[CANCERRESEARCH
37,2673-2679,
August1977]
Forms of Cytosolic Nicotinamide Adenine. Dinucleotide-linked
Glycerol-3-phosphate Dehydrogenase in Normal and
Neoplastic Mouse Tissues1
Lana S. Rittmann,2 Suzanne M. Johnston, and Thomas P. Fondy3
Departmentof Biology, SyracuseUniversity,Syracuse,New York 13210
SUMMARY
L-Glycerol-3-phosphate:NAD
2-oxidoreductase
(EC
1.1.1.8) in unfrachionated homogenates and in preparations
partially purified by affinity chromatography was examined
for potential differences among forms from normal and
neoplastic mouse tissues. Polyacrylamide gel isoelectnic
focusing, heat inactivation, and immunoelectrophoresis
with goat anti-mouse liver enzyme as antiserum applied to
separate and mixed preparations showed that the major
form of the enzyme, regardless ofhhe initial specific activity,
was indistinguishable by these criteria in all normal tissues
examined including skeletal muscle, liver, kidney, brain,
spleen, and heart as well as in BW7756 hepatoma. Several
less cationic, heat-labile forms specific for certain tissues
were also observed. Two strongly anionic forms of the
enzyme appeared in early stages of fetal development and
accounted for the major proportion of the enzyme activity in
L1210 leukemia. One of these forms was observed in trace
amounts in adult brain, kidney, and heart tissues. Li210
leukemia glycemol-3-phosphate dehydrogenase had elechno
phonetic, immunological, and heat stability properties dif
foment from the major form of the enzyme found in all
normal tissues. Analysis of forms of the enzyme in a spec
trum of tumors (including the ascites and solid forms of
L12i0 leukemia, Sarcoma 180, Ad755 adenocarcinoma, and
H129 hepaloma; the solid humors B16 melanoma and
BW7756 hepatoma; and the ascites form of the Ehnlich
tumor as well as a long-passage suspension culture of P388
leukemia in log- and plateau-phase growth) suggested that
the anionic forms may be characteristic of rapidly dividing
cells in populations with high growth fractions.
INTRODUCTION
G3PDH4 has a key position in metabolism, linking glycoly
sis to phospholipid and triglyceride pathways (2, 12, 14, 32).
I This work was supported
by USPHS
Research
Grant CA-10250
from the
National Cancer Institute.
2 Present
address:
Yale
University
School
of
Medicine,
Department
of
Pharmacology, New Haven, Conn. 06510.
3 Recipient
of
USPHS
Career
Development
Award
CA-70332
from
the
National Cancer Institute. To whom request for reprints should be addressed,
at Department of Biology, Syracuse University, Syracuse, N. V. 13210.
varsity, 130 Collage Place, Syracuse, N. V. 13210.
4 The
abbreviation
used
is: G3PDH,
glycerol-3-phosphate:NAD@
droganase.
Received December 6, 1976; accepted May 16, 1977.
2-dahy
Paradoxically, this enzyme, which functions at a key junc
tune between carbohydrate and phospholipid metabolism,
is known to be present in low amounts or not to be measum
able in a wide variety of cancer types in rats, mice, and
humans (4, 5, 26-30). The fact that the activity of the enzyme
is depressed in cells that require membrane synthesis for
support of cell growth and division suggests either that the
activity remaining in cancer cells represents a form of the
enzyme important in regulation of phospholipid synthesis
or that a distinct route to phospholipid synthesis, independ
ent of NAD-linked G3PDH, functions in many neoplastic
tissues. Agnanoff and Hajra (1) have established the exis
tence of such a pathway as a source of lysophosphahidic
acid for phospholipid synthesis in Ehrlich ascites tumor
cells as well as in several normal tissues. Howard et a!. (11)
have extended these observations to neoplastic cells in
culture and also to a dividing noncancenous cell line.
