Storage Protein Synthesis in Maize - NCBI

Plant Physiol. (1976) 57, 740-745

Storage Protein Synthesis in Maize .ISOLATION OF ZEIN-SYNTHESIZING POLYRIBOSOMES1 Received for publication October 29, 1975 and in revised form December 20, 1975

BRIAN A. LARKINS, CHARLES E. BRACKER, AND C. Y. TSAI Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mM Mg2+ contained unique classes of large polysomes. These large polysomes were sensitive to ribonudcease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the prindpal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels.

The major class of storage protein synthesized in the developing endosperm of cereal grains is a hydrophobic protein, prolamine. This protein is characterized by its solubility in 70% ethanol and its unique amino acid composition. In maize this protein, zein, is found mainly in structures called protein bodies (26). Zein, which can account for 50% of the total endosperm protein in normal maize varieties (19), is particularly rich in glutamic acid (glutamine), alanine, and leucine, and nearly devoid of the essential amino acids lysine and tryptophan (20). The discovery of opaque-2 and floury-2 endosperm mutants, which have reduced zein but increased non-zein protein (hence increased lysine and tryptophan), has greatly improved the nutritional quality of maize (17, 21). Although the reduced size of protein bodies in the mutant endosperm has been noted (26), the biochemistry of zein synthesis and the mechanisms by which the mutations reduce zein synthesis are unknown. In this communication we report optimal conditions for freezing and storing post-pollination kernels, and procedures for isolating polysomes which synthesize zein in vitro. A preliminary report of these results appeared elsewhere (12). MATERIALS AND METHODS Harvesting and Storage of Kernels. Maize kernels were harvested by two procedures. In the first, kernels were cut off the cob 22 days after pollination, placed in plastic bags, frozen on dry ice, and stored at -20 C. In the second, whole ears harvested at 22 days after pollination were frozen in liquid N2. The kernels were then removed and stored at -20 and -80 C. Only whole kernels were used for polysome isolation. Polyribosome Isolation. Free and membrane-bound polysomes were isolated by a procedure adapted from Larkins and

Davies (14). Kernels were ground in buffer A (0.2 M tris-HCl, pH 8.5, 0.2 M sucrose, 60 mM KCl, 50 mM MgCl2, and 5 mM dithiothreitol), strained through four layers of cheesecloth, and centrifuged at 5OOg for 5 min. The supernatant fraction was centrifuged at 37,000g for 10 min to separate free and membrane-bound polysomes. The supernatant containing free polysomes was decanted and layered over 4 ml of 600 mg/ml sucrose in buffer B (40 mm tris-HCl, pH 8.5, 20 mm KCl, and 10 mm MgCl2), while the 37,000g pellet was suspended in buffer A containing 1% (v/v) Triton X-100 to solubilize membranebound polysomes. Membrane-bound polysomes that were solubilized by Triton X-100 and recovered in the supernatant fraction after centrifugation at 37,000g for 10 min were also layered over 4 ml of 600 mg/ml sucrose in buffer B. Both free and initially membrane-bound polysomes were pelleted by centrifugation for 75 min at 229,000g in the 65 rotor of a Beckman L265 ultracentrifuge. Polysome pellets were suspended in buffer B, layered on 150-600 mg/ml sucrose gradients, and centrifuged at 189,000g for 45 min in a Beckman SW 50.1 rotor. Gradients were scanned at 254 nm with an ISCO Model UA-5 absorbance monitor. Areas of the polysome profiles were determined from the average of three measurements with a planimeter. The regions measured were polysomes, i.e. material sedimenting faster than monosomes, and large polysomes, i.e. material sedimenting faster than polysomes containing 5 ribosomes. Assay for Endolytic Messenger Ribonuclease Activity. Methods for preparing pea polysomes and their use for determining ribonuclease activity have been described (5). Polysome pellets were suspended in buffer B and aliquots incubated in the postribosomal supernatant from maize kernels for varying times. RNase activity was determined by the conversion of large polysomes to small polysomes (5). Negative Staining of Polyribosomes. Samples were prepared for electron microscopy by touching a carbon coated grid to the surface of a droplet of the polysome preparation. The grid was flotated on 2% (v/v) glutaraldehyde in 0.05 M sodium cacodylate, pH 7.2, for 2 to 5 min to fix the polysomes. The grids were touched to the surface of glass-distilled H20 for a few seconds, negatively stained with 2.5% (w/v) uranyl acetate, and examined with a Philips EM 200 at 60 kv. In Vitro Protein Synthesis. Protein synthesis was conducted by adding maize polysomes to an in vitro system derived from wheat germ (General Mills). The S-232 supernatant fraction was prepared according to the procedure of Marcus (16). Transfer RNA from 22-day pollinated maize kernels was isolated by a procedure adapted from Brunngraber (2). The reaction mixture contained in a final volume of 0.28 ml: 1 to 2 A260 units of polysomes, 20 ,ug of tRNA, 0.12 ml of S-23, 48 mm KCl, 4 mm Mg-acetate, 1 mm ATP, 35 /im GTP, 11 mm creatine phos-

