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Investigations have been conducted into the relationship between fatty acid and phenol
synthesis in submerged cultures of Aspergillus fumigatus. Both groups of metabolites are derived
from acetate but phenol synthesis only occurs a t a late stage in the incubation period and is de-
pendent on a change in the internal environment of the mycelium. Secretion of acetate-derived
phenols commenced many hours after exhaustion of ammonium ions from the culture medium, or
transfer of mycelium to glucose solution. Lipid synthesis, however, was maintained a t this stage
and continued into the phenol-secreting phase when sufficient glucose was present t o provide the
necessary substrates. The additional lipid formed was identified as triglyceride.
The composition of fatty acids in the total lipids was very similar to that found in other
, Aspergillus sp. and related Ascomycetes and consisted principally of C18:2 with c16:0, c16:1, C1s:o
and Cmi.
Addition of 4-fluorophenylalanine, but not cycloheximide, caused a rapid reduction in fatty
acid synthetase activity and lipid synthesis, and abolished formation of orsellinic acid and
trihydroxytoluene, the normal secreted products.
Certain enzymes examined, including the dehydrogenases of the pentose phosphate pathway,
maintained their specific activity during the “stationary” phase whereas fatty acid synthetase
was present at greatly reduced activity. The activity of ATP citrate lyase was severely reduced to
an undetectable level after approximately 40 h incubation; acetyl-CoA would therefore not be
made available for fatty acid, phenol or ergosterol synthesis after this time.
Initiation of phenol synthesis cannot be ascribed simply t o diversion of substrates from fatty
acid synthesis or to reduction in fatty acid synthetase activity.
Fatty acids together with a large group of natural- phase in bacteria. The metabolic routes concerned
ly-occurring phenols are formed by means of conden- utilize the same thioester substrates and depend
sation reactions between acetyl-CoA and malonyl- upon the presence of a synthetase complex of en-
CoA. The synthesis of the latter products, however, zymes. They presumably benefit the cell by helping
entails few if any reductive steps and the oxygen to maintain a suitable internal environment under
functions from the malonyl residues are therefore adverse conditions, e.g. by releasing coenzyme A.
often retained as phenolic groups. A phenomenon The acetate-derived phenols thus formed are general-
that is common t o the synthesis of fatty acids and ly secreted into the culture medium.
phenols in fungi lies in their formation as a response It has been established that the synthetases
t o deprivation of an essentialnutrient with a resultant responsible for the formation of fatty acid and a
disturbance to the internal environment of the cell single ring phenol, 6-methylsalicylic acid (I),(whose
[l]. Growth of the organism then ceases and the synthesis does involve one reductive step to
mycelium enters a stage that resembles the stationary remove the oxygen function a t C-4) are distinct
proteins in Penicillium (patulum [Z]. The two com-
Abbreviation. POPOP, p-bis,2-(5-phenyloxazolyl)ben- plexes, however, possess many properties in common
zene.
