[CANCER
RESEARCH
34, 3165-3
172, December
1974J
Half-life of N-Acetylneuraminic Acid in Plasma Membranes of
Rat Liver and Morris Hepatoma 77771
Erik Harms2and Werner Reuttor
Biochemisches Institut der Università 'tFreiburg, 78 Freiburg im Breisgau, Germany
SUMMARY
been measured in the plasma membranes with tracer amino
acids (1, 13, 16). This method is commonly used to investigate
The degradation rates of N-acetylneuraminic acid (NANA) the turnover of any protein. Introducing carbohydrates as
were investigated in plasma membranes and in the total precursors allows for the measurement of the half-life of
glycoproteins. Studies on the metabolism of NANA3 seem to
protein of rat liver and Morris hepatoma 7777.
The plasma membranes of Morris hepatoma 7777 could not be useful because of the high concentration of this compound
be isolated by known methods useful for isolation of rat liver in plasma membrane glycoproteins (I I , 35 , 40) and because of
plasma membranes. Therefore, a modified method was this amino sugar's key role in cell-cell interaction in normal
developed. The specific activity of 5'-nucleotidase as a plasma and transformed tissues (7, 18, 29, 33, 43, 45). In this report
membrane marker enzyme was enriched twelvefold relative to we describe the half-life of protein-bound NANA in plasma
homogenate.
membranes of liver and a hepatoma. Moreover, this study
of the plasma
N-Acetyl-D-mannosamine is the most suitable precursor of supports the concept that constituents
protein-bound NANA. When compared to the use of free membrane are synthesized and degraded at different rates.
Our previous findings (17, 36) revealed a high concentration
NANA of a comparable specific radioactivity, the rate of
incorporation is 6 times higher in liver and 20 times higher in of protein-bound NANA in Morris hepatomas when compared
hepatoma. ‘
“
C labeling in the acetyl group or in the hexose with liver, although the activity of the key enzyme of NANA
skeleton of N-acetyl-D-mannosamine
resulted in identical synthesis, the UDP-GlcNAc-2'-epimerase, was diminished in
measurement of the half-life of NANA, leading to a uniform the Morris hepatoma 7777 to 6% of that of the liver. This
discrepancy may be due to a decreased requirement of NANA
turnover of the NANA molecule.
There is no difference between the half-lives of NANA of caused by the lack of secretion of plasma glycoproteins (39) or
the liver membranes and hepatoma membranes. The same by a prolongation of the half-life of protein-bound NANA in
applies to total liver and hepatoma protein. Yet, in both Morris hepatomas; this NANA is localized primarily in the
tissues, the half-life of plasma membrane-bound NANA is plasma membrane.
The use of D-glucosamine to measure the half-life of NANA
shorter (liver, 22.2 to 24 hr; host liver, 25.5 hr; Morris
in plasma membranes is not advisable because D-glucosamrne is
hepatoma 7777; 23.7 hr) than the half-life of total protein
bound NANA (liver, 34 hr; host liver, 30.1 hr; Morris the source of 3 precursors for glycoprotein synthesis. This
difficulty is overcome by our previous observation (17) that
hepatoma 7777, 33.9 hr). This finding indicates that sialopro
teins with considerably longer half-lives of NANA exist in ManNAc is the most suitable in vivo precursor of both liver
and hepatoma sialoproteins. Comparative studies on both liver
subcellular structures other than plasma membranes.
In plasma membranes, the half-lives of NANA were much and hepatoma membranes require a comparable yield and
purity of plasma membranes of both tissues. The published
shorter than the half-lives of amino acids when a non
reutilizable amino acid precursor was used. It may be methods are not appropriate for these studies on hepatoma.
concluded that the mean turnover of NANA is more rapid Therefore, an isolation procedure for hepatoma plasma
membranes has been developed.
than the turnover of amino acids in plasma membranes.
