Vol. 66, No. 3, 1975
BIOCHEMICAL
--In Vitro
Synthesis
of Zein-like
Brian Department
Received
August
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Protein
A. Larkins of Botany
by Maize
Polyribosomes
and Arthur
Dalby
and Plant
Pathology
Purdue
University
West Lafayette,
Indiana
47907
11,1975 SUMMARY
Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes produced one main radioactive protein. This protein was soluble in --in vitro 70% ethanol and had the same mobility in electro horesis on sodium dodecyl sulfate-gels as a zein standard. ofp t dleucine top4d lysine The ratio incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membranebound polyribosomes. INTRODUCTION Two major endosperm zein,
classes
of maize
(1).
much of which
micrographs protein
rough
in distinct reveal
endoplasmic in the
synthesized
in
the developing
is a 70% ethanol-soluble
endosperm
maize
polyribosomes
protein a close
reticulum
synthesis
protein
(3),
of zein
bodies
known as (2).
association
Electron
between
however,
the role
or any other
plant
these
of storage
has not been demonstrated. The discovery
amounts
of zein
the nutritional is known
but
increase
how these
years
of "opaque"
the content
of maize
mutants
of non-zein
seed protein
mutations
alter
(4).
storage
which protein
However, protein
accumulate
small
has greatly virtually
synthesis,
improved nothing
nor how
zein
is controlled,
Protein endosperm
in recent
quality
about
synthesis
protein
are
is compartmentalized
and the
membrane-bound
protein
One of these
of developing
bodies
protein
of storage
synthesized has been
produced
by a protein
reported, (5).
body fraction
but
no attempt
An attempt
to repeat
derived
from developing
was made to analyze these
experiments
the types using
wheat of
a protein
BIOCHEMICAL
Vol. 66, No. 3, 1975
body
fraction This
communication
of a plant in zein
from corn
storage
endosperm is
protein,
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was unsuccessful
first
report
and demonstrates
(6).
of the successful the role
in vitro -___
of membrane-bound
synthesis polyribosomes
synthesis. MATERIALS
AND METHODS
Free and membrane-bound polyribosomes were isolated in an undegraded form from frozen 23-day post-pollination kernels of maize. Kernels were ground in 3-5 volumes of buffer A (0.2 M tris-HCl, pH 8.5; 0.2 M sucrose; 0.06 M KCl; 0.05 M MgC12; 0.005 M dithiothreitol), strained through 4 layers of cheesecloth, and centrifuged at 500 x g for 5 min. in a Sorvall SS-34 rotor. The supernatant was centrifuged at 37,000 x g to separate the free and membrane-bound polyribosomes The supernatant containing free polyribosomes was decanted and layered (7, 8). over 4 ml of 1.75 M sucrose in buffer B (0.04 M tris-HCl, pH 8.5; 0.02 M KCl; and 0.01 M M&12). The 37,000 g pellet was resuspended in buffer A containing 1% material was removed by centrifugation Triton X-100. After 15 min. the insoluble at 37,000 x g for 10 min. The supernatant containing detergent-solubilized membrane-bound polyribosomes was also layered over 4 ml of 1.75 M sucrose in were pelleted by centrifugation for 75 min. at 229,000 x g buffer B. Polyribosomes Polyribosome pellets were avg. in a 65 rotor of a Beckman L2-65 ultracentrifuge. resuspended in buffer B and layered on linear 15-60% sucrose gradients and centrifuged at 189,000 x g avg. for 45 min. in a Beckman SW 50.1 rotor (8, 9). The optical density at 254 nm was recorded with an ISCO Model UA-5 absorbance monitor. Protein synthesis was conducted by adding maize polyribosomes to