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Preparation and properties of a microsomal DPNH-cytochrome c reductase
ARCRIVES OF BIOCHEMISTRY AND BIOPHYSICS 71, loo-110
(19%)
Preparation and Properties of a Microsomal DPNH-Cytochrome c Reductase David GaSmkell From the
Johnson Foundation for Medical of Pennsylvania,
Philadelphia,
Physics,
University
Pennsylvania
Received December 11, 1956
In the course of studies (1) leading to the isolation of microsomal cytochrome (cytochrome bS), which is a participant of a DPNH-cytochrome c reductase system, it was dbserved that an appreciable part of the cytochrome c reductase activity could be separated from cytochrome bg by ammonium sulfate fractionation. A subsequent attempt to isolate a cytochrome b5 reductase yielded a soluble enzyme preparation which reduced cytochrome c but not cytochrome b6 . This report describes the preparation from pig liver microsomes of a relatively crude extract, and the properties of the enzyme contained therein. The relationship of this enzyme to the cytochrome b6 system will be discussed below. MATERIALS
AND METHODS
Horse-heart cytochrome c and DPNH2 were products of the Sigma Chemical Company. When it was found that the enzyme reduced 2,6-dichloroindophenol, the related dye 2,6-dichlorophenoL3’indophenol was selected as a preferable dye for diaphorase measurements, since the latter dye does not react readily with oxygen (2) and can therefore be handled aerobically. The Eastman Kodak product was used (this will be referred to as “dye” hereafter). Protamine sulfate was obtained from Nutritional Biochemicals Corporation. Pig livers were obtained from J. J. Felin and Company. The terms “reductase” and “diaphorase” will be used according to the definitions of Mahler et al. (3). 1 Public Health Service Research Fellow of the National Cancer Institute. 2 The following abbreviations are used in this paper: DPNH, reduced diphosFAD, flavine adenine phopyridine nucleotide; FMN, fl avine mononucleotide; dinucleotide; PCMBS, p-chloromercuribenzenesulfonic acid; Tris, tris(hydroxymethyl) aminomethane. 100
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
101
Microsomes were prepared from pig liver by the method described by Garfinkel (4). Perfusion of the livers is not necessary but does seem to improve the yield of enzyme. Precipitation of the microsomes with acid (5) or ammonium sulfate (6) materially decreases the yield of enzyme and should be avoided. The microsomes obtained may be stored frozen at -20”. An acetone powder was then prepared from the microsomes by the method of Morton (7). This may also be stored at -20”. To extract the enzyme, the powder was ground with a mortar and pestle, and then 20 ml. Tris buffer, pH 7.5,0.05 M, was added for every gram of powder. This was allowed to stand with intermittent stirring for 15 min., and then centrifuged in the Servall SS-1 centrifuge (10 min. at 8000 r.p.m.), the precipitate being discarded. To the supernatant was added enough saturated (at 0”) ammonium sulfate solution (adjusted to pH 8 with NHaOH) to make the solution 38% saturated in ammonium sulfate. It was allowed to stand one-half hour, then centrifuged in the Servall SS-1 for 10 min. at 8000 r.p.m., and the supernatant discarded. The precipitate was resuspended in Tris buffer (pH 7.5, 0.05 M) and again centrifuged at the same speed to remove fine particles which were not removed the first time. All operations were carried out at O-4” and the enzyme was kept cold thereafter. It has been the practice to keep it dark whenever possible, in view of the possibility (discussed below) that it is a flavoprotein. This preparation is stable for at least 2 days at 0’. Since the enzyme is destroyed by repeated freezing and thawing, it is preferable not to store it frozen. It survives lyophilization, but no attempt has been made to store it as a lyophilized powder. Activity of the enzyme was assayed as follows: For reductase activity, an amount of the enzyme yielding a conveniently measurable rate was added to a cuvette together with cytochrome c (to yield a final concentration of 26 PM) and 0.1 M Tris buffer, pH 7.5, to yield a final volume of 1.5 ml. The reaction was started by adding sufficient DPNH solution to make the final concentration 37 pM (effectively, this is “excess DPNH”). The optical density increase at 550 rnp was followed, either in the Beckman DU spectrophotometer (in which case readings were taken every 15 sec.) or in the split-beam spectrophotometer (8), which records automatically. Following Theorell (9), AErn~ 550 is taken as 19.1. To measure diaphorase activity, a final dye concentration of 26 pM was substituted for the cytochrome c, and the decrease in optical density at 645 rnp [with that there is a AE~M being taken as 25 (2)] is followed as above. It is noteworthy lag period of 34-2 min. before the reaction rate reaches a steady value, and this is prolonged by some of the inhibitors used. To check reduction of ferricyanide, 67 rmoles potassium ferricyanide was used as acceptor and followed at 420 rnp With ferricyanide, as with cytochrome c, CAEmu = 1.0, as determined directly). there is no lag period. The ratio of reductase to diaphorase activity did not seem constant from one batch of enzyme to another, but this may be the effect of the lag period, which sometimes makes it hard to judge when a steady state of diaphorase activity has been reached. A representative ratio of reductase to diaphorase to ferricyanide activity was 10:3:20. Enzymic activity is defined as micromoles acceptor reduced/min./mg. protein, and all activity assays were performed at room temperature. Most of the assays were performed in series which were compared to a stand-
102
GARFINKEL
ard activity. Here it is necessary that the same amount of eneyme be used throughout a series. This was usually about 0.1 mg. enzyme protein per measurement. The objection may be raised that this enzyme is actually an artifact resulting from the acetone-powder treatment. However, the same enzyme appears in pancreatin digests of microsomes, and it is even released from intact microsomes on sufficiently long standing (more than a week). It seems unlikely that these treatments would give rise to the same artifact.