For the first time, the measurements of the half-lives of
Glycosylation
by
plasma
membrane-bound
glycosyl
protein-bound
NANA allow a comparison of the cellular
transferases or different turnover rates of plasma membrane
compounds is regarded as responsible for the shorter half-life requirement and the synthetic capacity of NANA in liver and
in the Morris hepatoma.
of plasma membrane-bound NANA.
MATERIALS
INTRODUCTION
AND
METHODS
The synthesisand degradationof proteinconstituents
have Animalsand Tumors
I This
investigation
was
supported
by
the
Deutsche
Forschungsgemeinschaft (SFB 46, Molekulare Grundlagen der Ent
wicklung) and MuUer-Fahnenberg-Stiftung, Freiburg.
2Present address: Universitats-Kinderklinik, 69 Heidelberg, Hof
meisterweg
1-9, Germany.
Received March 4, 1974; accepted July 2, 1974.
DECEMBER
Male Buffalo rats weighing 150 to 200 g were bred in our•
3The abbreviations used are: NANA, N-acetylneuraminic acid or
sialic acid; GlcNAc, N-acetyl-D-glucosainine; ManNAc, N-acetyl
D-mannosamine; GIcN, glucosamine.
1974
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
3165
E. Harms and W. Reutter
laboratory and fed ad libitum with Altromin (Lage, Lippe,
Germany), a diet containing 19% protein. Morris hepatoma
7777 generations 90 to 100 (originally obtained from Dr. H. P.
Morris, Howard University, Washington, D. C.), were trans
planted i.m. to both rat hind legs every 3 weeks. The animals
were killed under light ether anesthesia by exsanguination.
Materials
ManNAc-U' 4C (labeled uniformly in the hexosamine
moiety@@3H-labeled ManNAc, acetic nh4
C, tol
uene-1 C, and toluene-3H were purchased from the Radio
chemical Centre (Amersham, England). ManNAc' 4C (labeled
in the acetyl group) was synthesized using a slight modifIca
tion of the method of Horton et a!. (20). In order to remove
excess acetate-l-' 4C and D-mannosamine, the acetyl-l-' 4Clabeled ManNAc was passed through a mixed-bed column
filled with Dowex 50W-X8H@ (50 to 100 mesh, 2 x 15 cm)
and Dowex l-X8-formate (50 to 100 mesh, 2 x 15 cm) and
eluted with water. For further purification, the eluate
containing the labeled ManNAc was applied to Whatman No.3
paper and developed by descending chromatography
in
Solvent System A (see below) for 18 hr to remove
peracetylated compounds. The area of labeled ManNAc was
cut out and eluted with water. The accompanying salt of
Solvent System A was removed by passing the eluate through a
mixed-bed resin column as described above. The purity of the
4 C
@
was
checked
by
paper
chromatography
System B) and paper electrophoresis (see below).
was synthesized in a cell-free system using
precursor as described earlier (17).
(Solvent
Sorvall
RC-2B centrifuge
equipped
with a GSA rotor
or an
55-34 rotor was used for low-speed centrifugation, and a
Beckman L2-65 B preparative ultracentrifuge equipped with an
SW-27 rotor was used for high-speed centrifugations. All
operations were carried out at 0—4°.
Enzyme Assays. The following enzyme markers were
assayed according to the methods given in the associated
references to check the purity of the membrane fractions
(Table
1 ):
S'-nucleotidase
(EC
3.1.3.5)
(27),
Mg2@:Ca2@—ATPase
(EC 3.6.1 .3) (9), Na@:K@—ATPase(EC
3.6.1 .4) (9), succinate dehydrogenase [succinate :2-(p-iodo
phenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium
oxidoreductase
(EC 13 .99.1)] (3 1), and glucose 6-phosphatase (EC 3.13.9)
(6). P, was determined by the method of Fiske and SubbaRow
(12).
Uptake and Distribution of ManNAc-3H and NANA-'4C
The concentration dependency of the amino sugar uptake
was measured by injecting 1 mCi ManNAc-3 H per kg body
weight into the tail vein of each tumor-bearing rat. The
specific radioactivity was varied for each rat and ranged from
0.5 to 500 mCi/mmole.
