Regulation of the Embden-Meyerhof Pathway in

[CANCER RESEARCH 32, 2793-2798,
December 1972]
Regulation of the Embden-Meyerhof Pathway in a Transplantable
Rat Thyroid Tumor
M. F. Meldolesi and V. Macchia
Centro di Endocrinologia e di Oncologia Sperimentale del C. N. R., Istituto di Patologia Generale, Università di Napoli, Naples, Italy
SUMMARY
The amount of lactate, pyruvate, and glycerol 1-phosphate
formed from each of the available intermediates of the
Embden-Meyerhof
pathway has been measured (by the
sequence method) in extracts of a transplantable thyroid
tumor and of normal rat thyroid.
The regulation of the rate-limiting step catalyzed by
phosphofructokinase
was modified in the tumor with respect
to the thyroid as evidenced by the concurrent enhancement of
phosphofructokinase
activity, by the increase in lactate
production from fructose 6-phosphate, and by the lack of the
inhibition of lactate production from glucose 6-phosphate by
adenosine triphosphate up to 6 mM. Moreover the partially
purified phosphofructokinase
of the tumor was less inhibited
by 4 mM citrate and less sensitive to the reversal of citrate
inhibition by cyclic 3',5'-adenosine monophosphate than was
the thyroid enzyme.
Therefore it seems possible that, besides the enhancement
of the phosphofructokinase
activity, a modification in the
control by allosteric effectors of such an enzyme may modify
the control of the glycolytic pathway in the thyroid tumor.
INTRODUCTION
Although several studies on the glycolytic enzyme activities
in various tumors have been reported (14), it is not yet clear
how these modifications may influence, in vivo or in vitro, the
overall rate and hence the regulation of the Embden-Meyerhof
pathway (13, 27). In normal tissues this pathway is controlled
by at least 4 rate-limiting steps catalyzed by hexokinase,
P-fructokinase,
glyceraldehyde
3-P dehydrogenase,
and
pyruvate kinase (21). Therefore it seems possible that in the
tumors the modification of the glycolytic pathway may be
related either to a different amount of 1 or more of these key
enzymes or to a variation in the control mechanism of such
enzymes by allosteric effectors.
For clarification of this possibility, it was considered
interesting to study sequentially (18, 21) the activities of the
key enzymes of the glycolytic pathway in a transplantable
thyroid tumor as compared to those of normal thyroid.
The rat thyroid tumor used throughout these studies was
one of a series of tumors developed in Fischer rats by Wollman
(25) and designated at line 1-8. This tumor resembles the
Received February 14, 1972; accepted September 13, 1972.
DECEMBER 1972
thyroid in that it elaborates a periodic acid-Schiff-positive
colloid material and is capable of trapping and organifying
iodide (5), although its growth is thyroid-stimulating hormone
independent (25) since it does not respond to the in vitro
addition of thyroid-stimulating hormone (16).
MATERIALS
AND METHODS
The auxiliary enzymes, the coenzymes, and the substrates,
including DL-glyceraldehyde 3-P diethylacetal, barium salt
(converted to the sodium salt by utilizing Dowex 50-H+), were
from Boehringer/Mannheim,
Mannheim, Germany; bovine
serum albumin and EDTA were from Sigma Chemical Co., St.
Louis, Mo.; and dithiothreitol was from Calbiochem, Los
Angeles, Calif., DEAE-cellulose (Whatman DE-52) had an
exchange capacity of 1.0 mEq/g. Male Fischer rats, weighing
approximately 200 g, were from Charles River (Laboratories,
Chicago, 111.).The thyroid tumor, 1-8, kindly supplied by Dr.
S. H. Wollman, NIH, Bethesda, Md., was transplanted s.c. in
Fischer rats and was excised 2 months after implantation. The
thyroid glands were removed from normal animals. The
thyroids and the tumors were rapidly weighed, minced, and
homogenized for 3 min in a Potter-Elvehjem homogenizer in 4
volumes of sucrose (250 mM) and Tris-HCl buffer (50 mM),
pH 7.4. The homogenate was then centrifuged at 22,000 X g
for 60 min, and the supernatant was collected and diluted with
another 2 volumes of the homogenization medium immedi
ately before the assay or centrifuged at 105,OOOX# for 60
min before dilution. All operations were performed at 4°.
