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Role of menaquinone in Corynebacterium diphtheriae electron transport
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT COMMUNICATIONS sc 63I67 Role of menaquinone in Corynebacterium diphtheriae electron transport
A strain of Corynebacterium diphtheriae was found by P APPENHEIMER, Howto grow slowly and to exhibit impaired electron transport between cytochromes band c. Spectroscopic studies showed the mutant to possess a normal complement of cytochromes, including the diphtherial cytochromes a, band c. However, succinate reduced only cytochrome b, while either NADH or dithionite LAND AND MILLERl
TABLE I
C. diphtheriae Menaquinone was isolated by hot methanol extraction, purified, and estimated as described by
MENAQUINONE CONTENT OF STRAINS OF BISHOP, PANDYA AND KING 2•
Strain
mgMKjIoog wet wt. +14 0.18
reduced a and c as well. This, together with low rates of electron transport in the mutant strain, suggested to the authors that cells of that strain lacked a non-heme electron carrier, acting between cytochromes band c in the parental strain. The present communication reports further studies of the mutant, and presents results TABLE II C 7 AND C7SC STRAINS OF C. diphtheyiae Respiration was measured manometrically at 25°. Cells were grown and harvested as described previously 1. Each Warburg flask contained 500 fLmoles phosphate buffer, and from 21-30 mg dry wt, cell suspension in a total volume of 2.5 ml. The pH was 7+ The reaction was initiated by the addition from the side arm of 120 fLmoles of succinate or 0.75 ,umoles TMPD + 90 ftmoles ascorbate. Values are corrected for endogenous respiration which was less than 10 % of respiration with substrate. OXYGEN CONSUMPTION BY INTACT CELLS OF THE
Additions
Oxygen consumption (fLatoms/30 min per mg dry wt.) Straiw
Succinate Succinate + 0.3 ,nmoles Succinate + 0-44 pmoles Succinate + 0.18 pmoles Succinate 0.18 pmoles menadione TMPD ascorbate
+
menadione hydrolapachol NQNO NQNO 0.3 ,Hmoles
C7 0.28 0.29 0.29
+
+
0.18 0.3 1 0.09 0.03 0.03 0,13
Abbreviation: NQNO, 2-nonyl-4-hydroxyquinoline-N-oxide.
Biochim. Biophys. Acta, rrS (1966) 189-191
19°
SHORT COMMUNICATIONS
suggesting that the lesion in electron transport involves a homologue of menaquinone (a-methyl-r.a-naphthoquinone, with an isoprenoid side chain at the 3-Position). Table I shows the menaquinone (MK) content of the parental C7 and the mutant C7SC strains, as measured by the method of BISHOP, PANDYA AND KING 2 who also detected MK-8 in a different strain of C. diphiheriae. It is clear that the mutant cells contain less than 5 % of the MK found in the wild-type strain, which, considered in light of its known role in bacterial electron transport", suggests that the respiratory defect may be due to a diminished synthesis of MR. Table II shows rates of respiration measured with intact cells of the two strains. It is seen that menadione (z-methyl-r.a-naphthoquinone) stimulates succinate oxidation in the C7SC strain about 70 % while having little effect on the parental C7 strain. Furthermore, C7SC respiration is inhibited by a-hydroxy-jfj-methylbutylj-ra-naphthoquinone (hydrolapachol), which under similar conditions is quite without effect on the C7 strain. In other (unpublished) studies both strains were found to be inhibited by z-nonyl-4-hydroxyquinoline-N-oxide (NQNO) and it is clear from Table II that NQNO inhibition is not released by added menadione. Finally, electron transport with a system consisting of ascorbate plus catalytic amounts of tetramethyl-p-phenylenediamine, which passes electrons into the respiratory chain at cytochrome C4,5, is considerably lower in the mutant C7SC strain. The differential effects of menadione and hydrolapachol upon the two strains suggest impairment of the respiratory chain involving a naphthoquinone, a conclusion consistent with the low lVIK content in the mutant (Table I). Menadione appears to bypass a rate-limiting step of electron transport in the mutant although itis not the natural quinone of the C. cliphtheriae respiratory chain and possibly differs from the natural MK in locus of action. Indeed both phylloquinone (z-methyl-g-phytyllA-naphthoquinone) and menaquinone were tested for effect on growth rate and succinate respiration in both strains but showed little