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On the mechanism of enzyme action. L. The influence of naphthoquinone on the mechanism of fat formation in Fusarium lycopersici
On the Mechanism of Enzyme Action. L. The Influence of Naphthoquinone on the Mechanism of Fat Formation in Fusarium Lycopersici J. A. Maselli and F. F. Nord From the Department
of Organic Chemistry and Enzymology,’ New York 58, New York Received
February
Fordhum
University,
4, 1952
INTRODUCTION
Fusarium lycopersici (Flyco) is one of the members of the genus Fusarium capable of growing in a pigmented state. The empirical formula assigned to lycopersin, one of the pigments isolated from Flyco is CzoHlsOs (1). A variety of reducing agents were without effect on this compound (2). This is in contrast to the easy reducibility of solanione, a naphthoquinone pigment isolated from Fusarium solani II2 Purple (3). The influence of both of these pigments on fat formation in certain members of the Fusarium family was investigated, and it was demonstrated that the fat content of Flyco obtained pigmented on RaulinThorn medium increased with a rising glucose content of the medium, while the reverse occurred when the organism was grown unpigmented on Czapek-Dox medium (4). Fusarium Zini Bolley (FlB), a nonpigment-forming organism, gre\\ unpigmented in both media and showed a decreasing fat content in relation to increased glucose concentrations. The addition of solanione (5) and related naphthoquinones to the culture medium of FIB resulted in considerable alterat,ion (6) of its metabolism. Thus, mat weights, as well as fat coefficients, were lowered, and the amount of lipides formed 1 Communication No. 257. This work was Office of Naval Research and was aided by a of the Research Corporation. Condensed from submitted to the Graduate School, Fordham the requirements for the Ph.D. degree. 219
carried out under the aegis of the grant from the F. G. Cottrell Fund a part of the dissertation of J. A.M. University, in partial fulfilment of
220
J. A. MASELLI
AND F. F. NORD
was decreased. With the napht’hoquinones, in addition to the above facts, the extent of sterol formed was diminished, while that of the unsaturation of the fatty acids was increased. In general, similar effects n-ere noted when components of codehydrogenases, such as riboflavin and nicotinic acid (7), mere introduced into the cult’ure media of FlB. This observation indicated that the quinones might participate in t’he dehydrogenat’ion of saturated t’o unsaturated fatty acids. Furt’her relationships of the effect of naphthoquinones on FlB grown on acet)ate arc discussed in a previous paper (8). On the other hand, the addition of solanione, riboflavin, and nicotinic acid to the media of the pigment’-forming Flyco, was found to have either the same or opposit’e effects as with FIB. These influences seemed to depend on the extent of pigmentation of Flyco. In order to improve our understanding of the last observation, we have st’udied the effect of naphthoyuinone, in \wying concentrations, on fat, formation in Flyco c*ont,aitling different amounts of Iycopersin. EXPERIMENTAL
Culture
Employed
The Flyco culture used in these investigations was Strain No. R-5-6, obtained through the courtesy of Dr. S. P. Doolittle, U. S. Dept. of Agriculture, Beltsville, Md. All Flyco stocks were maintained on potato dextrose agar. The medium used in the actual experiments was a Raulin-Thorn subst)rate of the following composition: Tartaric acid.......... Magnesium carbonate. Ammonium tartrate Potassium carbonate. Ammonium sulfate Xmc sulfate Ferrous sulfate Ammonium phosphate Glucose Tap water
dibasic
Sk.0 8.0 80.0 12.0 5.0 1.4 1.4 12.0 1500.0 to 30 1.
One liter of the above solution was used as the medium for each flask. Continued subculturing on this medium resulted in a loss of pigment-producing ability (7). We utilized this finding for obtaining two Flyco cultures, which, by visual observation, were found to contain widely different concentrations of lycopersin in the m,vcelia. These two cultures were inoculated into Fernbach flasks, in which were present, in addition to 1 1. of Raulill-Thorn medium, amounts (c.P.) varying from 1 to 3 mg. of naphthoquinone
MECHANISM
OF
FAT
FORMATION
221
Growing of the Organism. The growth period was, in all cases, 21 days. The procedures for growing the mold, collecting the fully grown mats, and preparing them for subsequent studies were the same as those reported previously. The naphthoquinone was dissolved in chloroform and the solution introduced into the flasks prior to the addition of the medium.