Assay of Glycolysis. Lactate, pyruvate, and glycerol-1-P
formation was measured from all the available substrates of
the glycolytic pathway. Each reaction mixture contained, in a
final volume of 1.45 ml: 150 mM KC1; 7 mM MgCl2 ; 25 mM
KHCO3 buffer, pH 7.5; 40 mM potassium phosphate buffer,
pH 7.5; 1.5 mM ATP or 4.0 mM ADP; 1.5 mM NAD* (unless
otherwise stated); one of the following substrates at saturating
concentrations: 20 mM glucose, 10 mM glucose-6-P, 10 mM
fructose-6-P, 10 mM fructose 1,6-di-P, 40 mM DL-glyceraldehyde-3-P, 15 mM 3-P-glycerate, 15 mM 2-P-glycerate, 15 mM
P-enolpyruvate, 0.25 ml of the supernatant at 22,000 X g or
105,000 X g (or alternatively 0.140 or 0.040 ml with
2-P-glycerate or P-enolpyruvate as substrate, respectively).
ATP was used with glucose, glucose-6-P or fructose-6-P as
substrate; ADP was used with fructose-1,6-di-P, glyceraldehyde-3-P, 3-P-glycerate, 2-P-glycerate or P-enolpyruvate. The
acidic solutions were previously neutralized with l N KOH and
2793
M. F. Meldolesi and V. Macchia
production by the 22,000 X g supernatant of the thyroid and
of the thyroid tumor was measured in the presence of
nonlimiting amounts of cofactors and substrates. In the
presence of glucose, lactate production is linear up to 30 min
was also for 15 min. Some experiments were carried out in the of incubation. With the other substrates, lactate, pyruvate, and
absence of KHCO3 with air as the gas phase, pH 7.5, as glycerol-1-P production is linear up to at least 10 min. The
indicated in the text. The extra enzymes added to the NADH required in vitro for lactate formation from pyruvate is
incubation mixture were previously dialyzed with 50 mM generated mostly at the glyceraldehyde-3-P dehydrogenase
Tris-HCl buffer, pH 7.5; when undialyzed enzymes were used, step. For this reason the principal end product formed from
a control was run with the corresponding amount of substrates such as 3-P-glycerate, 2-P-glycerate, and P-enolpyru(NH4)2S04. The incubation was stopped by adding 0.1 ml of vate is pyruvate. Therefore, in thyroid extracts, lactate
18% (w/v) HC1O4. After centrifugation, samples of the production from the various intermediates of the Embdenprotein-free supernatant were used for the determination of Meyerhof pathway increases progressively from glucose to
Jáctate, pyruvate, and glycerol-1-P by enzymatic methods (8). glyceraldehyde-3-P, whereas it diminishes when 3-P-glycerate
The amounts of lactate, pyruvate, or glycerol-1-P present or other substrates such as 2-P-glycerate or P-enolpyruvate are
before incubation were measured and subtracted from the added to the medium (Table 1). On the other hand, pyruvate
amounts present after incubation. Lactate formation from accumulation, which is very low in the 1st part of the
pyruvate was also measured at 36°,pH 7.5 (10).
sequence, greatly increases when the in vitro generation of
NADH becomes too low, i.e., after the glyceraldehyde-3-P
Hexokinase (6), P-fructokinase (19), glyceraldehyde-3-P
dehydrogenase (2), P-glycerate kinase (4), P-glycerate mutase dehydrogenase
step (Table 1). The total amount
of
(3), enolase (1), pyruvate kinase (22), and cytochrome oxidase pyruvate + lactate formed from the various substrates of the
(24) activities were determined at 22°and expressed as nmoles glycolytic pathway shows significant increases at 5 points of
of substrate utilized by the supernatant at 22,000 X g or at the sequence, i.e., between glucose and glucose-6-P; between
105,000 X^Xmg
(wet weight)"1 X min"1. The thyroid
fructose-6-P and fructose-1,6-di-P; between glyceraldehyde-3-P
and 3-P-glycerate;between 3-P-glycerate and 2-P-glycerate, and
glands from 20 to 35 animals were used for each experiment;
all experiments were repeated at least 5 times and the results finally between 2-P-glycerate and P-enolpyruvate (Table 1).