an --in vitro system derived from wheat germ (10). The reaction mixture contained: 1-2 A260 units of polyribosomes, 20 ug tRNA, 0.12 ml of S23l, 0.048 M KCl, 0.004 M Mgacetate, 0.001 M ATP, 35 uM GTP, 0.011 M creatine phosphate, 0.016 mg. creatine kinase, 0.035 M tris-acetate at pH 8.0, 0.003 M dithiothreitol, 45 uM each of 19 amino acids, and either 0.125 uCi of~4C~leucine (175 mCi/mM) orp4C]-lysine (50 mCi/mM) (Schwartz Bioresearch Inc.) in a final volume of 0.28 ml. Assays were incubated for 60 min. at 300 C. Hot 5% CC13COOH-insoluble and hot 70% ethanol-soluble protein from the in vitro assays were dialyzed in an SDS buffer (0.05 M tris-HCl, pH 6.9; 0.5% SE; 1% 2-mercaptoethanol) and analyzed by electrophoresis in 15% SDS-gels (15% acrylamide; 0.3% bisacrylamide; 0.1% SDS; 0,375 M tris-HCl, pH 8.5; 0.025% TEMED) at 4 ma/gel for 3 hr. (11). The hot acid-insoluble protein from reaction mixtures without polysomes was used as a control. A purified zein preparation (Nutritional Biochemicals Corporation) was solubilized in SDS buffer and used as a standard. The zein standard was stained with Coomassie blue and scanned at 600 nm. The incorporation ofo4C7leucine andp4 C1 lysine into protein was determined by filter paper disk techniques. One half of a reaction mixture was spotted on a cellulose disk and the hot acid-insoluble radioactivity determined (12). The other half of the reaction mixture was adjusted to 70% ethanol and extracted at The hot ethanol-soluble protein was spotted on cellulose disks 60° C for 90 min. and processed through a zein purification procedure (13). The assays were conducted in triplicate and radioactive samples were counted to 7% relative standard error. Lysine counts were multiplied by 3.5 to correct for the difference in specific activity of the two amino acids in the reaction mixtures.
germ:
IAbbreviations: Sodium dodecyl
S23 is sulfate,
a post 23,000 x g supernatant obtained SDS: N,N,N',N'-tetramethylenediamine,
1049
from wheat TEMED.
BIOCHEMICAL
Vol. 66, No. 3, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Typical
density-gradient
polyribosomes
are
gave A260JA280 free
size-class contained absorbance which
several
bound
TOP
showed
distinct
deeper
free
classes
of free 1.
Both
indicating
a normal
size (Fig.
will
but
be presented
BOTTOM
Fig.
were
size-classes
found
TOP
analysis
(manuscript
BOTTOM
as the
polyribosomes
peaks were
to ribonuclease
to actively
The
1-B shows a major
their
Further
(14).
the 10 or ll-mer
absorbance
separately
Figure Free and initially pollinated maize kernels. (A, 1.92 A260 units Of polyribosomes) or buffer units of membrane-bound The optical density at
with
large
susceptible
polyribosomes
of purification
The membrane-bound
not shown).
of polyribosomes
degree
distribution
other
were
membrane-bound
and membrane-bound
size-classes.
in the gradient,
(data
free
1-A).
Several
polyribosomes
and initially
a high
polyribosome
polyribosomes
polyribosomes Both
of 1.7,
peak at the 8-mer.
sedimented
the
in Fig.
of maximum absorbance
The membrane-bound were
depicted
ratios
polyribosomes
profiles
were
separated
not
resolved.
(Fig.
I-C),
as
of the memhranein preparation).
synthesize
TOP
protein
BOTTOM
1
membrane-bound polyribosomes from 23-day postPolyribosome pellets were resuspended in buffer B B, 2.1 A260 units of membrane-bound free polyribosomes; B plus 1 ug/ml pancreatic ribonuclease (C, 0.55 A260 polyribosomes) and separated on 15-60% sucrose gradients. 254 nm was scanned with an ISCO UA-5 absorbance monitor.