Ten mm after the injection
the blood
was collected by aortal puncture. The rats were decapitated,
and the livers and hepatomas were quickly removed and frozen
in liquid nitrogen. In order to obtain an acid-soluble fraction,
the tissues were vigorously homogenized in 3.5 volumes of 0.9
N ice-cold
HC1O4
(w/v)
and
centrifuged
at 35,000
X g for 15
C mm at 0°.The total uptake of ManNAc was calculated from
C as the radioactivity measured in the supernatant. The time
dependency of ManNAc uptake and incorporation
was
measured after the application of 1 mCi ManNAc-3 H per
kg body weight (specific radioactivity, 200 mCi/mmole). Five
Isolation of Plasma Membranes
to 60 mm later the livers and hepatomas were excised and
Liver. The plasma membrane fraction of the liver was treated as described above. To measure the total radioactivity
(acid-soluble and insoluble fraction), 3 .5 volumes of ice-cold
prepared by the method of Neville (28) using the modification
water were added to the tissue and homogenized with 30
of Emmelot et a!. (10) and Ray (35).
Hepatoma. A high loss of hepatoma membrane fraction was strokes at 3000 rpm using a Servodyn (Cole Palmer, Chicago,
Ill.). Using a l-ml NCS at 60°,0.5 ml of this homogenate was
observed when this method was applied to Morris hepatoma
7777. To obtain a yield comparable to that of liver, a new solubiized until a clear solution was achieved. The protein
method was developed. Under light ether anesthesia, the rats bound radioactivity was measured using the procedure of Mans
were exsanguinated and perfused via the inferior vena cava in and Novelli (26). The protein-bound radioactivity can also be
situ with at least 40 ml ice-cold 0.9% NaC1 solution containing determined by calculating the difference between acid-soluble
2 mr@i CaCl2 (9). The hepatomas were prepared almost and total radioactivity.
Three hundred j.zCi of
AN4
C (with a specific
necrosis free. The crude connective tissue was removed by
squeezing the hepatomas through a sieve with pores 0.8 mm in radioactivity of 29.7 mCi/mmole) per kg body weight were
diameter. The resulting brei was suspended in 1 volume (w/v) injected into the tail veins of 3 tumor-bearing rats that were
of the isotonic homogenization medium (0.15 M KC1, 1 [email protected] maintained in metabolic cages. Sixty, 120, and 240 mm after
NaHCO3 , and 2 mm CaC12, pH 7.6), passed through 3 layers injection, the rats were killed, as stated above, and the tissues
of cheesecloth to remove smaller particles of connective tissue were removed and stored in liquid nitrogen until the free and
NANA could be
and clumps, diluted to 10 volumes with the medium (see specific rathoactivities of the protein-bound
determined.
Additionally,
the
specific
radioactivity
of NANA
above), and homogenized by 25 strokes (per 2 to 3 g) of a
in
the
collected
urine
was
measured
after
chromatography
in
loosely fitting 30-ml Dounce-type homogenizer (B. Braun
Apparatebau, Melsungen, Germany). The homogenate was Solvent System A.
diluted with a hypotonic medium consisting of 1 mae NaHCO3
and 2 mr@iCaCl2, pH 7.6, to 1:100 (w/v) (9). The subsequent
Estimation of the Half-life of NANA
centrifugation steps of this horn c@enate were performed as
outlined in Chart 1. The resulting membrane subfractions were
Each of 16 normal and 16 tumor-bearing rats received a
collected and washed twice in the hypotonic medium. A single injection of ‘
“
C-labeled ManNAc. Four rats were killed
3166
AN4
CANCER
RESEARCH
VOL. 34
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
Half-life
ofNANA
inLiverand HepatomaMembranes
I_
HEPATOMA
HOMOGENATE]
500 x g
10 mm
+
peLLet
(discarded)
1. 500
x g
40 mm
supernatant
(discarded)
resuspendedin
________________ pellet
+
Chart I. Isolation
of plasma
membranes
from
32% sucrose
120 mm
Morris hepatoma 7777. For preparation of the hepatoma
homogenate, see “Materials and Methods.― The sucrose
solutions (w/v) have the following densities [p(g cm at 22°: 48%: 1.190; 43.5%: 1.181; 41.5%: 1.176; 38%:
1.153; 32%: 1.128. The densities of membrane fractions
M,, M,, and M4 correspond to rat liver plasma mem
brane fractions M1, M @,and M, isolated according to
the method of Ray (35).
