ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 196, No. 2, September, pp. 396-402, 1979
Inhibition JOHN Biological
of Bacterial MAKEMSON’ Laboratories,
Harvard
Bioluminescence AND J. WOODLAND University,
Cambridge,
by Pargylinel HASTINGS Massachusetts
02138
Received February 13, 1979; revised May 2, 1979 Pargyline (N-benzyl-N-methyl-2-propynylamine), an inactivator of mitochondrial monoamine oxidase, inhibits growth and in vivo and in vitro bioluminescence in Beneckea harueyi. The inhibition is competitive with the two substrates, FMNH, and aldehyde, and the inhibitor binds with a reaction intermediate of the the enzyme luciferase to form a stable, but reversible, adduct. Inhibition of in vivo bioluminescence is an apparently complex phenomenon, and may involve a block in the synthesis of aldehyde.
Pargyline (N-benzyl-N-methyl-2-propynylamine) is known to inactivate mitochondrial monoamine oxidase by forming a stable (covalent) adduct with the flavin residue on the enzyme (1). Since bacterial luciferase utilizes flavin mononucleotide (2), it seemed possible that pargyline might also be active as an inhibitor of the light-emitting reaction. Indeed, pargyline was found to be an inhibitor both in vivo and in vitro. An analysis of its action suggests that the inhibition involves a stable but reversible adduct with a reaction intermediate, and is competitive with the substrates FMNH, and long-chain aldehyde. MATERIALS
AND METHODS
The bacterium used was Beneckea harueyi, strain B-392 (3), designated as Lucibacterium harveyi in Bergey’s Manual of Determinative Bacteriology (4). Two media were used: a minimal medium containing NH&l (1 g/liter) as the nitrogen source and a complex medium containing Bacto-peptone (5 g/liter) as the nitrogen source, both with 3 ml glycerol/liter. Both media utilized Hepes3 buffer, with the following r Supported in part by National Science Foundation Grants 74-23651 and 77-19917 and National Institutes of Health Grant GM-19536. Data presented in part at the 78th Annual Meeting of the American Society for Microbiology, 1978. * Special Fellow, NIH. Present address: Department of Biological Sciences, Florida International University, Tamiami Campus, Miami, Fla. 33199. 3 Abbreviation used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. 0003-9861/79/100396-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
base salt composition: 20 g NaCl, 12.35 g MgSO,. ‘7H20, 11.9 g Hepes, 0.38 g KCl, 0.64 ml of 50% NaOH, and 14 mg ferric ammonium nitrate per liter of distilled water. To both media K,HPO, sterilized separately was added aseptically after autoclaving to make the medium 2.5 mM in phosphate; the final pH was 7.0. Stock cultures were maintained on the complex medium to which 15 g Bacto-agar and 0.1 g CaCO, per liter were added. The relationship between viable colony counts and Petroff-Hauser cell counts to optical density at 660 mm of broth cultures was linear from 0.05 to 0.7 Coleman optical density units (1.1 x lOa cells ml-’ = 0.1 OD units). Viable cell counts were made by dilution in cold sterile broth and plating on a complex medium; in all cases viable cell counts equaled the PetroffHauser cell counts. All experiments with living cells were done with cultures inoculated with exponentially growing cells, and aliquots for experiments were taken prior to stationary phase, at densities between 2 x IO* and 8 x lo8 cells ml-‘.. Luminescence was measured in tubes or scintillation vials using a calibrated photometer (5, 6) and was expressed in light units; one light unit was equal to 2.1 x 10”’ q sec.-’ except in Fig. 2. Luciferase activity was measured at 25°C in a reaction mixture containing 1.0 ml 0.02 M phosphate buffer, pH 7.0, containing 10 ~1 of purified luciferase (29.2 mg ml-’ and 6 x lOI quanta s-’ rng-‘), diluted in phosphate buffer before assay, and 2 yl of a 0.1% decanal sonicate. In other experiments o&anal, nonanal, and dodecanal were used. Reactions were initiated by injection of 1.0 ml of 50 pM FMNH, solution made in 0.02 M phosphate buffer (pH 7.0) and reduced by bubbling hydrogen in the presence of platinized asbestos. High concentrations of some aldehydes were known to inhibit (7, 8); in cases where high concentrations of aldehyde were utilized, the inhibition was circumvented by adding the aldehyde subsequent
396
PARGYLINE
INHIBITION
OF BACTERIAL
BIOLUMINESCENCE
397
