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Proc. Nat. Acad. Sci. USAVol. 71, No. 4, pp. 1322-1325, April
1974
The Synthesis of Human Placental Lactogen by Ribosomes
Derivedfrom Human Placenta
(reproduction/protein synthesis/hormone/pregnancy)
IRVING BOIME AND SOPHIE BOGUSLAWSKI
Departments of Obstetrics and Gynecology and Pharmacology,
Washington University School of Medicine, 660 S. Euclid Ave.,St.
Louis, Missouri 63110
Communicated by Oliver H. Lowry, December 7, 1973
ABSTRACT In a very active cell-free system containingpolysomes
derived from human placenta and a cell-sapfraction prepared from
ascites tumor cells, the syn-thesis of the hormone human placental
lactogen (HPL)was detected. The identification was based on the
follow-ing: (a) The in vitro synthesized protein labeled
withP5Simethionine migrated at the same rate as authenticHPL on
sodium dodecyl sulfate-polyacrylamide gels and(b) tryptic
fingerprint analysis of the labeled proteinyielded peptides having
the same mobilities as seen withthe same analysis of purified
HPL.The amount of HPL synthesized in a cell-free system
containing polysomes derived from term placenta wasabout 10% of
the total proteins synthesized aind in a com-parable system
containing first trimester ribosomes thelevel of synthesis was
about 5%. These data suggest thepotential for quantitating the HPL
mRNA activity as afunction of the period of gestation and for
isolating themRNA itself.
It is well established that the human placenta synthesizes
atleast two protein hormones, human chorionic gonadotrophin(HCG)
and human placental lactogen (HPL). The levels ofthese hormones
peak at different periods of gestation; HCGreaches its peak level
in serum and urine at 10-12 weeks,whereas, the highest titers of
HPL are attained at term. Infact, HPL seems to represent a major
protein synthesized inthe placenta at terrh (1-3). Therefore, it is
apparent that thesynthesis and/or secretion of these two hormones
is differen-tially coupled to gestation.
In order to investigate the factors involved in regulatingthe
synthesis of the placental protein hormones, it would behelpful to
examine their production from placental polysomesin vitro. Such a
cell-free system would also be useful for study-ing the
biosynthesis of placental peptide hormones as a func-tion of
gestation. Furthermore, the possession of polysomessynthesizing
these proteins is a prerequisite for isolatingmRNA's specific for
HPL and HCG.We have previously shown that ribosomes with
relatively
high endogenous activity can be prepared from first andthird
trimester placentas (4). In the present work, it was ob-served that
in cell-free systems containing either first or thirdtrimester
polysomes, the placental hormone HPL was syn-thesized and
represented a substantial portion of total pro-tein
synthesized.
MATERIALS AND METHODS
[I5S]Methionine was obtained from Amersham Searle.
Humanplacental lactogen (95% pure) was purchased from Nutri-
Abbreviations: HPL, human placental lactogen; HCG,
humanchorionic gonadotrophin.
tional Biochemicals. Rat liver tRNA was generously providedby
Dr. Dolph Hatfield.
Isolation of Placental Ribsomes. Ribosomes derived fromfirst and
third trimester placentas were prepared as describedpreviously (4).
The tissue, which was washed free of blood,was pressed through a
1.5-mm grid to remove connectivetissue and vasculature. The
preparations were further homog-enized on a 1:1 v/w basis in a
buffer containing 30 mMTris - HCl, (pH 7.5), 120 mM KCl, 7 mM
2-mercaptoethanol,5 mM magnesium acetate, and 0.5 mM EDTA.
Homogeniza-tion was carried out in the cold for about 3 min with
motor-driven Teflon and glass homogenizers (Thomas Co.,
Philadel-phia, Pa.). The homogenate was then centrifuged at 8500 Xg
for 10 min at 4°. The supernatant fluid was brought to
1%deoxycholate concentration with a 10% solution. This sus-pension
was then layered on a discontinuous sucrose gradientcomposed of 4
ml each of 40 and 45% sucrose solutions pre-pared in the
homogenizing buffer. The gradients were placedin a Beckman type 60
titanium rotor and centrifuged at200,000 X gfor3hr at 4'.The top
layers were then aspirated and the tubes containing
the pellets were gently rinsed with homogenizing buffer andthe
pellets were resuspended in this buffer with a small
handhomogenizer. (Occasionally, some white, fluffy
materialcollected around the ribosome pellet; much of this was
re-moved with a stirring rod.)