effect, probably due to extremely low solubility in aqueous solvents. It may be argued that menadione is acting in this case in a manner similar to intracellular lVIK, since menadione shows an important effect on respiration only in that strain which is deficient in lVIK. Furthermore, the failure of menadione to relieve NQNO inhibition may be regarded as evidence that menadione acts at the same restricted locus as the natural MK and does not span a greater portion of the chain. It was concluded- from spectroscopic observations that the major defect in electron transport by the diphtheria C7SC strain occurred between cytochromes b and c and the present communication regards that defect as a block in menaquinone synthesis. This conclusion should be contrasted with the report by BRODIE AND ADELSON 3 that, in the extensively studied Mycobacierius« phlei electron transport system, MK acts between a flavoprotein and cytochrome b. Finally, although the major block in respiration in the C7SC strain appears to be at the cytochrome b-c region, the low rate of electron transport with the TMPD-ascorbate system indicates that some component of electron transport between cytochrome c and oxygen is also defective. Since the mutant cells exhibit normal cytochrome spectra, the defect in the terminal portion ofthe respiratory chain may be examined in light of CRANE'S suggestions that quinones playa role in the terminal portion of the respiratory chain. This investigation was supported by the National Science Foundation Grant Biocbim, Biophys. Acta, u8 (1966) 189-191
SHORT COMMUNICATrONS
19 I
GB-r662. D. J. K. was the recipient of a Bowdoin College Undergraduate Research Fellowship. The authors wish to thank Dr. A. M. PAPPENHEIMER, Jr. both for the strains used in this study and for his helpful suggestions. The excellent technical assistance of Mrs. P. MORRISON is gratefully acknowledged. Department oj Biology, Bowdoin College,
J. KROGSTAD J. L. HOWLAND
D.
Bruesioick, Me. (U.S.A.) I
2 3 4 5
6
A. M. PAPPENHEIlvlER jr., J. L. HOWLAND AND P. M. MILLER, Biochim: Biophys. Acta, 64 (1962) 229· D. H. L. BISHOP, K. P. PANDYA AND H. K. KING, Biochem. J., 83 (1962) 606. A. F. BRODIE AND J. ADELSON, Science, 149 (1965) 265. J. L. HOWLAND, Biocliim: Biophys, Acta, 77 (1963) 4 19. D. R. SANADI, Federation P1'OC., 24 (1965) I'Z96. F. L. CRANE, Biochemistry, I (1962) 510.
Received November zand, 1965 Biochim. Biophys. Acta, 118 (1966) 189-191
SC 63164 Inability of rat-liver mitochondria to oxidize betaine aldehyde The question of the intra-cellular site for the oxidation of betaine aldehyde in rat liver has long been unsettled. WILLIAMS! and KAGAWA, WILKEN AND LARDy2 found a considerable amount of betaine aldehyde oxidase activity in mitochondria where BIANCHI AND AZZONE 3 also claimed to have isolated a NAD+·linked betaine aldehyde dehydrogenase (EC 1.2.1.8). GLENN AND VANK0 4 reported that the mitochondrial conversion of betaine aldehyde to betaine was catalyzed by a NAD+· dependent non-specific aldehyde dehydrogenase (Ee 1.2.1.3) which exerted its greatest activity with D-glyceraldehyde as a substrate. On the other hand, other workers'v ? supported the idea that almost all the betaine aldehyde dehydrogenase in liver was found in the cytoplasmic fraction and not in the mitochondria. In the interpretations of the effects of z-amino-z-methylpropanol on the oxidation of choline by rat-liver mitochondria in this Iaboratorys.", it was essential to know whether or not the oxygen uptake was a single-step conversion of choline to betaine aldehyde or whether betaine aldehyde was also further oxidized to betaine. This report presents direct evidence to SllOW that betaine aldehyde was not oxidized in rat-liver mitochondria and indicates how the artifact of betaine aldehyde oxidation by mitochondria may arise. As shown in Fig. I, betaine aldehyde was not oxidized by NAD+ in rat-liver mitochondria, whereas under the same conditions fi-hydroxybutyrate showed a rapid rate of NADH formation. This is direct evidence showing that mitochondria were unable to utilize betaine aldehyde. It is also shown in Fig. I that the protein-fraction betaine aldehyde dehydrogenase isolated from rat-liver supernatant-fluid fraction oxidized betaine aldehyde as indicated by the reduction of NAD+. Again, we failed to detect any significant oxygen uptake when betaine aldehyde Biochim, Biophys. Acta, !IS (1966) 191-194