(w/v)
27000 rom
Spmncorotor SW 27
(@ 9S000x9av)
diLuted in one voLof
buffer—
medium
(1mMNaHCO312mMCaCL2, pH 7.6)
@
12000 xg
peLlet resi@spended in
supernatant
@flmin
(discarded)
48% sucrose (w/v)
membrane
+
fractions
sucrose32.
sucrose 38.
sucrose 43.5
120mm
sucrose45@5
Spinco rotor
M1
-PM2
27000rom
INWIIIII
SW 27
SIOHhIIlI,ii
-PM3
—.P,Wl4
sucrose48.0
(sampLe)
at 12, 18, 24, and 36 hr, respectively, to remove blood, liver,
and, in the case of tumor-bearing rats, the hepatoma. From
each liver or hepatoma an aliquot of about 1.5 g was dissected
to estimate the specific radioactivity of NANA in the total
protein. The 4 livers or hepatomas from each time group were
pooled (giving about 20 g liver and 30 to 40 g hepatoma) for
the isolation of plasma membranes as described above. The
radioactivity was determined
ManNAc, the incorporated
according to the method of Mans and Novelli (26). However,
the heat step was omitted.
The half-life was estimated by plotting the logarithms of the
specific radioactivities (ordinate) against the time (abscissa),
and a regression line was calculated using the least-squares
dissected
yielded the half-life time.
aliquots
were homogenized
in 3.5 volumes
0.9 N
HC1O4 for the estimation of the specific radioactivity of
NANA in the total tissue protein. Following a 15-mm
centrifugation at 35 ,000 X g, the sediments were rehomog
enized with H2 0 and recentrifuged. After 2 additional H2 0
washings, the NANA was released by hydrolyzing the pellet in
0.2 N H2 504 for 75 min at 80°(17). The specific radioactivity
of NANA in the plasma membranes was measured after
hydrolyzing the isolated fractions in 0.1 N H2 SO4 for 60 mm
at 80°.After neutralization with saturated Ba(OH)2 solution
followed by a centrifugation at 11,000 X g, NANA was
purified by anion exchange and paper chromatography as
described earlier (1 7). The specific radioactivity of NANA was
calculated after measurement by the periodate:resorcinol
method (20) using an internal standard and counting the
radioactivity. In order to measure the half-life of protein
bound radioactivity in the plasma after injection of labeled
DECEMBER
1974
method.
The division
of log 2 by the regression
coefficient
Protein Determinations
Protein concentrations of membrane fractions were deter
mined by the method of Lowry et aL (24). The biuret method
(2) waspreferredfor serumandcrudehomogenates.
Crystal
line bovine serum albumin was used as a standard.
Paper Chromatography
and Electrophoresis
Two paper chromatography solvent systems were used:
Solvent A (23), n-propyl alcohol:water: 1M sodium acetate, pH
5.0 (7:2: 1, v/v/v),
paper;
and
in conjunction
Solvent
B (44),
with Whatman
No. 3 filter
ethyl acetate:pyridine:water
3167
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
@
@
.@
@n
.@
r@i
r@
E. Harms and W. Reutter
paper (Macheiy and Nagel, Düren,Germany), using 1.5%
sodium tetraborate solution, pH 9.0, mixed with 5 mr@iEDTA,
at 30 to 50 V/cm and 0 for 2 hr.
V
@
V
‘1,
0
V
V
0.
C'
o.c
@
@
0
@o
a'@©
%qre,
p
.@
,@. C... O\
4141414141
C'@O@
‘@.