1050
BIOCHEMICAL
Vol. 66, No. 3, 1975
when
incubated
from wheat protein zein
in a heterologous
germ (Table
1).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--in vitro
SDS-gel
electrophoresis synthesis
was done
to determine
if
isolated
on the basis
of its
is
zein
Protein
ofp4dLeucine
Synthesized
Polyribosome Type
synthesizing
had occurred in hot
derived
--in vitro.
70% ethanol
Since
(13),
both
1
andL14 C] Lysine
by Free
system
ofp4C]leucine-labeled
solubility
Table Comparison
protein
Incorporation
and Membrane-bound
into
Polyribosome
Fractions.
Hot 70% Ethanol-Soluble Radioactivity (CPM)
Hot Acid-Insoluble Radioactivity (CPM) Leucine
Lysine
Leucine
Lysine
Lysine/Leucine
Free
18,602
12,764
1,615
193
0.012
MembraneBound
35,358
7,469
18,810
332
0.0175
Distribution of radioactivity in hot acid-insoluble and hot 70% ethanolsoluble protein synthesized by free and membrane-bound polyribosome fractions. Assays and extraction procedures are described in Methods and Materials. The radioactive counts were the averages of triplicate assays, and were counted to a relative standard error of 7%. Lysine counts were multiplied by 3.5 to correct for the difference in specific activity between lysine and leucine in reaction mixtures. The counts per minute are expressed per total reaction mixture.
the
acid-insoluble
and hot
purified
zein
preparation
revealed
one intensely
bands migrating gel,
but
they
coincided
peak
represented
total
hot
region
faster
and slower
represented
only
the membrane-bound
than
and hot
the two main
the
radioactive
bands
of the
70% ethanol-soluble
bands
a major
total
radioactive
radioactive
1051
also
Both
standard protein the hot
A
2-C) Other
and minor
appeared
in the
protein.
protein
of the zein
protein.
(Fig.
of two bands.
two main
fraction
included
electrophoresed.
as a standard
34% of the acid-insoluble
ethanol-soluble
were
consisting
a small
polyribosomes
with
protein
was electrophoresed stained
The hot acid-insoluble
that
ethanol-soluble
synthesized peak
(Fig.
(Fig. 2-C).
by 2-B) This
and 57% of the acid-insoluble
and
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
ORIGINC->
RESEARCH COMMUNICATIONS
FRONT(+)
ORIGINL-)
FRONT
C+>
.
C.
1052
BIOCHEMICAL
Vol. 66, No. 3, 1975
hot
ethanol-soluble
molecular
weight
may represent
a smaller
results
Since
zein
only
labeled
the mole
Table hot
1 shows acid-insoluble
of either
free
insoluble
protein
bound
protein
porated
only
the protein
fraction typical
into
ratio
and hot
incorporated (18,810/35,358
9% (1615/18,062
for
Zein were
these
it
Fig.
2-B).
in vitro,
and
of identification
contains
20.2
mole %
the major
product
of
--in vitro
should
fraction
two amino
of the[14C]1
polyribosomes, eucine
into
in zein.
incorporation
produced
by equal
eucine
incorporation,
the should
acids
amounts
in the hot then
50% of theC14C]-leucine
The free
be
In addition,
Whenf14i]1
of total
products
andC14Cllysine protein
Figure
showed
2-A cf.
methods
byb4Cllysine.
polyribosomes.
x 100)
dialysis.
although
was synthesized
zein
ethanol-soluble
x 100).
2-A),
the 70% hot-ethanol
more than
during
the radioactive
of Cl4Cll eucine
was used as a measure
protein
polyribosomes
synthesized
reported
results
removed
(Fig.
other
If
than
weight
(Fig.
of zein.