by centrifugation (104g for 10 min) at 4°C and resuspension in fresh medium at 25°C recovered only about 2% of the luminescence activity. Similar results were obtained with cells harvested on Millipore filters as well as by dilution of pargylineinhibited cultures. The substrates of the in vitro luciferase RESULTS reaction are reduced flavin mononucleotide and long-chain fatty aldehyde. If Pargyline is an effective inhibitor of both (FMNH,) by pargyline is due to its growth and luminescence in B. harueyi. The the inhibition reaction or interference with one of these, percentage decrease in the rate of growth then inhibition might also be reversed by by 1, 2, 5, and 10 mM pargyline was 12.5, exogenous addition of one of these substrates. 24, 58, and lOO%, respectively. Similar Although FMN is not readily permeable, concentrations rapidly decreased the lumidue to its negative charge (phosphate), nescence of the cells (Fig. 1). At higher concentrations, the inhibition exhibits a neither oxidized nor reduced FMN had any with biphasic character and is not reversed by significant effect on the inhibition pargyline. But, neither did riboflavin, and removing the inhibitor. Cells treated with although it lacks the phosphate, it is not 20 InM pargyline for 2 min at 25°C followed an effective substrate in the luciferase reaction nor does it have a high affinity for 200 luciferase in vitro (9). In order to see if the pargyline was acting by interfering with or removing intracellular aldehyde, exogenous additions of decanal were made to cell suspensions at various times subsequent to the addition PARGYLINE.mM of an inhibitory level of pargyline (10 InM). In fact, a prompt reversal of the inhibition occurred (Fig. 2>, suggesting that pargyline inhibition is at least partly related to aldehyde function. An unusual feature is that the reversal is apparently less effective immediately after pargyline addition than later. Under these conditions decanal will not stimulate luminescence of uninhibited cells. The effect of other long-chain aldehydes and of long-chain fatty acids on the reversal of pargyline inhibition of bioluminescence is shown in Table I. The two other aldehydes tested (o&anal and dodecanal) were less effective than decanal. Long-chain fatty acids were mostly inactive. The exception was myristic acid which is also the most effective in stimulating bioluminescence in certain dark “aldehyde” mutants (10); the FIG. 1. Inhibition of B. harveyi bioluminescence by 14-carbon chain length is postulated to be pargyline. Aliquots (1 ml) of an exponentially growing the natural aldehyde and to cycle in the B. harueyi culture (5 x 10’ cells ml-‘) were pipetted into scintillation vials containing different amounts of reaction (11). However, myristic acid only pargyline as indicated and the bioluminescence was recovered less than half of the bioluminesrecorded with time. cence recoverable with decanal, and an to FMNH,. This is referred to as the double-injection procedure. The aldehydes were from Aldrich; FMN and pargyline were from Sigma Chemical Company. Pargyline was made up in the salt medium and sterilized by Millipore filtration (0.45 pm). Purified luciferase was the gift of T. Baldwin and M. Nicoli.
MAKEMSON
0
I -20
I 0
I 20
AND HASTINGS
I i , 40
/ I t
, 60 SECONDS
I so
1 100
It 120
t
/ 140
FIG. 2. Reversal of pargyline-inhibited in. wivo bioluminescence by decanal. Pargyline (0.1 ml) was added to O.S-ml aliquots of growing cells (5 x IO* cells ml-*) at zero time. One light unit is 1.05 x 1Ol2 quanta s-*. At the times indicated 1 ml of a 0.1% decanal sonicate in distilled water was injected into the inhibited cell suspension. The injection of distilled water alone also nonspecifically stimulated (bubble formation); after correction for this, the net effect is plotted (inset) as the percentage of initial bioluminescence prior to inhibition.
additional response was obtained by the subsequent addition of decanal. The nature of the in vitro bioluminescence reaction allows a distinction between TABLE PARGYLINE
INHIBITION
OF BIOLUMINESCENCE
inhibition due enzyme with which causes catalytic cycle
to blocking the reaction of substrate(s) and inhibition the turnover time of the to be altered. The in vitro
I
AND ITS REVERSAL
BY FATTY
ALDEHYDES
AND ACIDS
Reversal of inhibition Bioluminescence” Experiment 1 2
3
Bioluminescence
Prior to inhibition
After 1 min with 10 mM pargyline
29 27 13 14 14
0.25 0.25 0.8” 1.05 1.3
Decanal Dodecanal Decanal Dodecanal Octanal
41 39 39 40
0.3 0.3 0.3 0.3
Myristic acid Decanoic acid Dodecanoic acid Ethanol
Addition6
Immediate
Decanal at 20 s
20.3 4.8 8.3 1.9 2.9
-
8.0 0.2 0.3 0.3
22 20 19 21
n Light units per milliliter of a Hepes-peptone culture at 6 to 8 x lo* cells/ml, 1.0 ml/vial. b Aldehydes, 0.01% in buffer, 1 ml added; fatty acids, 0.2 M in 95% ethanol, 10 pl added. C30 s after pargyline addition.