00 ‘1@
‘.000
+1
+@ +1
(‘4‘@1@
r-@
—
0
v@ r@
0.
\03
@
Determination of Radioactivity
Radioactivities
.E
V
estimation
@
.@. ,@. a\
C'
0.
.E@o
r@i@
I,)
4141414141
@
@
,@. a@ r@
“@
@
m C
@.o00
@
@
V
.@;
Absolute
Activity
FHT780 A (Bertholdand Frieseke,Karlsruhe,Germany).
@.
<
0#@
C'- -
@.
4141414141
.@
C'
E
C'
V
,@.
@0 C'-
u
°.
e@ r@i
Concentration Depençlency. The uptake of ManNAc was
proportional to the concentration of the injected solution over
a range of 2 X 106 to 2 X l0@ M in both liver and hepatoma
C'
I.0
-@E
CO
,@3
E
u
C.,
V
0\
F-
1:
0.
0#@
0
+
,,@ —
‘.c
+1 +1 41 +1 41
111
A
— —
@H 4141
@q@r@oc@
L)
+
Uptake of ManNAc
— “0
‘fl —‘
C'
‘#,
The advantage of ManNAc was shown when the uptake of
labeled ManNAc was compared with NANA as a possible
precursor for incorporation into NANA-containing glyco
proteins.
+1 +1 +1
‘.0
z
C'
@,o
O\ O@—
EV
@
the
4@4+1
‘.0 —
m r—
a@
C'
F-
+
@
using
00
,c
+
@
quenching
RESULTS
@,
@
the
0
C'
@
of
0.
11
@
(5)
.@
0
—
V
using Bray's scintillator
Analyzer 544 system. Filter discs of the procedure of Mans
and Novelli were counted in a toluene scintillator (21). Paper
chromatograms were scanned in a radio paper chromatograph
V
V
@
@
— ‘,@
—
C― — C'
@‘4
r',@ C―l
00
© ‘0 r@ ‘/@C'1
— 0
0
‘V
@
@
were determined
for aqueous solutions in a Packard Tri-Carb Model 3390
scintillation spectrometer,
equipped with a Model 544
Absolute Activity Analyzer (Packard Instruments,
Inc.,
LaGrange, Ill.). The samples were corrected for quenching by
adding toluene-1 4C or toluene-3 H as internal standards or by
C'4
0
0―‘1)
r@ rI — —
‘,@,@.
3
;‘..
C'
.@
V
‘O
.@
u
@
0V
V
C'
,@.
0.
‘,@c\
z
44 +4 +1 +1 +1
@‘
r@
00
C
C.)
@
0
r'4
“@‘@
414141
(‘4
‘uS
-,
‘,@
@
V
‘@00
V
@8
N
r-@ r@ ‘@j
0
— C'a@o@t-..‘@.
I
,@. (@,
C
-a>'
3
V
-x
0
@
V
.n
@
0.
C—
C-C—
C'C'
010
E
.@ .E
C'
@.
C'V
0
0
@iiii
1O0•@ 200
V
Chart 2. Uptake of ManNAc into rat liver (.) and Morris hepatoma
7777 (A) measured by acid-soluble radioactivity 10 mm after a single
0
injection of ManNAc-3H (1 j@Ci/gbody weight), varying the specific
>
radioactivity
(12:5: 1, v/v/v), using Schleicher and Schull No. 2043 bMgl
paper impregnated with 2% sodium tetraborate solution.
Borate electrophoresis (25) was performed on MN No. 214
3168
50
I*cted nMoteManNAc/g body weight
V
from
500 to 0.5 mCi/mmole.