Preparation of Ribosome-Free Supernates (Cell-Sap) fromPlacenta
and Ascites Tumor Cells. The preparation of the cell-sap fraction
from the placenta was carried out as describedabove except for the
following: (a) there was no EDTA inthe homogenizing buffer and (b)
the post-mitochondrialsupernatant fluid was not treated with
deoxycholate, and itwas centrifuged at 250,000 X g for 2 hr in the
absence ofof sucrose solutions. The cell-sap fraction so obtained
wasdialyzed overnight against the homogenizing buffer withoutEDTA
and was stored in 200- to 300-Al aliquots in liquidnitrogen. The
ribosomal and cell-sap fractions derived fromKrebs II ascites tumor
cells, except for the omission of thepreincubation step, were
prepared as described elsewhere (5).
Assay for Protein Synthesis. Protein synthesis was assayedin
0.06 ml reaction mixtures composed of 30 mM TristHCl (pH 7.5), 3.3
mM magnesium acetate, 70 mM KCl, 7mM 2-mercaptoethanol, 1 mM ATP,
0.1 mM GTP, 0.6 mMCTP, 10 mM creatine phosphate, 0.16 mg/ml of
creatinekinase, 40 AM each of 19 nonradioactive amino acids and0.5
MM [35S]rnethionine (specific activity 200--300 Ci/mmol).
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Cell-Free Synthesis of Human Placental Lactogen 1323
TABLE 1. The incorporation of [tIS]methionine intoproteins
synthesized by placenta ribosomes
[35S]Metincorp
Ribosomes Cell-sap (cpm/0.06 ml)
None Ascites 9,500Placenta (F.T.) Ascites 625,000Placenta (T.T.)
Ascites 675,000Placenta (T.T.) Placenta (F.T.) 545,000Placenta
(T.T.) None 10,800
The assays were performed as described in Materials and
Meth-ods. Where indicated, each reaction mixture contained the
cell-sapequivalent of 80 and 130 ,ug of protein for ascites and
placenta,respectively, and 50 ,g of ribosomal RNA. The placentas
werefrom either the first trimester (F. T.) or third trimester (T.
T.)
In addition, 3 Ag of rat-liver tRNA was added to all
reactions.The amount of ribosomes and cell-sap added will be noted
inthe appropriate experiment. Incubation was at 330 for 90min. The
reactions were stopped by the addition of either0.2 ml of 0.1 N KOH
or 25 gg of pancreatic ribonuclease.Incubation was continued for 20
min and 1 ml of 10% coldCC13COOH was then added. The mixture was
cooled at 00for 5 min and the precipitate was collected on a
0.45-jum poresize Millipore filter, washed three times with 3 ml
each of 5%CC13COOH, dried and counted in a Packard liquid
scintilla-tion counter.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.A
five-fold scaled up reaction mixture containing [35S ]methio-nine
was treated with 0.2 ml of 0.1 N KOH per 0.06 ml as de-scribed
above. Following incubation, the mixture was ad-justed to 10% in
CClaCOOH, allowed to incubate for 10 minat 40, then centrifuged at
4000 rpm in a Sorvall SS-34 rotorfor 10 min. The precipitate was
washed once with cold 5%CC13COOH and twice with acetone to remove
residual CC13-COOH.The samples of in vitro products were prepared
for analysis
by dissolving them in 0.02 M Tris * HCl (pH 6.8) containing1%
sodium dodecyl sulfate, 1% 2-mercaptoethanol, 10%glycerol, and
0.001% bromphenol blue, followed by heating at100° for 1 min. The
proteins were electrophoresed at 200 Vfor 4-5 hr in a slab
containing a linear 7-28% gradient ofpolyacrylamide and
subsequently stained and destained ac-cording to procedures
previously described (5). The driedslabs were then exposed to x-ray
film (Kodak RPR-54) for1-3 days.
Tryptic Peptide Analysis. Ten to 20-fold scaled up
reactionmixtures were incubated for 90 min after which 25 jig
ofribonuclease per 0.06 ml was added. The mixtures were
thenincubated and processed for acrylamide gel electrophoresis
asdescribed above. A sample containing 2 to 4 X 106 cpm
wasdistributed to nine 0.6-cm slots extending across the top of
anSDS-polyacrylamide slab gel. In the two lanes near the
ex-tremities of the gel 10 jig of purified HPL was applied.
Im-mediately after electrophoresis the two end strips containingthe
HPL standard and one adjacent lane containing an aliquotof the
labeled mixture were sliced away from the main portionof the gel
and stained. The remaining untreated gel wasimmediately dried and
then an autoradiograph was obtainedfollowing a 10-hr exposure. The
band corresponding to HPL
Molecular weight
47,500
21,600-
A P
FIG. 1. Autoradiograph of [35S]methionine
labeled-proteinssynthesized in a cell-free system containing
ribosomes from thirdtrimester placenta (P) or Krebs II ascites
tumor cells (A). Thecell-sap used was prepared from ascites tumor
cells. Sodium do-decyl sulfate-polyacrylamide gradient (7-28%) gel
electrophoresiswas carried out as described in Materials and
Methods. Approx-imately 100,000 cpm of CCl3COOH-precipitable
material was ap-plied to each lane. The molecular weight standards
indicatedcorrespond to heavy chain of IgG (47,500) and HPL (21,600;
95%pure, Nutritional Biochemicals).