(15).
incorporated
or membrane-bound
polyribosomes
soluble
% lysine
of smaller
polyribosomes.
activity,
bycl4Clleucine
of lysine/leucine
approximate
protein
composition
polyribosomes,
more extensively
ratio
into
mole
free
bands
made to characterize
acid
amino
0.25
the membrane-bound
were
not
radioactivity
a zein-like
enzymatic
were
of the
total
material
low molecular
which
the membrane-bound
has no known
on the unusual but
of
This
products
of the
that
Attempts
front. chains
RESEARCH COMMUNICATIONS
radioactive
to the two zein
fraction
product
necessary.
leucine,
polypeptide
demonstrate
was the major
based
the
peak corresponding
represented
were
nearer
contained
of the ethanol-soluble
a radioactive
it
fractions
migrating incomplete
SDS-gels
These
protein
AND BIOPHYSICAL
acid-
the membraneinto
ethanol
however,
incor-
ethanol
soluble
protein
2
SDS-gel electrophoresis of P+ C1 leucine labelled products of free and membrane-bound polyribosomes. Figure 2-A depicts the hot acid-insoluble (@) and the hot 70% ethanol-soluble (U) protein synthesized by the free polyribosomes. Figure 2-B depicts the hot acid-insoluble (0) and the hot 70% ethanolsoluble (W) protein synthesized by the membrane-bound polyribosomes. The hot acid-insoluble protein from control mixtures (A> did not contain polyribosomes (Fig. 2-A, Fig. 2-B). Figure 2-C, the zein standard, was stained with Coomassie blue and scanned at 600 nm.
1053
Vol. 66, No. 3, 1975
These
results
BIOCHEMICAL
suggest
that
by the membrane-bound The ratio synthesized and 0.0175 fraction
ratio
these
ratios
leucine
free
indicate
ratio,
Experiments
values
present
expected
was synthesized
the protein of zein
to further
suggest characterize
(15).
as is
it
soluble
predominantly
protein
gave values
the If
produced
of 0.012
lysine/leucine is assumed
suggested
--in vitro
mole that
complete
by Fig.
2, then
had the
lysine
and
(15). identical
that
the ethanol
polyribosomes
in vitro,
in 70% ethanol, strongly
in
approximate
in zein
synthesized that
The solubility
are
These
were
composition
leucine
to C14C]Leucine
and membrane-bound
of 0.0125 chains
protein
polyribosomes.
respectively.
polypeptide
the ethanol-soluble
ofC14C]lysine by the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mobility
the protein the products
in SDS-gels,
synthesized based
--in vitro
and lysine/ is
zein.
on immunoprecipitation
in progress. ACKNOWLEDGEMENT This
This
is
research Journal
was supported Paper
number
in part
by a grant
5925 of the Purdue
from
University
the Lilly Agricultural
Foundation. Experi-
ment Station. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Moss&, J. (1966) Federation Proc., 25, 1663-1669. Wolf, M. J., Khoo, U., and Seckinger, H. L. (1967) Science, 157, 556-557. 57, 1042-1050. Khoo, U., and Wolf, M. J. (1970) Amer. J. Bot., Mertz, E. T., Bates, L. S., and Nelson, 0. E. (1964) Science, 145, 279-280. Morton, R. K., and Raison, J. K. (1964) Biochem. J., 91, 528-538. C. M. (1966) Plant Physiol., 41, 325-327. Wilsan, Davies, E., Larkins, B. A., and Knight, R. H. (1972) Plant Physiol. 50, 581-584. Larkins, B. A., and Davies, E. (1975) Plant Physiol., 55, 749-756. B. A. (1975) Submitted to Plant Physiol. Jackson, A. O., and Larkins, Marcus, A. (1974) in "Methods in Enzymology", K. Moldave and L. Grossman eds. Vol. 30, pp. 749-761, Academic Press, New York. Lee, K. H., Tsai, C. Y., and Dalby, A. (1975) (in preparation). G. D, (1961) Arch. Biochem. Biophys., 94, 48-53. Mans, R. J., and Novelli, Dalby, A. (1974) Cereal Chem., 51, 586-592. in Protein Biosynthesis", P. N. Campbell and Nell, H. (1966) in "Techniques .I. R. Sargent eds. Vol. 2, pp. 101-178. Murphy, J. J., and Dalby, A. (1971) Cereal Chem., 48, 336-349.