SHORT COMMUNICATIONS sc 63I67 Role of menaquinone in Corynebacterium diphtheriae electron transport
A strain of Corynebacterium diphtheriae was found by P APPENHEIMER, Howto grow slowly and to exhibit impaired electron transport between cytochromes band c. Spectroscopic studies showed the mutant to possess a normal complement of cytochromes, including the diphtherial cytochromes a, band c. However, succinate reduced only cytochrome b, while either NADH or dithionite LAND AND MILLERl
TABLE I
C. diphtheriae Menaquinone was isolated by hot methanol extraction, purified, and estimated as described by
MENAQUINONE CONTENT OF STRAINS OF BISHOP, PANDYA AND KING 2•
Strain
mgMKjIoog wet wt. +14 0.18
reduced a and c as well. This, together with low rates of electron transport in the mutant strain, suggested to the authors that cells of that strain lacked a non-heme electron carrier, acting between cytochromes band c in the parental strain. The present communication reports further studies of the mutant, and presents results TABLE II C 7 AND C7SC STRAINS OF C. diphtheyiae Respiration was measured manometrically at 25°. Cells were grown and harvested as described previously 1. Each Warburg flask contained 500 fLmoles phosphate buffer, and from 21-30 mg dry wt, cell suspension in a total volume of 2.5 ml. The pH was 7+ The reaction was initiated by the addition from the side arm of 120 fLmoles of succinate or 0.75 ,umoles TMPD + 90 ftmoles ascorbate. Values are corrected for endogenous respiration which was less than 10 % of respiration with substrate. OXYGEN CONSUMPTION BY INTACT CELLS OF THE
Additions
Oxygen consumption (fLatoms/30 min per mg dry wt.) Straiw
Succinate Succinate + 0.3 ,nmoles Succinate + 0-44 pmoles Succinate + 0.18 pmoles Succinate 0.18 pmoles menadione TMPD ascorbate
+
menadione hydrolapachol NQNO NQNO 0.3 ,Hmoles
C7 0.28 0.29 0.29
+
+
0.18 0.3 1 0.09 0.03 0.03 0,13
Abbreviation: NQNO, 2-nonyl-4-hydroxyquinoline-N-oxide.
Biochim. Biophys. Acta, rrS (1966) 189-191
19°
SHORT COMMUNICATIONS
suggesting that the lesion in electron transport involves a homologue of menaquinone (a-methyl-r.a-naphthoquinone, with an isoprenoid side chain at the 3-Position). Table I shows the menaquinone (MK) content of the parental C7 and the mutant C7SC strains, as measured by the method of BISHOP, PANDYA AND KING 2 who also detected MK-8 in a different strain of C. diphiheriae. It is clear that the mutant cells contain less than 5 % of the MK found in the wild-type strain, which, considered in light of its known role in bacterial electron transport", suggests that the respiratory defect may be due to a diminished synthesis of MR. Table II shows rates of respiration measured with intact cells of the two strains. It is seen that menadione (z-methyl-r.a-naphthoquinone) stimulates succinate oxidation in the C7SC strain about 70 % while having little effect on the parental C7 strain. Furthermore, C7SC respiration is inhibited by a-hydroxy-jfj-methylbutylj-ra-naphthoquinone (hydrolapachol), which under similar conditions is quite without effect on the C7 strain. In other (unpublished) studies both strains were found to be inhibited by z-nonyl-4-hydroxyquinoline-N-oxide (NQNO) and it is clear from Table II that NQNO inhibition is not released by added menadione. Finally, electron transport with a system consisting of ascorbate plus catalytic amounts of tetramethyl-p-phenylenediamine, which passes electrons into the respiratory chain at cytochrome C4,5, is considerably lower in the mutant C7SC strain. The differential effects of menadione and hydrolapachol upon the two strains suggest impairment of the respiratory chain involving a naphthoquinone, a conclusion consistent with the low lVIK content in the mutant (Table I). Menadione appears to bypass a rate-limiting step of electron transport in the mutant although itis not the natural quinone of the C. cliphtheriae respiratory chain and possibly differs from the natural MK in locus of action. Indeed both phylloquinone (z-methyl-g-phytyllA-naphthoquinone) and menaquinone were tested for effect on growth rate and succinate respiration in both strains but showed little effect, probably due to extremely low solubility in aqueous solvents. It may be argued