Download Effect of Zinc on Tricarboxylic Acid Cycle Intermediates and

Journal of General Microbiology (I 977), gg, 43-48
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43
Effect of Zinc on Tricarboxylic Acid Cycle Intermediates and
Enzymes in Relation to Matoxin Biosynthesis
By S . K. GUPTA, K. K. M A G G O N AND T. A. V E N K I T A S U B R A M A N I A N
Depdrtment of Biochemistry, Vdllabhbhai Patel Chest Institute, University of
Delhi, Delhi I 10007, India
(Received 7 September I 976 ; revised I 8 October 1976)
SUMMARY
The stimulatory action of zinc on aflatoxin production by Aspergilluspdrdsiticus
investigated by studying the levels of tricarboxylic acid (TCA)
cycle intermediates and related enzymes in the fungal mycelium. During the stationary phase of growth, the levels of a-keto acids declined in zinc-sufficient cultures
compared with those in zinc-deficient cultures. TCA cycle enzymes did not show
any significant changes due to zinc availability. In zinc-deficient cultures, enzymes
of the TCA cycle had maximum activity on the fourth day, after which their activity
declined. In zinc-sufficient cultures, some enzymes showed maximum activity on the
fourth day, others on the second day.
N R R L ~ ~has
~ Obeen
INTRODUCTION
Several reports have described the stimulatory action of zinc on aflatoxin biosynthesis
in Aspergillus PdrdSitiCW and A . $dVW (Mateles & Adye, 1965; Lee, Townsley & Walden,
1966; Gupta & Venkitasubramanian, 1975;Maggon, Gopal & Venkitasubramanian, 1g73),
but the mechanism of this stimulatory action is not understood at present. The effect of zinc
on the metabolism of A. pdrdsiticus has not been reported. It has been shown that soybean is
a poor substrate for aflatoxin production because of its low content of zinc which.is bound
to phytic acid (Gupta & Venkitasubramanian, 1975).
Acetyl-CoA is a precursor for aflatoxin biosynthesis and also enters the tricarboxylic
acid (TCA) cycle by condensing with oxaloacetate to form citrate. The study of TCA cycle
intermediates and enzymes during zinc deficiency may reveal the mechanism of action of
zinc in aflatoxin biosynthesis. We have therefore investigated the levels of TCA cycle
enzymes and intermediates of A . parasiticus grown in zinc-deficient medium.
METHODS
Organism. Aspergilluspdrusiticus N R R L ~ ~ (toxigenic)
~ O
obtained from Northern Regional
Research Laboratory, Peoria, Illinois, U.S.A., was used throughout and was maintained as
soil cultures (Hesseltine, Bradle & Benjamin, I 960).
Medium dnd growth conditions. Sucrose/low salt (SLS) synthetic medium contained
(per litre double-distilled water) :sucrose, 85 g; asparagine, 10 g; (NH,),SO,, 3.5 g; KH2P04,
10 g; MgSO,. 7H20,2 g; CaC1,.2H20,75 mg; ZnSO,. 7H20,10mg; MnCl,, 5 mg; ammonium
molybdate, 2 mg; Na2B40,, 2 mg; and FeS0,.7H20, 2 mg. The medium was adjusted to
pH 4.5 with HCl.
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S. K. GUPTA AND OTHERS
44
A spore suspension in sterile double-distilled water was prepared from 5- to 6-day-old
cultures of A . parasiticus grown on medium containing 2 :h (w/v) glucose, 2 yo(w/v) agar and
I % (v/v) peptone. Flasks containing 100 ml SLS medium were inoculated with 3 x 106 to
3-4 x 1oSspores, and incubated without shaking at 26 5 I "C. After various periods of growth,
mycelium was harvested either for determination of dry weight and aflatoxin content, or for
the estimation of TCA cycle intermediates and enzymes. All operations were done below 4 "C.
Extraction and estimation of qfiatoxins. Aflatoxins were extracted from the medium and
mycelium with chloroform and separated by t.1.c. using the solvent system toluene/isoamyl
alcohol/methanol (go :32 :3, by vol.) (Reddy, Viswanathan & Venkitasubramanian, 1970).
Individual bands were eluted with methanol and estimated spectrophotometrically using
the extinction coefficients reported by Nabney & Nesbitt (1965).
Extraction and estimation of TCA cycle intermediates. Intermediates of the TCA cycle
were extracted from mycelium as described by Williamson & Corkey (1973). Mycelium was
quickly separated from the' medium by decanting and immediately frozen by pressing
between plates of aluminium foil precooled in liquid nitrogen. Frozen mycelium was stored
at -20 "C until used. The mycelium (500 mg) was ground with acid-washed sea sand in a
precooled mortar (4 "C) containing 5 ml 8 % (w/v) perchloric acid in 40% (v/v) ethanol.