In addition to its role in glycerol 3-phosphate formation in
vivo, forms
of cyhosolic
G3PDH may be involved
in the
oxidation of glycerol 3-phosphate formed by phosphoryla
lion of glycerol in the metabolism of triglycerides (31), in the
oxidation of NADH in conjunction with lactic dehydrogen
ase for support of anaerobic glycolysis (18, 35), and in the
glycerol 3-phosphate shuttle for transport of reducing
equivalents across the milochondnial membrane (22). In
deed, Dionisi et a!. (7) have evidence that suggests that
some tumor strains including the Ehrlich-Lettré ascites tu
mom lack the glycerol 3-phosphate shuttle and have me
placed it with the malate-aspartate shuttle. Other wofkens
(3) cite evidence for enhanced G3PDH shuttle activity in
virally transformed cells. This work is predicated on the
assumption that G3PDH activity in transformed cells is a
property of the same protein responsible for the activity in
adult rabbit muscle tissue. It is conceivable that the com
plex roles of cytosolic G3PDH and its altered behavior in
neoplasia may be due to the functioning of multiple forms of
the enzyme. It becomes important to determine whether
there exists a form of the enzyme functionally important in
dividing cells and distinct from the enzyme present in differ
enliated tissues.
Conclusions concerning the presence of multiple forms
of an enzyme in normal and neoplastic differentiation me
quine comparison of forms by several sensitive, independ
ent criteria applied to as high a proportion of the activity
present in the original tissues as is experimentally possible.
In addition, such conclusions require comparison of con
centraled, purified forms. In this paper we have examined
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2673
L. S. Rittmann
et
@l.
by a variety of techniques enzyme activity from normal and
neoplashic tissues immediately after solubilization and also
after concentration and partial purification by affinity chro
matography (15). These criteria establish that G3PDH activ
ity in L1210 leukemia consists of 2 anionic forms that differ
from the major cationic forms found in all normal tissues
examined. These L1210 leukemia enzyme forms are heat
labile and in this respect resemble a heat-labile form of the
enzyme observed in fetal tissues and in embryoid bodies in
mouse tematocarcinoma (16). However, other heat-labile
forms of the enzyme characteristic of adult differentiated
tissues (particularly brain and heart) have been observed in
this and related studies on rabbit tissues (20), demonstrat
ing that a complex pattern of multiple forms exists in normal
adult, fetal, and neoplashic tissues.
Preliminary reports of this work have been presented by
Rittmann et a!. (21). An independent parallel study (17) has
recently appeared in which heat denaturation and ion-ex
change chromatography in an analysis of G3PDH activity
from a wide variety of solid and ascites tumor cells were
applied to cultured tumor and normal cells.
MATERIALS AND METHODS
The 1-mi
system
with
a 1-cm
light
path contained 0.1 mM dihydroxyacetone phosphate and
0.1 mM NADH. The change in absorbance was monitored at
340 nm. One enzyme unit was defined as the amount of
enzyme that would catalyze the oxidation of 1 j.@molecoen
zyme pen mm (1 IU).
2674
i010) were
harvested
from
80 B6D2FI
mice
on Day 4 after
passage of 1 x 10@cells. Cells were flushed out of the
penitoneal cavity into 0.85°/o
NaCI solution containing 0.1°/o
hepanin, centrifuged at 500 x g for 5 mm, and resuspended
in 8 ml of 0.85%
Mice. DBA/2 and C57BL/6 x DBA/2 F, (hereafter called
B6D2F1)mice were obtained from Dr. William Bradnen, Bnis
tol Laboratories, Syracuse, N. Y., and C57L/J mice were
obtained from The Jackson Laboratory, Bar Harbor, Maine.