PROPERTIES
Enzymes having the type of activity described above are usually flavoproteins or hemoproteins, and the presence of these constituents was therefore investigated. The enzyme preparation is slightly yellowish and contains flavine [total flavine is about 7 X 1O-5 pmole/mg. protein, as determined by the decrease in optical density at 450 rnp on reduction, assuming A EmM = 11.3, the value for free flavine (lo)]. Most of the flavine present is reduced by DPNH (as compared with dithionite reduction). If the preparation is analyzed for FMN and FAD (ll), the flavine is found to be about 80% FMN. Additional evidence on this point will be brought out below. Addition of DPNH to the preparation causes no change that could be due to heme compounds, and in view of the lack of effect of heme enzyme inhibitors, to be shown below, it is unlikely that this is a heme enzyme. The enzyme was found to absorb on C, alumina gel and on calcium phosphate gel (12)) from which it was not eluted by phosphate buffers or phosphate buffers with added ammonium sulfate, but it was eluted by 5 % sodium pyrophosphate, pH 9.5. It is apparently not precipitated by dilute ethanol, but when an ethanol fractionation was carried out in sodium phosphate buffer, it absorbed on the phosphate crystals that were precipitated out. It combines strongly with protamine, and this is a useful method of removing it from solutions of other enzymes, where it, is present as a contaminant, but no method of taking advantage of this for preparative purposes has yet been found. It is most stable around pH 8 but is moderately stable in the pH range from 5 to 10. The fact that this enzyme combines with protamine indicates that it has a low isoelectric point. However, the fact that it is digested (with loss of activity) by pancreatic proteases (in pancreatin) indicates that at least part of the molecule satisfies the specificity requirements of such enzymes as trypsin and chymotrypsin. It is also noteworthy that such of this enzyme as survives the digestion readily loses its activity on exposure to high ionic strength, such as concentrated ammonium sulfate
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
103
solutions. Part of this activity returns slowly when the ionic strength is lowered again. The enzyme is thermolabile, with the extent of inactivation by heat being shown in Fig. 1. It is to be noted that the points on the diaphorase curve at 30” and 40” have no lag period associated with them. This may be related to the fact that in the unfractionated extract [or in the pancreatin digest of microsomes (l)] heating for 1 min. at these temperatures actually activates the enzyme. This may be due to destruction of an inhibitor, which is removed by ammonium sulfate fractionation. The effect of pH in various buffers on the reductase and diaphorase activity is shown in Figs. 2 and 3, respectively. The inhibition by phosphate, pyrophosphate, and borate ions suggests that the enzyme may be generally inhibited by multiply charged anions. It will be seen from Table I that sulfate ion also inhibits. The similarity of the properties of the reductase and diaphorase activities (particularly their reactions to temperature and to the various buffers tested) indicates that they are in fact due to activities of the same enzyme. The effect of various metal ions on activity is shown in Table I. Sodium sulfate is included to check the effect of sulfate ion, as some of these metals were available only as the sulfate. The inhibitory effects of such ions as Ca++, Mg++, and Co++ are probably due to generalized binding
30
40 50 60 Temp. “C
70
80
FIG. 1. Effect of heat. Samples of the enzyme were heated for 1 min. to the temperature indicated, then promptly cooled in ice, and subsequently assayed for both reductase and diaphorase activity under standard conditions. The enzyme which has been kept at 0” throughout is taken as standard. Solid circles represent reductase activity; open circles, diaphorase activity.
104
GARFINKEL
PH FIQ. 2. Reductase activity in various buffers. These points are determined as in the standard activity assay, except that the buffer indicated is substituted for 0.1 44, pH 7.5 Tris buffer. 0, 0.1 M sodium acetate; X, 0.1 M Tris; 0, 0.1 M sodium phosphate; 0, 0.1 M sodium pyrophosphate; a, 0.1 M sodium borate.
0 6
7
8
9
IO
PH FIG. 3. Diaphorase activity in various buffers. This is determined in the same way as for Fig. 2. The dye is unstable below pH 6, and no readings were taken in this region. 0,O.l M sodium acetate; 0,O.l M Tris; X, 0.1 M sodium phosphate; 0, 0.1 M sodium pyrophosphate; a, 0.1 M sodium borate. to the protein
rather than to specific binding to groups in the active center. It is interesting that Mg++ activates reductase activity and inhibits diaphorase, whereas Mn ++ has the opposite effect. The relatively slight effect of Pb++ on reductase activity is somewhat puzzling, particularly in view of the behavior of Hg++ and Ag+.