PARGYLINE
INHIBITION
OF BACTERIAL
reaction of h&erase, initiated with FMNH2, involves only a single catalytic cycle (12) due to the fact that the duration of one catalytic cycle (about 5-10 s) is long compared to the lifetime of the substrate, which is autooxidized rapidly (less than 0.5 s) by molecular oxygen (13). The reaction of reduced flavin, oxygen, and luciferase occurs during the first fraction of a second to form an active intermediate, postulated to be a reduced flavin-peroxy adduct (14). Over the course of the next 5 to 10 s (dependent upon temperature, aldehyde chain length, and some other factors) this intermediate reacts further to form products and emit light. Inhibitors may thus act either by reducing the amount of intermediate formed, or by altering the rate at which the reaction intermediates turn over, i.e., the catalytic cycle. Thus, with a given amount of substrate, the amount of product (photons) formed would be less in the first case, but the reaction would go to completion in the same time. In the latter type of inhibition (alteration of turnover time), the photon yield would be the same, but a different (longer) time would be required. These distinctions can be seen in the experiments of Fig. 3, where different amounts of pargyline were present in the assay mixture prior to the initiation of the reaction with FMNH,. The initial rate (I,,,) is very much reduced as the pargyline concentration increases. But, there is also an effect on the kinetics of the decay resulting in an increase in the turnover time. It might be that the increased turnover time would compensate for the decreased initial intensity resulting in little effect on the quantum yield. But, in fact, the quantum yield of the reaction is decreased so this kinetic effect is not sufficient to compensate for the decreased initial intensity. Therefore, it is suggested that both (i) the amount of intermediate formed is less at higher pargyline concentrations, and (ii) the luciferase turnover rate is slower in the presence of pargyline. It might be expected that the first effect would be subject to competitive inhibition with the substrates FMNH, and aldehyde. With FMNH, this was indeed found to be so;
BIOLUMINESCENCE
I.0 I 0
1 5
IO SECONDS
15
I 20
FIG. 3. Pargyline inhibition of the FMNH,initiated in vitro luciferase reaction with decanal. Different amounts of pargyline as indicated (final concentrations) were added to reaction mixtures prior to the injections of FMNH2. The effect of pargyline at different concentrations on the initial intensity Ulll,,, l ), the quantum yield (O), and the half-life (tJ of the light decay (A), is shown in the inset.
the K, FMNH, was 1.6 x lo-+ M and the K, pargyline was 1.1 x 10m3 M. With decanal the inhibition appeared to be fully reversible by aldehyde at lower pargyline (1 mM) concentrations but not completely so at higher (10 mM) levels (Fig. 4). However, this inhibition is fully reversed upon dilution, both after mixing with luciferase prior to a reaction, and with a reaction mixture subsequent to reaction in the presence of 10 mM pargyline. The effect of pargyline on the kinetics during the course of the reaction of the luciferase intermediate, and the reversibility of this effect, can be shown in both the presence and the absence of aldehyde. Pargyline inhibits if added secondarily, subsequent to the initiation of the luciferase reaction by FMNH, (Fig. 5), and this inhibition can be overcome by the later addition of more aldehyde, at concentrations higher than used in the initial reaction. It can be shown that the luciferase intermediate is formed in reactions initiated with FMNH,
MAKEMSON
400
r
AND HASTINGS
I/Imox 0 07. 006
/
P*RGYLINE mM
0 05 IOh 004. 003 /---/p IOh
002.
l&&Ll+T -1 0
2
FIG. 4. Decanal competition in the pargyline inhibition of B. hmueyi luciferase. A Lineweaver-Burk plot of the initial intensity l/Z,,, as related to the decanal concentration, assayed by the double-injection procedure. Reaction mixtures were made up without aldehyde and with different concentrations of pargyline as indicated and then initiated by the injection of 1 ml of FMNH2, immediately followed by the injection of 1 ml of FMNH,, immediately followed by the injection of 1 ml of aldehyde (decanal suspension at different concentrations), as indicated. Microliters of sonicate per assay are used for decanal concentration since decanal does not form a true solution at these concentrations.
without aldehyde by the fact that later, secondary addition of aldehyde results in luminescence (Fig. 6). This intermediate, designated II and postulated to be the luciferase-bound peroxy-reduced flavin (14, 15) also has a long lifetime and reacts slowly to form H,O, and oxidized FMN without light emission (16, 17). The quantity of the intermediate present can be inferred from the bioluminescence obtained upon secondary injection of aldehyde. As shown in Fig. 6, pargyline has a strong effect on the lifetime of the intermediate in the absence of aldehyde. This effect is more pronounced than in the reaction with aldehyde present, presumably because aldehyde competes with pargyline. Several other long-chain aldehydes were examined, notably o&anal, nonanal, and dodecanal. With the first two the results were essentially the same as with decanal in Figs. 4 and 5 (although with different
rates characteristic of each aldehyde). The results with dodecanal were quite different. Pargyline added secondarily neither inhibited nor altered the half-decay time. Added prior to the initiation of the reaction the initial intensity was decreased, but there was very little effect on the decay time. These results can be explained by the fact that dodecanal apparently binds more strongly and apparently irreversibly compared to decanal and octanal (8). Consequently, secondary addition of pargyline may not readily be able to displace dodecanal and thereby inhibit the reaction, whereas secondary addition of pargyline was able to inhibit the decanal reaction. DISCUSSION
Unlike the case of monoamine oxidase, the inhibition of luciferase by pargyline I
I
I
SECONDS
FIG. 5. Inhibition of in vitro luminescence by pargyline added secondarily (after the I,,,) and reversal by aldehyde. The reactions were initiated by the injection of 1 ml of FMNH, to reaction mixtures with decanal. (0) Uninhibited reaction. The addition of pargyline approximately 1 s after the beginning of the reaction resulted in a prompt inhibition and a decrease in the rate of decay of luminescence (A), parallel to that observed with pargyline added prior to the initiation of the reaction (0). The subsequent addition of excess decanal at 3 (A) and 9 (17) s reversed the inhibition and resulted in the more rapid decay of luminescence.