Tumor. Ll2l0leukemiawasobtained in ascitesform from
Dr. William Bradner. It was maintained by weekly passage of
1 x 106 cells i.p. into DBA/2 mice. BW7756 hepatoma was
purchased from The Jackson Laboratory and maintained by
s.c. passage of fragments of tumor into C57L/J mice. Bi6
melanoma grown in C57BL/6 mice was also obtained from
Dr. William Bradner. Sarcoma 180 was maintained in CD-i
mice, Ad755 adenocarcinoma was maintained in AKR x
DBA/2 F, (hereafter called AKD2F,) mice, and Hi29 hepa
toma was maintained in C3H mice. All of these tumors and
the P388 cell line were obtained from Dr. Alan Sartomelli,
New Haven, Conn. Ehrlich ascites tumor grown in HAICR
mice was a hyperdiploid Lettné
line with modal chromosome
numbers of 45 to 47 obtained from Dr. T. Hauschka, Roswell
Park Memorial Institute, Buffalo, N. Y.
Chemicals. NADH sodium salt (chromatographically pure
grade) was purchased from P-L Biochemicals, Inc. , Milwau
kee, Wis. , and dihydmoxyacetone phosphate, prepared from
the dimethylketal di-monocyclohexylamine salt, was ob
tamed from Sigma Chemical Co. , St. Louis, Mo. Agamgels
used in immunoelectnophomesis were prepared with lona
gar. Acrylamide gels for isoelectnic focusing were pun
chased as Pagplates from LKB, Stockholm, Sweden.
Glycerol-3-phosphate Dehydrogenase Assay. The assay
system used for heat inactivation has been described previ
ously by Fondy et a!. (9). All other assays were done at room
temperature in 0.05 M Tnis-chlonide (pH 7.5)-i mM EDTA-1
mM 2-mercaptoethanol.
Preparations of Homogenates. All buffers contained 50
mM tniethanolamine acetate (pH 7.5)-i mM EDTA-1 mM 2mercaptoethanol. All operations were performed at 0-4°.
Mice were killed by cervical dislocation. Liver, muscle, and
kidney were excised and immediately homogenized in 3
volumes of buffer. Heart, brain, and spleen tissues were
excised, quick frozen in dry ice, and used 2 to 5 weeks later.
These tissues were homogenized in a volume-to-weight ma
tb of 2:1 . Liver, kidney, and brain were homogenized with
Thomas hand homogenizers. Muscle, heart, and spleen
were homogenized for 1.5 mm with a Bninkmann PlO Poly
tron homogenizer at a dial setting of 4. BW7756 hepatoma
was excised from mice on Day 20 after s.c. passage and
homogenized with a Thomas hand homogenizer in 3 vol
umes of tniethanolamine acetate buffer. B16 melanoma,
Sarcoma 180, Ad755 adenocancinoma, H129 hepatoma, and
L1210 leukemia solid tumors were removed from mice on
Day 15 after s.c. passage and homogenized in 2 volumes of
tniethanolamine acetate buffer. L1210 ascites cells (3.2 x
NaCI solution.
RBC were
osmotically
lysed
by adding 24 ml of distilled water. After 30 sec the cells were
returned to isotonicity by addition of 8 ml of 3.6°/sNaCI
solution followed by centnifugation at 150 x g for 7 mm.
This procedure was repeated if the cell pellet was not free of
RBC. Cell count and in vivo tumorigenicity demonstrated
that no significant loss of L1210 cells occurred during hypo
tonic lysis of RBC. Cells were resuspended in 2 volumes of
tniethanolamine acetate buffer and disrupted on a Parr ni
trogen bomb apparatus at 1400 psi for 20 mm. Sarcoma 180
and Ad755 ascites cells were harvested on Day 7 and treated
similarly. Ehrlich ascites cells were harvested on Day 8 after
passage with 2.5 x 10@ascites cells. All tissue preparations
were
centrifuged
at 31 000 x g for 1 hr after
cell
rupture
to
produce the crude homogenates.
Affinity Column. To obtain tissue preparations with simi
lamenzyme activities, crude homogenates were added to
Sepharose 4B-hexamethylenedmammne-tnmnitmophenyl
affinity
columns as described by Komnbluth et a!. (15) and eluhed
with
0.5
mM
NADH.
Eluents
were
subsequently
concen
trated by ultrafiltration to a volume of 5 ml with, in succes
sion, the Amicon Model 52 to 10 ml and the Model MMC to 5
ml. All concentration steps used Amicon PM-10 mem
branes.