was cut out with scissors and placed in a centrifuge tubewith
5-10 ml of H20. The suspension was incubated first for2 hr at 370
and then overnight at 4°. The mixture was centri-fuged at 10,000 X
g for 15 min and the supernatant fluid wasdecanted and saved. This
fraction was lyophilized and theresidue was taken up with 1 ml of
water. Seven milligrams ofpurified unlabeled HPL were added; this
mixture was dena-tured, treated with trypsin, and then the digests
were chroma-tographed and electrophoresed to yield tryptic maps as
de-scribed previously (5). The maps were exposed to x-ray filmand
stained with ninhydrin to localize the unlabeled peptides.
RESULTS
The high endogenous protein synthetic activity of
placentaribosomes is shown by the incorporation of
[35S]methionine(Table 1). As found previously (4), the cell-sap
fraction fromascites tumor cells is somewhat more active than
homologouscell-sap. It can also be seen that the endogenous
activitiesof the polysomes from first trimester and term are
comparable.To investigate the nature of the proteins synthesized,
ribo-
somes derived from third trimester tissue were incubated
withcell-sap from ascites cells. The [3S]methionine labeled
prod-ucts were analyzed by sodium dodecyl sulfate-acrylamide
gelelectrophoresis (Fig. 1). There were apparently several pro-
Proc. Nat. Acad. Sci. USA 71 (1974)
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1324 Biochemistry: Boime and Boguslawski
..;
.j
..f
.:'.'4
I..~~~~Ae2~~~~~~~~~~~~~~~~~~~~~~~~~~
5
e4}w ~* B
FIG. 2. Two-dimensional tryptic fingerprint analyses of 7 mgof
unlabeled carrier HPL and of a mixture of the labeled proteinthat
electrophoresed at the same rate as HPL. The equivalent ofabout
200,000 cpm was mixed with the carrier. Panel A is
theautoradiograph of panel B, which has been stained with
ninhy-drin. The ninhydrin-positive peptides of HPL which show
thesame mobility as the labeled peptides are denoted by the
dottedrings.
teins synthesized in the cell-free system, many of which
weredifficult to resolve from the background. Some of the samebands
appeared when proteins synthesized by ribosomes fromascites cells
were also examined. However, at least one majordistinct protein was
synthesized only by the placental ribo-somes. This protein did not
appear when pancreatic ribonu-clease was used to inhibit protein
synthesis and migrated atthe same rate as purified HPL.More direct
identification was obtained as follows: The
band containing the protein was eluted from a preparativegel,
mixed with purified unlabeled HPL, digested with trypsin,and the
resulting peptides analyzed by two dimensionalchromatography and
electrophoresis. The fingerprints weresubjected to autoradiography
and then sprayed with ninhy-drin in order to localize the peptides
derived from the purifiedcarrier. The exposed x-ray film was then
compared with theninhydrin-stained fingerprint. HPL contains six
methionineresidues (6, 7) each of which is distributed in a single
trypticpeptide. Two of these methionine containing peptides
contain21 and 29 amino acids and it is probable they would not
mi-grate in the solvent systems employed. Based on the amino-
Molecular weight
47,500 -
21,600-
Proc. Nat. Acad. Sci. USA 71 (1974)
A._ .. :..... .. .- ..
_h _ Al.. :
A.*_ __- '.-
i:
--- ---- -w - -
T.T. F.T. F.T.
Opp,or VW._ ._-W
T.T.
FIG. 3. Autoradiograph of [35S]methionine-labeled
proteinssynthesized in the cell-free system containing ribosomes
from firsttrimester (F.T.) or third trimester (T.T.) placenta.
Approxi-mately 100,000 cpm of CCl3COOH-precipitable material was
ap-plied to lanes denoted F.T. and T.T. The third lane
contained50,000 cpm each of the F.T. and T.T. samples.
acid sequence of HPL (6, 7), tryptic hydrolysis should
theoret-ically yield 21 peptides. The ninhydrin-stained map
displays19 major peptides and about 5 minor ones (Fig. 2B).As can
be seen in Fig. 2A, there are more than six [13S]-
methionine-containing peptides present on the autoradio-graph.
However, four labeled peptides have identical mobili-ties with
peptides of purified HPL. These are denoted inFig. 2B by the dotted
lines surrounding the correspondingninhydrin positive peptides.
This pattern was consistentlyobserved on maps of this protein in
each of three independentexperiments.