These steps are catalyzed, respectively, by hexokinase,
from a typical experiment are presented.
P-fructokinase, glyceraldehyde-3-P dehydrogenase + P-glycer
Partial Purification of Thyroid and Tumor P-fructokinase.
The enzyme was partially purified according to the method of ate kinase, P-glycerate mutase, and enolase. The last reaction,
Layzer and Conway (11) by centrifugation at 22,000 X g and catalyzed by enolase, could be also considered rate-limiting in
by fractionation on a DEAE-cellulose column with a linear the Embden-Meyerhof pathway (12). Further evidence that
and P-fructokinase are rate-limiting steps in
gradient of Tris-phosphate buffer (pH 8) formed by mixing 50 hexokinase
thyroid
extracts
was obtained by adding 3.6 fig of yeast
ml each of 50 mM and 600 mM buffer containing 0.2 mM
hexokinase
(final
concentration,
0.34 unit/ml) to glucose or 9
EDTA and 0.2 mM ATP and 0.7 mM dithiothreitol. The
to 18 fig of rabbit muscle P-fructokinase (final concentration,
enzyme was eluted with an approximately
300 mM
0.34 to 0.7 units/ml) to fructose-6-P; this addition resulted in
Tris-phosphate buffer.
Citrate inhibition of P-fructokinase was measured at pH 7.4 the increase of the rates obtained with saturating concentra
tions of glucose or fructose-6-P to those obtained with
in a reaction mixture containing (final concentrations): 35 mM
glucose-6-P or fructose-1,6-di-P as substrate, respectively
triethanolamine
buffer, pH 7.4; 1.5 mM MgCl2; 7 mM
(NH4)2SO4;0.3 mM fructose-6-P; 1 mM ATP; 0.1 mM NADH; (Table 1).
In the thyroid tumor lactate production from glucose
aldolase (35 jug/ml); triose-P isomerase (2.0 /¿g/ml);
glycerol-1-P dehydrogenase (10 Mg/ml); bovine serum albumin, (Table 1) as well as hexokinase activity (Table 2) are both
0.01%. Rates of decrease in absorbance at 340 nm were higher than those of normal thyroid. Moreover the addition of
determined 3 to 5 min after the reaction was started by yeast hexokinase to glucose enhances lactate production to the
addition of the P-fructokinase preparation in the absence or in levels obtained from glucose-6-P.
the presence of citrate. Cyclic 3',5'-AMP (0.6 mM final
In the thyroid tumor extracts, lactate formation from
glucose-6-P and from fructose-6-P is as high as that obtained
concentration), pH 7.4, was added 4.5 min after the reaction
from fructose-1,6-di-P.
The addition
of rabbit muscle
was started, and the rate of decrease in absorbance was
P-fructokinase enhances only slightly lactate production from
determined 1.5 to 2.5 min after its addition. NADH oxidase
fructose-6-P (Table 1). It seems therefore possible, as shown
activity was measured in the absence of ATP and subtracted
by
the sequence method, that at least in vitro P-fructokinase
from P-fructokinase activity.
