reported above L., ASD HOBERYAN, H. I>., Biochim. Bioon the electron chain-linked YXADH dephys. Ada 105, 397 (1965). 5. WOHCEL, A., .AND GOLDMAN, 1). S., Biochenl. hydrogenase from AR. tuberculosis, a 4-B Biophys. Res. Commun. 17, 559 (1964). specific enzyme which does not catalyze H 6. WORCEL, A., C:OLUX\N, L). S., AND CLEIAND, exchange between NADH and HzO. W. W ., J. Biol. (‘hem. 240, 3399 (1965). The slower rate of detritiation observed 7. WORCEL, A., Riochim. Biophys. Acta 113, 178 as compared to the rate of NADH oxidation (1966). is probably due to an isotope discrimination 8. MONOD, J., WYMIIN,J., AND CHANGBLX,J. P., effect. This enzyme, like ot,her KADHJ. Mol. Hiol. 12, 88 (1905). linked dehydrogenases (14)) oxidizes NADH 9. HEINEN, W., KXI~NOSE, M., KIJSUXOSE, E., preferentially over NADT. The AMP GOLDMAN, I). S., ANDW.ZGNER,M. J., drch. activation of the NADH oxidase and Biochem. Biophys. 104, 448 (1964). dehydrogenase does not change the stereo- 10. SAN PIETI~O, A., J. Niol. Chem. 217, 579 (1955). 11. K~xAK~\Y, G., LIJD~~IEG, J., M.\THER, J. II., specificity of the H removal from NADH, NOKMORE,W. N., Tosr, L., UDAKA, S., AND and similarly has no effect on the lack of VENNESL.IND, B., Biochemistr~g 2, 1009 NADH-Hz0 H-exchange reaction. (1963). NADH : (acceptor) oxidoreductases, either 12. STRECKER, H. J., ANI) KORKES, S., J. Hiol. solubilized from the NADH oxidase particles Chem. 196, 769 (1952). or found already soluble in the cell-free 13. ODA, T., AND GOLDMAN, 1). S., li’iochim. Bioextract’ of ‘11. tuberculosis are also 4-B phys. =Lcta 59, 601 (1962). NADH specific and show no NADH-HZ0 14. KH.\KO\~,(+., UDAK.\, S., AND VENNESL.IND, 13., H-exchange. This is taken as a further Biochemistry 1, 251 (1962).
OF
BIOCHEMISTRY
Stereospecificity
AND
BIOPHYSICS
42&423
118,
of the Aliosteric Mycobacterium A. WORCEL
Tuberculosis
AND
(1967)
NADH
Dehydrogenase
tuberculosis’
DEXTER
S. GOLDMAN
Research Laboratory, Veterans Administration Hospital, and the Institute Research, Ciniversity of Wisconsin, Madison, Wisconsin 53706 Received
from
for Enzyme
June 17, 196G
The electron chain-linked, allosteric NADH dehydrogenase from Mycobacterium tuberculosis is a 4-B NADH specific enzyme, and does not catalyze NADH-II20 H-exchange. AMP activation of this dehydrogenase does not affect the stereospecificity of the H removal from NADH and has no effect on the lack of NADIIGHZO H-exchange. A soluble, AMP-sensitive NADH-menadione oxidoreductase can be extracted from the particulate NADH oxidase complex. This soluble diaphorase, as well as the soluble NADH-nitro-blue tetrazolium oxidoreductase present in cell-free extracts of this microorganism are also 4-B NADH specific enzymes and do not catalyze the NADH-H20 H-exchange reaction; this is taken as further proof that the soluble diaphorases from M. tuberculosis are derived from the membrane-bolrnd NADH oxidase complex.
Drysdale and Cohn (1) showed that the mitochondrial KADH oxidase system specifically removes the 4-B hydrogen (2) of NADH and also catalyzes an exchange reaction between the 4-B hydrogen of KADH and HzO. Ernster et al. (3, 4), in a confirmation and extension of these findings, reported that soluble KADH dehydrogenases derived from the mitochondria.1 electron chain-linked KADH dehydrogenase are also 4-B NADH specific and catalyze NADH-HZ0 H-exchange. The microsomal NADH-cytochrome b5 oxidoreductase, the DT diaphorase2 and the NADH-NADP transhydrogenase, on the other hand, were found to be 4-A NADH specific and devoid of the exchange reaction.