2 Abbreviations: I S23: post 23,000g supernatant; TEMED: This research was supported in part by a grant from the Lilly Endowment to C. Y. T. Journal paper no. 6074 of the Purdue University N,N,N',N'-tetramethylenediamine; EGTA: ethyleneglycol-bis-(2-aminoethylether)tetraacetic acid; DEP: diethylpyrocarbonate. Agricultural Experiment Station. 740

Plant Physiol. Vol. 57, 1976

-

phate, 16 ,ug of creatine kinase, 35 mm tris-acetate at pH 8, 3 mM dithiothreitol, 45 um each of 19 amino acids, and 0.125 IACi of '4C-leucine (175 mCi/mmole) (Schwarz Bioresearch Inc.). Assays were conducted at 30 C for 30 min. The hot 5% trichloroacetic acid-insoluble and hot 70% ethanol-soluble protein were prepared according to the procedures of Marcus (16) and Dalby (3), respectively. Sodium Dodecyl Sulfate Gel Electrophoresis. Hot 5% (w/v) trichloroacetic acid-insoluble and hot 70% (v/v) ethanol-soluble protein from the in vitro assays were dialyzed in an SDS buffer (0.05 M tris-HCl, pH 6.9, 0.5% [w/v] SDS, and 1% [v/vJ 2mercaptoethanol) and analyzed by electrophoresis in 15% SDS gels (15% [w/v] acrylamide, 0.3% [w/v] bisacrylamide, 0.1% [w/v] SDS, 0.375 M tris-HCI, pH 8.5, and 0.025% [vlv] TEMED) (K. H. Lee et al., unpublished data). Gels 8 cm long were cast in Plexiglas tubes, and electrophoresis was at 4 mamp/ gel for 3 hr. Gels were frozen on dry ice, sliced into 2-mm sections, incubated overnight at 37 C in Omnifluor containing 3% (v/v) Protosol (New England Nuclear), and counted in a Beckman LS-100 scintillation counter. A purified zein sample (Nutritional Biochemicals Corp.) was solubilized in SDS buffer and used as a standard. The zein standard was stained with 4 1

.U

n

n~~~~~

B

A

U-~~~~~~~ BOTTOM TOP

741

ZEIN SYNTHESIS

TOP

FIG. 1. Free and membrane-bound polyribosomes from 22-day postpollination kernels frozen on dry ice. Maize kernels previously cut off the cob and frozen with dry ice were ground in 5 volumes of buffer A. Free and membrane-bound polysomes were prepared by the procedures described under "Materials and Methods."

Coomassie blue and scanned at 600 nm with a Gilford linear gel scanner.