of thyroid tumor has lost its rate-limiting function and that
the control may be transferred to another site in the glycolytic
RESULTS AND DISCUSSION
chain, as shown in other tissues and in other experimental
conditions (18, 23). For clarification of this hypothesis a series
Thyroid tumor extracts (supernatant at 22,000 X g) contain of experiments has been done. Some of them have been
an amount of endogenous lactate slightly but significantly performed in the presence of various concentrations
of
higher (p < 0.05) than does normal thyroid (10.8+1.0
and fructose-1,6-di-P, ADP, and NAD+ (Table 3), in order to show
8.0 ±0.5 nmoles X mg (wet weight)"1, respectively). Lactate that a maximal lactate production from fructose-1,6-di-P was
the pH was finally adjusted to 7.5 after addition of cofactors
and substrate. The reaction was carried out in a Dubnoff
metabolic incubator, with N2 :C02 (95:5) as the gas phase at
35°,pH 7.5, for 5 min. With glucose as substrate incubation
2794
CANCER RESEARCH VOL. 32
Regulation ofGlycolysis
in a Thyroid Tumor
3',5'-AMP was not included in the elution mixture to avoid
obtained in our experimental conditions and that lactate
production in the tumor extracts from fructose-6-P reached
the same optimal levels obtained from fructose-1,6-di-P.
Moreover some differences between thyroid and tumor, as far
as the effects produced by ATP on lactate production from
glucose and glucose-6-P are concerned, have been shown. In
fact, when increasing the ATP concentration in the presence of
1 of these 2 substrates, a progressive inhibition of lactate
production from thyroid extracts has been observed (Chart 1).
This inhibition is probably due to the effect produced by ATP
at the step catalyzed by P-fructokinase which is sensitive to
the allosteric control by ATP. In fact the limiting effect is
partially overcome when fructose-6-P is added as substrate
(Chart 1). These data are in agreement with observations
previously reported in other tissues (17). On the contrary, in
tumor extracts, there is a progressive increase in lactate
production with increasing ATP concentrations. Such increase
is evident with ATP up to 6 mM, when glucose is used as
substrate (Chart 2). The P-fructokinase activity is also slightly
higher in the tumor than in the thyroid extracts (Table 2).
In order to investigate whether the tumor enzyme shows a
different control by allosteric effectors such as citrate and
cyclic 3',5'-AMP (9, 11, 17, 20, 26), P-fructokinase from
interference with citrate inhibition. The thyroid enzyme was
strongly inhibited by 4 mM citrate, whereas the tumor enzyme
activity was only slightly depressed under the same
experimental
conditions
(Table 4). The percentage of
inhibition by citrate is decreased slightly by increasing enzyme
concentration. However, the differences between thyroid and
tumor P-fructokinase are quite evident. The reversal of citrate
inhibition by cyclic 3',5'-AMP addition was less effective on
the tumor than on the thyroid enzyme (Table 4). Therefore, it
seems possible that the allosteric regulation of P-fructokinase
by such substances is less effective in the tumor than in the
thyroid. These modifications may influence the control of the
glycolytic pathway. In fact, like in yeast extracts, conformational changes may be an effective controlling factor in
P-fructokinase regulation more than the total amount of the
enzyme (7).
Table 2
Enzymatic activities of the glycolytic pathway in the supernatant at
22,000 X g of rat thyroid and thyroid tumor homogenates
Activity [nmoles substrate utilized
X mg (wet wt) "' X min "' ]
tumor and from thyroid extracts was partially purified (about
18-fold and 11-fold, respectively) on DEAE-cellulose column
with a linear gradient of Tris-phosphate buffer (11). The
degree of purification was low because the enzyme loses some
activity during purification, even in the presence of ATP and
dithiothreitol. Moreover the thyroid P-fructokinase was not
completely separated from thyroglobulin, which behaves as a
broad peak. The different degree of purification between
thyroid and tumor enzyme may be related to the lowest
amount of thyroglobulin present in the tumor (5). Cyclic
Rat thyroid
Thyroid tumor
0.14.55
±
HexokinaseP-fructokinaseGlyceraldehyde-3-P
0.421.201
+
dehydrogenaseP-glycerate
1.578.00
kinaseP-glycerate
3.028.80
±
mutaseEnolasePyruvate
2.011.50+
±
1.028.50+
1.53.35
kinase1.98
0.36.75
+
0.437.50±
i
2.090.00
6.546.80
±
3.914.40
+
1.026.901
+
1.5
Table 1
Lactate and pyruvate formation in the 22,000 g supernatant of rat thyroid and thyroid tumor.