Previous work from this laboratory showed that the particulate NADH dehydrogenase of Mycobacteriuw tuberculosis is an allosteric enzyme activated by AMP (5). The AMP activat,ion, which causes a marked kinetic (6) and thermodynamic (7) change in the dehydrogenase is probably due to a conformational change in the enzyme (S). It was of interest to us to see if this allosteric activation had any effect on the stereospecificity of the enzyme. Studies on the stereospecificity of t#he particulate and soluble NADH dehydrogenases would also be of importance in the continuing investigations on the question of the derivation of the soluble NADH dehydrogenases from the membrane-bound ,\ADH dehydrogenases (9).
1 This research was supported, in part, by research grant, A102416.08 from the National Institute of Allergy and Infectious Diseases, USPHS. 2 Abbreviations: DT, diaphorase, NADH, NADPH: (acceptor) oxidoreductase, dicoumarol sensitive flavoenzyme; NBT, nitro-blue tetrazolium chloride, 2,2’-di-p-nitrophenyl-5,5’-diphenyl - 3,3’(3,3’ - dimethoxy - 4,4’ - biphenylene) dit,etraeolium chloride.
MATERIALS
AND
METHODS
TzO was purchased from New England Nuclear Corp., Boston, Massachusetts. (T)NAD+, prepared by the method of San Pietro (10) as modified by Krakow et al. (II), was 70yo pure when assayed spectrophotometrically by NADH formation wit,h glllcose and glucose dehydrogenase, and had a 420
NADH
DEHYI)ROGENASE TABLE
STEREOSPECIFICITY OF T REMOV.IL
FI~OM NADT
421
STEREOSPECIFICITY I BY THE ALLOSTERIC NADH
OXID:SE
FROM
.%I. TUBERCULOSIP Substrate Experimental
-AMP
Expt. 1 AA&mitt cpm in Hz0 To T removed Expt. 2 cpm in Hz0 yc T removed
0.219 599 24 262 9
A-NADT +4MP
0.291 479 19 241 8
--Enzyme
0 358 14 254 9
B-NADT +AMP
-AMP
0.219 2,156 8(i 2,638 93
--Enzyme
0.291 2,450 97 2,778 97
0 622 25 108 4
(1The incubation mixtttre contained 5 rmoles of NADT (total cpm in Expt. 1 was 1.0 X IO5 aud in Expt. 2 was 1.13 X 105), 40 @moles of phosphate buffer, pH 7.5, 1.2 mg of oxidase protein aud water to a final volume of 4.0 ml. AMP, when added, was at a final concentration of 1 mM. The reaction proceeded for 5 minutes at 30”; the reaction was stopped by immersing the tubes in a boiling wat,er bath for 2 minutes. Radioactivity was det,ermined on 0.1.ml aliquots as described above. specific activity of 2 X lo5 counts per minttte per pmole at 20% counting efficiency. 4-A NADT [with the tritium on the A-side of the nicotinamide plane (2)] was prepared by reducing the (T)NAD+ with the B-specific glucose dehydrogenase purified from beef liver (12) and 4-B NADT was prepared by reducing (T)NAD+ with the A-specific yeast alcohol dehydrogenase (Sigma Chemical Co., St. Lotus, Missouri) essentially as described by Krakow et al. (11). The preparation of the particttlate NADH oxidase and the soluble NAI)H-nitroblue tetrazolium oxidoredttctase from extracts of hf. tttberculosis has been previortsly described [(l(i), (13)l. The soluble NADH-menadione oxidorednctase of JI. fubercdosis was obtained from t,he particulate N.4DH oxidase. The particles were ext,racted twice at -2O”, each time with 20 volrtmes of acet,one. The suspension was filtered under suction, washed twice with ethyl ether, air dried, and ground to a fine powder. The dry powder was extracted with 0.1 M Tris buffer (pH 7.5) equal in volume to the original suspension of particles. The homogenized srtspension was centrifttged at, 105,OtMg for A0 minutes. The restdting yellow supernatant solution contained the NADHmenadione oxidoredttctase activity. NAD+ reduct,ion and NAI)H oxidation were followed spectrophotometrically (6). The reaction was run in two steps; in the first, (T)NAl>+ was incttbated wit)h the appropriate enzyme and sttbstrate until completely redriced, the enzyme was denatured by heating the reaction mixture in boiling water for 2 minutes, and after cooling, the NADT was reoxidized in t.he secotid step by addition of the NAI)H dehydrogenase or oxidase and fitrther incttbation (11). After heatiiig for 2 millrites to stop the oxidation reaction, II20 was re-
covered from the reaction mixture by sublimation (4). Tritium in the H& was measured with a Packard Instrument Co. scintillation counter. RESULTS
When t#heparticulate NADH oxidase from M. tuberculosis is incubated with t,he 4-R
Y
20 Minutes
J 40
FIG. 1. Time course of B-NADT oxidation and T removal by particulate NADII oxidase. Conditions as in Table I, except t,hat 0.4 mg NADH oxidase protein was ttsed. Incubation temperatrtre was 20”. At times indicat,ed, aliquot samples were removed and analyzed for t,ritiat.ed water. Radioactivity has been corrected for a zero-time control. (a---@) counts/mm. in water; (X-X) ratio betweeii the rates of removal of tritium and the oxidation of stthstrate (isotope etl’ect).