Analytical
Procedures
Lycopersin Determinations. The concentrations of lycopersin in the mycelia of the various series of experiments were determined by a previously described method (7). A control curve with pure lycopersin was established, using a 520 rnp filter with the Coleman Nepho-Calorimeter, Residual Glucose, Fat, and Sterol Determinations. The medium was analyzed for residual glucose by the Munson-Walker gravimetric procedure (9). The total fat and sterol determinations were carried out, as reported earlier (lo), with modifications. In the total fat analyses, chloroform was used as the extracting solvent, and since lycopersin is chloroform-soluble, corrections were made for this factor. These corrections amounted to a subtraction of the number of milligrams of lycopersin found in the sample of mycelium from the weight of chloroform-extractable material finally obtained from it. Duplicate determinations employing this procedure showed excellent agreement. The fat, subjected to spectrophotometric analyses and determinations of iodine values and sterol contents, was extracted from the mycelium with petroleum ether (b.p. 30-60°C.). The oils were obt)ained by drying the extracts with anhydrous sodium sulfate, filtering, and evaporating off the solvent in vaczLo in a stream of nitrogen. The fats were stored in an atmosphere of nitrogen at -15°C. to prevent possible changes in composition. The petroleum ether extracts were found to contain small amounts of pigment resulting from the slight solubility of lycopersin in this solvent. Consequently, prior to the sterol and other determinations involving light absorption, these t,races of pigment were removed to prevent interference. This was accomplished by shaking anhydrous et.hyl ether solutions of the fats with Florisil three times. This procedure was found to have no effect, on the fat or sterol because of the following considerations: (a) The iodine value of the fat before pigment removal was 72.0. (b) After shaking with Florisil three times, the iodine value of the fat (after filtrnt,ion and evaporation of the solvent in vac~o in a stream of nitrogen) was found to be 71.5, and the sterol content amounted to 1.4ye of the fat. (c) After shaking this same fat sample with Florisil three times more, the amount, of sterol was shown to be 1.3Ye. The sterol present in the depigmented fat was then determined in the usual manner (10). il control curve using pure ergostcrol previously isolated from FIB was prepared using the Coleman Eepho-Colorimetcr. Fat Coe$cienf. The fat, coefficient or carbohydrate conversion factor is defined as the number of grams of total lipide oht,nined from 100 g. of glucose utilized. Iodine Values. Prior to further studies, the fat was saponified under conditions which would not cause alteration of t,he iodine values; i.e., such as might occur if conjugation of the double bonds wcrc permitted to take place. The procedure (11) employed involved the refluxing of about 2 g. of fat in 10 ml. of a solution of 3 g. of KOII in 50 g. of 95v0 ethanol for 1.5 hr. The fn.tt)y acids mere then separated
222
.I. A.
MASELLI
AND
F.
F.
NORD
as usual. A sample of fatty acids with an iodine value of 148 after being subjected to this procedure was found to have an iodine value of 147, thereby undergoing no change. The iodine values of the fatty acids were determined according to the Hanus method. Spectrophotometric Analyses. Spectrophotometric analyses of the fatty acids were carried out as hitherto (12). Control curves were drawn up using pure linoleic and linolenic acids purchased from the Hormel Foundation. RESULTS
In Table I are listed some of the data obtained in the course of these It is apparent that, the addition of the quinone had considerable influence on the formation of lycopersin by the enzymatic system present in Flyco. In culture 1, in which the lycopersin concentration was originally quite high, the effect of the naphthoquinone was to experiments.
TABLE Effect of Naphthoquinone Culture
I
on Some Aspects of Growth and Fat Formation in Flyco
Naph~hdodqeudinoinone Mat weights mg./l. medum g.
Residual g.,f!?lZLl
Lycopersin concentration mg./g. mat
stem1 in fat %
Total li ide k
1
Control 1 2 3
10.84 10.89 13.99 10.30
0.14 0.53 0.48 8.56
26.4 22.0 25.1 15.8
1.35 1.03 1.10 1.21
16.2 17.7 21.4 20.0
2
Control 1 2 3
7.16 8.78 12.66 10.17
0.07 0.34 0.95 1.90
7.7 12.0 27.1 15.3
1.15 1.03 0.64 0.72
13.2 17.3 21.6 19.1
reduce the amount of the pigment formed, especially when 1 and 3 mg. of quinone were present. In culture 2, where the amount of lycopersin available in the control is much less, the effect seems to have been an enhancement of lycopersin formation. It, is also noticeable that the greatest
amounts
of glucose residues
were found
in the media to which
had been added 3 mg. of quinone. Mat weights also vary, with maxima in both cultures at the 2-mg. level of added pigment. Data relative to two aspects of the lipides synthesized by Flyco are also included in Table I. The percentage of total lipides in all caBes in which quinone was present is higher than in the controls. In both cultures, maxima occur at the 2-mg. level of added quinone, concurrently with mat weights. The sterol contents, in general, are lowered with the addition of quinone.
MECEIANISM
OF
FAT
FORMATION
223
Figure 1 depicts the relationship between the iodine values of the fatty acids and the lycopersin contents of the mats. The amount of desaturation of the fatty acids formed was lower in all cases except one, when compared with the controls. In Fig. 2 is depicted the parallelism between lycopersin concentrations and fat coefficients. Since iodine values, per se, can serve only in the most general sense as an indication of the extent of desaturation of a mixture of fatty acids, another method was resorted to for a more exacting analysis of
Naphthoquinone
added,
FIG. 1. Parallelism between lycopersin the fatty acids of Flyco. Pigment concentrations --------- Iodine values
mg./l.
of medium
concentrations
and iodine values of
0 Culture 1 q Culture 2
the same. Thus, isomerization of the fatty acids, followed by ultraviolet spectrophotometry, was carried out (12). By means of spectrophotometric analyses, the presence of linoleic acid, characterized by a maximum absorption at 234 rnp, and linolenic acid with a maximum absorption at 268 rnp was established. No maxima were observed in the region of 320 rnp, thus indicating the absence of any tetraene acid, such as arachidonic acid. According to reported data (12), the heights of these maxima, when compared to those obtained with pure diene and t,riene fatty acids, afford a quantitative
224
J. .4. MASELLI
AND
F.