Lactate and pyruvate formation [nmoles X mg (wet wt)"1 X hr"1 ]
Normal thyroid
Substrate"
Cofactors6
Lactate
NAD*ATP,
GlucoseGlucose-6-PFructose-6-PFructose-1,
NAD*ATP,
NAD*ADP,
NAD*ADP,
6-di-PGlyceraldehyde-3-P3-P-glycerate2-P-glycerateP-enolpyruvateGlucose
NAD*ADP,
NAD*ADP,
NAD*ADP,
NAD*ATP,
unit/ml)Fructose-6-P
+ hexokinase (0.34
unit/ml)Fructose-1
+ P-fructokinase (0.7
glyceraldehyde-3-Pdehydrogenase
, 6-di-P +
(6.1 units/ml)ATP,
NAD*ATP,
NAD*ADP,
NAD*6.0
2"23.5±
230.5+
350.0±
453.0+
+
520.5
421.51622.5
±
722.3i
246.5 +
4140.0±
i 70.5
Pyruvate
0.25.310.55.5
t
0.620.5
t
2.022.5
i
3.0480.01
+
15.01200.0+
22.04400.0
60.03.1
+
0.323.2
i
2.0IS.Oi
±
0.427.9
Thyroid tumor
Lactate
2115.0+
t
9123.01
10132.0+
10138.0
1538.8
±
442.0 +
645.0±
7143.0i
±9170.01
11160.01
120.7
Pyruvate
0.21.1
+
tO.21.5
0.11.7
+
0.21.5
i
0.2755.0t
i
20.01250.0+
30.04400.0
100.01.1
i
±0.21.3+
0.22.0
i 0.4
"Substrate concentration: 20 mM glucose; 10 mM glucose-6-P, fructose-6-P, frucióse-1,6-di-P2; 40 mM glyceraldehyde-3-P; 15 mM
3-P-glycerate, 2-P-glycerate and P-enolpyruvate.
Cofactor concentration: 1.5 mM NAD*; 1.5 mM ATP; 4 mM ADP. Incubation was carried out at 35°for 5 min, final pH 7.5, in the presence
of 150 mM KC1, 7 mM MgCl,, 25 mM KHCO3 buffer (pH 7.5) and 40 mM potassium phosphate buffer (pH 7.5) with 95% N, + 5% CO, as the gas
phase.
c Mean ±S.E.
DECEMBER 1972
2795
M. F. Meldolesi and V. Macchia
Table 3
Láclateproduction by thyroid tumor supernatant at 22,000 X g in the
presence of various concentrations ofcofactors and substrate
Incubation was carried out for 5 min as described in Table 1.
Láclateproduction Jnmoles X
mg (wet wt)'1 X hr'1 ]
Thyroid extracts contain an amount of endogenous
glycerol-1-P (2.1 ±0.3 nmoles X mg (wet weight)'1) quite
Concentration
(mM)0.51.01.52.03.04.08.010.0NAD*089
795±
898±
898±
999+
8101±
9103±
presence of thyroid or tumor extracts, resulted also in the
increase of the rates of pyruvate production obtained from
2-P-glycerate to those obtained with P-enolpyruvate
as
substrate (Tables 5 and 6, respectively). Moreover lactate
production from pyruvate is higher in the tumor than in the
thyroid (14.8 ±0.5 and 4.9 ±0.3 /uniólesof substrate utilized
X mg (wet weight)"1 X hr"1, respectively).
1,6-di-Pc50
±470
597±
±465
±696
892±
898±
±870
±897
898±
±9ADPb45 ±6Fructose±7
"Incubation was carried out in the presence of 10 mM fructose-1,
6-di-Pand 4 mM ADP.
6 Incubation was carried out in the presence of 10 mM fructose-1,
6-di-Pand 1.5 mMNAD*.
c Incubation was carried out in the presence of 4.0 mM ADP and 1.5
mMNAD*.
similar to that of the tumor (2.3 ±0.3 nmoles X mg (wet
weight)"').