422
WORCEL
AND
TABLE II ABSENCE OF NADT-Hz0 T-EXCHANGE DURING B-NAI)T OXIDATION BY THE NADH OXID~ISE OF M. TUBERCULOSIS~ Incubation Conditions Aerobic Anaerobic
cpm in Hz0 y. T removed
-AMP
+AMP
3224 100
3208 100
;;$
-AMP
193 6
692 22
+AMP 843 26
a Conditions as in Table I except that incubation was carried out for 30 minutes at 30”, the final volume was 3.0 ml, and the total cpm was 0.96 X 105. Anaerobiosis was obtained by flushing 02-free Nz through Thunberg-type reaction tubes for 10 minutes prior to enzyme addition.
NADT, more than 90 % of the T is recovered in the Hz0 (Table I). On the other hand, no T removal occurs when the enzyme oxidizes 4-A NADT. There is no change in the stereospecificity of the T removal when the oxidase is activated by AMP. The time course of T removal from the 4-B NADT is shown in Fig. 1. The rate of detritiation measured by the T recovered in the Hz0 is slower than the rate of NADH oxidation followed spectrophotometrically at TABLE B-NADT
OXIDATION
III
AMP
<0.005 420 15
AA340 mr/min cpm in Hz0 y0 T removed
340 rnp, which results in a time-dependent increase in the specific activity of both the residual NADT as well as the TzO formed. The same pattern was observed in the presence of AMP. The loss of T from NADT could be due either to the oxidation of NADT or to a T exchange reaction between NADT and Hz0 catalyzed by the NADH dehydrogenase. The exchange reaction was ruled out by incubating the enzyme with 4-B NADT under anaerobic conditions. As seen in Table II after 30 minutes at 30” only 20 % of the T could be recovered in the HzO. Aerobically, oxidation and T removal were completed in 5 minutes. The small anaerobic T removal was due to oxidation of NADT during the heating and handling of the mixture. The soluble NADH-menadione oxidoreductase extracted from the NADH oxidase particles a,lso shows 4-B specificity. T removal and NADH oxidation occur only in the presence of menadione (Table III). These data also show that the dehydrogenase does not catalyze T exchange between NADT and H,O. This soluble NADH dehydrogenase is activated by AMP; as
BY NADH-MENBDIONE
NOW2
Additions to enzyme
GOLDMAN
REDCCTASE~
Menadione
<0.005 451 16
0.180 2335 83
Menadione AMP
-Enzyme
0.230 2482 87
0 376 13
a Conditions as in Table I except that 200 pg of soluble NADH-menadione oxidoreductase and 30 pg of menadione used in 1.0 ml final volume. Total cpm was 2.84 X 104. TABLE NADT
OXIDATION
IV
BY SOLUBLE NADH-NBT
L>I.~PHOR.GE~ B-NADT
A-NADT -NBT AASO mM/min cpm in Hz0 y0 T removed
0 268 8
protein
+NBT
-Enzyme
0.340 370 11
0.080” 244 8
-NBT 0 260 8
+NBT 0.260 3,064 96
a Conditions as in Table I, except that 200 gg of soluble NADH-NBT oxidoreductase protein and 0.2 pmole NBT were used in 1.0 ml final volume. The pH of t,he reaction mixture was adjusted to 10.0 with N KOH. Total cpm was 3.18 X 104. b Reduction of NBT due to the glucose remaining from the prior reaction forming A-NADT.