F.
NORD
measure of the amounts of these acids present in fatty acid mixtures. The remainder of the unsaturated acids, as characterized by the iodine values, may be considered as oleic acid. In Table II are recorded t)he results of these ana~lyses. It, can be seen that the amounts of oleic acid vary considerably in all the samples. Thus, in culture 1, in the fatt)y acids obtained from fat formed in the presence of quinones, the amount of olcic acid was higher than in the c*ontrol. In culture 2, the contrary effect is looted. ‘I’he linoleic and lino-
Ndphthoqulnone
added. tmg /I. of nledlum
FIG. 2. Relationship between lycopersin Flyco. Pigment concentrations --------Fat coefficients
concentrations 0 Culture q Culture
and fat coefficients
of
1 2
lenic acid contents do not vary as much. However, when the total amounts of unsaturated acids are taken into account, it can be seen that in culture 2 the control fat possesses more of these acids. 1hSCUSSION
It should be pointed out, t,hat in its natural habitat, Flyco grows with an abundance of pigment, probably approximating that of the control of culture 1. Apparently, there arc several intermediate reactions in the
formation of this pigment. It is possible t,hat in t.his sequence some of the steps are blocked by quinones and others promoted, because of the reversible reducibility of these compounds. It follows, therefore, that in the case of a high lycopersin-forming ability, as wibh culture 1 (Table I), the system is probably saturated with respect to one of the intermediates; consequently, the quinone action might be deviated toward a prevention of a later step in the synthesis of lycopersin. When, in the control cult’ure, only a slight amount of this pigment becomes noticeable, the effect of enhancement of its formation seems to t’ake precedenre upon t,he addition of naphthoquinone. Since tlhe lycopersin concentration is lower when 3 mg. of quinone is present in the medium than when 2 mg. is, this fact might be indicTABLE 13Ject oj Naphthoquinone Naph~~~;;inone Culture ng./l. medium 1 Control 1 2 3 2
Control 1 2 3
6 Values expressed
on Fornaation Oleic acid 70 20.18 31.31 27.42 31.34 39.51 26.39 23.40 27.94
as percent,ages
II sf limatwaled Linoleic acid 7% 25.84 23.97 23.38 22.95
Fatty Acids Liytnic
21.68 23.68 17.77 17.55 of fatty
acids in f&t>-
by Flyco” Unsaturqted
R 1.68 1.25 0.84 0.79
fattkac’ds 49.70 56.53 51.64 55.08
2.31 1.99 1.36 1.33
63.50 52.06 42.53 46.82
acid mixtures.
ative of an increased inhibitory effect caused’ by the higher concent,ration of the effector. This is corroborated by the amount of residual glucose present in the media at the end of the growing period. As recorded in Table I, with both cultures, the amounts of glucose left behind when 3 mg. of quinone is present are highest, in culture 1 amounting to about 17yo of the added carbohydrat’e. The mat weights in culture 1 are comparatively higher than those in culture 2, suggesting t,hat# larger amounts of lycopersin are physiologically more beneficial to the growth of the organism. A lowering of the amount, of sterol in Flyro, as brought out by the data in Table I, was also observed in determinations involving the addition of several naphthoquinones to t>he culture media of the non-
226
J. A.
MASELLI
AND
F.
F.