Heat Inactivation of Crude Homogenates. All crude ho
mogenates were adjusted to similar protein concentrations.
Mixed tissue homogenates were prepared by adding equal
units of enzyme from the separate homogenates. Protein
was determined by the method of Warburg and Christian
(33).
At 0 time
a 0.5-mi
aliquot
of crude
homogenate
was
added to 4.5 ml of tniethanolamine acetate buffer preequili
brated to the desired temperature in a temperature-con
trolled water bath. The solution was agitated, and a 0.1-mi
sample was immediately withdrawn and assayed. At 1-mm
intervals, aliquots were assayed. After 10 mm, aliquots were
taken every 5 mm, and after 30 mm aiiquots were taken
every 30 mm. Heat inactivation of the enzyme from heart,
spleen, brain, and Li210 required increasing the sensitivity
CANCER RESEARCHVOL. 37
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G3PDH in Norma! and Neop!astic
of the spectrophotometer to a full-scale absonbance of 0.2.
The presence of endogenous substrates for enzymes that
oxidize NADH in L1210 and spleen preparations required a
5-mm preincubation of enzyme with NADH and buffer at 24°
for elimination of this contribution to coenzyme oxidation
activity independent of G3PDH. After a 5-mm incubation,
dihydroxyacehone phosphate was added ho the cuvet to 0.1
mM,
and
the activity
was
measured.
Heat
inactivation
Mouse Tissues
Partially purified enzyme preparations were appropriately
diluted so that enzyme from various tissues would be corn
pared at identical activity levels. In cases in which staining
arcs for enzyme activity were desired, gels were washed for
48 hr in 0.15 M NaCI and then stained for enzyme activity (8).
RESULTS
was
carried out at 48 and 50°.
Flat-Plate Isoelectric Focusing. Isoelechnicfocusing was
performed with LKB Pagplahes containing 1% (pH 3 ho 10)
ampholytes on an LKB Multiphon flat-plate system at 4°.
Samples of 15 @l
were applied to the gel via 5- x 10-mm
pieces of filler paper. The anode buffer was 1 M H:3PO:i,and
the cathode buffer was 1 M NaOH. The voltage was set
initially at 210 and increased every 10 mm until the final
voltage of 1150 was reached at 60 mm. At 60 mm the sample
application paper was removed, and focusing was contin
ued for an additional 30 mm. When focusing was complete,
the gel was immediately stained for enzyme activity accord
ing to the method of Fondy et a!. (8).
Heat Inactivation Followed by Isoelectric Focusing. In
several cases it was necessary to follow visually the disap
pearance of forms with heating. Samples were heal mach
vated as described above and examined by gel elechnofo
cusing at lime intervals of 0, 10, 20, 30, 60, and 90 mm.
Antibody Preparation. Approximately 500 j@gof partially
purified mouse liven glycenol-3-phosphate dehydrogenase
were mixed with 1 ml of a 50-mg/mI solution of goat anti
rabbit muscle G3PDH as prepared previously by Holohan
(10). The mixture was kept at 37°for 1 hr and then was
removed to 4°for 48 hr. The precipitate was washed repeat
edly with cold 0.85% NaCI solution until the supennatant
had an A2,4)reading of 0. The precipitate was resuspended in
1 ml of 0.85% NaCI solution and emulsified in 1 ml of
complete Freund's adjuvanh on a vortex mixer at high speed
for 1.5 mm. The emulsion was injected s.c. in 1-mI aliquols
at several sites on the back of an adult goat. Three weeks
later the goat was bled (200 ml) from the jugular vein with a
standard blood donor kit. Additional booster injections of
precipitates containing G3PDH were given once a month for
3 months with intermittent testing of antibody titer. The
serum was purified by 3 ammonium sulfate precipitations as
described by Campbell et a!. (6). The final ‘y-globulmn
precip
ihatewas dissolved in 0.15 M NaCl-0.2 M borate (pH 8.3) to a
volume one-half that of the original serum sample, dia
lyzed against the same buffer for 3 days at 4°,and centni
fuged at 1400 x g for 30 mm. This antiserum was used for
immunoelectrophonesis. All partially purified preparations
from normal tissues except that from liven gave only a
single, detectable precipitin arc with this antiserum after
immunoelectrophoresis.