Trivial Name. Ergosterol, 5,7,22-ergostatrien-3B-01. and may be elaborated from similar enzyme compo-
Enzymes. Glucose 6-phosphate dehydrogenase (EC nents. The present investigation examines possible
1.1.1.40):6-phosphogluconate dehydrogenase (EC 1.1.1.44); interrelationships between fatty acid and trihydroxy-
rnalate dehydrogenase (EC 1.1.1.37); malate dehydrogenase toluene (11) biosynthesis in submerged cultures of
(decarboxylating) (NADP) (EC 1.1.1.40); isocitrate dehydro-
genase (NADP+)(EC 1.1.1.42);ATP citrate lyase (EC 4.1.3.8); Aspergillus furnigatus, with particular attention to
hexokinase (EC2.7.1.1). factors t h a t may be responsible for regulation of
fatty acid synthesis and initiation of phenol forma- approximately 10' conidialml. This gave rise to
tion after cell growth has stopped. Preliminary growth that was satisfactorily reproducible and
results describing some of this work have been homogeneous. A lag phase occurred followed by
reported [3]. swelling of the conidia and emergence of germ tubes
OH over the initial 8-10 h incubation. The cultures
then grew exponentially (but populations in various
stages of growth were probably present a t any given
time) and the rate of increase in protein and DNA
C0,H content and dry weight were very similar. They
""QCH3
6H 6H exhibited a sharp transition t o the stationary phase
(I) R = H, 6-methylsalicylic acid (11) trihydroxytoluene (Fig.1) after exhaustion of nitrogen which was
available originally as ammonium ions. This took
(111) R = OH, orsellinic acid
place around 24 h after which net protein and DNA
synthesis ceased. Formation of cell-wall material
EXPERIMENTAL PROCEDURE continued, however, and secretion of the phenols,
orsellinic acid (111) and trihydroxytoluene typically
Cultural Conditions commenced some 8 h later [3,4]. I n the [14C]ace-
A . fumigatw (I.M.I. 39353) was obtained from tate experiments, colonies o f mycelium were trans-
the Commonwealth Mycological Institute (Kew, ferred t o glucose solution (5O/,, w/v) before phenol
Surrey). Stock cultures of this organism were main. secretion. They were then distributed (50 ml)
tained a t 4 "C on modified Czapek-Dox agar (Oxoid, into conical flasks.
London) and preparations of conidia were obtained
as described previously [a]. Submerged cultures Estimation
were incubated in conical flasks of 250-ml capacity of Total Protein, DNA and Ammonium Ions
containing 50ml of Raulin-Thom medium [5] and
were silanized before use t o prevent formation of Protein was isolated from the mycelium after
surface mycelium around the flask sides [4].Mycelium grinding with acid-washed sand and extraction
was harvested by filtration and washed with 0.2 M with 1 M NaOH a t 40 "C €or 30 min. Protein samples
potassium phosphate (pH 7.0) or Tris-HC1 (pH 8.0) were precipitated from the supernatants of the
buffer as required. centrifuged homogenates with loo/, trichloroacetic
acid solution prior t o estimation by the method of
Lowry et al. [6], using bovine serum albumin as
Growth Characteristics standard. DNA was extracted from a ground suspen-
Growth was based on the cultural conditions sion of mycelium with 0.3 M NaOH a t 30 "C over-
described previously [4] using inocula containing night. The supernatant was neutralized with acetic
3.5
3 .0
-p 2.5 -
1 w
0
0
-B
Y
.-c 2.0-
0
r 1 .0
*:Jfj;: (
I
2
z
4
.-m
1.5 - 0.5 -F
5 1 .
m
D
2 1 .0 -0 e<
z
0.5 -0.5
0 , + r-1 .O
acid and treated with CdC1, [7]. DNA was determined while triglycerides were dissolved in benzene and
by the diphenylamine reaction as modified by Burton reacted for 30 min [la].
[8] except that perchloric acid was not added t o the Fatty acids were analysed by gas-liquid chro-
sample. Results gained from assay of organic phos- matography using a Varian aerograph series 1520
phate in the DNA were confirmatory. Determination instrument with a polyethylene glycol adipate
of the residual ammonium ion content of the medium column on Phasesep N I carrier (6'/0, w/w) (Phase
has been described previously [a]. Separations Ltd., Rock Ferry, Cheshire). They were
identified by their retention times relative t o stan-
dard mixtures [15], and behaviour on silver nitrate-
Isolation of Lipids and Their Assay impregnated plates.