NORD
pigmented FlB (6). The sterol values indicate that lycopersin formation is not paralleled by sterol formation, since there is not much relation between the final concentrations of these metabolic products. A consideration of the curves in Fig. 1 points to the fact that there is a greater dependence of iodine values on lycopersin concentration than on the amount of quinone added to the media. Thus, as the quantities of pigment formed increase, the iodine values decrease. In general, a similar relationship exists between fat coefficients and pigment concentrations. Accordingly, as recorded in Fig. 2, as lycopersin concentrations vary, so do the fat coefficients. These findings suggest an interrelationship between fat formation and lycopersin synthesis. The decrease in the amounts of unsaturated fatty acids determined in Flyco grown in the presence of quinones in culture 2 (Table II) suggests a decreased activity of the fatty acid dehydrogenase system of Flyco. This is contrary to the findings obtained with the nonpigmented FlB. In this organism it was demonstrated t,hat added quinones increased the desaturation (6) of the fatty acids formed. However, if, as in other aspects of fat formation in Flyco, the concentration of lycopersin is considered, an explanation can be offered for t’hese observations, Thus, as is shown in Fig. 3, there seems to be a relationship in both cultures between the amounts of saturated acids formed and the quantities of lycopersin synthesized. The values for the saturated fatty acids were obtained by difference. By analogy, as in the case of FIR, the saturated fatty acids of Flyco may be assumed to contain mainly palmitic (10) and stearic (13) acids. The aspects of fat formation studied in these experiments with Flyco show parallelism with lycopersin concentrations. However, t)hese relationships are not exactly proportional, suggesting further interactions of the added quinone. These reversibly reducible compounds have been reported to be active in different vital metabolic processes (I-C, IFi, 16). Therefore, interpretations of the effect of quinones on the mechanism of enzyme action in living cells are, obviously, not specific. However, in the experiments reported here, these effeck may bc considered secondary, with the greatest influence of t’he quinones being on lycopersin format,ion. The presence of lycopersin, in turn seems t,o cause distinct changes in the composition of the fat, formed by t,he organism. The participat,ion of lycopersin appears to have followed two pat,hs in its influence on fat formation. Thus, the finding that, there is more saturation in the fat formed as a result of increased pigment con-
MECHANISM
227
OF FAT FORMATION
centration can be related to the fact that this pigment is not quinoid in nature and cannot be hydrogenated. It would seem, therefore, that the dehydrogenases operative in Flyco interact in part with an intermediate phase in the sequence of lycopersin formation. Since the lat’ter is nonreducible (2), further transfer of hydrogen does not take place. One of these paths might involve t,his competition with the saturated fatty acids for the action of the dehydrogenase systems (13) which are probably operative in the formation of unsaturated fats. 60
55
35
Naphthoqulnone added, mg./ 1. of medwm FIG. 3. Parallelism between lycopersin concentrations and saturated acid contents of Flyco fatty acids. _____ Pigment concentrations 0 Culture 1 --------- Saturated fatty acid contents q Culture 2
fatty
Another interpretation is that, since lycopersin is a normal part of the mycelium of Flyco, its presence affords a finer balance in the enzymatic processes of the cells. This possibility is borne out by the increase of fat coefficients with greater pigment concentrations. Therefore, it is conceivable that when lycopersin concentrations and fat coefficients are lower, the dehydrogenase systems act more extensively on the fat already formed and caonvert it. t,o a more unsaturated fat. This influence would afford the same result as the above interpretation. It is also possible that both effects might take place concurrently.
228
J. -4. YASELLI
The results of natural pigments therefore general these compounds
.4ND F. F. NORD
these experiments point to the integral function of in the enzymatic reactions of their hosts, and have int’erest, in contradiction to conceptIs which relegate to waste products. SUMMARY
FusarizLm lycopersici was continually subcultured giving rise to wide variance in the quantities of lycopersin formed by this organism. Two cultures were selected (one with about 25 mg. lycopersin/g. mycelium and the other with about + of this amount) in experiments in which different quantities of naphthoquinone were added to the media prior to inoculation. There was noted a relationship of mat n-eights, fat coefficients, iodine values of fatty acids, and composition of the fat,s formed with lycopersin concentrations. These findings point, to an interaction of lycopersin with t,he dehydrogenase systems operative in fat formation and, also, to the distinetj action of t>his natural pigment in the enzymatic reactions of its host,.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
KREITIVIAS, G., AND NoRD, 8'. F., Arch. Hiochem. 21, 457 (1949). KREITMAS, G., SEBEK, O., AXD NORI), F. F., ,,I&. Riochenr. 28, 77 (1950). WEISS, S., ARTDNom, F. F., iirch. ~iochem. 22, 288 (1949). NORD, F. F., FIORE, J. V., ASD WEISS, S., Arch. Hiochem. 17, 345 (1948). WEISS, S., FIORE, J. V., ASD NORD, F. F., dt,ch. Hiochem. 22, 314 (1949). MASELLI, J. A., AXI) NORD, F. F., Arch. Riorh,et/l. 24, 235 (1949). NORD, F. F., FIORE, J. T., KREITJIAR., (:., ANI) Wr:rss, S., .4rch. Hiochm. 23, 480 (1949). COLEMAX, It. J., AND NORD, F. F., drch. Hiochevz. Hiophys. 38, 385 (1952). ASSOCIATION OF OFFIPIAL AGRICULTURAL CHEMISTS, Official and Trntativr Methods of Analysis, 6th Ed. Washington, II. C., 1946. FIORE, J. V., Arch. Riochem. 16, 161 (1948). HILDITCH, T. F’., The Chemical Constitution of Natural Fats, 2nd ed., 1,. 46.5. Wiley & Sons, New York, 1947. BRICE, B. A., ANI) STAIR-,M. I,., J. Optical &x. .~Tx. 36, 532 (1945). ~TASELLI, J. A., ASU NORD, F. F., Arch. Biochem. Biophys. 36, 377 (1952). HOFFMANN, N.,<)STENHOF, O., .~ND LEE, W. H., Monafsh. 76, 180 (1946). KUHX, R., ASU BEIR.ERT, H., Chem. Be?. 60, 101 (1947). HELLERMAX, I,., ANI) PERKINS, M. Ti:., -1. Rio/. Chern. 107, 241 (1934).