In normal thyroid glycerol-1-P production
increases, like lactate formation, when the control points of
the pathway are overcome by the addition of the substrate
further down in the sequence (Table 5). NADH required for
glycerol-1-P formation at the step catalyzed by glycerol-1-P
dehydrogenase is probably generated mostly at the glyceraldehyde-3-P dehydrogenase step (Table 1) which, in the thyroid,
as well as in other tissues (21) and in vitro, has a rate-limiting
.?
<u
<U
cn
E
IO
4o
(O
QJ
10
3.0
O
6.0
ATP
C
3.0
6.0
9.0
ATP (mM)
Chart 1. Láclateproduction by thyroid supernatant at 22,000 X g in
Ihe presence of various concenlralions of ATP wilh Ihe following
subslrales: •¿â€”»,
20 mM glucose; •¿ »,10 mM glucose-6-P; o o, IO
mM fructose-6-P. Incubation was carried out at 35°,pH 7.5, in Ihe
presence of 150 mM KC1, 7 mM MgCl2, 40 mM potassium phosphate
buffer (pH 7.5), and 1.5 mM NAD*, wilh air as Ihe gas phase.
9.0
(mM)
Chart 2. Lactate production by thyroid tumor supernalanl at
22,000 X g in the presence of various concentralions of ATP wilh the
following subslrales: •¿â€”•,
20 mM glucose; •¿-•,
10 mM glucose-6-P;
o—o, 10 mM fruclose-6-P. Incubalion was carried oui as described in
Charl 1.
Table 4
Effects of citrate and of cyclic 3',5'-AMP on P-fructokinase activity0
fcontrol =1001
Addition
In normal thyroid extracts, pyruvate production from
3-P-glycerate is efficiently increased by addition of P-glycerate mutase (12.5 units/ml) (Table 5). The same results are
obtained in tumor extracts (Table 6), where lactate and
pyruvate production
from 3-P-glycerate and P-glycerate
mutase activity are enhanced with respect to normal thyroid
(Table 2).
The addition of enolase (10 units/ml) to 2-P-glycerate in the
2796
(4 mM)
and cyclic 3',5'-
3' ,5'-AMP
TissueRat
thyroid
Thyroid tumorCyclic
(0.6mM)102
101Citrate
(4 mM)20
AMP(0.6mM)10382
66Citrale
a P-fructokinase activity was measured at pH 7.4 as previously
described under "Materials and Melhods."
CANCER RESEARCH VOL. 32
Regulation ofGlycolysis in a Thyroid Tumor
activity in the glycolytic pathway. In fact the addition of
glyceraldehyde-3-P
dehydrogenase to fructose-1,6-di-P, en
hances the production of lactate in thyroid extracts (Table 1).
In the tumor, glycerol-1-P production from the different
substrates (Table 6) and glyceraldehyde-3-P dehydrogenase
activity (Table 2) are both higher than in the thyroid.
Moreover the addition of glyceraldehyde-3-P dehydrogenase to
fructose-1,6-di-P
does not influence remarkably lactate
production in tumor extracts (Table 1).
No significant differences have been shown when the
sequence data obtained with 22,000 X g supernatant were
compared with those obtained with 105,000 X g supernatant
(Tables 5 and 6). The same results have been obtained also as
Table 5
Lactate, pyruvate, and glycerol-I-P formation in the 22,000 X g and
105,000 X g supernatant of the same homogenate of rat thyroid
Experimental conditions are identical to those of Table 1.
far as enzymatic activities are concerned (Table 7). Supernatants at 22,000 X g and 105,OOOXg of both thyroid and
tumor do not show detectable cytochrome oxidase activity
(Table 7).
On the basis of these experimental results it seems possible
to conclude that the regulation of the glycolytic pathway
seems probably to be modified in thyroid tumor extracts
particularly at the step catalyzed by P-fructokinase. The
evidence supporting this fact is (a) the enhancement of lactate
production from fructose-6-P in the tumor; (b) the difference
in response between tumor and thyroid when extra
P-fructokinase is added to fructose-6-P as substrate; (c) the
Table 6
¡retate, pyruvate, and glycerol-1-P formation in the 22,000 X g and
105,000 X g supernatant of the same homogenate
of thyroid Tumor 1-8
Experimental conditions are identical to those of Table 1.