This arc stained positively for
G3PDH activity. The partially purified preparation from
mouse liver gave this same enzymahically active precipitin
arc along with a 2nd arc that did not contain G3PDH and
presumably represents a liver protein contaminant present
in the immune complex used to immunize the goat.
lmmunoelectrophoresis. Electrophoresis was carried out
in 1% agar plates in 0.05 M phosphate buffer (pH 7.2) at 7 V/
cm for 2.5 hr. Troughs were then cut in the gel, filled twice
with antibody, and allowed ho develop overnight at 24°.
Forms of NAD-linked G3PDH on Polyacrylamide Gel Iso
electric Focusing. Multiple forms of NAD-linked G3PDH
appeared in all tissues examined. The major form of the
enzyme migrated identically in all normal tissues examined
including liven, brain, heart, and spleen as well as the
BW7756 hepatoma (Fig. 1). Muscle and kidney, which are
not shown in this figure, shared this same pattern of cahi
onic forms. L1210 leukemia enzyme possessed these forms
but only as a small proportion of the total activity. Almost all
of the L1210 leukemia G3PDH activity was found in 2 an
ionic forms. At enzyme concentrations between 1 and 6 IU/
ml, only the heart enzyme appeared ho have trace activity in
positions corresponding to the major L1210 forms. Heart
also appeared to have forms with intermediate isoelechnic
points. These intermediate forms were seen in muscle at
higher enzyme concentrations and in brain in several en
zyme preparations.
At increased enzyme concentrations (10 lU/mI), at least 1
of the anionic forms appeared in brain and kidney. Fig. 2
shows that the anionic forms in brain and kidney focus at
the same pH as do the anionic forms in L1210 and do not
resolve from the Li 210 forms when coelectrofocused.
Early in development on approximately Day 10 of gesta
lion, whole-embryo homogenates expressed a major por
lion of their enzyme activity in 2 anionic forms indishin
guishable from the Li210 anionic forms (Fig. 3). The me
mainder of the embryonic G3PDH is expressed as the cati
onic pattern characteristic of differentiated adult tissues.
Fig. 4 shows that an anionic form is seen in the ascites
humor of Sarcoma 180 and Ad755 adenocarcinoma as well
as in log-phase P388 leukemia cells. The same pattern is
seen in early stationary-phase P388 leukemia cells. Both
anionic forms are present in substantial amounts in the
ascites H129 cells along with some of the major cationic
forms and weak representations of the minor cationic en
zyme forms. The anionic forms are not seen in the enzyme
activity from Ehrlich ascites cells (not shown) or in the solid
tumors examined, including BW7756 hepatoma (Fig. 1) and
B16 melanoma (Fig. 5). Implantation s.c. of Sarcoma 180,
H129 hepatoma, Ad755 adenocancinoma, on L1210 leuke
mia produced solid forms of neoplasms that grew more
slowly than did the p. ascites tumors. Sarcoma 180,
Ad755, and L1210 solid tumors exhibited the cahionic pat
tern of G3PDH activity characteristic of adult differentiated
mouse tissues with little on no evidence of the anionic
forms. H129 solid hepatoma had a faint representation of
the anionic forms and a much stronger normal cationic
pattern.
Reaction with Goat Antibody to Mouse Liver G3PDH.
When partially purified enzyme from various tissues was run
on agar gel electrophoresis (pH 7.2), all normal tissues as
well as BW7756 hepahoma had at leash 1 major, mutually
indistinguishable cationic form of G3PDH activity (Fig. 6B).
AUGUST 1977
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2675
L. S. Rittmann
et a!.