of Organic Chemistry and Enzymology,’ New York 58, New York Received
February
Fordhum
University,
4, 1952
INTRODUCTION
Fusarium lycopersici (Flyco) is one of the members of the genus Fusarium capable of growing in a pigmented state. The empirical formula assigned to lycopersin, one of the pigments isolated from Flyco is CzoHlsOs (1). A variety of reducing agents were without effect on this compound (2). This is in contrast to the easy reducibility of solanione, a naphthoquinone pigment isolated from Fusarium solani II2 Purple (3). The influence of both of these pigments on fat formation in certain members of the Fusarium family was investigated, and it was demonstrated that the fat content of Flyco obtained pigmented on RaulinThorn medium increased with a rising glucose content of the medium, while the reverse occurred when the organism was grown unpigmented on Czapek-Dox medium (4). Fusarium Zini Bolley (FlB), a nonpigment-forming organism, gre\\ unpigmented in both media and showed a decreasing fat content in relation to increased glucose concentrations. The addition of solanione (5) and related naphthoquinones to the culture medium of FIB resulted in considerable alterat,ion (6) of its metabolism. Thus, mat weights, as well as fat coefficients, were lowered, and the amount of lipides formed 1 Communication No. 257. This work was Office of Naval Research and was aided by a of the Research Corporation. Condensed from submitted to the Graduate School, Fordham the requirements for the Ph.D. degree. 219
carried out under the aegis of the grant from the F. G. Cottrell Fund a part of the dissertation of J. A.M. University, in partial fulfilment of
220
J. A. MASELLI
AND F. F. NORD
was decreased. With the napht’hoquinones, in addition to the above facts, the extent of sterol formed was diminished, while that of the unsaturation of the fatty acids was increased. In general, similar effects n-ere noted when components of codehydrogenases, such as riboflavin and nicotinic acid (7), mere introduced into the cult’ure media of FlB. This observation indicated that the quinones might participate in t’he dehydrogenat’ion of saturated t’o unsaturated fatty acids. Furt’her relationships of the effect of naphthoquinones on FlB grown on acet)ate arc discussed in a previous paper (8). On the other hand, the addition of solanione, riboflavin, and nicotinic acid to the media of the pigment’-forming Flyco, was found to have either the same or opposit’e effects as with FIB. These influences seemed to depend on the extent of pigmentation of Flyco. In order to improve our understanding of the last observation, we have st’udied the effect of naphthoyuinone, in \wying concentrations, on fat, formation in Flyco c*ont,aitling different amounts of Iycopersin. EXPERIMENTAL
Culture
Employed
The Flyco culture used in these investigations was Strain No. R-5-6, obtained through the courtesy of Dr. S. P. Doolittle, U. S. Dept. of Agriculture, Beltsville, Md. All Flyco stocks were maintained on potato dextrose agar. The medium used in the actual experiments was a Raulin-Thorn subst)rate of the following composition: Tartaric acid.......... Magnesium carbonate. Ammonium tartrate Potassium carbonate. Ammonium sulfate Xmc sulfate Ferrous sulfate Ammonium phosphate Glucose Tap water
dibasic
Sk.0 8.0 80.0 12.0 5.0 1.4 1.4 12.0 1500.0 to 30 1.
One liter of the above solution was used as the medium for each flask. Continued subculturing on this medium resulted in a loss of pigment-producing ability (7). We utilized this finding for obtaining two Flyco cultures, which, by visual observation, were found to contain widely different concentrations of lycopersin in the m,vcelia. These two cultures were inoculated into Fernbach flasks, in which were present, in addition to 1 1. of Raulill-Thorn medium, amounts (c.P.) varying from 1 to 3 mg. of naphthoquinone
MECHANISM
OF
FAT
FORMATION
221
Growing of the Organism. The growth period was, in all cases, 21 days. The procedures for growing the mold, collecting the fully grown mats, and preparing them for subsequent studies were the same as those reported previously. The naphthoquinone was dissolved in chloroform and the solution introduced into the flasks prior to the addition of the medium.