PARGYLINE
INHIBITION
OF BACTERIAL
SECONDS
FIG. 6. Pargyline interaction with the luciferase reaction intermediate II. Intermediate II was formed by initiating the reaction with FMNH, in the absence of aldehyde in the presence of 0.3 mM (O), 1.0 mM (A), 10 mM (A), or no (0) pargyline. At various times after the injection of FMNHz, 1 ml of 0.01% decanal suspension was injected; the resulting initial intensity (I,,,) is plotted as a function of the time when decanal was injected. The half-life of intermediate II as a function of pargyline concentration is shown in the inset.
appears not to involve the formation of a covalent adduct because the inhibition of luciferase can be reversed. The results suggest that instead it complexes with the luciferase-peroxy-flavin intermediate, but this complex is readily reversed by the addition of excess aldehyde or by dilution. When complexed with pargyline, the dark decay of the intermediate is effectively prevented, thereby providing a potential method for trapping and characterizing the intermediate at room temperature, alternative to the low-temperature method of Hastings et al. (14). Pargyline can also inhibit the initial formation of the reduced flavin enzyme intermediate; this inhibition is competitive both with the reduced flavin substrate (& = 1.3 x 10e3 M) and with decanal (Ki = 3.7 X lo-4 M). The interaction of pargyline in viva is more difficult to interpret. In certain respects its action is explicable simply in terms of the inhibition of intracellular luciferase, since the inhibition is reversible under certain conditions with exogenously added aldehyde. However, it is difficult to explain why this is not immediately reversible after the pargyline addition. It may indicate
BIOLUMINESCENCE
401
that pargyline has more than one mode of inhibition, and/or that cellular compartmentalization is involved. For example, it is possible that pargyline initially binds with luciferase upon entry into the cell, but then equilibrates with other binding sites (proteins), possibly in other cellular phases, to which the pargyline binding is stronger. Thus pargyline inhibition of luminescence might involve both its direct interaction with luciferase and reaction with other components of the bioluminescent system. Pargyline does not remain bound to the luciferase of inhibited cells; upon lysis they release a luciferase which is as fully active as that obtained from control cells (data not shown). At higher concentrations and with longer times of exposure to pargyline the inhibition of in wivo bioluminescence is not reversed by dilution of the cells. By contrast, the in vitro inhibition of luciferase is readily reversed by dilution under all conditions examined. The “irreversibly” inhibited cells may still be caused to emit maximal luminescence by addition of aldehyde. Based on this, the “irreversible” effect could be attributed to an inhibition of aldehyde synthesis. The inhibition can apparently be overcome by adding exogenous myristic acid or long-chain aldehyde. However, decanal addition to these partially reversed cells resulted in a bioluminescent flash. This may result in the inability of exogenous myristic acid to fully saturate luciferase with aldehyde, presumably tetradecanal (myristic aldehyde), due to slow transport and/or conversion to the aldehyde. An alternative explanation is that the myristic acid transport and conversion to tetradecanal operates at its optimal levels but is only one of several aldehyde synthetic pathways. The amount of myristic acid added in the experiment shown in Table I was the optimal amount for these cells and for bioluminescence in an aldehyde mutant (M-17) grown under identical conditions. The data extant cannot distinguish between these possibilities. REFERENCES 1. CHUANG, H. Y. K., PATEK, D. R., HELLERMAN, L. (1974) J. Biol. Chm. 2381-2384.
AND
249,
402
MAKEMSONANDHASTINGS
2. HASTINGS, J. W., AND NEALSON, K. H. (1977) Annu. Rev. Microbial. 31, 549-595. 3. REICHELT, J. L., AND BAUMANN, P. (1973) Arch. Mikrobiol. 94, 283-330. 4. BAUMANN, P., AND BAUMANN, L. (1977) Annu. Rev. Microbial. 31, 39-61. 5. HASTINGS, J. W., AND WEBER, G. (1963) J. Opt. Sot. Amer. 53, 1411-1415. 6. MITCHELL, G., AND HASTINGS, J. W. (1971) Anal. Biochem. 39, 243-250. 7. HASTINGS, J. W., WEBER, K., FRIEDLAND, J., EBERHARD, A., MITCHELL, G. W., AND GUNSALUS, A. (1969) Biochemistry 8,4681-4639. 8. BAUMSTARK, A. L., CLINE, T. W., AND HASTINGS, J. W. (1979) Arch. Biochem. Biophys. 192, 449-455. 9. BALDWIN, T. O., NICOLI, M. Z., BECVAR, J. E., AND HASTINGS, J. W. (1975) J. Biol. Chem. 250, 2X3-2768.
10. ULITZUR, S., AND HASTINGS, J. W. (1978) Nat. Acad. Sci. USA 75, 266-269.
Proc.
11. ULITZUR, S., AND HASTINGS, J. W. (1979) Proc. Nat. Acad. Sci. USA 76, 265-267. 12. HASTINGS, J. W., AND GIBSON, 0. H. (1963) J. Biol. Chem. 238, 2537-2554. 13. GIBSON, Q. H., AND HASTINGS, Biochem. J. 83, 368-377.
J.
W.
(1962)
14. HASTINGS, J. W., BALNY, C., LE PEUCH, C., AND DOUZOU, P. (1973) Proc. Nat. Acad. Sci. USA 70, 3468-3472. 15. HASTINGS, J. W. (1978) Critical Reviews of Biochemistry (Fasman, G., ed.), Vol. 5, pp. 163-184, CRC Press, Cleveland, Ohio. 16. HASTINGS, J. W., AND BALNY, C. (1975) J. Biol. Chem. 250, 7288-7293. 17. BECVAR, (1978)
J. E., Tu, Biochemistry
S.-C., AND HASTINGS, 17, 1807-1812.