MICROSOMAL
DPNH-CYTOCHROME
TABLE I Effect of Metal-Ion Inhibitors Assayed under standard conditions with the indicated hibitor present. Substance
105
C REDUCTASE
Concentration
concentration
of in-
Inhibition Reductase Diaphorase
Y
Lead acetate Calcium chloride Cobalt acetate Sodium sulfate Manganous sulfate Zinc acetate Zinc acetate Magnesium sulfate Mercuric chloride Silver acetate
7 3.5 7 3.5 3.5 3.5 7 3.5 1 3
x x x x x x x x x x
lo-’ lo-3 lo-* 10-a 10-a 10-a 1tF’ 10-a 10-a 10-a
TABLE II E#ect of Suljhydryl Inhibitors Assayed under standard conditions with the indicated hibitor present. Inhibitor
Concentration
Todoacetic acid= Iodoacetamidea Iodoacetamidea Phenyl mercury acetate p-Chloromercuribenzoate PCMBS PCMBS
0625 0.125 0.0915 lo-’ 10-b 10-4 10-G
3”i 23 19 37 70 74 30 9 100 90
concentration
7” 87 95 84 10 53 39 85 100 93
of in-
Inhibition Reductase Diaphorase
iii 100 85 98 97 98 97
2 89 19 87 85 85 85
QEnzyme incubated with the indicated concentration of inhibitor for at least one-half hour prior to assay, in Tris buffer, pH 7.5,O. 1 M, at 0”. No additional inhibitor was added at the time of assay.
To determine whether intact sulfhydryl groups are necessary for activity, experiments were carried out as shown in Table II. A possible explanation for the fact that all of these inhibitors have a stronger effect on reductase activity than on diaphorase activity is that there are several sulfhydryl groups in or near the active center, more of which are required to be intact for reductase activity than for diaphorase activity. To prove that the sulfhydryl groups of the enzyme are what is attacked by these inhibitors, it is desirable to show that addition of other sulf-
106
GARFINKEL
TABLE Effect of Various
Assayed under standard conditions, hibitor present. Substance
Atebrin Atebrin Atebrin FMN” Versene Versene Antimycin A Amytal “bg inhibitor” “ba inhibitor” co KCN NaNa Hydroxylamine Hydroxylamine
III Inhibitors
with the indicated
Concentration
1.6 X 1O-6 M 8 x 10-e &.f 1.6 X lo-4M 1.3 X lo-4M 2 x 10-2 M 6.7 X lo-2 M 0.0033 mg./ml. 6.7 X lo-‘M 0.4 mg./ml. 2 mg./ml. (Bubbled through) 1 X 10-2 M 1 X 10-2 M 3.3 x 10-z M 1 x 10-r M
concentration
of in-
Inhibition Reductase Diaphomse
NZne None None None 25 28 None None 86 99 None None None None 30
OFMN neither inhibits nor activates and did not seem to counteract when both were tried together.
No%ne 51 66 None 26 None 11 None 26 44 38 17 None 13 41 atebrin
hydryl compounds protects the enzyme. The physiologically important compounds of this type, cysteine and glutathione, reduce both the dye and cytochrome c, but it was found that, under the conditions used, the reduction of cytochrome c by glutathione was slow enough to permit assay of enzyme activity. In the presence of 4 X 1O-4 M glutathione, 1 X 10e4M PCMBS inhibited reductase activity 61% and 1 X 1O-6 M PCMBS inhibited it 15%. In the presence of 8 X 10msM glutathione, 1 X 1O-4 M PCMBS inhibited 99 %. These results show definitely that the enzyme does require intact sulfhydryl groups for activity. The effects of various other inhibitors are shown in Table III. One can generalize that the effects of hemoprotein inhibitors (CO, cyanide, azide) and cytochrome-system inhibitors (antimycin, amytal) are at most slight. The “bb inhibitor”3 is an exception to this. Its profound effect on reductase activity may be due to combination with the cyto3 The “bs inhibitor” is an antibiotic type inhibitor of unknown structure isolated by Dr. A. M. Pappenheimer, Jr. It is a potent inhibitor of respiratory and cytochrome systems, particularly the cytochrome bg system.
MICROSOMAL
DPNH-CYTOCHROME
107
C REDUCTASE
chrome c, as appreciable precipitation takes place when the inhibitor is added to the cuvette for reductase assay. To check this matter further, the effect of this inhibitor on ferricyanide reduction was studied, and it was found that there was no inhibition at all. It seems unlikely, therefore, that this inhibitor strongly affects the enzyme. The effects of atebrin [which competes with flavines for the apoenzyme part of many flavoproteins (13, 14)] and of FMN are not sufficient to prove that this enzyme is a flavoprotein, particularly as the atebrin inhibition is hard to reproduce, and this matter must therefore be considered undecided. Affinities of the enzyme for DPNH and cytochrome c are determined DPNH
>
0
IO Substrate
Substrate
(PM)
Cytochrome
5
IO
Substrate FIG. 4. Affinity under the standard being determined. the left: velocity Lineweaver-Burk
I5 (PM)
20
(,uM)
c
25
0
5 Substrate
IO
15
20
25
(pM)
constants of DPNH and cytochrome c. These were determined assay conditions, except for the constituent whose affinity is For each determination 0.09 mg. enzyme protein is used. On (V) as a function of substrate (8) concentration. On the right: plots.