that menadione is acting in this case in a manner similar to intracellular lVIK, since menadione shows an important effect on respiration only in that strain which is deficient in lVIK. Furthermore, the failure of menadione to relieve NQNO inhibition may be regarded as evidence that menadione acts at the same restricted locus as the natural MK and does not span a greater portion of the chain. It was concluded- from spectroscopic observations that the major defect in electron transport by the diphtheria C7SC strain occurred between cytochromes b and c and the present communication regards that defect as a block in menaquinone synthesis. This conclusion should be contrasted with the report by BRODIE AND ADELSON 3 that, in the extensively studied Mycobacierius« phlei electron transport system, MK acts between a flavoprotein and cytochrome b. Finally, although the major block in respiration in the C7SC strain appears to be at the cytochrome b-c region, the low rate of electron transport with the TMPD-ascorbate system indicates that some component of electron transport between cytochrome c and oxygen is also defective. Since the mutant cells exhibit normal cytochrome spectra, the defect in the terminal portion ofthe respiratory chain may be examined in light of CRANE'S suggestions that quinones playa role in the terminal portion of the respiratory chain. This investigation was supported by the National Science Foundation Grant Biocbim, Biophys. Acta, u8 (1966) 189-191
SHORT COMMUNICATrONS
19 I
GB-r662. D. J. K. was the recipient of a Bowdoin College Undergraduate Research Fellowship. The authors wish to thank Dr. A. M. PAPPENHEIMER, Jr. both for the strains used in this study and for his helpful suggestions. The excellent technical assistance of Mrs. P. MORRISON is gratefully acknowledged. Department oj Biology, Bowdoin College,
J. KROGSTAD J. L. HOWLAND
D.
Bruesioick, Me. (U.S.A.) I
2 3 4 5
6
A. M. PAPPENHEIlvlER jr., J. L. HOWLAND AND P. M. MILLER, Biochim: Biophys. Acta, 64 (1962) 229· D. H. L. BISHOP, K. P. PANDYA AND H. K. KING, Biochem. J., 83 (1962) 606. A. F. BRODIE AND J. ADELSON, Science, 149 (1965) 265. J. L. HOWLAND, Biocliim: Biophys, Acta, 77 (1963) 4 19. D. R. SANADI, Federation P1'OC., 24 (1965) I'Z96. F. L. CRANE, Biochemistry, I (1962) 510.
Received November zand, 1965 Biochim. Biophys. Acta, 118 (1966) 189-191
SC 63164 Inability of rat-liver mitochondria to oxidize betaine aldehyde The question of the intra-cellular site for the oxidation of betaine aldehyde in rat liver has long been unsettled. WILLIAMS! and KAGAWA, WILKEN AND LARDy2 found a considerable amount of betaine aldehyde oxidase activity in mitochondria where BIANCHI AND AZZONE 3 also claimed to have isolated a NAD+·linked betaine aldehyde dehydrogenase (EC 1.2.1.8). GLENN AND VANK0 4 reported that the mitochondrial conversion of betaine aldehyde to betaine was catalyzed by a NAD+· dependent non-specific aldehyde dehydrogenase (Ee 1.2.1.3) which exerted its greatest activity with D-glyceraldehyde as a substrate. On the other hand, other workers'v ? supported the idea that almost all the betaine aldehyde dehydrogenase in liver was found in the cytoplasmic fraction and not in the mitochondria. In the interpretations of the effects of z-amino-z-methylpropanol on the oxidation of choline by rat-liver mitochondria in this Iaboratorys.", it was essential to know whether or not the oxygen uptake was a single-step conversion of choline to betaine aldehyde or whether betaine aldehyde was also further oxidized to betaine. This report presents direct evidence to SllOW that betaine aldehyde was not oxidized in rat-liver mitochondria and indicates how the artifact of betaine aldehyde oxidation by mitochondria may arise. As shown in Fig. I, betaine aldehyde was not oxidized by NAD+ in rat-liver mitochondria, whereas under the same conditions fi-hydroxybutyrate showed a rapid rate of NADH formation. This is direct evidence showing that mitochondria were unable to utilize betaine aldehyde. It is also shown in Fig. I that the protein-fraction betaine aldehyde dehydrogenase isolated from rat-liver supernatant-fluid fraction oxidized betaine aldehyde as indicated by the reduction of NAD+. Again, we failed to detect any significant oxygen uptake when betaine aldehyde Biochim, Biophys. Acta, !IS (1966) 191-194