Analytical
Procedures
Lycopersin Determinations. The concentrations of lycopersin in the mycelia of the various series of experiments were determined by a previously described method (7). A control curve with pure lycopersin was established, using a 520 rnp filter with the Coleman Nepho-Calorimeter, Residual Glucose, Fat, and Sterol Determinations. The medium was analyzed for residual glucose by the Munson-Walker gravimetric procedure (9). The total fat and sterol determinations were carried out, as reported earlier (lo), with modifications. In the total fat analyses, chloroform was used as the extracting solvent, and since lycopersin is chloroform-soluble, corrections were made for this factor. These corrections amounted to a subtraction of the number of milligrams of lycopersin found in the sample of mycelium from the weight of chloroform-extractable material finally obtained from it. Duplicate determinations employing this procedure showed excellent agreement. The fat, subjected to spectrophotometric analyses and determinations of iodine values and sterol contents, was extracted from the mycelium with petroleum ether (b.p. 30-60°C.). The oils were obt)ained by drying the extracts with anhydrous sodium sulfate, filtering, and evaporating off the solvent in vaczLo in a stream of nitrogen. The fats were stored in an atmosphere of nitrogen at -15°C. to prevent possible changes in composition. The petroleum ether extracts were found to contain small amounts of pigment resulting from the slight solubility of lycopersin in this solvent. Consequently, prior to the sterol and other determinations involving light absorption, these t,races of pigment were removed to prevent interference. This was accomplished by shaking anhydrous et.hyl ether solutions of the fats with Florisil three times. This procedure was found to have no effect, on the fat or sterol because of the following considerations: (a) The iodine value of the fat before pigment removal was 72.0. (b) After shaking with Florisil three times, the iodine value of the fat (after filtrnt,ion and evaporation of the solvent in vac~o in a stream of nitrogen) was found to be 71.5, and the sterol content amounted to 1.4ye of the fat. (c) After shaking this same fat sample with Florisil three times more, the amount, of sterol was shown to be 1.3Ye. The sterol present in the depigmented fat was then determined in the usual manner (10). il control curve using pure ergostcrol previously isolated from FIB was prepared using the Coleman Eepho-Colorimetcr. Fat Coe$cienf. The fat, coefficient or carbohydrate conversion factor is defined as the number of grams of total lipide oht,nined from 100 g. of glucose utilized. Iodine Values. Prior to further studies, the fat was saponified under conditions which would not cause alteration of t,he iodine values; i.e., such as might occur if conjugation of the double bonds wcrc permitted to take place. The procedure (11) employed involved the refluxing of about 2 g. of fat in 10 ml. of a solution of 3 g. of KOII in 50 g. of 95v0 ethanol for 1.5 hr. The fn.tt)y acids mere then separated
222
.I. A.
MASELLI
AND
F.
F.
NORD
as usual. A sample of fatty acids with an iodine value of 148 after being subjected to this procedure was found to have an iodine value of 147, thereby undergoing no change. The iodine values of the fatty acids were determined according to the Hanus method. Spectrophotometric Analyses. Spectrophotometric analyses of the fatty acids were carried out as hitherto (12). Control curves were drawn up using pure linoleic and linolenic acids purchased from the Hormel Foundation. RESULTS
In Table I are listed some of the data obtained in the course of these It is apparent that, the addition of the quinone had considerable influence on the formation of lycopersin by the enzymatic system present in Flyco. In culture 1, in which the lycopersin concentration was originally quite high, the effect of the naphthoquinone was to experiments.
TABLE Effect of Naphthoquinone Culture
I
on Some Aspects of Growth and Fat Formation in Flyco
Naph~hdodqeudinoinone Mat weights mg./l. medum g.
Residual g.,f!?lZLl
Lycopersin concentration mg./g. mat
stem1 in fat %
Total li ide k
1
Control 1 2 3
10.84 10.89 13.99 10.30
0.14 0.53 0.48 8.56
26.4 22.0 25.1 15.8
1.35 1.03 1.10 1.21
16.2 17.7 21.4 20.0
2
Control 1 2 3
7.16 8.78 12.66 10.17
0.07 0.34 0.95 1.90
7.7 12.0 27.1 15.3
1.15 1.03 0.64 0.72
13.2 17.3 21.6 19.1
reduce the amount of the pigment formed, especially when 1 and 3 mg. of quinone were present. In culture 2, where the amount of lycopersin available in the control is much less, the effect seems to have been an enhancement of lycopersin formation. It, is also noticeable that the greatest
amounts
of glucose residues
were found
in the media to which
had been added 3 mg. of quinone. Mat weights also vary, with maxima in both cultures at the 2-mg. level of added pigment. Data relative to two aspects of the lipides synthesized by Flyco are also included in Table I. The percentage of total lipides in all caBes in which quinone was present is higher than in the controls. In both cultures, maxima occur at the 2-mg. level of added quinone, concurrently with mat weights. The sterol contents, in general, are lowered with the addition of quinone.
MECEIANISM
OF
FAT
FORMATION
223
Figure 1 depicts the relationship between the iodine values of the fatty acids and the lycopersin contents of the mats. The amount of desaturation of the fatty acids formed was lower in all cases except one, when compared with the controls. In Fig. 2 is depicted the parallelism between lycopersin concentrations and fat coefficients. Since iodine values, per se, can serve only in the most general sense as an indication of the extent of desaturation of a mixture of fatty acids, another method was resorted to for a more exacting analysis of
Naphthoquinone
added,
FIG. 1. Parallelism between lycopersin the fatty acids of Flyco. Pigment concentrations --------- Iodine values
mg./l.
of medium
concentrations
and iodine values of
0 Culture 1 q Culture 2
the same. Thus, isomerization of the fatty acids, followed by ultraviolet spectrophotometry, was carried out (12). By means of spectrophotometric analyses, the presence of linoleic acid, characterized by a maximum absorption at 234 rnp, and linolenic acid with a maximum absorption at 268 rnp was established. No maxima were observed in the region of 320 rnp, thus indicating the absence of any tetraene acid, such as arachidonic acid. According to reported data (12), the heights of these maxima, when compared to those obtained with pure diene and t,riene fatty acids, afford a quantitative
224
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F.