108
GARFINKEL
as shown in Fig. 4. The value for cytochrome c (7 X 10-r M) is similar to those for the DPNH-cytochrome c reductase systems of mitochondria (15) and intact microsomes,* 1.75 X 10e6 and 8.3 X lo-6 1M, respectively, but appreciably different from that for the soluble DPNH-cytochrome c reductase of Mahler et al. (3, 16), which is 1.2 X lo-* M. DISCUSSION In a situation of this type, where there are present two enzyme systems having similar properties, it is necessary to show that we are dealing with only one of them. In this case the absence of cytochrome bs and of DPNH-cytochrome bg reductase must be demonstrated. Reduced difference spectra of the unfractionated extract (on reduction with dithionite but not with DPNH) show peaks at 424 rnp, which are displaced on addition of CO. This is behavior typical of protein hemochromogens; cytochrome bg , however, does not react with CO. On reduction with DPNH, the optical density change at 424 rnp is less than 0.0005, indicating that the cytochrome b6 content is less than 10~~ M (6). As mentioned above, an attempt was originally made to prepare DPNHcytochrome bg reductase by this method, and this was unsuccessful. The addition of cytochrome bg did not reproducibly increase the rate of cytochrome c reduction, even after a wide range of treatments, such as exposure to high ionic strength, incubation with cytochrome br, , heating, and digestion with ribonuclease and pancreatin. When the concentration of cytochrome br, here used was added to cytochrome bS reductase, cytochrome c, and DPNH, under the same conditions, reduction of cytochrome c did take place. Additional items of evidence are (a) the curve for the effect of pH on activity in Tris buffer is not identical for this enzyme and for DPNH-cytochrome b6 reductase (5) ; (b) this enzyme occurs as a contaminant in preparations of DPNH-cytochrome bg reductase, from which it can be removed completely. Therefore it may be concluded that the properties observed are not those of the cytochrome bg system. This enzyme is quite similar to the DPNH-cytochrome c reductase isolated by Mahler and co-workers (3, 16, 17) from pig heart sarcosomes. The question of whether these might be the same enzyme may be settled immediately. Not only are the sources of the enzymes different, but the enzyme here described absorbs on calcium phosphate gel and alumina gel whereas that of Mahler does not. The behavior toward metal-ion 4M. Klingenberg, in preparation.
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
109
inhibitors and toward phosphate and pyrophosphate differs appreciably, and the affinity constants differ quite widely (3.5 X 10e6 M as against 1.9 X 1O-5 M for DPNH, and T X lo-’ M as against 1.2 X 1O-4 M for cytochrome c). It is therefore concluded that these are not the same enzyme. Although not identical, these enzymes are similar in many ways (this would be even more strongly true if the enzyme here described is indeed a flavoprotein). For that matter, the DPNH-cytochrome b6 reductase of Strittmatter and Velick (5) is also quite similar in many properties, even though it does reduce a different cytochrome. All of these enzymes, for instance, are at least moderately stable to 10% ethanol at 40”. All reduce two molecules of acceptor, but the velocity depends on the first power only of the acceptor concentration. This suggests that they are part of a general class of enzymes. The actual function of this enzyme in the microsomes is open to question. It has been shown to reduce several substances, none of which occurs in its natural environment (cytochrome c does not occur in microsomes and its presence in appreciable concentration in the cytoplasm has not been demonstrated). The same question can of course be asked of the DPNH-cytochrome bscytochrome c reductase system of microsomes. As yet no satisfactory answer is available. ACKNOWLEDGMENTS The advice and encouragement gratefully acknowledged.
of Dr. R. W. Estabrook
and Dr. B. Chance are
SUMMARY
The preparation of a microsomal DPNH-cytochrome c reductase has been described. Its susceptibility to heat and to various inhibitors is shown, and also the effect of various buffers in the pH range 5-10. This enzyme is demonstrated to require intact sulfhydryl groups for activity, but it is doubtful if it is a flavoprotein. The Michaelis constants are 3.5 X lows M for DPNH and 7 X lo-’ M for cytochrome c. The relationships of this enzyme with other similar enzymes are discussed. REFERENCES 1. GARFINKEL, D., Biochim. et Biophys. Acta 21, 199 (1956). 2. SMITH, F. G., ROBINSON, W. B. AND STOTZ, E., J. Biol. Chem. 179, 881 (1949). 3. MAHLER, H. R., SARKAR, N. K., AND VERNON, L. P., J. Biol. Chem. 199, 585 (1952).
110
GARFINKEL
4. GARFINKEL, D., Arch. Biochem. 5. STRITTMATTER, P., AND VELICK, 6. STRITTMATTER, P., AND VELICK, 7. MORTON, R. K., in “Methods
71, 111 (1957). S. F., J. Biol. Chem. 221, 277 (1956). S. F., J. Biol. Chem. 221, 253 (1956). in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1. Academic Press, New York, 1955. 8. YANG, C. C., AND LEGALLAIS, V., Rev. Sci. Instr. 26, 801 (1954). 9. THEORELL, H., Biochem. 2.286, 207 (1936). 10. DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1952). 11. BESSEY, 0. A., LOWRY, O..H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 12. COLOWICK, S. P., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1. Academic Press, New York, 1955. 13. HAAS, E., J. Biol. Chem. 166, 321 (1944). 14. HELLERMAN, L., LINDSAY, A., AND BOVARNICK, M. R., .I. Biol. Chem. 163.553 (1946). 15. ESTABROOK, R. W., J. Biol. Chem., in press. 16, VERNON, L. P., MAHLER, H. R., AND SAREAR, N. K., J. Biol. Chem. 199, 599 (1952). 17. EDELHOCH, H., HAYISHI, O., AND TEPLEY, L. J., J. Biol. Chem. 197,97 (1952). Biophys.