(The injection
of 1000
(2000) nmoles ManNAc per g body weight lead to an uptake of 965
(1705)
nmoles
ManNAc
per g liver and 1002 (2180)
nmoles ManNAc
per g hepatoma.) For preparation and determination of acid-soluble
radioactivity, see “Materials
and Methods.―
CANCER RESEARCH VOL. 34
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
Half-life ofNANA
with
after
into
host
After the injection
of labeled
Membranes
when compared with ManNAc. A continuous increase of the
specific radioactivity of protein-bound NANA in hepatoma
was measured within 4 hr of the injection of NANA. In host
liver, the specific radioactivity already was decreased 4 hr after
the label (Table 2). Two hr after the injection about 60% of
radioactivity was excreted via the urine as NANA. Chroma
tographic analysis (Solvent System A) of the urine did not
show a metabolic conversion or a decomposition of the
injected NANA. The specific radioactivity of the excreted
NANA was identical to the injected substance, which indicates
the absence of an extracellular pool of free NANA. This is in
contrast to the known existence of an intracellular pool.
regard to the perchloric acid-soluble radioactivity 10 min
the injection. Under these conditions, the uptake of label
the hepatoma is 1.5 times higher than the uptake in the
liver (Chart 2).
Time Dependency.
in Liver and Hepatoma
ManNAc
the blood was quickly cleared of radioactivity (Chart 3). Sixty
min after the injection only 11% of the acid-soluble
radioactivity found at S min could be detected in plasma.
Therefore, a pulse-labeling procedure was not necessary.
Pulse-labeling procedures using ManNAc may be followed by
disturbances of the steady state of NANA metabolism. This
was very important in view of the previous supposition that
the rate of NANA synthesis was regulated primarily by the
activity of the UDP-GlcNAc@2'-epimerase (17).
Within 1 hr after the injection of labeled ManNAc, the total
radioactivity increased in hepatoma and decreased in host
liver. The acid-soluble radioactivities (Chart 4) decreased,
whereas the protein-bound radioactivity increased in both
tissues.
Measurement
of the Half-lives of NANA
ManNAc is the most suitable precursor for protein-bound
NANA in in vivo studies when compared with free NANA as
demonstrated
above, and when compared with GlcN as
previously shown. The use of ManNAc, labeled either in the
acetyl group or in the carbohydrate chain, disclosed no
difference in the resulting half-lives of protein-bound NANA
Uptake of NANA
of rat liver plasma membranes
NANA, when given i.v., penetrates into the host liver as well
as into the hepatoma tissue; however, to a very low extent
(Table
3), indicating
that the
1500
V
In
@- 1500
In
ID
E
In
0)
ID
0.
-@
1000
U
C
1000
>‘
>
U
C
C-,
ID
0
>‘
@—
500
@0
CD
500
U
ID
0
@0
ID
I
0
I-
0
I
I
10
20
-I-
30
I
e
I
40
50
60
10
S
20
I
I
I
I
30
40
50
60
time after injection(mm)
Chart 4. Total and acid-soluble radioactivity of rat liver and Morris
time after injection(min)
Chart 3. Disappearance of radioactivity from plasma during the 1st
hepatoma 7777 during the 1st hr after a single injection of ManNAc-3 H
(1 @Ci/gbody weight; specific radioactivity,
200 mCi/mmole):
., total
hr after a single injection of ManNAc-3H (1 @iCi/gbody weight; radioactivity of rat liver; o, acid-soluble radioactivity of rat liver; @,
specific radioactivity,
200 mCi/mmole).
For preparation
and deter
mination of total (.) and acid-soluble ((o) radioactivity,
see “Materials
and Methods.―
total
radioactivity
of Morris hepatoma
activity of Morris hepatoma
“Materials
and Methods.―
7777;
@,acid-soluble
7777. For experimental
radio
details, see
Table 2
Uptake and incorporation
AN4
C (speafic radioactivity, 29. 7 mCi/mmole) aftera single infection
of 0.3 pCi/g body weight into rumor-bearing rats.