Enzyme from L1210 leukemia migrated as a single anionic
form. Fig. 6A shows the reaction of enzyme from various
tissues with goat anti-mouse liver G3PDH. Enzyme from
heart, liver, and muscle (Fig. 6A) as well as that from brain,
spleen, and kidney produced precipitin arcs with goat anti
mouse liver G3PDH. These arcs subsequently stained for
enzyme activity and were localized also by protein stain
after thorough washing in 0.85% NaCI solution. L1210 leu
kemia enzyme failed to form a precipitate with the anhise
rum.
Subsequent
staining
with
enzyme
or
protein
stain
failed to produce a visible precipitin arc in 5 separate expen
iments.
Heat Inactivation. Muscle, liven, brain, spleen, and
BW7756 hepatoma crude homogenates had G3PDH heat
inactivation curves that were biphasic at 50°.L1210 leuke
mia G3PDH inactivated as a single heat-sensitive form. At
48°,G3PDH activity in liver and muscle (Chant 1) as well as in
brain, heart, spleen, and BW7756 hepatoma exhibited mul
tiphasic heat inactivation curves, whereas L1210 leukemia
enzyme inactivated rapidly as a single form. When partially
purified brain enzyme was heat inactivated for 0, 10, 20, 30,
60, and 90 mm and aliquots were focused on polyacryl
amide gels, there was a rapid loss of the anionic forms
corresponding to those forms observed in L1210 cells and a
much slower decrease in intensity of the cationic forms.
Cationic forms characteristic of adult, differentiated tissues
were still observable after 90 mm, whereas anionic forms
were notvisible
after10 mm at50°.
DISCUSSION
Differences in isozymes in normal and neoplastic states
have been used in cancer diagnosis and monitoring of
treatment (19, 25, 36) and may become important in chemo
therapy and immunotherapy. Moreover, with increasing in
terest in agents that promote nonmalignant differentiation
of malignant cells, differences in isozyme forms between
neoplastic and adult differentiated tissues become poten
tially useful markers of normal and neoplastic differentia
lion.Ithas been suggested that,in cancer cells,isozymes
favoring storage biosynthetic pathways such as gluconeo
0'
C
C
0
E
a,
>
C)
4
Minutes
of
48°
Chart 1. Heat inactivation of normal and neoplastic mouse tissue crude
homogenates at 48°.x , liver G3PDH; 0, muscle G3PDH; •,L12i0 leukemia
G3PDH from ascites cells taken on Day 4 after p. inoculation of i0@cells.
2676
genesis or triglyceride synthesis are replaced by isozymes
that function to promote the efficient use of metabolic fuel
in support of increased cell division (34). Our results show
that significant differences exist between NAD-linked
G3PDH found in ascites tumors including L1210 leukemia
and enzyme from normal tissue preparations. In L1210
ascites leukemia, themeis a dramatic reduction in the cati
onic pattern found in all normal tissues. Two bands that
focus at much lower pH's are the major enzyme forms in
L1210 leukemia. Examination of other rapidly dividing as
cites tumors also reveals the presence of at leash 1 of these
forms at the expense of the normal cationic pattern with the
exception of the ascites form of Ehrlich carcinoma as it
approaches plateau phase. Implantation s.c. of those tu
moms,which exhibit anionic enzyme forms in their ascites
state, produced solid, more slowly growing neoplasms and
resulted in the reestablishment of the normal cationic pat
tern and loss or great reduction in the anionic forms.
BW7756 hepatoma, 1 of the few neoplastic tissues in which
G3PDH reaches normal levels (13), also displayed the nor
mal cationic pattern. Loss of the anionic forms in switching
from a rapidly dividing ascites tumor to a more slowly grow
ing solid tumor suggests that the anionic forms may be a
property of rapidly dividing cells in populations with high
growth fraction. Some contribution of normal adult differ
entiated tissue forms may be expected from host cell infil
tration of solid tumors, but the virtual absence of the an
ionic forms in the solid tumors suggests that the tumor cells
themselves no longer exhibit the anionic enzyme forms if
the cells are harvested from solid rather than ascihes tu
moms.