(19%)
Preparation and Properties of a Microsomal DPNH-Cytochrome c Reductase David GaSmkell From the
Johnson Foundation for Medical of Pennsylvania,
Philadelphia,
Physics,
University
Pennsylvania
Received December 11, 1956
In the course of studies (1) leading to the isolation of microsomal cytochrome (cytochrome bS), which is a participant of a DPNH-cytochrome c reductase system, it was dbserved that an appreciable part of the cytochrome c reductase activity could be separated from cytochrome bg by ammonium sulfate fractionation. A subsequent attempt to isolate a cytochrome b5 reductase yielded a soluble enzyme preparation which reduced cytochrome c but not cytochrome b6 . This report describes the preparation from pig liver microsomes of a relatively crude extract, and the properties of the enzyme contained therein. The relationship of this enzyme to the cytochrome b6 system will be discussed below. MATERIALS
AND METHODS
Horse-heart cytochrome c and DPNH2 were products of the Sigma Chemical Company. When it was found that the enzyme reduced 2,6-dichloroindophenol, the related dye 2,6-dichlorophenoL3’indophenol was selected as a preferable dye for diaphorase measurements, since the latter dye does not react readily with oxygen (2) and can therefore be handled aerobically. The Eastman Kodak product was used (this will be referred to as “dye” hereafter). Protamine sulfate was obtained from Nutritional Biochemicals Corporation. Pig livers were obtained from J. J. Felin and Company. The terms “reductase” and “diaphorase” will be used according to the definitions of Mahler et al. (3). 1 Public Health Service Research Fellow of the National Cancer Institute. 2 The following abbreviations are used in this paper: DPNH, reduced diphosFAD, flavine adenine phopyridine nucleotide; FMN, fl avine mononucleotide; dinucleotide; PCMBS, p-chloromercuribenzenesulfonic acid; Tris, tris(hydroxymethyl) aminomethane. 100
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
101
Microsomes were prepared from pig liver by the method described by Garfinkel (4). Perfusion of the livers is not necessary but does seem to improve the yield of enzyme. Precipitation of the microsomes with acid (5) or ammonium sulfate (6) materially decreases the yield of enzyme and should be avoided. The microsomes obtained may be stored frozen at -20”. An acetone powder was then prepared from the microsomes by the method of Morton (7). This may also be stored at -20”. To extract the enzyme, the powder was ground with a mortar and pestle, and then 20 ml. Tris buffer, pH 7.5,0.05 M, was added for every gram of powder. This was allowed to stand with intermittent stirring for 15 min., and then centrifuged in the Servall SS-1 centrifuge (10 min. at 8000 r.p.m.), the precipitate being discarded. To the supernatant was added enough saturated (at 0”) ammonium sulfate solution (adjusted to pH 8 with NHaOH) to make the solution 38% saturated in ammonium sulfate. It was allowed to stand one-half hour, then centrifuged in the Servall SS-1 for 10 min. at 8000 r.p.m., and the supernatant discarded. The precipitate was resuspended in Tris buffer (pH 7.5, 0.05 M) and again centrifuged at the same speed to remove fine particles which were not removed the first time. All operations were carried out at O-4” and the enzyme was kept cold thereafter. It has been the practice to keep it dark whenever possible, in view of the possibility (discussed below) that it is a flavoprotein. This preparation is stable for at least 2 days at 0’. Since the enzyme is destroyed by repeated freezing and thawing, it is preferable not to store it frozen. It survives lyophilization, but no attempt has been made to store it as a lyophilized powder. Activity of the enzyme was assayed as follows: For reductase activity, an amount of the enzyme yielding a conveniently measurable rate was added to a cuvette together with cytochrome c (to yield a final concentration of 26 PM) and 0.1 M Tris buffer, pH 7.5, to yield a final volume of 1.5 ml. The reaction was started by adding sufficient DPNH solution to make the final concentration 37 pM (effectively, this is “excess DPNH”). The optical density increase at 550 rnp was followed, either in the Beckman DU spectrophotometer (in which case readings were taken every 15 sec.) or in the split-beam spectrophotometer (8), which records automatically. Following Theorell (9), AErn~ 550 is taken as 19.1. To measure diaphorase activity, a final dye concentration of 26 pM was substituted for the cytochrome c, and the decrease in optical density at 645 rnp [with that there is a AE~M being taken as 25 (2)] is followed as above. It is noteworthy lag period of 34-2 min. before the reaction rate reaches a steady value, and this is prolonged by some of the inhibitors used. To check reduction of ferricyanide, 67 rmoles potassium ferricyanide was used as acceptor and followed at 420 rnp With ferricyanide, as with cytochrome c, CAEmu = 1.0, as determined directly). there is no lag period. The ratio of reductase to diaphorase activity did not seem constant from one batch of enzyme to another, but this may be the effect of the lag period, which sometimes makes it hard to judge when a steady state of diaphorase activity has been reached. A representative ratio of reductase to diaphorase to ferricyanide activity was 10:3:20. Enzymic activity is defined as micromoles acceptor reduced/min./mg. protein, and all activity assays were performed at room temperature. Most of the assays were performed in series which were compared to a stand-
102
GARFINKEL
ard activity. Here it is necessary that the same amount of eneyme be used throughout a series. This was usually about 0.1 mg. enzyme protein per measurement. The objection may be raised that this enzyme is actually an artifact resulting from the acetone-powder treatment. However, the same enzyme appears in pancreatin digests of microsomes, and it is even released from intact microsomes on sufficiently long standing (more than a week). It seems unlikely that these treatments would give rise to the same artifact.