F.
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measure of the amounts of these acids present in fatty acid mixtures. The remainder of the unsaturated acids, as characterized by the iodine values, may be considered as oleic acid. In Table II are recorded t)he results of these ana~lyses. It, can be seen that the amounts of oleic acid vary considerably in all the samples. Thus, in culture 1, in the fatt)y acids obtained from fat formed in the presence of quinones, the amount of olcic acid was higher than in the c*ontrol. In culture 2, the contrary effect is looted. ‘I’he linoleic and lino-
Ndphthoqulnone
added. tmg /I. of nledlum
FIG. 2. Relationship between lycopersin Flyco. Pigment concentrations --------Fat coefficients
concentrations 0 Culture q Culture
and fat coefficients
of
1 2
lenic acid contents do not vary as much. However, when the total amounts of unsaturated acids are taken into account, it can be seen that in culture 2 the control fat possesses more of these acids. 1hSCUSSION
It should be pointed out, t,hat in its natural habitat, Flyco grows with an abundance of pigment, probably approximating that of the control of culture 1. Apparently, there arc several intermediate reactions in the
formation of this pigment. It is possible t,hat in t.his sequence some of the steps are blocked by quinones and others promoted, because of the reversible reducibility of these compounds. It follows, therefore, that in the case of a high lycopersin-forming ability, as wibh culture 1 (Table I), the system is probably saturated with respect to one of the intermediates; consequently, the quinone action might be deviated toward a prevention of a later step in the synthesis of lycopersin. When, in the control cult’ure, only a slight amount of this pigment becomes noticeable, the effect of enhancement of its formation seems to t’ake precedenre upon t,he addition of naphthoquinone. Since tlhe lycopersin concentration is lower when 3 mg. of quinone is present in the medium than when 2 mg. is, this fact might be indicTABLE 13Ject oj Naphthoquinone Naph~~~;;inone Culture ng./l. medium 1 Control 1 2 3 2
Control 1 2 3
6 Values expressed
on Fornaation Oleic acid 70 20.18 31.31 27.42 31.34 39.51 26.39 23.40 27.94
as percent,ages
II sf limatwaled Linoleic acid 7% 25.84 23.97 23.38 22.95
Fatty Acids Liytnic
21.68 23.68 17.77 17.55 of fatty
acids in f&t>-
by Flyco” Unsaturqted
R 1.68 1.25 0.84 0.79
fattkac’ds 49.70 56.53 51.64 55.08
2.31 1.99 1.36 1.33
63.50 52.06 42.53 46.82
acid mixtures.
ative of an increased inhibitory effect caused’ by the higher concent,ration of the effector. This is corroborated by the amount of residual glucose present in the media at the end of the growing period. As recorded in Table I, with both cultures, the amounts of glucose left behind when 3 mg. of quinone is present are highest, in culture 1 amounting to about 17yo of the added carbohydrat’e. The mat weights in culture 1 are comparatively higher than those in culture 2, suggesting t,hat# larger amounts of lycopersin are physiologically more beneficial to the growth of the organism. A lowering of the amount, of sterol in Flyro, as brought out by the data in Table I, was also observed in determinations involving the addition of several naphthoquinones to t>he culture media of the non-
226
J. A.
MASELLI
AND
F.
F.