NADH
DEHYDROGENASE
with the particulate enzyme, AZUP activation changes neither the stereospecificity nor the lack of the T exchange reaction. Finally, the NADH-NBT oxidoreductase of M. tubenxlosis (13) is also 4-B specific and lacks the NADT-Hz0 T-exchange reaction (Table IV). DISCUSSION
STEREOSPECIFICITY
423
proof that t’he soluble NADH diaphornses from ext’racts of M. tuberculosis are derived from the membrane-bound i\‘ADH dchydrogenase and are probably released by the mechanical disruption of the bacillus. ACKNOWLEDGMENTS Radioactivity counting ey\lipment was generously provided by I>rs. John W. Porter and A. ,4. MacKinney, Jr. of this hospital. Meat byprodrlcts were generously furnished by Oscar Mayer & Co., Madison, Wisconsin.
Ernster et al. (3) reported that the 4-B specific mitochondrial electron chain-linked NADH dehydrogenase, as well as the various REFERENCES soluble NADH dehydrogenases derived from it, catalyze H exchange between 1. I)KYSUALE, Cr. It., .IKD COHN, M., Biochinz. Biophys. Acta 21, 397 (1956). NADH and HzO, while the 4-A specific 2. CORNFORTH,J. W., RYBACK, G., P~P.J.IK, G., NADH dehydrogenases (NADH-NADP I~ONNINGER, C., .\ND SCHROEPFER,G., Riotranshydrogenase, DT diaphorase, microthem. Biophys. Res. Commun. 9, 371 (1962). somal NADH-cytochrome bg reductase) do 3. EILNSTER, L., HoBERM.~N, 11. I)., Ho\x\m), not catalyze H exchange. They suggested R. L., KING, T. E., LEE, C. I'., MXKLER, that this may reflect a general difference in B., .',ND sOTTOC.\SA, c:., ;VUtUJX 207, 940 the mechanism of action between A and B (IQ&i). specific dehydrogenases. That this is not so 4. LEE, C. P., SIX~AI~D-I)IIQUESNE,N., EIINSTEII, can be seen from the results reported above L., ASD HOBERYAN, H. I>., Biochim. Bioon the electron chain-linked YXADH dephys. Ada 105, 397 (1965). 5. WOHCEL, A., .AND GOLDMAN, 1). S., Biochenl. hydrogenase from AR. tuberculosis, a 4-B Biophys. Res. Commun. 17, 559 (1964). specific enzyme which does not catalyze H 6. WORCEL, A., C:OLUX\N, L). S., AND CLEIAND, exchange between NADH and HzO. W. W ., J. Biol. (‘hem. 240, 3399 (1965). The slower rate of detritiation observed 7. WORCEL, A., Riochim. Biophys. Acta 113, 178 as compared to the rate of NADH oxidation (1966). is probably due to an isotope discrimination 8. MONOD, J., WYMIIN,J., AND CHANGBLX,J. P., effect. This enzyme, like ot,her KADHJ. Mol. Hiol. 12, 88 (1905). linked dehydrogenases (14)) oxidizes NADH 9. HEINEN, W., KXI~NOSE, M., KIJSUXOSE, E., preferentially over NADT. The AMP GOLDMAN, I). S., ANDW.ZGNER,M. J., drch. activation of the NADH oxidase and Biochem. Biophys. 104, 448 (1964). dehydrogenase does not change the stereo- 10. SAN PIETI~O, A., J. Niol. Chem. 217, 579 (1955). 11. K~xAK~\Y, G., LIJD~~IEG, J., M.\THER, J. II., specificity of the H removal from NADH, NOKMORE,W. N., Tosr, L., UDAKA, S., AND and similarly has no effect on the lack of VENNESL.IND, B., Biochemistr~g 2, 1009 NADH-Hz0 H-exchange reaction. (1963). NADH : (acceptor) oxidoreductases, either 12. STRECKER, H. J., ANI) KORKES, S., J. Hiol. solubilized from the NADH oxidase particles Chem. 196, 769 (1952). or found already soluble in the cell-free 13. ODA, T., AND GOLDMAN, 1). S., li’iochim. Bioextract’ of ‘11. tuberculosis are also 4-B phys. =Lcta 59, 601 (1962). NADH specific and show no NADH-HZ0 14. KH.\KO\~,(+., UDAK.\, S., AND VENNESL.IND, 13., H-exchange. This is taken as a further Biochemistry 1, 251 (1962).