For experimental details, see “Materials
and Methods―
7777Total
Time
radioactivity
radioactivity
after
of protein-bound
NANA
radioactivity
radioactivity
radioactivity
(nCi/ml)Protein-bound
injection
(nCi/ml)Acid-soluble
(mCi/mole)60
(nCi/g)Specific (mCi/mole)Acid-soluble (nCi/g)Specific
(mm)PlasmaLiverMorrishepatoma
120
24066.6
DECEMBER
34.9
12.57.5
10.1
9.416.2
11.3
7.42.77
3.67
2.4733.4
19.6
7.12.74
radioactivity
of protein-bound
NANA
3.26
3.90
1974
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
3169
E. Harms and W. Reutter
Table 3
Half-lives ofprotein-bound NANA in rat liver and Morris hepatoma 7777
after labeling with ManNAc
Regression and correlation coefficients are calculated
For experimental details see “Materials
and Methods.―
PrecursorPreparationRegression
coefficient
(Kr)Normal
by the least-squares method.
coefficientHalf-life
(log)Correlation
liverManNAc@(Ua.l
rat
protein0.008860.85634.0ManNAc-(U-1
4C)Total
membrane
4C)
22.2Host
0.01350.989
Plasma membrane0.0126
ManNAc@(1l@@14C)Plasma
0.99724.0
liverManNAc-(1-'
protein0.01000.99830.1ManNAc-(1-'
4C)Total
180.97925.5Morris
4C)Plasma
membrane0.01
7777ManNAc-(1-'
hepatoma
protein0.008880.81733.9ManNAc-(1-'
4C)Total
membrane0.01270.99223.7
4C)Plasma
a u, uniformly
@
labeled
in the hexose part.
b , c-i of the acetyl group labeled.
acetyl group of NANA remains attached to the carbohydrate
chain of this acid. In the subsequent experiments ManNAc
@i06
14 C was used (see “Materialsand Methods―).
The half-life of protein-bound NANA was measured in both
the plasma membranes and the unfractionated tissue of normal
liver, host liver, and hepatoma. The half-lives of protein-bound
NANA in the total livers (normal livers as well as host livers)
and hepatomas do not differ greatly. The half-lives of plasma
membrane NANA (Chart 5; Table 3) are shorter than in the
unfractionated tissues, but nearly identical in hepatoma and
host liver. The amount of radioactivity incorporated into
glycolipids (chloroform:methanol
extraction) ranged below
5% compared to glycoproteins and is therefore negligible.
Additionally, the half-life of protein-bound radioactivity was
measured in the serum of normal and tumor-bearing rats.
Nearly identical values were obtained for all sera ranging
between 22 and 23 hr.
In
io@
0
12
24
36
hours after injection of ManNAc-1-14C
@
DISCUSSION
The results demonstrate that ManNAc is the precursor
preferred for metabolic studies of protein-bound NANA,
which is in accordance with ganglioside-labeling experiments
using hepatocytes and hepatoma cell lines (4). CMP-NANA
cannot be used because injected nucleotide sugars are split by
nucleotide hydrolases localized in plasma membrane (10). Free
NANA is not suitable because this amino sugar acid is taken up
only to a negligible extent by liver and hepatoma (Table 2).
The advantages of ManNAc are established first by its high rate
of uptake into both liver and hepatoma cells (17) and its
reversible biosynthetic conversion to NANA (14, 37, 41, 42).
This is in contrast to findings using Sarcoma 180 cells, which
took up only very little ManNAc (34). After injection of
labeled ManNAc, the specific radioactivity of protein-bound
NANA increases to a higher level in hepatoma than in the liver.
This may be due to a higher uptake of ManNAc and the
3170
Chart 5. Decay of specific radioactivity of protein-bound NANA
after a single injection of
C (0.3 @Ci/gbody weight;
specific radioactivity, 6 1 mCi/mmole). ‘@,
plasma membranes of Morris
hepatoma
7777; A, total protein
of Morris hepatoma
7777 (each point,
N = 4); 0, plasma membranes of host livers; ., total protein of host
livers (each point, N 4). For experimental details, see “Materials
and
Methods.―
lack of secretion of glycoproteins by hepatoma cells (39).