PROPERTIES
Enzymes having the type of activity described above are usually flavoproteins or hemoproteins, and the presence of these constituents was therefore investigated. The enzyme preparation is slightly yellowish and contains flavine [total flavine is about 7 X 1O-5 pmole/mg. protein, as determined by the decrease in optical density at 450 rnp on reduction, assuming A EmM = 11.3, the value for free flavine (lo)]. Most of the flavine present is reduced by DPNH (as compared with dithionite reduction). If the preparation is analyzed for FMN and FAD (ll), the flavine is found to be about 80% FMN. Additional evidence on this point will be brought out below. Addition of DPNH to the preparation causes no change that could be due to heme compounds, and in view of the lack of effect of heme enzyme inhibitors, to be shown below, it is unlikely that this is a heme enzyme. The enzyme was found to absorb on C, alumina gel and on calcium phosphate gel (12)) from which it was not eluted by phosphate buffers or phosphate buffers with added ammonium sulfate, but it was eluted by 5 % sodium pyrophosphate, pH 9.5. It is apparently not precipitated by dilute ethanol, but when an ethanol fractionation was carried out in sodium phosphate buffer, it absorbed on the phosphate crystals that were precipitated out. It combines strongly with protamine, and this is a useful method of removing it from solutions of other enzymes, where it, is present as a contaminant, but no method of taking advantage of this for preparative purposes has yet been found. It is most stable around pH 8 but is moderately stable in the pH range from 5 to 10. The fact that this enzyme combines with protamine indicates that it has a low isoelectric point. However, the fact that it is digested (with loss of activity) by pancreatic proteases (in pancreatin) indicates that at least part of the molecule satisfies the specificity requirements of such enzymes as trypsin and chymotrypsin. It is also noteworthy that such of this enzyme as survives the digestion readily loses its activity on exposure to high ionic strength, such as concentrated ammonium sulfate
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
103
solutions. Part of this activity returns slowly when the ionic strength is lowered again. The enzyme is thermolabile, with the extent of inactivation by heat being shown in Fig. 1. It is to be noted that the points on the diaphorase curve at 30” and 40” have no lag period associated with them. This may be related to the fact that in the unfractionated extract [or in the pancreatin digest of microsomes (l)] heating for 1 min. at these temperatures actually activates the enzyme. This may be due to destruction of an inhibitor, which is removed by ammonium sulfate fractionation. The effect of pH in various buffers on the reductase and diaphorase activity is shown in Figs. 2 and 3, respectively. The inhibition by phosphate, pyrophosphate, and borate ions suggests that the enzyme may be generally inhibited by multiply charged anions. It will be seen from Table I that sulfate ion also inhibits. The similarity of the properties of the reductase and diaphorase activities (particularly their reactions to temperature and to the various buffers tested) indicates that they are in fact due to activities of the same enzyme. The effect of various metal ions on activity is shown in Table I. Sodium sulfate is included to check the effect of sulfate ion, as some of these metals were available only as the sulfate. The inhibitory effects of such ions as Ca++, Mg++, and Co++ are probably due to generalized binding
30
40 50 60 Temp. “C
70
80
FIG. 1. Effect of heat. Samples of the enzyme were heated for 1 min. to the temperature indicated, then promptly cooled in ice, and subsequently assayed for both reductase and diaphorase activity under standard conditions. The enzyme which has been kept at 0” throughout is taken as standard. Solid circles represent reductase activity; open circles, diaphorase activity.
104
GARFINKEL
PH FIQ. 2. Reductase activity in various buffers. These points are determined as in the standard activity assay, except that the buffer indicated is substituted for 0.1 44, pH 7.5 Tris buffer. 0, 0.1 M sodium acetate; X, 0.1 M Tris; 0, 0.1 M sodium phosphate; 0, 0.1 M sodium pyrophosphate; a, 0.1 M sodium borate.
0 6
7
8
9
IO
PH FIG. 3. Diaphorase activity in various buffers. This is determined in the same way as for Fig. 2. The dye is unstable below pH 6, and no readings were taken in this region. 0,O.l M sodium acetate; 0,O.l M Tris; X, 0.1 M sodium phosphate; 0, 0.1 M sodium pyrophosphate; a, 0.1 M sodium borate. to the protein
rather than to specific binding to groups in the active center. It is interesting that Mg++ activates reductase activity and inhibits diaphorase, whereas Mn ++ has the opposite effect. The relatively slight effect of Pb++ on reductase activity is somewhat puzzling, particularly in view of the behavior of Hg++ and Ag+.