The homogenate was centrifuged a t 25 ooo g for I o min, and the supernatant was decanted
and stored at o to 4 "C. The residue was again extracted with 2 to 5 ml 6 % perchloric acid
and centrifuged, and the supernatant was combined with the first extract. The combined
extracts were brought to pH 6.0to 7.0 by slowly adding a solution of 3 M-K,CO, containing
0.5 M-triethanolamine or 0.5 M-KOH, and then centrifuged to remove precipitated KC10,.
a-Keto acids, citric acid and malic acid were estimated in this supernatant. a-Keto acids,
acetaldehyde, pyruvic acid, a-ketoglutaric acid and oxaloacetate, were estimated by preparing their phenylhydrazine derivatives and separating them by t.1.c. using the modified
method of Friedemann & Haugen (I 943) : yellow phenylhydrazones were scraped from the
plates and estimated quantitatively by the method of El Hawary & Thompson (1953).
Citric acid was estimated by the method of Ettinger, Goldbaum & Smith (1952) and malic
acid by the fluorometric method of Hummel (1949).
Extraction and assay of TCA cycle enzymes. Mycelium was decanted from the medium
after various periods of growth and washed with cold 0.05 M-Tris/HCl buffer, pH 7.3.
Washed mycelium was ground with acid-washed sea sand in the same buffer in a precooled
mortar and centrifuged at 15000 g for 10min. The supernatant was used as the source of
enzymes. Malate dehydrogenase (EC. I . I . I .37) was assayed by the procedure of Yoshida
(1973); malatedehydrogenase (decarboxylating; EC. I . I .I .40) and isocitrate dehydrogenase
(NADP+,EC. I . I .I .42) accordingtoOchoa( I 955) ;succinate dehydrogenase (EC. I . 3 .99 . 1 )
by the method of Kalra, Krishna Murti & Brodie (1971); pyruvate dehydrogenase (EC.
I . 2 . 4 . I .) and a-ketoglutarate dehydrogenase (EC. I . 2 . 4 . 2 ) as described by Stumpf,
Zarudnaya & Green (1947).
The activities reported are the means of six separate experiments.
RESULTS AND DISCUSSION
Preliminary observations indicated there was no fungal growth or aflatoxin production
in the absence of zinc. To investigate the role of zinc in the metabolism of A . pdrdsiticus and
the mechanism of its stimulatory action on aflatoxin production, two concentrations of
zinc were used: 10 mg 1-1 which was optimum for aflatoxin production, and 0.25 mg 1-1
which was the minimum required for fungal growth. The growth of A . pdrdSitiCUS and
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45
TCA cycle in a$atoxin biosynthesis
Table I.Eflect of zinc on growth and @atoxin synthesis by A . PardSitiCUS
The results for mycelial dry wt and total aflatoxin are expressed as g (100 ml medium)-l and mg
(IOO ml medium)-' respectively.
Zinc-deficient SLS medium
SLS medium
h
I
f
7
Days of
growth
Mycelial dry wt Total aflatoxin Mycelial dry wt Total aflatoxin
I -07
I '04
2-04
2
0.68
5-70
3'24
15-61
4
2.66
6.97
4-06
21-59 .
6
3'36
6-05
4'74
27'94
8
4'45
h
\
Table 2. Eflect of zinc on the content of TCA cycle intermediates in A . parasiticus
All results are expressed as pmol (g dry wt mycelium)-'.
Days of
growth
Acetaldehyde
Zinc-deficient SLS medium
2
3'63
64'59
4
6
31-60
8
17-19
SLS meQium
2
4
6
8
57'67
29'55
24'05
19-55
Pyruvic acid
3'76
5'38
2-57
1-81
4'17
3-05
2-51
1-61
a-KetoOxaloacetate glutaric acid
2.69
4'34
2.86
I -26
I '92
3-41
2-39
1-19
Citric acid
Malic acid
0.65
0.57
0.17
0.42
0.39
0.96
5-89
3'55
6.39
6.3I
1-01
0.05
0.82
0'12
6-02
3'36
4'67
0.68
1'22
0.87
0.53
0.25
0.62
3-12
aflatoxin production were both inhibited by zinc deficiency, but aflatoxin synthesis more
so than growth (Table I). This agrees with the reports of previous workers (Lee et al., 1966;
Maggon et d.,1973).
The effect of zinc on pyruvic acid and TCA cycle intermediates is shown in Table 2. In
zinc-deficient medium, the levels of a-keto acids reached a maximum on the fourth day of
growth. In zinc-sufficient medium, the levels of acetaldehyde, pyruvate and a-ketoglutarate
were maximal on day 2, during the late-exponential phase of growth (Detroy & Hesseltine,
1g70),and oxaloacetate was maximal on day 4. Thus, the zinc-sufficient cultures had higher
levels of these intermediates during growth and lower levels in the stationary phase than did
the zinc-deficient cultures. The only exceptions were oxaloacetate and citrate which showed
lower levels in zinc-sufficient medium. In the stationary phase, A. pdrdsiticus had lower
levels of a-keto acids in zinc-sufficient medium.