J. W.
Inhibition JOHN Biological
of Bacterial MAKEMSON’ Laboratories,
Harvard
Bioluminescence AND J. WOODLAND University,
Cambridge,
by Pargylinel HASTINGS Massachusetts
02138
Received February 13, 1979; revised May 2, 1979 Pargyline (N-benzyl-N-methyl-2-propynylamine), an inactivator of mitochondrial monoamine oxidase, inhibits growth and in vivo and in vitro bioluminescence in Beneckea harueyi. The inhibition is competitive with the two substrates, FMNH, and aldehyde, and the inhibitor binds with a reaction intermediate of the the enzyme luciferase to form a stable, but reversible, adduct. Inhibition of in vivo bioluminescence is an apparently complex phenomenon, and may involve a block in the synthesis of aldehyde.
Pargyline (N-benzyl-N-methyl-2-propynylamine) is known to inactivate mitochondrial monoamine oxidase by forming a stable (covalent) adduct with the flavin residue on the enzyme (1). Since bacterial luciferase utilizes flavin mononucleotide (2), it seemed possible that pargyline might also be active as an inhibitor of the light-emitting reaction. Indeed, pargyline was found to be an inhibitor both in vivo and in vitro. An analysis of its action suggests that the inhibition involves a stable but reversible adduct with a reaction intermediate, and is competitive with the substrates FMNH, and long-chain aldehyde. MATERIALS
AND METHODS
The bacterium used was Beneckea harueyi, strain B-392 (3), designated as Lucibacterium harveyi in Bergey’s Manual of Determinative Bacteriology (4). Two media were used: a minimal medium containing NH&l (1 g/liter) as the nitrogen source and a complex medium containing Bacto-peptone (5 g/liter) as the nitrogen source, both with 3 ml glycerol/liter. Both media utilized Hepes3 buffer, with the following r Supported in part by National Science Foundation Grants 74-23651 and 77-19917 and National Institutes of Health Grant GM-19536. Data presented in part at the 78th Annual Meeting of the American Society for Microbiology, 1978. * Special Fellow, NIH. Present address: Department of Biological Sciences, Florida International University, Tamiami Campus, Miami, Fla. 33199. 3 Abbreviation used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. 0003-9861/79/100396-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
base salt composition: 20 g NaCl, 12.35 g MgSO,. ‘7H20, 11.9 g Hepes, 0.38 g KCl, 0.64 ml of 50% NaOH, and 14 mg ferric ammonium nitrate per liter of distilled water. To both media K,HPO, sterilized separately was added aseptically after autoclaving to make the medium 2.5 mM in phosphate; the final pH was 7.0. Stock cultures were maintained on the complex medium to which 15 g Bacto-agar and 0.1 g CaCO, per liter were added. The relationship between viable colony counts and Petroff-Hauser cell counts to optical density at 660 mm of broth cultures was linear from 0.05 to 0.7 Coleman optical density units (1.1 x lOa cells ml-’ = 0.1 OD units). Viable cell counts were made by dilution in cold sterile broth and plating on a complex medium; in all cases viable cell counts equaled the PetroffHauser cell counts. All experiments with living cells were done with cultures inoculated with exponentially growing cells, and aliquots for experiments were taken prior to stationary phase, at densities between 2 x IO* and 8 x lo8 cells ml-‘.. Luminescence was measured in tubes or scintillation vials using a calibrated photometer (5, 6) and was expressed in light units; one light unit was equal to 2.1 x 10”’ q sec.-’ except in Fig. 2. Luciferase activity was measured at 25°C in a reaction mixture containing 1.0 ml 0.02 M phosphate buffer, pH 7.0, containing 10 ~1 of purified luciferase (29.2 mg ml-’ and 6 x lOI quanta s-’ rng-‘), diluted in phosphate buffer before assay, and 2 yl of a 0.1% decanal sonicate. In other experiments o&anal, nonanal, and dodecanal were used. Reactions were initiated by injection of 1.0 ml of 50 pM FMNH, solution made in 0.02 M phosphate buffer (pH 7.0) and reduced by bubbling hydrogen in the presence of platinized asbestos. High concentrations of some aldehydes were known to inhibit (7, 8); in cases where high concentrations of aldehyde were utilized, the inhibition was circumvented by adding the aldehyde subsequent
396
PARGYLINE
INHIBITION
OF BACTERIAL
BIOLUMINESCENCE
397