1054
BIOCHEMICAL
--In Vitro
Synthesis
of Zein-like
Brian Department
Received
August
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Protein
A. Larkins of Botany
by Maize
Polyribosomes
and Arthur
Dalby
and Plant
Pathology
Purdue
University
West Lafayette,
Indiana
47907
11,1975 SUMMARY
Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes produced one main radioactive protein. This protein was soluble in --in vitro 70% ethanol and had the same mobility in electro horesis on sodium dodecyl sulfate-gels as a zein standard. ofp t dleucine top4d lysine The ratio incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membranebound polyribosomes. INTRODUCTION Two major endosperm zein,
classes
of maize
(1).
much of which
micrographs protein
rough
in distinct reveal
endoplasmic in the
synthesized
in
the developing
is a 70% ethanol-soluble
endosperm
maize
polyribosomes
protein a close
reticulum
synthesis
protein
(3),
of zein
bodies
known as (2).
association
Electron
between
however,
the role
or any other
plant
these
of storage
has not been demonstrated. The discovery
amounts
of zein
the nutritional is known
but
increase
how these
years
of "opaque"
the content
of maize
mutants
of non-zein
seed protein
mutations
alter
(4).
storage
which protein
However, protein
accumulate
small
has greatly virtually
synthesis,
improved nothing
nor how
zein
is controlled,
Protein endosperm
in recent
quality
about
synthesis
protein
are
is compartmentalized
and the
membrane-bound
protein
One of these
of developing
bodies
protein
of storage
synthesized has been
produced
by a protein
reported, (5).
body fraction
but
no attempt
An attempt
to repeat
derived
from developing
was made to analyze these
experiments
the types using
wheat of
a protein
BIOCHEMICAL
Vol. 66, No. 3, 1975
body
fraction This
communication
of a plant in zein
from corn
storage
endosperm is
protein,
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was unsuccessful
first
report
and demonstrates
(6).
of the successful the role
in vitro -___
of membrane-bound
synthesis polyribosomes
synthesis. MATERIALS
AND METHODS
Free and membrane-bound polyribosomes were isolated in an undegraded form from frozen 23-day post-pollination kernels of maize. Kernels were ground in 3-5 volumes of buffer A (0.2 M tris-HCl, pH 8.5; 0.2 M sucrose; 0.06 M KCl; 0.05 M MgC12; 0.005 M dithiothreitol), strained through 4 layers of cheesecloth, and centrifuged at 500 x g for 5 min. in a Sorvall SS-34 rotor. The supernatant was centrifuged at 37,000 x g to separate the free and membrane-bound polyribosomes The supernatant containing free polyribosomes was decanted and layered (7, 8). over 4 ml of 1.75 M sucrose in buffer B (0.04 M tris-HCl, pH 8.5; 0.02 M KCl; and 0.01 M M&12). The 37,000 g pellet was resuspended in buffer A containing 1% material was removed by centrifugation Triton X-100. After 15 min. the insoluble at 37,000 x g for 10 min. The supernatant containing detergent-solubilized membrane-bound polyribosomes was also layered over 4 ml of 1.75 M sucrose in were pelleted by centrifugation for 75 min. at 229,000 x g buffer B. Polyribosomes Polyribosome pellets were avg. in a 65 rotor of a Beckman L2-65 ultracentrifuge. resuspended in buffer B and layered on linear 15-60% sucrose gradients and centrifuged at 189,000 x g avg. for 45 min. in a Beckman SW 50.1 rotor (8, 9). The optical density at 254 nm was recorded with an ISCO Model UA-5 absorbance monitor. Protein synthesis was conducted by adding maize polyribosomes to an --in