ENZYMES IN CANDIDA ALBICANS' I. PATHWAYS OF GLUCOSE DISSIMILATION
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ENZYMES IN CANDIDA ALBICANS'
I. PATHWAYS OF GLUCOSE DISSIMILATION
G. RAMANANDA RAO, T. RAMAKRISHNAN, AND M. SIRSI
Pharmacology Laboratory, Indian Institute of Science, Bangalore
Received for publication April 6, 1960
Among the several yeastlike fungi of the genus of the fact that diabetic patients, in whose sys-
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Candida, only Candida albicans is potentially tems the carbohydrate metabolism has been
pathogenic to laboratory animals and to humans. altered, are more prone than normal persons to
The study of the metabolism of this organism infection by C. albicans, it was decided to in-
has attained importance in view of the observa- vestigate the pathways of glucose dissimilation
tion that treatment with broad spectrum anti- in this organism. This paper presents the results
biotics would destroy even the normal nonpatho- of these investigations employing both whole
genic bacterial flora of the body and bring in its cells and cell-free extracts of C. albicans.
train the adverse effects of this altered ecological MATERIALS AND METHODS
relationship of the microorganisms in the host,
chief among which is the overgrowth of C. Test organism. C. albicans strain Z248, ob-
albicans. tained from the London School of Hygiene and
A few studies have been carried out, mainly Tropical Medicine, has been used. The patho-
with whole cells, to investigate the detailed genicity of this organism was established by in-
metabolism of this fungus. Van Niel and Cohen fecting mice and noting the mortality and typical
(1942) showed that C. albicans fermented sucrose lesions in the kidneys. Stock culture was main-
more slowly than glucose in anoxia. Thjotta and tained on slants of Sabouraud's glucose agar at
Thorheim (1955) dealt with the fermentation of room temperature.
various carbohydrates such as lactose, maltose, Medium employed. The medium used for grow-
glucose, sucrose, and galactose by various ing C. albicans was essentially that of Bradley
strains of different species of Candida. (1958) with the following composition: glucose,
Ramachandran and Walker (1957) showed the 20 g; yeast extract, 2.0 g; potassium nitrate,
growth of Candida species in a simple medium 2.0 g; K2HPO4, 2.0 g; MgSO4 7H20, 0.50 g;
containing glucose as carbon source. A detailed calcium carbonate, 0.25 g; deionized water to
study of the respiratory metabolism of normal 1.0 liter. The medium was adjusted to pH 6.5
and divisionless strains of C. albicans was con- before sterilization.
ducted by Ward and Nickerson (1958). Respira- Preparation of cells for manometric studies. The
tion studies with Candida stellatoidea with various organism was grown in the above medium by
substrates, which included sugars, amino acids, incubating at 37 C for 20 hr. Cells were harvested
fatty acids, and intermediates of the citric acid by centrifugation and washed three times with
cycle, were conducted by Bradley (1958). In view ice cold 0.03 M phosphate buffer, pH 7. Finally
the washed cells were suspended in the same
1 This work was supported in part by a grant buffer and used in Warburg experiments.
from Messrs. Sarabhai Chemicals, Baroda. Preparation of cell-free extracts. The harvested
Abbreviations employed: G-6-P, glucose 6-phos- cells were pressed between filter paper folds and
phate (B. D. H.); F-6-P, fructose 6-phosphate; mixed with twice their weight of Alumina 301
F-1,6-P, fructose 1,6-diphosphate; R-5-P, ribose
5-phosphate; ATP, adenosine triphosphate (Nu- (325) and ground in a precooled mortar for 10
tritional Biochemical Corporation); 6-PG, 6 to 15 min. The enzvmes were extracted with
phosphogluconate (Schwartz); TPN, triphos- 0.03 M phosphate buffer, pH 7.0, and centrifuged
phopyridine nucleotide (Sigma Chemical Com- at 13,780 X g for 30 min in a Servall centrifuge.
pany); DPN, diphosphopyridine nucleotide The cell-free supernatant, dialyzed against
(Pabst Laboratories); G-3-P, glyceraldehyde 0.003 M phosphate buffer, pH 7, at 2 to 5 C for
3-phosphate. 10 hr, was used in the present study.