The build up of TCA cycle intermediates in zinc-sufficient cultures at the end of the
exponential growth phase may play a role in triggering the onset of aflatoxin synthesis.
Bu'Lock (I 9-75), Weinberg (I 971)and Demain (I 972)have postulated that the accumulation
of primary precursors at the end of exponential growth is necessary for the initiation of
secondary metabolism. The results in Table 2 seem to provide evidence for this. The
decrease in TCA cycle intermediates in zinc-sufficient cultures during the stationary phase
(days 4 to 8) indicates the diversion and possible utilization of these intermediates into
secondary biosynthesis, particularly that of aflatoxin. During the stationary phase in zincdeficient cultures, TCA cycle intermediates accumulated and there was reduced aflatoxin
formation.
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S. K. G U P T A A N D OTHERS
Table 3. Eflect of zinc on the speciJic activities of TCA cycle enzymes of A. parasiticus
Specific activities [units (mg protein)-']
A
f
Days of
growth
Pyruvate
dehydrogenase
Isocitrate
dehydrogenase
a-Ketoglutarate
dehydrogenase
Succinate
dehydrogenase
Malate
dehydrogenase
\
Malate dehydrogenase (decarboxylating)
Zinc-deficient SLS medium
2
4
6
8
'
SLS medium
2
4
6
8
I -80
7'14
2.3 I
I -70
4'74
I *g8
3-04
I -96
3'25
I -65
2-48
3'56
2-80
2-97
I '00
2.00
3-14
1-55
2.14
3.20
3-70
1.38
I -03
1 '55
2-32
3'97
3-80
5.00
1.52
2-57
I -05
1.38
63-30
100~00
66-60
2 I -30
242'50
67'44
57'94
47'04
9-60
7.10
5'70
5'1 5
8.60
5-60
6-00
I 0.50
In zinc-deficient cultures these precursors are not used to form secondary metabolites at
the same rate as in zinc-sufficient cultures. There have been many reports of the accumulation
of citric and malic acids during zinc deficiency (Foster, 1949; Trumpy & Mills, 1963; Shu &
Johnson, 1948; Martin, 1955; Sanchez-Marroquin, Carreno & Ledezma, 1970; Lockwood,
1975). Foster (1949) suggested that zinc plays a part in the utilization of carbohydrate by
fungi because, under conditions of partial zinc deficiency, less carbohydrate is completely
oxidized and a greater proportion is diverted to form organic acids. Foster & Waksman
(1939) concluded that in RJzizopus nigricans zinc has a specific role in carbohydrate metabolism : they reported accumulation of fumaric acid during zinc deficiency. Romano,
Bright & Scott (1967) observed that addition of zinc increased cell synthesis with a more
efficient utilization of glucose and a corresponding decrease in fumaric acid yields. Chester &
Rolinson (195I) reported that zinc-deficient cultures of A . niger produced large quantities of
citric acid. The results of this study are supported by the findings of Bryzgalova & Orlova
( I975) who concluded that conditions which favour the synthesis of secondary metabolites
decrease the accumulation of organic acids and vice versa.
The activities of the enzymes of the TCA cycle of A . parasiticus in zinc-deficient media
reached a maximum on the fourth day of growth but declined as the cultures aged (Table 3).
In zinc-sufficient medium the activities of pyruvate dehydrogenase, isocitrate dehydrogenase
and a-ketoglutarate dehydrogenase reached a maximum on the fourth day of growth;
those of succinate dehydrogenase, malate dehydrogenase and malate dehydrogenase (decarboxylating) remained low throughout. The activities of other enzymes were high in zincsufficient cultures during the exponential growth phase, but variable during the stationary
phase. Bu'Lock et ul. (1965) observed a marked decrease in the activity of the TCA cycle at
the end of the exponential growth phase in Penicillium urticae. A decline in TCA cycle
enzyme activity can lead to accumulation of acetyl-CoA, glycolytic intermediates and citrate
(Bu'Lock, 1965, 1975; Demain, 1968). Similar results have been obtained in this study
(Table 2). The activation of acetyl-CoA carboxylase by citrate and a-glycerophosphate
would result in the formation of more malonyl-CoA (Resmussen & Klein, 1960). Increased
formation of malonyl-CoA would lead to enhanced aflatoxin formation as observed in zincsufficient cultures.
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TCA cycle in aflatoxin biosynthesis
47
Part of this work was supported by a PL-480 grant no. FG-111-438.S. K. G. is grateful to
Dr D. N. Gupta, University College of Medical Science, New Delhi, for his keen interest
and constant encouragement. K. K. M. is grateful to the Council of Scientific and Industrial
Research, New Delhi, for placement in the Scientists Pool.
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