Peptide number 2 in Fig. 2 does not correspond preciselyto a
ninhydrin positive peptide although it was always presenton maps of
the labeled protein. Peptide numbers 1 and 3 to 5apparently overlap
with ninhydrin-stained peptides; how-ever, this correspondence is
not reproducible and thus mayreflect nonspecific tryptic cleavages.
The other labeled pep-tides do not have ninhydrin counterparts and
must reflectpeptides derived from other proteins eluted from the
gel. -The peptide spots were cut out of the map and their
radio-
activity determined. The four labeled coincident
peptidesconstituted 50-60% of the total radioactivity among
thelabeled areas detected by the autoradiograph. (The
radio-activity at the origin was not included in this
calculation.)The intensity of the labeling of the peptides probably
re-
flects their position in the HPL molecule since the amount
ofreinitiation in this system is not great (unpublished
obser-vation). Therefore, peptides nearest the carboxyl end of
the
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Cell-Free Synthesis of Human Placental Lactogen 1325
protein will have the greatest radioactivity. This assumesthat
the yield of each tryptic peptide is the same. Preliminarysequence
analyses performed in collaboration with Dr. RuthHogue Angeletti
suggest that the least radioactive peptide ofthe four coincident
ones seen in Fig. 2A is the peptide closestto the amino
terminus.The ninhydrin-stained fingerprint in Fig. 2B
represents
carrier HPL plus a much smaller mass of protein eluted fromthe
gel. It is unlikely that the eluted protein was sufficient togive
rise to visible spots (which might account for the coin-cident
peptides). This conclusion is supported by a finger-print of the
carrier by itself; this was indistinguishable fromthat in Fig. 2B.
As a further control, labeled proteins syn-thesized by ascites
polysomes were eluted from the sameregion of a preparative
polyacrylamide gel as the labeledprotein of Fig. 2 and
fingerprinted. There was no corre-spondence between the labeled
areas and the ninhydrinpositive peptides. These data strongly
suggest that the bandmigrating at the same rate as HPL on the
polyacrylamide gelwas in fact HPL.
Polyacrylamide gel analysis of the proteins synthesized byfirst
trimester ribosomes also revealed a band migrating asHPL (Fig. 3).
Trypsin treatment of this protein yielded aradioactive fingerprint
similar to that obtained with termribosomes.
For quantitating levels of the protein in the HPL region,
theband was cut out from the dried gel and the radioactivity
de-termined. In the case of third trimester ribosomes the
bandcontained about 10% of the total amount of radioactivityapplied
to the gel; in the case of first trimester ribosomes thefigure was
about 5%. These values somewhat overestimatethe HPL synthesized
since, while the band is very discrete, acertain amount of other
labeled protein comigrates in thisregion, as shown above.
DISCUSSION
The (overestimated) value for the percentage of HPL syn-thesized
in the cell-free system is in satisfactory agreementwith the data
obtained by Friesen, et al. (8) who showed thatin term placenta
slices, HPL constitutes 2-4% of the totalprotein synthesized. The
results are also consistent with invivo findings which indicate
that in the third trimester ofpregnancy the placenta secretes
larger quantities of the proteininto maternal serum than in the
first trimester (3, 9).
It has been proposed that HPL might be derived from aprecursor
protein (8, 10). This was based on the finding thatin a mixture of
labeled proteins from term placenta slices, some
large molecular-weight proteins were precipitated with
HPLantibody. While the predominant protein synthesized in
thecell-free system is HPL, it is possible that a precursor
isgenerated which is rapidly cleaved to HPL. Furthermore, aprotein
with a small variation in molecular weight might notbe detected on
polyacrylamide gels. Perhaps a more defini-tive answer regarding
this point can be obtained by iso-lating the HPL mRNA and
translating it in a nonplacentalcell-free system. This approach
might be fruitful since thecleavage activity for some precursors
may reside in the micro-somal fraction of the cell (12). It was
demonstrated that themRNA encoding for the light chain derived from
a myelomatumor was translated in a heterologous cell-free system
andan apparent precursor of this protein was detected (11, 12).
Since polysomes from placenta can synthesize a significantamount
of a specific human placental hormone, HPL, theypresumably contain
correspondingly high levels of the specificmRNA, which can be
isolated and used as a reagent to studythe regulation of the
biosynthesis of this peptide hormone.
We are grateful to Dr. Dolph Hatfield for his generous gift
oftRNA and to Dr. Ruth Hogue Angeletti for performing some
se-quence analyses on the tryptic peptides and to Charles
Lawrencefor his helpful discussion. The authors would also like to
thankKathy Neely for her excellent assistance in preparing this
manu-script. This work was aided in part by a grant from the
Popula-tion Council (#M73, 135), and NIH Grant #Am-16865.
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Proc. Nat. Acad. Sci. USA 71 (1974)
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