Lipids were generally obtained from fresh myce-
lium by extraction with chloroform -methanol Prevaration of Cell-Free Extracts
(2:1, v/v) [9] containing butylated hydroxytoluene
Cell-free extracts were prepared from fresh
(50 mg/l) as antioxidant. Solvent extraction of myce-
mycelium (1 g) by grinding with acid-washed sand
lium which had been rapidly frozen and freeze-dried
(2 g) and Tris-HC1 buffer, p H 7.0 (2 ml; I = 0.05 M)
gave essentially similar results. The light petroleum-
at 4 "C. Cell-debris and sand were removed by cen-
soluble fraction of this material was assayed for trifugation at 37000 x g for 20 min and then re-
total fatty acid esters by the hydroxamate method extracted with buffer. Low molecular weight compo-
[lo]. Neutral lipids were eluted with chloroform
nents were removed by gel filtration chromatography
after chromatography on columns of silicic acid and on Sephadex G-25 using Tris-HC1 buffer. Partial
determined as free [ill or esterified fatty acids (tri- purification of fatty acid synthetase from the initial
glycerides) or ergosterol. Ergosterol was measured
buffer extract (0.2 M potassium phosphate contain-
spectrophotometrically (A:'?, 270 at A,, 282 nm). ing 3 mM K-EDTA and 1 mM dithiothreitol, p H 6.8)
Phospholipids were eluted with methanol and com- was achieved by ammonium sulphate fractionation
prised essentially of phosphatidylcholine and phos- (20-35O/,) or centrifugation a t 151000 x g (Bav
phatidylethanolamine with the latter predominant.
5.4 cm) for 2.2 h in a Beckman L2 65B preparative
They were assayed as esterified fatty acids [lo] and
ultracentrifuge using a titanium 50 rotor. Protein
sometimes additionally by phosphate determination
content of all extracts was determined as described
after digestion.
above [6].
I n the [Wlacetate incorporation experiments,
the lipid extract was saponified with 2 M KOH in
ethanol-water (1 :1, v/v) and acidified t o generate Enzyme Assays
the constituent fatty acids. These were further Total and specific activities of various enzymes
purified by chromatography on silieic acid and thin- concerned with intermediary metabolism were in-
layer chromatography on Kieselgel G . They were vestigated at different stages of incubation. The
estimated by the copper soap method [ll]. All rationale behind these studies lay in the considera-
assays were performed in triplicate using triolein, tion that enzymic or substrate limitation might be
phosphatidylcholine and oleic acid as the relevant imposed on the formation of fatty acids after growth
standards. (as determined by net protein synthesis) had ceased,
with a consequent diversion of acetyl-CoA and
malonyl-CoA into phenol synthesis. Both sequences
Gas-Liquid Chromatographic Analysis require cytoplasmic acetyl-CoA [2] but a major
of Fatty Acids variation between the pathways is their requirement
Total fatty acids were extracted from freeze-dried for NADPH. Particular attention was therefore
mycelium as their methyl esters with 1 M methanolic paid t o the enzymes responsible for generating this
HC1 in a sealed tube under nitrogen, a t 110 "C for 12 h reduced cofactor and for the provision of acetyl-CoA.
1121. The methyl esters were separated according to The nature of the mycelium from which extracts
their degree of unsaturation by thin-layer chromatog- were prepared changed markedly over the period
raphy on plates of silver nitrate-Kieselgel G - studied. At the time of phenol synthesis, the dry
water (1:20:40, by wt) [13], with light petroleum- weight increased considerably relative t o protein
ether (9 :1, v/v) as solvent. Lipid regions were located and the amount of soluble, extractable protein
by spraying with dichlorofluorescein in ethanol diminished. Moreover, the mycelium became pig-
(O.lO/o, w/v) and viewing under ultraviolet light. mented, probably due t o the presence of a melanin-
Constituent fatty acids within individual lipids were type substance, The possibility that these changes
also methylated using boron trifluoride methanol might affect the results was checked by reextracting
complex containing 140/,, boron trifluoride. Free the cell-debris and by mixing mycelium from different
fatty acids and phospholipids were dissolved in stages of growth. These procedures con&med that
methanol and reacted for 2 and 15 min respectively the physical nature of the mycelium and the presence
or absence of inhibitors or activators were not lene ( loo/,, w/v) and 2,5-diphenyloxazole (0.5O/,,
responsible for changes in activity of any of the w/v)*
enzymes tested.