by centrifugation (104g for 10 min) at 4°C and resuspension in fresh medium at 25°C recovered only about 2% of the luminescence activity. Similar results were obtained with cells harvested on Millipore filters as well as by dilution of pargylineinhibited cultures. The substrates of the in vitro luciferase RESULTS reaction are reduced flavin mononucleotide and long-chain fatty aldehyde. If Pargyline is an effective inhibitor of both (FMNH,) by pargyline is due to its growth and luminescence in B. harueyi. The the inhibition reaction or interference with one of these, percentage decrease in the rate of growth then inhibition might also be reversed by by 1, 2, 5, and 10 mM pargyline was 12.5, exogenous addition of one of these substrates. 24, 58, and lOO%, respectively. Similar Although FMN is not readily permeable, concentrations rapidly decreased the lumidue to its negative charge (phosphate), nescence of the cells (Fig. 1). At higher concentrations, the inhibition exhibits a neither oxidized nor reduced FMN had any with biphasic character and is not reversed by significant effect on the inhibition pargyline. But, neither did riboflavin, and removing the inhibitor. Cells treated with although it lacks the phosphate, it is not 20 InM pargyline for 2 min at 25°C followed an effective substrate in the luciferase reaction nor does it have a high affinity for 200 luciferase in vitro (9). In order to see if the pargyline was acting by interfering with or removing intracellular aldehyde, exogenous additions of decanal were made to cell suspensions at various times subsequent to the addition PARGYLINE.mM of an inhibitory level of pargyline (10 InM). In fact, a prompt reversal of the inhibition occurred (Fig. 2>, suggesting that pargyline inhibition is at least partly related to aldehyde function. An unusual feature is that the reversal is apparently less effective immediately after pargyline addition than later. Under these conditions decanal will not stimulate luminescence of uninhibited cells. The effect of other long-chain aldehydes and of long-chain fatty acids on the reversal of pargyline inhibition of bioluminescence is shown in Table I. The two other aldehydes tested (o&anal and dodecanal) were less effective than decanal. Long-chain fatty acids were mostly inactive. The exception was myristic acid which is also the most effective in stimulating bioluminescence in certain dark “aldehyde” mutants (10); the FIG. 1. Inhibition of B. harveyi bioluminescence by 14-carbon chain length is postulated to be pargyline. Aliquots (1 ml) of an exponentially growing the natural aldehyde and to cycle in the B. harueyi culture (5 x 10’ cells ml-‘) were pipetted into scintillation vials containing different amounts of reaction (11). However, myristic acid only pargyline as indicated and the bioluminescence was recovered less than half of the bioluminesrecorded with time. cence recoverable with decanal, and an to FMNH,. This is referred to as the double-injection procedure. The aldehydes were from Aldrich; FMN and pargyline were from Sigma Chemical Company. Pargyline was made up in the salt medium and sterilized by Millipore filtration (0.45 pm). Purified luciferase was the gift of T. Baldwin and M. Nicoli.
MAKEMSON
0
I -20
I 0
I 20
AND HASTINGS
I i , 40
/ I t
, 60 SECONDS
I so
1 100
It 120
t
/ 140
FIG. 2. Reversal of pargyline-inhibited in. wivo bioluminescence by decanal. Pargyline (0.1 ml) was added to O.S-ml aliquots of growing cells (5 x IO* cells ml-*) at zero time. One light unit is 1.05 x 1Ol2 quanta s-*. At the times indicated 1 ml of a 0.1% decanal sonicate in distilled water was injected into the inhibited cell suspension. The injection of distilled water alone also nonspecifically stimulated (bubble formation); after correction for this, the net effect is plotted (inset) as the percentage of initial bioluminescence prior to inhibition.
additional response was obtained by the subsequent addition of decanal. The nature of the in vitro bioluminescence reaction allows a distinction between TABLE PARGYLINE
INHIBITION
OF BIOLUMINESCENCE
inhibition due enzyme with which causes catalytic cycle
to blocking the reaction of substrate(s) and inhibition the turnover time of the to be altered. The in vitro
I
AND ITS REVERSAL
BY FATTY
ALDEHYDES
AND ACIDS
Reversal of inhibition Bioluminescence” Experiment 1 2
3
Bioluminescence
Prior to inhibition
After 1 min with 10 mM pargyline
29 27 13 14 14
0.25 0.25 0.8” 1.05 1.3
Decanal Dodecanal Decanal Dodecanal Octanal
41 39 39 40
0.3 0.3 0.3 0.3
Myristic acid Decanoic acid Dodecanoic acid Ethanol
Addition6
Immediate
Decanal at 20 s
20.3 4.8 8.3 1.9 2.9
-
8.0 0.2 0.3 0.3
22 20 19 21
n Light units per milliliter of a Hepes-peptone culture at 6 to 8 x lo* cells/ml, 1.0 ml/vial. b Aldehydes, 0.01% in buffer, 1 ml added; fatty acids, 0.2 M in 95% ethanol, 10 pl added. C30 s after pargyline addition.