MICROSOMAL
DPNH-CYTOCHROME
TABLE I Effect of Metal-Ion Inhibitors Assayed under standard conditions with the indicated hibitor present. Substance
105
C REDUCTASE
Concentration
concentration
of in-
Inhibition Reductase Diaphorase
Y
Lead acetate Calcium chloride Cobalt acetate Sodium sulfate Manganous sulfate Zinc acetate Zinc acetate Magnesium sulfate Mercuric chloride Silver acetate
7 3.5 7 3.5 3.5 3.5 7 3.5 1 3
x x x x x x x x x x
lo-’ lo-3 lo-* 10-a 10-a 10-a 1tF’ 10-a 10-a 10-a
TABLE II E#ect of Suljhydryl Inhibitors Assayed under standard conditions with the indicated hibitor present. Inhibitor
Concentration
Todoacetic acid= Iodoacetamidea Iodoacetamidea Phenyl mercury acetate p-Chloromercuribenzoate PCMBS PCMBS
0625 0.125 0.0915 lo-’ 10-b 10-4 10-G
3”i 23 19 37 70 74 30 9 100 90
concentration
7” 87 95 84 10 53 39 85 100 93
of in-
Inhibition Reductase Diaphorase
iii 100 85 98 97 98 97
2 89 19 87 85 85 85
QEnzyme incubated with the indicated concentration of inhibitor for at least one-half hour prior to assay, in Tris buffer, pH 7.5,O. 1 M, at 0”. No additional inhibitor was added at the time of assay.
To determine whether intact sulfhydryl groups are necessary for activity, experiments were carried out as shown in Table II. A possible explanation for the fact that all of these inhibitors have a stronger effect on reductase activity than on diaphorase activity is that there are several sulfhydryl groups in or near the active center, more of which are required to be intact for reductase activity than for diaphorase activity. To prove that the sulfhydryl groups of the enzyme are what is attacked by these inhibitors, it is desirable to show that addition of other sulf-
106
GARFINKEL
TABLE Effect of Various
Assayed under standard conditions, hibitor present. Substance
Atebrin Atebrin Atebrin FMN” Versene Versene Antimycin A Amytal “bg inhibitor” “ba inhibitor” co KCN NaNa Hydroxylamine Hydroxylamine
III Inhibitors
with the indicated
Concentration
1.6 X 1O-6 M 8 x 10-e &.f 1.6 X lo-4M 1.3 X lo-4M 2 x 10-2 M 6.7 X lo-2 M 0.0033 mg./ml. 6.7 X lo-‘M 0.4 mg./ml. 2 mg./ml. (Bubbled through) 1 X 10-2 M 1 X 10-2 M 3.3 x 10-z M 1 x 10-r M
concentration
of in-
Inhibition Reductase Diaphomse
NZne None None None 25 28 None None 86 99 None None None None 30
OFMN neither inhibits nor activates and did not seem to counteract when both were tried together.
No%ne 51 66 None 26 None 11 None 26 44 38 17 None 13 41 atebrin
hydryl compounds protects the enzyme. The physiologically important compounds of this type, cysteine and glutathione, reduce both the dye and cytochrome c, but it was found that, under the conditions used, the reduction of cytochrome c by glutathione was slow enough to permit assay of enzyme activity. In the presence of 4 X 1O-4 M glutathione, 1 X 10e4M PCMBS inhibited reductase activity 61% and 1 X 1O-6 M PCMBS inhibited it 15%. In the presence of 8 X 10msM glutathione, 1 X 1O-4 M PCMBS inhibited 99 %. These results show definitely that the enzyme does require intact sulfhydryl groups for activity. The effects of various other inhibitors are shown in Table III. One can generalize that the effects of hemoprotein inhibitors (CO, cyanide, azide) and cytochrome-system inhibitors (antimycin, amytal) are at most slight. The “bb inhibitor”3 is an exception to this. Its profound effect on reductase activity may be due to combination with the cyto3 The “bs inhibitor” is an antibiotic type inhibitor of unknown structure isolated by Dr. A. M. Pappenheimer, Jr. It is a potent inhibitor of respiratory and cytochrome systems, particularly the cytochrome bg system.
MICROSOMAL
DPNH-CYTOCHROME
107
C REDUCTASE
chrome c, as appreciable precipitation takes place when the inhibitor is added to the cuvette for reductase assay. To check this matter further, the effect of this inhibitor on ferricyanide reduction was studied, and it was found that there was no inhibition at all. It seems unlikely, therefore, that this inhibitor strongly affects the enzyme. The effects of atebrin [which competes with flavines for the apoenzyme part of many flavoproteins (13, 14)] and of FMN are not sufficient to prove that this enzyme is a flavoprotein, particularly as the atebrin inhibition is hard to reproduce, and this matter must therefore be considered undecided. Affinities of the enzyme for DPNH and cytochrome c are determined DPNH
>
0
IO Substrate
Substrate
(PM)
Cytochrome
5
IO
Substrate FIG. 4. Affinity under the standard being determined. the left: velocity Lineweaver-Burk
I5 (PM)
20
(,uM)
c
25
0
5 Substrate
IO
15
20
25
(pM)
constants of DPNH and cytochrome c. These were determined assay conditions, except for the constituent whose affinity is For each determination 0.09 mg. enzyme protein is used. On (V) as a function of substrate (8) concentration. On the right: plots.