Glucose-6-phosphate dehydrogenase was assayed Purification of Solvents
spectrophotometrically [16] a t p H 8.0; it was found
that Mg2+ were unnecessary for this enzyme from Light petroleum (b.p. 40-60 "C), ether and etha-
A . fumigatus. 6-Phosphogluconate dehydrogenase nol were prepared as described previously 11241.
was assayed in a similar system containing 7 mM
Mga+. Malate dehydrogenase and malate dehydro- Materials
genase (decarboxylating) were assayed by procedures Coenzymes, malonyl-CoA, glucose 6-phosphate,
involving reduction of NAD+ and NADPf respec- 6 - phosphogluconate, tris,(hydroxymethyl) amino-
tively [17,18]. Isocitrate dehydrogenase activity was methane(Tris), cycloheximide and 4-fluorophenyl-
similarly determined [19]. ATP citrate lyase was as- alanine were obtained from Sigma London Chemical
sayed by measuring the fall in absorbance a t 340 nm Co. Ltd, and bovine serum albumin, dithiothreitol
after coupling to malate dehydrogenase [20]. Hexo- and other chemicals of AnalaR standard from
kinase was determined by coupling the reaction t o B.D.H. Ltd (Poole, Dorset). Acetyl-CoA was prepar-
glucose-6-phosphate dehydrogenase [21]. The pres- ed by the method of [26]. Sephadex G-25 was obtain-
ence of 6-phosphogluconate dehydrogenase was ed from Pharmacia (Uppsala, Sweden), Kieselgel G
corrected for in the blank which did not contain from Merck (Darmstadt, Germany) and methyl
ATP. Fatty acid synthetase was assayed spectro- esters of fatty acids from Applied Science Laborato-
photometrically [22]. ries (Richmond, Surrey). Malate dehydrogenase and
Proteinase activity was tested for [23] using glucose 6-phosphate dehydrogenase were purchased
casein or protein present in the extract as substrate. from Boehringer Mannheim GmbH (Mannheim,
The increase in absorbance of the supernatant at Germany). Sodium [1-l4C]acetate was obtained from
280 nm after incubation for 1 h at 37 "C, on precipita- the Radiochemical Centre (Amersham, Bucks.).
tion with loo/, trichloroacetic acid, was less than
0.05 absorbance unit; no increase was observed at
0 "C showing that peptide hydrolytic activity was RESULTS
absent. Relationship between Total Fatty Acid,
Phenol and ETgosteTol PToduction
The results from several experiments giving the
Isolation of Phenols range of values obtained are shown in Fig.2. Nitro-
Trihydroxytoluene and orsellinic acid were isolat- gen exhaustion in the medium became apparent at
ed from the medium immediately after harvesting 23.5-25 h (see also Fig. 1) and this correlated with
by extraction with ether and resolved by chromatog- the cessation of exponential growth and net protein
raphy on silicic-acid-Celite [24,25]. They were synthesis. Lipid synthesis, as measured by the in-
estimated spectrophotometrically and purified by crease is esterified fatty acids, continued into the
crystallization [4]. stationary phase at a slower rate but this decreased
sharply a t the onset of trihydroxytoluene secretion
which occurred several hours later.> Ergosterol
~
mentation with [14C]acetate or inhibitors. I n the feature which may be correlated with the onset of
first experiments, total fatty acid and ergosterol phenol secretion. Production of lipids including
content increased throughout the test period (Table 3). ergosterol was particularly high in this experiment
Incorporation of [l4C]acetate was maximal in the and increased during the test period (Table4).
first two groups (up t o 7 h after transfer) with some Again, orsellinic acid and trihydroxytoluene were
loss of activity in the later groups, probably due t o not detected in significant amounts in groups 1
turnover of the lipids. Phenols were not secreted in and 2. Incorporation into these phenols was determin-
assayable amounts in groups 1 and 2. Conversion ed after addition of carrier material to the ether
into phenols, as measured after addition of carrier extract of the medium and the lipid extract from the
material, was insignificant in all groups (approxi- mycelium; in both cases this value was very low.
mately 0.1 O/,). Formation of phenols in the later groups, however,
Next, [14C]acetate was added to mycelium a t was considerable with the major portion of the radio-
various time intervals after transfer (Tables 4 and 5). activity residing in orsellinic acid (Table 5).