One of the anionic forms is present in both the log and
early stationary phases of the P388 cell line. However, the
early stationary-phase cells were in that state for only 1 day.
It is not certain how much time is required to switch to the
production of the normal cationic pattern of G3PDH that is
representative of the more differentiated tissues and how
long it should take for the anionic forms present in the
rapidly dividing culture cells ho decay.
An anionic form of G3PDH similar ho that observed in
L1210 leukemia has also been found in the Browne-Pierce
carcinoma in rabbits (20). This anionic form in rabbits is
decidedly distinct from other heat-labile forms of the en
zyme isolated from brain and heart.5 The presence of the
anionic forms and the significant loss of the cationic pattern
in fetal mice establish these anionic forms of G3PDH as
another example of “oncofetal―
enzyme forms related po
tentially to cell growth and division.
Kozak and Jensen (16) have shown that mice possess 2
isozymic forms of G3PDH that can be distinguished from
each other by their heat inactivation and elechnophoretic
properties. These researchers found an embryonic form in
fetal brain and skeletal and heart muscle although all adult
tissues contained essentially only the adult form. Our isoe
lectnic focusing data show the presence of a much more
complex pattern of G3PDH than that obtained previously
with lower resolution techniques. This multiple band pat
tern
S R.
more
Kornbluth
accurately
and
T.
describes
P.
Fondy,
the multiphasic
manuscript
in
heat
mach
preparation.
CANCER RESEARCH VOL. 37
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G3PDH in Norma! and Neoplastic
vation curves for G3PDH obtained by this laboratory and
others, especially at temperatures below 50°.It is apparent
that differentiated tissues in young adult mice and rabbits
do share a cationic form of the enzyme that is indistinguish
able among the tissues examined. However, there are 5ev
emalother forms of the enzyme, distinct from the extremely
anionic oncofetal forms, that are expressed preferentially in
different tissues.
High-resolution analysis by flat-plate gel isoelectnic fo
cusing in conjunction with heat inactivation at temperatures
below 50°in our work has shown that most of the heat-labile
activity found in muscle, brain, and heart resides in forms
more anionic than does the major form common to all
normal tissues. However, these forms are much less anionic
than the oncofetal forms described in this paper. Indeed,
DEAE-cellulose chromatography of the embryonic form
from mouse brain (Ref. i6, p. 7778) shows that it is in fact
less anionic than is the enzyme from ascites tumors under
the same conditions (Ref. 17, p. 3715). Thus, the heat-labile
enzyme observed in neonatal tissues (16) should not be
equated with the heat-labile oncofetal forms observed in
our work and in parallel studies (17).
We found a small amount of the oncofetal anionic forms
in several adult tissues. Whether this has a functional signif
icance is not known. Their presence may merely represent
the incomplete switching off of genes that have been active
in enzyme synthesis in the fetal state. In any event it is a
well-documented phenomenon that an isozyme that is pre
dominant in a neoplasm is always expressed in some adut
or fetal tissue of the same species (24).
The presence of an oncofetal form of G3PDH in adult
brain and kidney is not unique to this enzyme. Sato and
Weinhouse (23) have shown that the fetal form of glycogen
phosphorylase is also present as a major form in matkidney
and brain. Besides the existence of the oncofetal form of
G3PDH in adult brain, kidney, and heart, other differences
among adult forms also exist. For example, heart, muscle,
and brain have additional cationic forms, which the other
adult tissues do not express. The possible existence of
tissue-specific isoenzymes of G3PDH is the subject of de
tailed studies in rabbit tissues (20).@
Immunological and heat inactivation properties of L1210
leukemia G3PDH were also examined in comparison to the
normal enzyme. The failure of the Li210 enzyme to form a
precipitin reaction with either the goat anti-mouse liven
G3PDH or the goat anti-rabbit muscle G3PDH suggests that
the enzyme in ascites L1210 leukemia is structurally differ
ent from the major enzyme forms present in adult diffenen
tiated tissues. The rapid, monophasic heat inactivation of
the L1210 enzyme in comparison to the multiphasic, slower
inactivation curves of the normal enzymes also suggests
structural differences. Whether L1210 enzyme is a gene
product distinct from any of the more cationic enzyme
forms functioning in other tissues will require biochemical
and genetic characterization of both oncofetal and normal
forms. Kinetic comparison of the homogeneous forms may
provide insight into the different roles of the enzyme. Ulti
mately, differences in the enzyme between normal and neo
plastic tissues, whether these differences are genetically or
epigenetically based, may be exploitable for chemotherapy
or immunotherapy.
