Chromatographic analysis of polypeptides from polyribonucleotide stimulated enzymatic synthesis

Vol. 13, No. 4, 1963

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CBBOMATOGRAPBIC ANALYSIS OFpOLYPR'TlDESFROMPOLYRIBONUCLEOTIDE STIMUL4TEDENZYMATICSYNTBESIS

by Marvin A. Smith and Mark A. Stahmann

Department of Biochemistry,

University

of Wisconsin

Madison 6, Wisconsin

Received

August

29,

1963

In this communication we report the use of co-chromatography for fractionating

into polypeptides,

and the identity

peptiaes obtained from a partial Samples of the lysine-C laboratories

of lysine-C 14

products from the enzymatic incorporation

of these products with known poly-

hydrolyzate

14 polypeptides,

of synthetic

enzymatically

of Dr. S. Ochoa (Gardner gal.,

1962;

using a polyadenylic

acid (poly-A)

isolated

, were added to a partial

from E.

preparation

polylysine.

synthesized in the Kaziro et al.,

1963)

and an enzyme system acid hydrolyzate

of poly-

lysine and subjected to chrcmatography on carboxymethyl-cellulose All the radioactivity

in the enzymatically

recovered in the first

14 polylysine

recovered as dilysine,

but significant

polypeptides up to the tetradecamer. not significantly

greater

In such biosynthetic for the incorporation

columns.

synthesized products was

peaks.

The highest activity

was

was found in all

lysine

activity Activities

in larger peptiaes were

than background. systems, poly-A is considered to be the template

of lysine residues into polypeptldes.

Assuming that

the amino acid code requires a fixed nuniber of nucleotides for the incorporation

of each amino acid residue, one w0ula expect a correlation 251

Vol. 13, No. 4, 1963

BIOCHEMICAL

between the size of the po4-A

AND BIOPHYSICAL

and the size of the resulting

Consequently, a determination

of the size distribution

to stimulate the synthesis and of the po44sine the number of nucleotide residue.

Materials

These reaction inactivate

synthesized should reveal

and Methods.

of the polypeptides.

Aliquots

of the cell-free

provided by Drs. Y. Kaziro,

mixtures were boiled after

enzymes, and frozen until

described (Stewart,

J. W., et al.,

analyzed.

was prepared by partial

using techniques already

One ml of the reaction

columns. The carrier

acid hydrolysis

hydsolyzate

peptides from 4sine

220 mj.4was measured and

containing

passed through a flar

po44sine

acid for 80 minutes. of poly-

cell.

about 2O 4sine The absorbancy at

Aliquots were collected

recorded.

14 for determination of C activity.

Packard TM-Carb Scintlllatlon

Spectrometer.

was that described by Bray (1960).

was corrected for background, a quantitative

at s-minute

This was measured with a The scintillation

One nil. aliquots

were sdded to 10 ml of scintillation

added to the column was obtained.

mixture

po44sine

contained a continuous distribution

through polypeptides

The effluent

of the

of high molecular weight synthetic

hyarobromiae at 7O'C in 6 Ii hydrochloric

The resulting

residues.

Analysis was madeby

with 10 ml of water containing 10 mg of carrier

and added to carboxymethyl-cellulose

po44sine

A. Grossman and S. Ochoa.

1962) which allows resolution

2O homologous 4sine polypeptides.

was diluted

1. coli reaction

the incubation period to

co-chromatograplw on carboxymethyl-cellulose

fractions

of the poly-A used

residues required to code a single amino acid

of the size distribution

mixtures were kind4

intervals

poly-peptide.

In this communication we are concerned with the chromatographic

determination

first

RESEARCH COMMUNICATIONS

mixture

from each of the

mixture.

When the activity

recovery of the lysine-C

The operating efficiency

14

of the instrument

was about 47$. Results and Discussion. l(A)

The results are presented in Fig. 1.

shows that no incorporation

poly-A,

Figure

of lysine-C 14 occurred in the absence of

and that chromatography allowed separation of the first 252

2O 4sine

\‘ol. 13, No. 4, 1963

polypeptides

BIOCHEMICAL

in the carrier

AND BIOPHYSICAL

polylysine

from the incubation mixture.

hydrolyzate

RESEARCH COMMUNICATIONS

xith

Over 99$ of the radioactivity

lysine which did not interfere

no interference was eluted as

with separation of the higher peptides.

Less than l$ passed through with the frontal

peak as an acidic component.

!Chis may represent the maximumamount of lysine-C 14 that was linked to carrier

RNA. No incorporation

poly-A indicating initiation

into peptides occurred in the absence of

that peptide synthesis by routes other than poly-A

did not occur.