Incorporation of radioactivity into lipids was rapid Further studies were conducted to test the effect
with maximum conversion around 5 h (group 2). of cycloheximide and 4-fluorophenylalanine on lipid
This value was considerably reduced in group3, a and phenol synthesis. These inhibitors of protein
synthesis act differently (see [4])and it would be
expected that multienzyme complexes are especially
susceptible t o incorporation of 4-fluorophenylala-
'-.
nine. Indeed, fatty acid synthetase activity was
reduced t o a low value within a few hours of supple-
mentation with this analogue but to a much lesser
60 extent with cycloheximide (Table 6 ) . These vaIues
were confirmed by direct measurement of lipids and
additionally after their subsequent conversion into
h
the constituent fatty acids. Lipid synthesis as deter-
4 40 7
Y mined by incorporation of [14C]acetate, however,
A
._
e
a,
was not reduced significantly over the value in the
2
c
control group or in that supplemented with cyclo-
20 a heximide. These results are presented in Table 6
and indicated that the rate of conversion of [14C]-
acetate into lipid (that is, as expressed over the
+I
20
' I
39 40 50
0 30-min incubation period) was more efficient in
each case in the first group, tested 5 h after transfer,
Incubation period ( h )
when the activity of fatty acid synthetase was much
Fig.3. Variation o n protein content and specific activity of higher. Formation of ergosterol was affected in the
fatty acid synthetase with incubation period. The values inhibited groups. The ether-extractable material
shown were obtained from the experimental material used
for Table 1. ( 0 )Specific activity of fatty acid synthetase; of the medium treated with 4-fluorophenylalanine
(m) total protein; (A)soluble protein (180OOxg supernatant) was colourless and secretion of acetate-derived
Table 2. Distribution of the major fatty acids in triglyceride, phospholipid and free fatty acids
Experimental details relating to the isolation of lipids are given in the text. The chromatography was conducted at 170 "C with
a gas %owof 25 ml/min. The results are expressed as rno1/100 mol total fatty acid recovered; components present above
of the total were determined with a standard deviation of & 7O/, and those between l-lOo/o, & 15*/,
~~ ~ ~ ~~~ ~ ~ ~~
Table 7. Effect of variation in incubation period o n the activities of enzymes from A. fumigatus
Enzymes were isolated and assayed in Tris-HC1or phosphate buffer as described in the text. The values shown give the mean
plus the stacdard error of the mean for the amount of nucleotide reacted for duplicate assays performed on separate extracts
from duplicate cultures (6-10). The maximum value is underlined
Incubation Total activity 10 x Total activity
period
Malate Glucose-6- 6-Phospho- Hexokinase Isocitrate ATP citrate Fatty acid Malate
dehydro- phosphate gluconate dehydro- lyase syntlietase dehydro-
genase dehydro- dehydro- genase genase
genase genase (decarboxyl-
atine)
h pmol/min ymol/min
17 2.7 f0.6 2.6 f0.3 6.1 0.4 1.1 f0.3 - - - -
20 11.1 f 0 . 8 7.1 f 0.7 - 2.8 f0.7 4.5 f0.3 2.5 f 0.3 1.5 f 0.3 0.1
21.5 22.1 f 1.3 12.9 f 1.0 12.1 f0.8 4.2 f 0.9 8.7 f 0.5 6.7 f 0.4 4.2 f 0.4 0.4 & 0.2
24 35.0 & 2.3 26.2 f 1.5 13.0 & 0.9
_.