PARGYLINE
INHIBITION
OF BACTERIAL
reaction of h&erase, initiated with FMNH2, involves only a single catalytic cycle (12) due to the fact that the duration of one catalytic cycle (about 5-10 s) is long compared to the lifetime of the substrate, which is autooxidized rapidly (less than 0.5 s) by molecular oxygen (13). The reaction of reduced flavin, oxygen, and luciferase occurs during the first fraction of a second to form an active intermediate, postulated to be a reduced flavin-peroxy adduct (14). Over the course of the next 5 to 10 s (dependent upon temperature, aldehyde chain length, and some other factors) this intermediate reacts further to form products and emit light. Inhibitors may thus act either by reducing the amount of intermediate formed, or by altering the rate at which the reaction intermediates turn over, i.e., the catalytic cycle. Thus, with a given amount of substrate, the amount of product (photons) formed would be less in the first case, but the reaction would go to completion in the same time. In the latter type of inhibition (alteration of turnover time), the photon yield would be the same, but a different (longer) time would be required. These distinctions can be seen in the experiments of Fig. 3, where different amounts of pargyline were present in the assay mixture prior to the initiation of the reaction with FMNH,. The initial rate (I,,,) is very much reduced as the pargyline concentration increases. But, there is also an effect on the kinetics of the decay resulting in an increase in the turnover time. It might be that the increased turnover time would compensate for the decreased initial intensity resulting in little effect on the quantum yield. But, in fact, the quantum yield of the reaction is decreased so this kinetic effect is not sufficient to compensate for the decreased initial intensity. Therefore, it is suggested that both (i) the amount of intermediate formed is less at higher pargyline concentrations, and (ii) the luciferase turnover rate is slower in the presence of pargyline. It might be expected that the first effect would be subject to competitive inhibition with the substrates FMNH, and aldehyde. With FMNH, this was indeed found to be so;
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I 20
FIG. 3. Pargyline inhibition of the FMNH,initiated in vitro luciferase reaction with decanal. Different amounts of pargyline as indicated (final concentrations) were added to reaction mixtures prior to the injections of FMNH2. The effect of pargyline at different concentrations on the initial intensity Ulll,,, l ), the quantum yield (O), and the half-life (tJ of the light decay (A), is shown in the inset.
the K, FMNH, was 1.6 x lo-+ M and the K, pargyline was 1.1 x 10m3 M. With decanal the inhibition appeared to be fully reversible by aldehyde at lower pargyline (1 mM) concentrations but not completely so at higher (10 mM) levels (Fig. 4). However, this inhibition is fully reversed upon dilution, both after mixing with luciferase prior to a reaction, and with a reaction mixture subsequent to reaction in the presence of 10 mM pargyline. The effect of pargyline on the kinetics during the course of the reaction of the luciferase intermediate, and the reversibility of this effect, can be shown in both the presence and the absence of aldehyde. Pargyline inhibits if added secondarily, subsequent to the initiation of the luciferase reaction by FMNH, (Fig. 5), and this inhibition can be overcome by the later addition of more aldehyde, at concentrations higher than used in the initial reaction. It can be shown that the luciferase intermediate is formed in reactions initiated with FMNH,
MAKEMSON
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r
AND HASTINGS
I/Imox 0 07. 006
/
P*RGYLINE mM
0 05 IOh 004. 003 /---/p IOh
002.
l&&Ll+T -1 0
2
FIG. 4. Decanal competition in the pargyline inhibition of B. hmueyi luciferase. A Lineweaver-Burk plot of the initial intensity l/Z,,, as related to the decanal concentration, assayed by the double-injection procedure. Reaction mixtures were made up without aldehyde and with different concentrations of pargyline as indicated and then initiated by the injection of 1 ml of FMNH2, immediately followed by the injection of 1 ml of FMNH,, immediately followed by the injection of 1 ml of aldehyde (decanal suspension at different concentrations), as indicated. Microliters of sonicate per assay are used for decanal concentration since decanal does not form a true solution at these concentrations.
without aldehyde by the fact that later, secondary addition of aldehyde results in luminescence (Fig. 6). This intermediate, designated II and postulated to be the luciferase-bound peroxy-reduced flavin (14, 15) also has a long lifetime and reacts slowly to form H,O, and oxidized FMN without light emission (16, 17). The quantity of the intermediate present can be inferred from the bioluminescence obtained upon secondary injection of aldehyde. As shown in Fig. 6, pargyline has a strong effect on the lifetime of the intermediate in the absence of aldehyde. This effect is more pronounced than in the reaction with aldehyde present, presumably because aldehyde competes with pargyline. Several other long-chain aldehydes were examined, notably o&anal, nonanal, and dodecanal. With the first two the results were essentially the same as with decanal in Figs. 4 and 5 (although with different
rates characteristic of each aldehyde). The results with dodecanal were quite different. Pargyline added secondarily neither inhibited nor altered the half-decay time. Added prior to the initiation of the reaction the initial intensity was decreased, but there was very little effect on the decay time. These results can be explained by the fact that dodecanal apparently binds more strongly and apparently irreversibly compared to decanal and octanal (8). Consequently, secondary addition of pargyline may not readily be able to displace dodecanal and thereby inhibit the reaction, whereas secondary addition of pargyline was able to inhibit the decanal reaction. DISCUSSION
Unlike the case of monoamine oxidase, the inhibition of luciferase by pargyline I
I
I
SECONDS
FIG. 5. Inhibition of in vitro luminescence by pargyline added secondarily (after the I,,,) and reversal by aldehyde. The reactions were initiated by the injection of 1 ml of FMNH, to reaction mixtures with decanal. (0) Uninhibited reaction. The addition of pargyline approximately 1 s after the beginning of the reaction resulted in a prompt inhibition and a decrease in the rate of decay of luminescence (A), parallel to that observed with pargyline added prior to the initiation of the reaction (0). The subsequent addition of excess decanal at 3 (A) and 9 (17) s reversed the inhibition and resulted in the more rapid decay of luminescence.