vitro system derived from wheat germ (10). The reaction mixture contained: 1-2 A260 units of polyribosomes, 20 ug tRNA, 0.12 ml of S23l, 0.048 M KCl, 0.004 M Mgacetate, 0.001 M ATP, 35 uM GTP, 0.011 M creatine phosphate, 0.016 mg. creatine kinase, 0.035 M tris-acetate at pH 8.0, 0.003 M dithiothreitol, 45 uM each of 19 amino acids, and either 0.125 uCi of~4C~leucine (175 mCi/mM) orp4C]-lysine (50 mCi/mM) (Schwartz Bioresearch Inc.) in a final volume of 0.28 ml. Assays were incubated for 60 min. at 300 C. Hot 5% CC13COOH-insoluble and hot 70% ethanol-soluble protein from the in vitro assays were dialyzed in an SDS buffer (0.05 M tris-HCl, pH 6.9; 0.5% SE; 1% 2-mercaptoethanol) and analyzed by electrophoresis in 15% SDS-gels (15% acrylamide; 0.3% bisacrylamide; 0.1% SDS; 0,375 M tris-HCl, pH 8.5; 0.025% TEMED) at 4 ma/gel for 3 hr. (11). The hot acid-insoluble protein from reaction mixtures without polysomes was used as a control. A purified zein preparation (Nutritional Biochemicals Corporation) was solubilized in SDS buffer and used as a standard. The zein standard was stained with Coomassie blue and scanned at 600 nm. The incorporation ofo4C7leucine andp4 C1 lysine into protein was determined by filter paper disk techniques. One half of a reaction mixture was spotted on a cellulose disk and the hot acid-insoluble radioactivity determined (12). The other half of the reaction mixture was adjusted to 70% ethanol and extracted at The hot ethanol-soluble protein was spotted on cellulose disks 60° C for 90 min. and processed through a zein purification procedure (13). The assays were conducted in triplicate and radioactive samples were counted to 7% relative standard error. Lysine counts were multiplied by 3.5 to correct for the difference in specific activity of the two amino acids in the reaction mixtures.
germ:
IAbbreviations: Sodium dodecyl
S23 is sulfate,
a post 23,000 x g supernatant obtained SDS: N,N,N',N'-tetramethylenediamine,
1049
from wheat TEMED.
BIOCHEMICAL
Vol. 66, No. 3, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Typical
density-gradient
polyribosomes
are
gave A260JA280 free
size-class contained absorbance which
several
bound
TOP
showed
distinct
deeper
free
classes
of free 1.
Both
indicating
a normal
size (Fig.
will
but
be presented
BOTTOM
Fig.
were
size-classes
found
TOP
analysis
(manuscript
BOTTOM
as the
polyribosomes
peaks were
to ribonuclease
to actively
The
1-B shows a major
their
Further
(14).
the 10 or ll-mer
absorbance
separately
Figure Free and initially pollinated maize kernels. (A, 1.92 A260 units Of polyribosomes) or buffer units of membrane-bound The optical density at
with
large
susceptible
polyribosomes
of purification
The membrane-bound
not shown).
of polyribosomes
degree
distribution
other
were
membrane-bound
and membrane-bound
size-classes.
in the gradient,
(data
free
1-A).
Several
polyribosomes
and initially
a high
polyribosome
polyribosomes
polyribosomes Both
of 1.7,
peak at the 8-mer.
sedimented
the
in Fig.
of maximum absorbance
The membrane-bound were
depicted
ratios
polyribosomes
profiles
were
separated
not
resolved.
(Fig.
I-C),
as
of the memhranein preparation).
synthesize
TOP
protein
BOTTOM
1
membrane-bound polyribosomes from 23-day postPolyribosome pellets were resuspended in buffer B B, 2.1 A260 units of membrane-bound free polyribosomes; B plus 1 ug/ml pancreatic ribonuclease (C, 0.55 A260 polyribosomes) and separated on 15-60% sucrose gradients. 254 nm was scanned with an ISCO UA-5 absorbance monitor.