Lactate, pyruvate, and glycerol-1-P formation
[nmoles X mg (wet wt)"1 Xhr"1]
Lactate, pyruvate, and glycerol-1-P formation
[nmoles X mg (wet wt)~' Xhr"']
Substrate
Lactate
Pyruvate
Glycerol-1-P
Lactate
Pyruvate
Glycerol-1-P
gGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-P-gly
Supernatant at 22,000 X
gGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvate3-Pglycerate
Supernatant at 22,000 X
227.5
2102.0±
0.20.8
3188±
±0.38.0
1.539
±
9116.0±
+
9480+
0.11.5
±
348.0±
0.722.5
±
2.067
±
1169.0
0.3720.0
±
1340
±
434.0±
1.5447.0
±
4.08.1
±
768.0±
20.01280.0
±
434.0±
10.01 ±
538.0+
871.0±
±22.04500.0
23.04500.0
390.0 ±
536.0+
772.0±
75.01080.0+
±
65.01090.0
±
P-glycerate +
P-glycerate +
20.040
42-P-glycerate
+
72-P-glycerate
+
18.04250.0
±
mutase12.5
mutase(12.5
units/ml)24.0
units/ml)4.8+
8(10
+ enolase
73.0 ± ±80.0,000
5(10
+ enolase
37.0 ± 70.0105,000 ±
10.0
units/ml)GlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateS
units/mlGlucoseFructose-6-PFructose-l,6-di-P3-P-glycerate2-P-glycerateP-enolpyruvateSupernatant
10519.8
at
X g1.5
296.0±
±0.22.0
8100.0±
0.42.0
±
1065 ±
0.3685.0+25.01
±
568.0
.0±
867.0±
23.04700.0
260.0 ±
+ 95.042
±91.0±
at4.5
g1.0+0.26.2
X
125.0±
1.833.2
±
346.0 ±
0.420.0+
±
2.067.0
±
+ 3.5
1.9435.0
435.0±
335.0±
11.01460.0
±
537.0±
22.04280.0
±
+ 80.010
±41.3
4195+
i
10450
±14
Table 7
Enzymatic activities" in the 22,000 X g and 105,000 X g supernatant of rat thyroid and
of thyroid tumor homogena tes f nmoles of substrate utilized X mg
fwetwt)'1 X min'1 ]
Activity
Rat thyroid
gsupernatant1.563.8520.80
X
Thyroid tumor
gsupernatant2.655.5032.00
X
HexokinaseP-fructokinaseGlyceraldehyde-3-P
dehydrogenase
P-glycerate mutase
Cytochrome oxidase22,000
26.30
26.00
40.80
41.70
N.D.b105,OOOX£supernatant1.383.8520.30
N.D.22,000
N.D.105,OOOX£supernatant2.305.4530.80
N.D.
a Standard errors were less than 5%.
b N.D., not detectable.
DECEMBER 1972
2797
M. F. Meldolesi and V. Macchia
lack of inhibition by ATP up to 6 mM when glucose-6-P is
used as substrate in tumor extracts, and (d) the altered
response to citrate and to cyclic 3',5'-AMP of the partially
12. Lea, M. A., and Walker, D. G. Factors Affecting Hepatic Glycolysis
and Some Changes That Occur during Development. Biochem. J.,
94:655-665,1965.
purified tumor P-fructokinase with respect to the thyroid 13. Lee, I. Y., Strunk, R. C., and Coe, E. L. Coordination among
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ACKNOWLEDGMENTS
15. Lorenson, M. Y., and Mansour, T. E. Studies on Heart
The authors are indebted to Dr. S. H. Wollman and to Dr. F.
Phosphofructokinase. Binding Properties of Native Enzyme and of
Eisenberg, NIH, Bethesda, Md., for helpful editorial suggestions.
Enzyme Desensitized to AUosteric Control. J. Biol. Chem., 244:
6420-6431,1969.
16. Macchia, V., Meldolesi, M. F., and Chiariello, M. Effect of TSH and
TSH-like Substances on Some Properties of a Transplantable
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