6541960] ENZYMES IN C. ALBICANS 655
TABLE 1
Utilization of intermediate of glycolytic and hexose-
monophosphace shunt pathways by
Candida albicans*
Oxygen Uptake:
at Time, in Min:
Substratet _____________
60 120 180
Glucose ..................... 18 32 40
Glucose 6-phosphate ........ 2 3 3
Fructose 6-phosphate ........ 4 6 6
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Fructose 1,6-diphosphate 1 2 2 MINUTES
Ribose 5-phosphate ......... 13 20 22 Figure 1. Hexokinase activity. Additions:
6-Phosphogluconate ......... 15 24 26 glucose (0.025 M), 0.2 ml; ATP (0.02 M), 0.7 ml;
MgSO4 * 7H20 (0.1 M), 0.2 ml; phenol red (0.1 per
*
Each flask contained 6.7 mg of whole cells cent), 0.4 ml; cell-free extract (0.32 mg protein),
(dry weight). 0.2 ml; H20 to 3.0 ml. 0 *, Enzyme omitted;
t Each flask contained 5 jumoles of the sub- X X, glucose omitted; 0(i , complete
strate. system.
t In,ul per mg whole cells, dry weight, corrected
for endogenous activity, at 30 C. di- or triphosphopyridine nucleotide in the pres-
ence of the appropriate substrate. Increase in
Protein determination. The protein content of optical density due to the reduction of DPN or
the cell-free extract was determined according to TPN was measured at 340 m,u with Beckman
the Biuret method (Gornell, Burdawill, and model DU spectrophotometer using 3-ml quartz
David, 1949). euvettes; light path, 1 cm.
Determination of enzyme activities. The photo- Measurement of oxidation. Conventional War-
metric method of Wajzer (1949) was used for burg (Umbreit et al., 1957) technique was used
the detection of hexokinase. Slein, Cori, and to measure the utilization of various substrates.
Cori's (1950) spectrophotometric determination Final volume in each flask was 3.0 ml; gas phase,
of the reaction product of the hexokinase reac- air; temperature, 30 C. The central well con-
tion, hexose 6-phosphate, was also used for test- tained 0.2 ml of 20 per cent potassium hydroxide.
ing hexokinase activity of the cell-free extract.
The resorcinol method of Roe (1934) as used by RESULTS
Slein (1955) was employed to detect the presence Studies with whole cells: glucose, 6-PG, and
of phosphoglucose isomerase. Aldolase activity R-5-P were very well utilized by the organism,
was measured by the colorimetric method of and G-6-P, F-6-P, and F-1,6-P were not utilized
Sibley and Lehninger (1949). The presence of to any significant extent (table 1).
phosphohexokinase was demonstrated by the Studies for the hexokinase activity of the cell-
method of Kuo-Huang Ling, Birne, and Lardy free extract showed a marked activity as tested
(1955). The method of DeMoss (1955) was used by both the methods (figures 1 and 2).
for testing the glucose 6-phosphate and 6-phos- The cell-free extract showed phosphoglucose
phogluconic dehydrogenase activity of the cell- isomerase activity. Under the test conditions
free extract. Pentose phosphate isomerase occur- 0.71 ,umole of F-6-P:min:mg of protein was
rence was demonstrated according to the method formed.
of Dische and Borenfreund (1951) as modified The presence of phosphofructokinase was
by Axelrod and Jang (1954). The formation of shown by the reduction of TPN in presence of
sedoheptulose, the product of transketolase ac- F-6-P (figure 3, curve A). The activity was ob-
tivity, was shown by the method of Dische, served only with TPN and not with DPN, and
Shettles, and Osnos (1949) as modified by since in this reaction TPN oxidizes glyceralde-
Axelrod et al. (1953). Lactic, glycerol, and hyde 3-phosphate formed during incubation, the
a-glycerophosphate dehydrogenases were meas- G-3-P dehydrogenase is TPN-specific. From
ured by determining the reduction of either figure 3, curves B and C, it appears that there656 RAO, RAMAKRISHNAN, AND SIRSI [VOL. 80
0.3;
S
, 0.240
E 0.240
;.