5.2 f 1.1 16.1 f 0.6
- 7.6 f 0.5 -f0.3
4.6 0.7 -& 0.4
28 45.8 2.9 27.1 f2.2 12.2 f 1.1 -
5.3 f 0.8 14.2 f 0.8 f0.5 1.4 0.4 65f0.2
37 G f 2 . 5 Ef1.5 6.2 f 0.4 2.7 i0.7 3.2 f 0.5
-
0.8 f 0.2
-
0.3 f 0.2
-
0
-
39 21.5 & 1.1 12.6 f 1.7 - 2.1 f 0.6
42 18.5 f 1.2 13.2 f 1.1 4.8 f0.8 1.4 & 0.5 - 0.4 & 0.2 0.3 -
48 16.4f 1.3 11.7 f 1.3 - 1.3 3 0.5 1.6 f 0 . 2 0 - -
66 18.5 f 1.0 11.4 f 1.2 4.9 f 0.8 1.4 f 0.7 0.8 f 0.2 0 0.2 0
phenols, with their quinone oxidation products, results indicated that the enzymes examined might
was completely prevented by the addition of this be divided into two groups: those which retained a
inhibitor. Addition of cycloheximide abolished the relatively constant specific activity throughout the
synthesis of the modified phenol, tetrahydroxytolu- incubation period including the dehydrogenases of
ene but permitted production of orsellinic acid and the pentose phosphate pathway and malate dehydro-
trihydroxytoluene. Over one-third of the 4-fluoro- genase, and others whose specific activity declined
phenylalanine was recovered as its transaminated during the stationary phase (with a more rapid fall
product, 4-fluorophenylpyruvic acid. in total activity), including ATP citrate lyase,
malate dehydrogenase (decarboxylating) (which
possessed a very low initial activity) and fatty acid
Activities of Enzymes Concerned with Fatty Acid synthetase (Table 8). The behaviour of ATP citrate
and Phenol Synthesis lyase was noteworthy since its activity dropped
The total activity ofthe enzymes studied (Table 7) rapidly a t around 4 0 h incubation. This enzyme is
and the amount of protein extracted from the myce- concerned with the production of cytoplasmic
lium decreased during the stationary phase. The acetyl-CoA [29,30].
Table 8. Effect of variation in incubation period on the specific activities of enzymes from A. fumigatus
The values shown are based on the mean values of the enzyme activities given in Table 7
ammonium ions from normal culture medium. Its primary factor responsible for the expression of
production cannot therefore arise as a n immediate phenol synthesis in A . fumigatus is the sudden
response to lack of nutrients and must therefore availability of aromatic synthetase activity rather
depend on metabolic events that ensue later as than switching off of fatty acid synthesis. No
secondary phenomena. Possibly these affect the fatty information is available as t o the source of the
acid synthetase or elaboration of the enzymic compo- aromatic synthetase but it may possibly be derived
nents that constitute it with the resultant formation from the fatty acid synthetase complex by disaggre-
of a modified complex [l]. Data from the inhibition gation under suitable metabolic conditions [l].
studies (Table 6) confirmed a possible relationship 6-Methylsalicylate synthetase isolated from P.patu-
between the synthetase complexes. Addition of lum possesses a molecular weight (1.3 x los) which is
cycloheximide resulted, as expected, in some reduc- exactly half the value given by fatty acid synthetase
tion of fatty acid synthetase activity since there (2.6 x lo6)in this organism [40].
was no replacement of the individual enzymes from
A. C. W. is grateful to the Science Research Council
endogenous sources of nitrogen. Phenol synthesis for a Research Studentship. N. M. P. thanks Mm P. Hick
remained expressed but a t a lower rate. On the for skilful technical assistance during part of this work.
other hand, addition of the analogue, 4-fluorophenyl-
alanine caused severe inhibition of this activity and
aromatic synthesis, presumably after incorporation REFERENCES
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