1050
BIOCHEMICAL
Vol. 66, No. 3, 1975
when
incubated
from wheat protein zein
in a heterologous
germ (Table
1).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--in vitro
SDS-gel
electrophoresis synthesis
was done
to determine
if
isolated
on the basis
of its
is
zein
Protein
ofp4dLeucine
Synthesized
Polyribosome Type
synthesizing
had occurred in hot
derived
--in vitro.
70% ethanol
Since
(13),
both
1
andL14 C] Lysine
by Free
system
ofp4C]leucine-labeled
solubility
Table Comparison
protein
Incorporation
and Membrane-bound
into
Polyribosome
Fractions.
Hot 70% Ethanol-Soluble Radioactivity (CPM)
Hot Acid-Insoluble Radioactivity (CPM) Leucine
Lysine
Leucine
Lysine
Lysine/Leucine
Free
18,602
12,764
1,615
193
0.012
MembraneBound
35,358
7,469
18,810
332
0.0175
Distribution of radioactivity in hot acid-insoluble and hot 70% ethanolsoluble protein synthesized by free and membrane-bound polyribosome fractions. Assays and extraction procedures are described in Methods and Materials. The radioactive counts were the averages of triplicate assays, and were counted to a relative standard error of 7%. Lysine counts were multiplied by 3.5 to correct for the difference in specific activity between lysine and leucine in reaction mixtures. The counts per minute are expressed per total reaction mixture.
the
acid-insoluble
and hot
purified
zein
preparation
revealed
one intensely
bands migrating gel,
but
they
coincided
peak
represented
total
hot
region
faster
and slower
represented
only
the membrane-bound
than
and hot
the two main
the
radioactive
bands
of the
70% ethanol-soluble
bands
a major
total
radioactive
radioactive
1051
also
Both
standard protein the hot
A
2-C) Other
and minor
appeared
in the
protein.
protein
of the zein
protein.
(Fig.
of two bands.
two main
fraction
included
electrophoresed.
as a standard
34% of the acid-insoluble
ethanol-soluble
were
consisting
a small
polyribosomes
with
protein
was electrophoresed stained
The hot acid-insoluble
that
ethanol-soluble
synthesized peak
(Fig.
(Fig. 2-C).
by 2-B) This
and 57% of the acid-insoluble
and
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
ORIGINC->
RESEARCH COMMUNICATIONS
FRONT(+)
ORIGINL-)
FRONT
C+>
.
C.
1052
BIOCHEMICAL
Vol. 66, No. 3, 1975
hot
ethanol-soluble
molecular
weight
may represent
a smaller
results
Since
zein
only
labeled
the mole
Table hot
1 shows acid-insoluble
of either
free
insoluble
protein
bound
protein
porated
only
the protein
fraction typical
into
ratio
and hot
incorporated (18,810/35,358
9% (1615/18,062
for
Zein were
these
it
Fig.
2-B).
in vitro,
and
of identification
contains
20.2
mole %
the major
product
of
--in vitro
should
fraction
two amino
of the[14C]1
polyribosomes, eucine
into
in zein.
incorporation
produced
by equal
eucine
incorporation,
the should
acids
amounts
in the hot then
50% of theC14C]-leucine
The free
be
In addition,
Whenf14i]1
of total
products
andC14Cllysine protein
Figure
showed
2-A cf.
methods
byb4Cllysine.
polyribosomes.
x 100)
dialysis.
although
was synthesized
zein
ethanol-soluble
x 100).
2-A),
the 70% hot-ethanol
more than
during
the radioactive
of Cl4Cll eucine
was used as a measure
protein
polyribosomes
synthesized
reported
results
removed
(Fig.
other
If
than
weight
(Fig.
of zein.