108
GARFINKEL
as shown in Fig. 4. The value for cytochrome c (7 X 10-r M) is similar to those for the DPNH-cytochrome c reductase systems of mitochondria (15) and intact microsomes,* 1.75 X 10e6 and 8.3 X lo-6 1M, respectively, but appreciably different from that for the soluble DPNH-cytochrome c reductase of Mahler et al. (3, 16), which is 1.2 X lo-* M. DISCUSSION In a situation of this type, where there are present two enzyme systems having similar properties, it is necessary to show that we are dealing with only one of them. In this case the absence of cytochrome bs and of DPNH-cytochrome bg reductase must be demonstrated. Reduced difference spectra of the unfractionated extract (on reduction with dithionite but not with DPNH) show peaks at 424 rnp, which are displaced on addition of CO. This is behavior typical of protein hemochromogens; cytochrome bg , however, does not react with CO. On reduction with DPNH, the optical density change at 424 rnp is less than 0.0005, indicating that the cytochrome b6 content is less than 10~~ M (6). As mentioned above, an attempt was originally made to prepare DPNHcytochrome bg reductase by this method, and this was unsuccessful. The addition of cytochrome bg did not reproducibly increase the rate of cytochrome c reduction, even after a wide range of treatments, such as exposure to high ionic strength, incubation with cytochrome br, , heating, and digestion with ribonuclease and pancreatin. When the concentration of cytochrome br, here used was added to cytochrome bS reductase, cytochrome c, and DPNH, under the same conditions, reduction of cytochrome c did take place. Additional items of evidence are (a) the curve for the effect of pH on activity in Tris buffer is not identical for this enzyme and for DPNH-cytochrome b6 reductase (5) ; (b) this enzyme occurs as a contaminant in preparations of DPNH-cytochrome bg reductase, from which it can be removed completely. Therefore it may be concluded that the properties observed are not those of the cytochrome bg system. This enzyme is quite similar to the DPNH-cytochrome c reductase isolated by Mahler and co-workers (3, 16, 17) from pig heart sarcosomes. The question of whether these might be the same enzyme may be settled immediately. Not only are the sources of the enzymes different, but the enzyme here described absorbs on calcium phosphate gel and alumina gel whereas that of Mahler does not. The behavior toward metal-ion 4M. Klingenberg, in preparation.
MICROSOMAL
DPNH-CYTOCHROME
C REDUCTASE
109
inhibitors and toward phosphate and pyrophosphate differs appreciably, and the affinity constants differ quite widely (3.5 X 10e6 M as against 1.9 X 1O-5 M for DPNH, and T X lo-’ M as against 1.2 X 1O-4 M for cytochrome c). It is therefore concluded that these are not the same enzyme. Although not identical, these enzymes are similar in many ways (this would be even more strongly true if the enzyme here described is indeed a flavoprotein). For that matter, the DPNH-cytochrome b6 reductase of Strittmatter and Velick (5) is also quite similar in many properties, even though it does reduce a different cytochrome. All of these enzymes, for instance, are at least moderately stable to 10% ethanol at 40”. All reduce two molecules of acceptor, but the velocity depends on the first power only of the acceptor concentration. This suggests that they are part of a general class of enzymes. The actual function of this enzyme in the microsomes is open to question. It has been shown to reduce several substances, none of which occurs in its natural environment (cytochrome c does not occur in microsomes and its presence in appreciable concentration in the cytoplasm has not been demonstrated). The same question can of course be asked of the DPNH-cytochrome bscytochrome c reductase system of microsomes. As yet no satisfactory answer is available. ACKNOWLEDGMENTS The advice and encouragement gratefully acknowledged.
of Dr. R. W. Estabrook
and Dr. B. Chance are
SUMMARY
The preparation of a microsomal DPNH-cytochrome c reductase has been described. Its susceptibility to heat and to various inhibitors is shown, and also the effect of various buffers in the pH range 5-10. This enzyme is demonstrated to require intact sulfhydryl groups for activity, but it is doubtful if it is a flavoprotein. The Michaelis constants are 3.5 X lows M for DPNH and 7 X lo-’ M for cytochrome c. The relationships of this enzyme with other similar enzymes are discussed. REFERENCES 1. GARFINKEL, D., Biochim. et Biophys. Acta 21, 199 (1956). 2. SMITH, F. G., ROBINSON, W. B. AND STOTZ, E., J. Biol. Chem. 179, 881 (1949). 3. MAHLER, H. R., SARKAR, N. K., AND VERNON, L. P., J. Biol. Chem. 199, 585 (1952).
110
GARFINKEL
4. GARFINKEL, D., Arch. Biochem. 5. STRITTMATTER, P., AND VELICK, 6. STRITTMATTER, P., AND VELICK, 7. MORTON, R. K., in “Methods
71, 111 (1957). S. F., J. Biol. Chem. 221, 277 (1956). S. F., J. Biol. Chem. 221, 253 (1956). in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1. Academic Press, New York, 1955. 8. YANG, C. C., AND LEGALLAIS, V., Rev. Sci. Instr. 26, 801 (1954). 9. THEORELL, H., Biochem. 2.286, 207 (1936). 10. DIMANT, E., SANADI, D. R., AND HUENNEKENS, F. M., J. Am. Chem. Sot. 74, 5440 (1952). 11. BESSEY, 0. A., LOWRY, O..H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 12. COLOWICK, S. P., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1. Academic Press, New York, 1955. 13. HAAS, E., J. Biol. Chem. 166, 321 (1944). 14. HELLERMAN, L., LINDSAY, A., AND BOVARNICK, M. R., .I. Biol. Chem. 163.553 (1946). 15. ESTABROOK, R. W., J. Biol. Chem., in press. 16, VERNON, L. P., MAHLER, H. R., AND SAREAR, N. K., J. Biol. Chem. 199, 599 (1952). 17. EDELHOCH, H., HAYISHI, O., AND TEPLEY, L. J., J. Biol. Chem. 197,97 (1952). Biophys.