Mouse Tissues
ACKNOWLEDGMENTS
We thank Linda Cunningham, Lucille Cosby, and Dr. Alan Sartorelli of
Yale Medical School and Dr. William Bradner of Bristol Laboratories for their
generous gifts of mice and tumors. We also gratefully acknowledge experi
mental assistance by John Crane.
REFERENCES
1 . Agranoff,
B. W., and Hajra,
A. K. The Acyl Dihydroxyacetone
Phosphate
Pathway for Glycerolipid Biosynthesis in Mouse Liver and Ehrlich Ascites
Tumor Cells. Proc. NatI. Acad. Sci. U. 5., 68: 411-415, 1971.
2. Baranowski, T. Crystalline Glycerophosphate Dehydrogenase from Rab
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2678
CANCER RESEARCHVOL. 37
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G3PDH in Norma! and Neoplastic Mouse Tissues
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Fig. 1. Isoelectric focusing of partially purified G3PDH from normal and neoplastic mouse tissues. Enzymes were focused in a pH range of 3 to 10 and
stained for G3PDH activity. Liver, spleen, brain, and heart G3PDH's were partially purified from normal adult mouse tissues. BW7756 hapatoma G3PDH was
extracted from Day 20 solid tumors, and L1210 G3PDH was extracted from cells taken from the peritoneal cavity on Day 4 after i.p. inoculation of 10@cells. All
enzyme preparations contained 1.3 to 2.8 lU/mI.
Fig. 2. Isoelactric focusing of partially purified G3PDH from normal and neoplastic mouse tissues at 10 lU/mI. Mixtures contained equal units of both
enzyme preparations. Other conditions are as described in Fig. 1.
Fig. 3. Isoelactric focusing of partially purified G3PDH from fetal and leukemic cells. Other conditions are as described in Fig. 1. Upper track, G3PDH from
Li210 ascites cells; lower track, G3PDH from whole fetus on Day 10 of gestation. Both preparations contained 1 IU G3PDH per ml.
Fig. 4. Isoelectric focusing of partially purified G3PDH from neoplastic calls (0.3 to 1.0 lU/mI). Other conditions are as described in Fig. 1. S,,,, G3PDH
from Sarcoma 180 ascites tumor cells; Ad755, G3POH from Ad755 adenocarcinoma ascites tumor cells; P,.,,,,G3PDH from long-passage P388 leukemia cell
culture in log phase.
Fig. 5. Isoelectric focusing of partially purified G3PDH from solid B16 (B,6) melanoma cells (2 lU/mI). Other conditions are as described in Fig. 1.
Fig. 6. A, immunoelactrophoresis of partially purified G3PDH from normal and neoplastic mouse tissues against goat anti-mouse liver G3PDH. Precipitin
arcs were stained with G3PDH activity after thorough washing of gals in 0.85% NaCI solution. Enzymes were prepared as in Fig. 1. B, agar electrophoresis of
G3PDH from normal and neoplastic mouse tissues stained for G3PDH activity.
AUGUST
1977
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1977 American Association for Cancer Research.
2679
Forms of Cytosolic Nicotinamide Adenine Dinucleotide-linked
Glycerol-3-phosphate Dehydrogenase in Normal and Neoplastic
Mouse Tissues
Lana S. Rittmann, Suzanne M. Johnston and Thomas P. Fondy
Cancer Res 1977;37:2673-2679.
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