(15).
incorporated
or membrane-bound
polyribosomes
soluble
% lysine
of smaller
polyribosomes.
activity,
bycl4Clleucine
of lysine/leucine
approximate
protein
composition
polyribosomes,
more extensively
ratio
into
mole
free
bands
made to characterize
acid
amino
0.25
the membrane-bound
were
not
radioactivity
a zein-like
enzymatic
were
of the
total
material
low molecular
which
the membrane-bound
has no known
on the unusual but
of
This
products
of the
that
Attempts
front. chains
RESEARCH COMMUNICATIONS
radioactive
to the two zein
fraction
product
necessary.
leucine,
polypeptide
demonstrate
was the major
based
the
peak corresponding
represented
were
nearer
contained
of the ethanol-soluble
a radioactive
it
fractions
migrating incomplete
SDS-gels
These
protein
AND BIOPHYSICAL
acid-
the membraneinto
ethanol
however,
incor-
ethanol
soluble
protein
2
SDS-gel electrophoresis of P+ C1 leucine labelled products of free and membrane-bound polyribosomes. Figure 2-A depicts the hot acid-insoluble (@) and the hot 70% ethanol-soluble (U) protein synthesized by the free polyribosomes. Figure 2-B depicts the hot acid-insoluble (0) and the hot 70% ethanolsoluble (W) protein synthesized by the membrane-bound polyribosomes. The hot acid-insoluble protein from control mixtures (A> did not contain polyribosomes (Fig. 2-A, Fig. 2-B). Figure 2-C, the zein standard, was stained with Coomassie blue and scanned at 600 nm.
1053
Vol. 66, No. 3, 1975
These
results
BIOCHEMICAL
suggest
that
by the membrane-bound The ratio synthesized and 0.0175 fraction
ratio
these
ratios
leucine
free
indicate
ratio,
Experiments
values
present
expected
was synthesized
the protein of zein
to further
suggest characterize
(15).
as is
it
soluble
predominantly
protein
gave values
the If
produced
of 0.012
lysine/leucine is assumed
suggested
--in vitro
mole that
complete
by Fig.
2, then
had the
lysine
and
(15). identical
that
the ethanol
polyribosomes
in vitro,
in 70% ethanol, strongly
in
approximate
in zein
synthesized that
The solubility
are
These
were
composition
leucine
to C14C]Leucine
and membrane-bound
of 0.0125 chains
protein
polyribosomes.
respectively.
polypeptide
the ethanol-soluble
ofC14C]lysine by the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mobility
the protein the products
in SDS-gels,
synthesized based
--in vitro
and lysine/ is
zein.
on immunoprecipitation
in progress. ACKNOWLEDGEMENT This
This
is
research Journal
was supported Paper
number
in part
by a grant
5925 of the Purdue
from
University
the Lilly Agricultural
Foundation. Experi-
ment Station. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Moss&, J. (1966) Federation Proc., 25, 1663-1669. Wolf, M. J., Khoo, U., and Seckinger, H. L. (1967) Science, 157, 556-557. 57, 1042-1050. Khoo, U., and Wolf, M. J. (1970) Amer. J. Bot., Mertz, E. T., Bates, L. S., and Nelson, 0. E. (1964) Science, 145, 279-280. Morton, R. K., and Raison, J. K. (1964) Biochem. J., 91, 528-538. C. M. (1966) Plant Physiol., 41, 325-327. Wilsan, Davies, E., Larkins, B. A., and Knight, R. H. (1972) Plant Physiol. 50, 581-584. Larkins, B. A., and Davies, E. (1975) Plant Physiol., 55, 749-756. B. A. (1975) Submitted to Plant Physiol. Jackson, A. O., and Larkins, Marcus, A. (1974) in "Methods in Enzymology", K. Moldave and L. Grossman eds. Vol. 30, pp. 749-761, Academic Press, New York. Lee, K. H., Tsai, C. Y., and Dalby, A. (1975) (in preparation). G. D, (1961) Arch. Biochem. Biophys., 94, 48-53. Mans, R. J., and Novelli, Dalby, A. (1974) Cereal Chem., 51, 586-592. in Protein Biosynthesis", P. N. Campbell and Nell, H. (1966) in "Techniques .I. R. Sargent eds. Vol. 2, pp. 101-178. Murphy, J. J., and Dalby, A. (1971) Cereal Chem., 48, 336-349.
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