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pigmented FlB (6). The sterol values indicate that lycopersin formation is not paralleled by sterol formation, since there is not much relation between the final concentrations of these metabolic products. A consideration of the curves in Fig. 1 points to the fact that there is a greater dependence of iodine values on lycopersin concentration than on the amount of quinone added to the media. Thus, as the quantities of pigment formed increase, the iodine values decrease. In general, a similar relationship exists between fat coefficients and pigment concentrations. Accordingly, as recorded in Fig. 2, as lycopersin concentrations vary, so do the fat coefficients. These findings suggest an interrelationship between fat formation and lycopersin synthesis. The decrease in the amounts of unsaturated fatty acids determined in Flyco grown in the presence of quinones in culture 2 (Table II) suggests a decreased activity of the fatty acid dehydrogenase system of Flyco. This is contrary to the findings obtained with the nonpigmented FlB. In this organism it was demonstrated t,hat added quinones increased the desaturation (6) of the fatty acids formed. However, if, as in other aspects of fat formation in Flyco, the concentration of lycopersin is considered, an explanation can be offered for t’hese observations, Thus, as is shown in Fig. 3, there seems to be a relationship in both cultures between the amounts of saturated acids formed and the quantities of lycopersin synthesized. The values for the saturated fatty acids were obtained by difference. By analogy, as in the case of FIR, the saturated fatty acids of Flyco may be assumed to contain mainly palmitic (10) and stearic (13) acids. The aspects of fat formation studied in these experiments with Flyco show parallelism with lycopersin concentrations. However, t)hese relationships are not exactly proportional, suggesting further interactions of the added quinone. These reversibly reducible compounds have been reported to be active in different vital metabolic processes (I-C, IFi, 16). Therefore, interpretations of the effect of quinones on the mechanism of enzyme action in living cells are, obviously, not specific. However, in the experiments reported here, these effeck may bc considered secondary, with the greatest influence of t’he quinones being on lycopersin format,ion. The presence of lycopersin, in turn seems t,o cause distinct changes in the composition of the fat, formed by t,he organism. The participat,ion of lycopersin appears to have followed two pat,hs in its influence on fat formation. Thus, the finding that, there is more saturation in the fat formed as a result of increased pigment con-
MECHANISM
227
OF FAT FORMATION
centration can be related to the fact that this pigment is not quinoid in nature and cannot be hydrogenated. It would seem, therefore, that the dehydrogenases operative in Flyco interact in part with an intermediate phase in the sequence of lycopersin formation. Since the lat’ter is nonreducible (2), further transfer of hydrogen does not take place. One of these paths might involve t,his competition with the saturated fatty acids for the action of the dehydrogenase systems (13) which are probably operative in the formation of unsaturated fats. 60
55
35
Naphthoqulnone added, mg./ 1. of medwm FIG. 3. Parallelism between lycopersin concentrations and saturated acid contents of Flyco fatty acids. _____ Pigment concentrations 0 Culture 1 --------- Saturated fatty acid contents q Culture 2
fatty
Another interpretation is that, since lycopersin is a normal part of the mycelium of Flyco, its presence affords a finer balance in the enzymatic processes of the cells. This possibility is borne out by the increase of fat coefficients with greater pigment concentrations. Therefore, it is conceivable that when lycopersin concentrations and fat coefficients are lower, the dehydrogenase systems act more extensively on the fat already formed and caonvert it. t,o a more unsaturated fat. This influence would afford the same result as the above interpretation. It is also possible that both effects might take place concurrently.
228
J. -4. YASELLI
The results of natural pigments therefore general these compounds
.4ND F. F. NORD
these experiments point to the integral function of in the enzymatic reactions of their hosts, and have int’erest, in contradiction to conceptIs which relegate to waste products. SUMMARY
FusarizLm lycopersici was continually subcultured giving rise to wide variance in the quantities of lycopersin formed by this organism. Two cultures were selected (one with about 25 mg. lycopersin/g. mycelium and the other with about + of this amount) in experiments in which different quantities of naphthoquinone were added to the media prior to inoculation. There was noted a relationship of mat n-eights, fat coefficients, iodine values of fatty acids, and composition of the fat,s formed with lycopersin concentrations. These findings point, to an interaction of lycopersin with t,he dehydrogenase systems operative in fat formation and, also, to the distinetj action of t>his natural pigment in the enzymatic reactions of its host,.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
KREITIVIAS, G., AND NoRD, 8'. F., Arch. Hiochem. 21, 457 (1949). KREITMAS, G., SEBEK, O., AXD NORI), F. F., ,,I&. Riochenr. 28, 77 (1950). WEISS, S., ARTDNom, F. F., iirch. ~iochem. 22, 288 (1949). NORD, F. F., FIORE, J. V., ASD WEISS, S., Arch. Hiochem. 17, 345 (1948). WEISS, S., FIORE, J. V., ASD NORD, F. F., dt,ch. Hiochem. 22, 314 (1949). MASELLI, J. A., AXI) NORD, F. F., Arch. Riorh,et/l. 24, 235 (1949). NORD, F. F., FIORE, J. T., KREITJIAR., (:., ANI) Wr:rss, S., .4rch. Hiochm. 23, 480 (1949). COLEMAX, It. J., AND NORD, F. F., drch. Hiochevz. Hiophys. 38, 385 (1952). ASSOCIATION OF OFFIPIAL AGRICULTURAL CHEMISTS, Official and Trntativr Methods of Analysis, 6th Ed. Washington, II. C., 1946. FIORE, J. V., Arch. Riochem. 16, 161 (1948). HILDITCH, T. F’., The Chemical Constitution of Natural Fats, 2nd ed., 1,. 46.5. Wiley & Sons, New York, 1947. BRICE, B. A., ANI) STAIR-,M. I,., J. Optical &x. .~Tx. 36, 532 (1945). ~TASELLI, J. A., ASU NORD, F. F., Arch. Biochem. Biophys. 36, 377 (1952). HOFFMANN, N.,<)STENHOF, O., .~ND LEE, W. H., Monafsh. 76, 180 (1946). KUHX, R., ASU BEIR.ERT, H., Chem. Be?. 60, 101 (1947). HELLERMAX, I,., ANI) PERKINS, M. Ti:., -1. Rio/. Chern. 107, 241 (1934).