(A)

NO POLY

(B)

POLY

-A

-A

i ‘!l.,,j ,.,, ~~~~~,_..,,._.,...,_.,,, I’......:,% 50

100 FRACTION

150

NUMBER

Figure 1. Co-chromatography of lysine-C 14 incorporation products and known lysine polypeptides. (A) In the absence of poly-A stimulation. (B) With poly-A stimulation. (C) !Cungstate precipitate of poly-A stimulated system. The bars represent radioactivity in CPM, and the dots absorbancy at 220 q. Lysine polypeptides were eluted from carboxymethylcellulose columns with an exponential sodium chloride gradient (Stewart et al., 1962). Peaks following the large frontal peals are lysine and consecutive oligomers, except the small peak following dimer in (A) and (B) which originates from the incubation mixture. The last large broad peak represents high molecular weight material eluted by increasing the gradient rate. 253

Vol. 13, No. 4, 1963

BIOCHEMICAL

The results in Fig. l(B).

AND BIOPHYSICAL

obtained when poly-A was added to this system are shown

Radioactivity

was recovered with lysine and all

containing from 2-14 lysine residues. dilysine.

peptides

Almost half was recovered as

This may arise from the cleavage of poly-A by ribonucleases

to fragments that code for dilysine, try-ptic-like

(Stewart,

or the cleavage of polylysine

enzymes. However, the distribution

was different

exactly

RESEARCH COMMUNICATIONS

from that obtained on tryptic

N. IL, 1962).

The position

with the position

significant

radioactivity

of the radioactivity

hydrolysis

of polylysine

of the radioactivity

of peptides in the partial

coincided No

acid hydrolyzate.

was detected in peptides containing more than 14

The increase in activity

lysine residues.

by

octa-lysine

polypeptides,

significant

and may be a reflection

as

recovered in the hepta- and

compared with neighboring polypeptiaes, of the size distribution

is

of the p&y-A

used to stimulate the biosynthesis. The products from a similar trichloroacetic precipitate

acid-sodium tungstate

was extracted

products analyzed after in Fig. l(C) indicate

1962).

This

with j$ ammonium hydroxide and the soluble

that the tungstate does not precipitate of the lower lysine polypeptiaes

dilysine

enzymatic

co-chromatographed with known lysine polypeptides,

conclude that poly-A

stimulation

results

shown

in incomplete.

Since products from goly-A stimulated

Conclusions.

lysine.

(Gardner et al.,

with

removing the excess ammonia. The results

and that precipitation

biosynthesis

experiment were precipitated

in the biosynthesis

we

of poly-

!Pryptic degradation of these products have been shownto form

the samepeptides as those from polylysine Chromatography on carbovthyl-cellulose the species formed in the biosynthetic Polynucleic

was adequate to separate all reactions.

(Khorana et al.,

between the size of the initiating

of the resulting

1963).

acids have been separated by chromatography on

diethylaminoethyl-cellulose correlation

(Kaziro et al.,

polypeptide

1961).

If there is a

nucleic acid and the size

it would follow that chromatographic analysis 254

Vol. 13, No. 4, 1963

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the nucleic acid and the resulting relationship.

polypeptide

should reveal this

However, such experiments are complicated by the cleavage

of nucleic acids or the peptides during the experiment. results

indicate

as dilysine,

that more than half of the radioactive

and as the distribution

this is not entirely dilysine active

due to tryptic

hydrolysis,

we suggest that the

stimulated by the most abundant

poly-A species in the reaction mixture.

important to determine the size distribution

produced.

lysine was found

of the poly-peptides suggests that

may arise from a biosynthesis

and to correlate

Inasmuch as our

It would therefore of the poly-A after

this with the size distribution

of the polylysine

seem incubation peptides

This may reveal the number of nucleotide residues required to

code single amino acid residues.

Acknowledmnts. The authors wish to express their gratitude to Drs. S. Ochoa. Y. Kaziro and A. Grossmanfor the E. coli incubation mixtures used'in this work. The investigation was supported by grants E 101 and GPM-14, 6724~~ from the National Institutes of Health, U. S. Public Health Service.

References Bray, G. A., Anal. Biochem., ?., 279 (1960). Gardner, R. S., Wahba, A. J., Basilio, C., Miller, R. S., Lengyel, P., and Speyer, J. F., Proc. Nat. Acad. Sci., 48, 2087 (1962). Grossman, A., and Ochoa, S., Proc. Nat. Acad. Sci., so, 54 K-iry&. Worana, H. G., cznd Vizsolyi, J. P., J. Am. Chem. sot., 83, 675 (1961). Stewart, J. W., and Stahmann, M. A., J. Chromatography, 2, 233 (1962); Polysmino Acids, Polypeptides and Proteins, Stahmann, M. A., ea., p. 95, University of Wisconsin Press, Madison, Wis., 1962. Stewart, N. K., M.S. Thesis, University of Wisconsin, Madison, Wis., 1962.