Secondly, the injection of ManNAc provides only CMP-NANA
in the nucleotide sugar pools needed for glycoprotein
synthesis. This is in contrast to GlcN, which is a precursor for
3 nucleotide sugar pools of different sizes (1 7), UDP-GlcNAc,
UDP-N-acetyl-D-galactosamine,
and CMP-NANA. Conse
quently, the half-life of measured NANA could be longer than
the true half-life of NANA measured by Kawasaki and
Yamashina (22) using GlcN as precursor.
The use of ManNAc, labeled either in its acetyl group or in
the carbon skeleton, yields identical half-lives of NANA. This
indicates that the turnovers of the acetyl group of NANA and
CANCER RESEARCH VOL. 34
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
Half-life
ofNANA
the whole amino sugar acid are identical. The half-life of
protein-bound NANA in the plasma membrane is shorter than
the half-life of the unfractionated cell. Since the main part of
the liver cell-bound NANA is localized in the plasma
membrane
(about
two-thirds;
E. Harms
and
W. Reutter,
unpublished results), it can be assumed that sialoproteins with
a slower turnover rate than in the plasma membrane exist in
other cell compartments.
The half-life of protein-bound NANA is not identical with
its turnover because a possible reutilization of the precursor
leads to a lengthening of the half-life (32). Therefore, it can be
concluded that in plasma membranes the turnover of
NANA is more rapid than the turnover of amino acids, as is
shown with guanidino-labeled arginine (7). This fmding raises
2 questions. (a) Are there constitutive sialoproteins that are
incorporated
into the plasma membrane
attachment
of NANA
sialytransferase
located
would
then
in plasma
without
NANA? The
be performed
membranes.
by a
Differences
between carbohydrate and protein turnover in plasma mem
brane glycoproteins necessitate the existence of glycosyltrans
ferases located in plasma membranes as shown for galactosyl
transferase in single cells by Roth et a!. (38). However, with
respect to plasma membranes of liver cells, the existence of
galactosyltransferase could not be measured (C. Bauer and W.
Reutter, unpublished results) and sialytransferase has not been
described. (b) Do sialoproteins have a more rapid turnover in
plasma membranes than do asialoproteins? That sialoproteins
do in fact have a more rapid turnover is supported by the
findings
of a faster degradation
of glycoproteins
in mouse liver
plasma membranes compared to aglycoproteins (16). This
higher turnover rate of sialoproteins indicates that plasma
membranes
are not degraded as an entity. Their constituents
are synthesized
and degraded at heterogeneous
rates as has
been found for other cellular membranes (1 , 3, 8, 13, 30). The
role of sialidase for this difference remains to be investigated.
No striking
difference
could be measured
either in the total
cell protein or in the isolated plasma membrane when the
half-life of protein-bound NANA was compared in liver and
the Morris hepatoma 7777. The half-life of NANA provides
information regarding the de novo synthesis of NANA for the
hepatoma and liver cells. A sufficient synthetic rate from
ManNAc can be calculated for NANA synthesis in Morris
hepatoma 7777 despite the low enzyme activity of the
UDP-G1cNAc-2'-epimerase with respect to the Km value and
the pool size of UDP-GlcNAc ( 17). Therefore, this decreased
hepatoma enzymic activity may be due to the lack of secretion
of serum glycoproteins (39), which are known to contain an
appreciable percentage of NANA ( 15). The higher synthetic
rate of NANA in the liver seems to be connected with the
specific function of plasma protein secretion. The relationship
between secretion of sialoproteins and activity of the
UDP-GlcNAc-2'-epimerase is not yet known.
We thank Dr. Joane Blaskovics, Dr. Milan Blaskovics, and Dr. C.
for reading
the manuscript
and
Barbara
Hassels for technical
assistance.
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CANCER
RESEARCH
VOL. 34
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1974 American Association for Cancer Research.
Half-life of N-Acetylneuraminic Acid in Plasma Membranes of
Rat Liver and Morris Hepatoma 7777
Erik Harms and Werner Reutter
Cancer Res 1974;34:3165-3172.
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