Z 0.160
0 0.160I
II I z
g 0.080
4 0.080 B
0
0~o
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0.000 000
10 20 30 40 o-ooo r
10 20 30
MINUTES MINUTES
Figure 2. Hexokinase activity. Additions: Figuire 4. (Glycerol (A, B, C) and a-glycerophos-
glucose (0.5 M), 0.2 ml; M\gSO4 7H20 (0.1 M), 0.15 phate (D) dlehydrogenases activity. Additions:
ml; ATP (0.02 M), 0.5 ml; tris(hydroxymethyl)- glycerol or a-glycerophosphate (0.5 iu), 0.5 ml;
aminomethane buffer (0.05 M), pH 7.9, 0.8 ml; phosphate buffer 0.2 M) pH 7, 1.0 ml; cell-free ex-
TPN (1.0 mg per ml), 0.2 ml; cell-free extract tract (0.27 mg protein) 0.2 ml; TPN or DPN (1.0
(0.08 mg protein) 0.5 ml. Volume adjusted to 3.0 mg per ml) 0.2 ml. Volume adjusted to 3.0 ml with
ml with water. water. A. TPN; B. DPN; C. TPN with added
0.400 _ ATP (0.02 Mi), 0.3 ml and MNgS04.7H20, (0.1 M),
0.15 ml; D. TPN.
A
0.400 -
0.320 -
0
0.320 -
E
°0.240
0.2000 F
w
a
~-5
z~0106 2
MINUTES
Figure 3. 6-Phosphofructokinase activity. Addi-
tions: fructose 6-phosphate (0.025 Mi), 0.2 ml; MINUTES
cystein.e
MgSO47H20 (0.1 M), 0.1 ml; HC (0.2 M), F'igurle 5. Glucose 6-phosphate (A) anld 6-phos-
0.2 ml; sodium arsenate (0.4 M), 0.2 mnl; ATP phogluconic (B) (lehydrogenase activity. Addi-
(0.02 Mt), 0.3 ml; tris (hydroxymethvl)amino- tions: glucose 6-phosphate or 6-phosphogluconate
methane buffer (0.05 M) pH 7.9, 0.9 ml; cell-free (0.025 M), 0.4 ml; MgSO4-7H2O (0.1 MI), 0.2 ml;
extract (0.08mg protein), 0.5 ml; TPN (1.0 mg per tris (hydroxymnethyl)aminomethane buffer (0.05
ml), 0.2 ml. Volume adjusted
2ith to 3.0 ml water. M) pH 7.9, 0.9 ml; cell-free extract. (0.08 mg pro-
Lactic dehydrogenase activity with TPN (B) and tein), 0.5 ml; TPN (1.0 mg per ml), 0.2 ml. Volulme
DPN (C) as coenzymes. Additions: sodium lactate adjusted to 3.0 ml with water.
Mg), 0.15 ml; phosphate buffer (0.2 M) pH 7,
(0.5
1.0 ml; cell-free extraet (0.27 mg protein), 0.2 ml;
TPN or DPN (1.0 mg per ml), 0.2 ml. Volume The cell-free extract had an aldolase activity
adjusted to 3.0 ml with water. of 16 units when tested by the colorimetric
method of Sibley and Lehninger (1949). One
(Ire tw o distinct enzymes wvhich function in unit is defined as that which produces an absorp-
dehydrogenation of lactatc. The one which rc- tion of 0.100:minmg of protein under the assay
quirCs TPN is highly active compared to the conditions.
othertwhich needs DPen. Glycerol anda-glycero(Bhosphat dehydroge-1960] ENZYMES IN C. ALBICANS 657
observed at 415 and 520 m,u correspond to hexose
phosphate and sedoheptulose respectively (figure
6), thus indicating the presence of ribose 5-phos-
phate isomerase, transketolase, and transaldo-
lase. It is clear from the graph that prolonged
incubation lowered the heptulose content which
1.000 might be due to the conversion of the latter into
z
w
hexose phosphate in presence of transaldolase.
a658 RAO, RAMAKRISHNAN, AND SIRSI [VOL. 80
the TPN by glycerol dehydrogenase. It is also DISCHE, Z., L. B. SHETTLES, AND M. OSNOS
possible that the same enzyme is able to act on 1949 New specific color reactions of hexoses
both glycerol and a-glycerophosphate and that and spectrophotometric micromethods for
the difference in the rates of metabolism of the their determination. Arch. Biochem., 22,
substrates is due to the difference in the affinlity 169-184.