PARGYLINE
INHIBITION
OF BACTERIAL
SECONDS
FIG. 6. Pargyline interaction with the luciferase reaction intermediate II. Intermediate II was formed by initiating the reaction with FMNH, in the absence of aldehyde in the presence of 0.3 mM (O), 1.0 mM (A), 10 mM (A), or no (0) pargyline. At various times after the injection of FMNHz, 1 ml of 0.01% decanal suspension was injected; the resulting initial intensity (I,,,) is plotted as a function of the time when decanal was injected. The half-life of intermediate II as a function of pargyline concentration is shown in the inset.
appears not to involve the formation of a covalent adduct because the inhibition of luciferase can be reversed. The results suggest that instead it complexes with the luciferase-peroxy-flavin intermediate, but this complex is readily reversed by the addition of excess aldehyde or by dilution. When complexed with pargyline, the dark decay of the intermediate is effectively prevented, thereby providing a potential method for trapping and characterizing the intermediate at room temperature, alternative to the low-temperature method of Hastings et al. (14). Pargyline can also inhibit the initial formation of the reduced flavin enzyme intermediate; this inhibition is competitive both with the reduced flavin substrate (& = 1.3 x 10e3 M) and with decanal (Ki = 3.7 X lo-4 M). The interaction of pargyline in viva is more difficult to interpret. In certain respects its action is explicable simply in terms of the inhibition of intracellular luciferase, since the inhibition is reversible under certain conditions with exogenously added aldehyde. However, it is difficult to explain why this is not immediately reversible after the pargyline addition. It may indicate
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that pargyline has more than one mode of inhibition, and/or that cellular compartmentalization is involved. For example, it is possible that pargyline initially binds with luciferase upon entry into the cell, but then equilibrates with other binding sites (proteins), possibly in other cellular phases, to which the pargyline binding is stronger. Thus pargyline inhibition of luminescence might involve both its direct interaction with luciferase and reaction with other components of the bioluminescent system. Pargyline does not remain bound to the luciferase of inhibited cells; upon lysis they release a luciferase which is as fully active as that obtained from control cells (data not shown). At higher concentrations and with longer times of exposure to pargyline the inhibition of in wivo bioluminescence is not reversed by dilution of the cells. By contrast, the in vitro inhibition of luciferase is readily reversed by dilution under all conditions examined. The “irreversibly” inhibited cells may still be caused to emit maximal luminescence by addition of aldehyde. Based on this, the “irreversible” effect could be attributed to an inhibition of aldehyde synthesis. The inhibition can apparently be overcome by adding exogenous myristic acid or long-chain aldehyde. However, decanal addition to these partially reversed cells resulted in a bioluminescent flash. This may result in the inability of exogenous myristic acid to fully saturate luciferase with aldehyde, presumably tetradecanal (myristic aldehyde), due to slow transport and/or conversion to the aldehyde. An alternative explanation is that the myristic acid transport and conversion to tetradecanal operates at its optimal levels but is only one of several aldehyde synthetic pathways. The amount of myristic acid added in the experiment shown in Table I was the optimal amount for these cells and for bioluminescence in an aldehyde mutant (M-17) grown under identical conditions. The data extant cannot distinguish between these possibilities. REFERENCES 1. CHUANG, H. Y. K., PATEK, D. R., HELLERMAN, L. (1974) J. Biol. Chm. 2381-2384.
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2. HASTINGS, J. W., AND NEALSON, K. H. (1977) Annu. Rev. Microbial. 31, 549-595. 3. REICHELT, J. L., AND BAUMANN, P. (1973) Arch. Mikrobiol. 94, 283-330. 4. BAUMANN, P., AND BAUMANN, L. (1977) Annu. Rev. Microbial. 31, 39-61. 5. HASTINGS, J. W., AND WEBER, G. (1963) J. Opt. Sot. Amer. 53, 1411-1415. 6. MITCHELL, G., AND HASTINGS, J. W. (1971) Anal. Biochem. 39, 243-250. 7. HASTINGS, J. W., WEBER, K., FRIEDLAND, J., EBERHARD, A., MITCHELL, G. W., AND GUNSALUS, A. (1969) Biochemistry 8,4681-4639. 8. BAUMSTARK, A. L., CLINE, T. W., AND HASTINGS, J. W. (1979) Arch. Biochem. Biophys. 192, 449-455. 9. BALDWIN, T. O., NICOLI, M. Z., BECVAR, J. E., AND HASTINGS, J. W. (1975) J. Biol. Chem. 250, 2X3-2768.
10. ULITZUR, S., AND HASTINGS, J. W. (1978) Nat. Acad. Sci. USA 75, 266-269.
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11. ULITZUR, S., AND HASTINGS, J. W. (1979) Proc. Nat. Acad. Sci. USA 76, 265-267. 12. HASTINGS, J. W., AND GIBSON, 0. H. (1963) J. Biol. Chem. 238, 2537-2554. 13. GIBSON, Q. H., AND HASTINGS, Biochem. J. 83, 368-377.
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14. HASTINGS, J. W., BALNY, C., LE PEUCH, C., AND DOUZOU, P. (1973) Proc. Nat. Acad. Sci. USA 70, 3468-3472. 15. HASTINGS, J. W. (1978) Critical Reviews of Biochemistry (Fasman, G., ed.), Vol. 5, pp. 163-184, CRC Press, Cleveland, Ohio. 16. HASTINGS, J. W., AND BALNY, C. (1975) J. Biol. Chem. 250, 7288-7293. 17. BECVAR, (1978)
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