GORNELL, A. G., C. S. BARDAWILL, AND M. M.
of the enzyme for these substrates. Experiments DAVID 1949 In Methods in enzymology,
are under way to isolate and purify the enzymes Vol. III, pp. 450-451. Edited by S. P. Colo-
which catalyze these reactions and to study their wick and N. 0. Kaplan. Academic Press,
kinetics. It is also proposed to obtain the actual Inc., New York.
equilibrium constants in these reactions by carry- KUO-HUANG LING., W. L. BIRNE, AND H. LARDY
ing out the reactions in the opposite direction 1955 Phosphohexokinase. In Methods in
Downloaded from http://jb.asm.org/ on March 22, 2021 by guest
using reduced TPN. The observed equilibrium enzymology, Vol. I, pp. 306-309. Edited by
for both the lactic and a-glycerophosphate de- S. P. Colowick and N. 0. Kaplan. Academic
hydrogenases toward the formation of the Press, Inc., New York.
oxidized substrates may also be due to the highly RAMACHANDRAN, K., AND T. K. WALKER 1957
Glucose metabolism in Candida species.
aerobic nature of C. albicans. Biochem. J., 65, 20-24.
SUMMARY ROE, J. H. 1934 A colorimetric method for the
Candida albicans possesses the enzymes of both determination of fructose in blood and urine.
J. Biol. Chem., 107, 15-22.
Embden-Meyerhof and hexose monophosphate SIBLEY, J. A., AND A. L. LEHNINGER 1949 De-
shunt pathways. Most of the enzymes were found termination of aldolase in animal tissues.
to be highly specific for triphosphopyridine J. Biol. Chem., 177, 859-872.
nucleotide. High activity of the glycerol and SLEIN, M. M. 1955 Phosphohexoisomerase from
lactic dehydrogenases with equilibrium favorable muscle. In Methods in enzymology, Vol. III,
to the formation of keto compounds has been pp. 299-306. Edited by S. P. Colowick and
observed. N. 0. Kaplan. Academic Press, Inc., New
York.
REFERENCES SLEIN, M. W., G. T. CORI, AND C. G. CORI 1950
AXELROD, B., R. S. BANDURSKI, C. WI. GREINER, A comparative study of hexokinase from yeast
AND R. JANG 1953 The metabolism of and animal tissues. J. Biol. Chem., 186, 763-
hexose and pentose phosphates in higher 780.
plants. J. Biol. Chem., 202, 619-634. THJOTTA, TH., AND B. J. TORHEIM 1955 StUdies
AXELROD, B., AND R. JANG 1954 Purification on the fermentation of sugars in the Candida
and properties of phosphoriboisomerase from group. Acta Pathol. Microbiol. Scand., 36,
alfalfa. J. Biol. Chem., 209, 847-855. 237-249.
BRADLEY, S. G. 1958 Effect of nystatin on UMBREIT, W. W., R. H. BURRIS, AND J. F.
Candida stellatoidea. Antibiotics & Chemo- STAUFFER 1957 Manoinetr ic techniques, 3rd.
therapy, 8, 282-286. ed., p. 158. Burgess Publishing Co., Min-
DEMoss, R. D. 1955 Glucose-6-phosphate and neapolis.
6-phosphogluconic dehydrogenases from Leu- VAN NIEL, C. B., AND A. L. COHEN 1942 The
conostoc mesenter-oides. In Methods in enzy- metabolism of Candida albicans. J. Cellular
mology, Vol. I, pp. 328-334. Edited by S. P. Comp. Physiol., 20, 95-112.
Colowick and N. 0. Kaplan. Academic WAJZER, J. 1949 Test spectrophotometrique de
Press, Inc., New York. l'hexokinase. Compt. rend., 229, 1270-1272.
DISCHE, Z., AND E. BORENFREUND 1951 A new WARD, J. M., AND W. J. NICKERSON 1958 Re-
spectrophotometric method for the detection spiratory metabolism of normal and division-
and determination of keto suigars and trioses. less strains of Candida albicans. J. Gen.
J. Biol. Chem., 192, 583-587. Physiol., 41, 703-724.You can also read