RESULTS Freezing and Storage of Kernels. Polysomes isolated from maize kernels frozen on dry ice were degraded. Degradation was more pronounced in the free (Fig. 1A) than in the membranebound polysomes (Fig. 1B). Procedures which inhibited RNase activity in other tissues such as high KCI concentration (1), high pH and high KCl concentration (11), EGTA (1 1, 13), exogenous RNA (18), DEP (25), or these in combination failed to increase polysome size. As assay for endolytic messenger

ribonuclease activity (5) was conducted to determine if polysome degradation occurred before or during the isolation procedure. Purified pea polysomes were added to post-ribosomal supernatant from maize kernels and incubated for varying times. A comparison of the control polysomes (Fig. 2A) and polysomes in maize extract at 0 C for 30 min (Fig. 2B) indicated that the large polysomes were stable under these conditions. When the mixture was incubated at room temperature for 60 min (Fig. 2C), endogenous RNase degraded the large polysomes into smaller polysomes. These results demonstrate that maize RNase is essentially inactive in buffer A at 0 C. Polyribosomes isolated'from fresh kernels, or from kernels of ears frozen in liquid N2, were undegraded. These profiles showed no evidence of degradation by RNase, although there were qualitative and quantitative differences between the free and membrane-bound polysomes (Fig. 3, E and F). The free polysomes contained a large proportion of monosomes, and the polysomes of maximum absorbance contained 10 to 11 ribosomes. The membrane-bound polysomes contained a smaller proportion of monosomes and showed three distinct size classes of large polysomes. The smallest major size class contained 8 ribosomes, but the number of ribosomes in the larger size classes was not resolved by the gradient. The A26oA280 ratio of these polysomes was 1.7, indicating a high degree of purification (22). Both types of polysomes were susceptible to exogenous RNase (data not shown). Effect of Magnesium Polysome Isolation. Maize kernels were ground in buffers of varying Mg2+ concentration to determine the effect of the Mg2+ concentration on polysome isolation. Figure 3 depicts the free and membrane-bound polysomes isolated in grinding buffers that contained 1, 5, and 50 mM Mg2+. Although the distinct membrane-bound polysome classes were on

t0

TOP B~OTTO TOP BOTTOM TOP BOTTOM FIG. 2. Stability of pea polysomes in post-ribosomal supernatant from maize kernels. Pea polysomes were either suspended in buffer B and applied directly to sucrose gradients (A), or added to post-ribosomal supernatant from maize kernels and incubated for 30 min at 0 C (B), or 60 min

at room temperature

(C).

742

.


LARKINS, BRACKER, AND TSAI

1.0 I A

C

E

0.5

mi In

IB

0 (I) U4

0.!

-

MmI

D D

B

IL

n u.

U

U

TOP

BOTTOM

F

TO P

BOTTOM

TOP

BOTTOM

FIG. 3. Free and initially membrane-bound polysomes from frozen 22-day post-pollination maize kernels ground in buffers of varying Mg2+ concentration. Kernels were ground in 5 volumes of buffer A containing 1, 5, or 50 mM MgCl2. Free and membrane-bound polysomes were prepared by the procedures described under "Materials and Methods." The number of A260 units of polyribosomes and the Mg2+ concentration in the grinding buffers were: A, 0.74 A260 free polysomes; B, 1.6 A260 membrane-bound polysomes in 1 mM Mg2+; C, 0.95 A250 free polysomes; D, 2.4 A260 membrane-bound polysomes in 5 iM Mg2+; and E, 1.62 A260 free polysomes; F, 3.1 A260 membrane-bound polysomes in 50 mM Mg2+.

Table I. Ratio of Large Polysomes to Total Polysomes Obtained from Maize Kernels Ground in Buffers of Varying Mg2+ Concentrations Large Polyribosomes/Total Polyribosomes

Mg2+ 1 mM 5 mM 50 mM

Free Polysomes

Membrane-bound Poly-

0.37 0.59 0.80

0.45 0.67 0.94

somes

Table II. Recovery of Maize Polysomes from Grinding Buffers of Varying Mg2+ Concentrations Maize kernels were ground in buffer A containing 1 mM Mg2+. The homogenate was strained through four layers of cheesecloth and centrifuged at 5OOg for 5 min. The supernatant fraction was divided into 3 aliquots, and the Mg2+ concentration was adjusted to 1, 5, and 50 mM. Free and membrane-bound polysomes were prepared by the procedures described under "Materials and Methods." Polysome pellets were suspended in 0.5 ml of buffer B, and 0.1-ml samples were diluted to 1 ml. A260 and A280 was determined with a Gilford Model 240 spectrophotometer.

not obtained with buffers containing

1

mm (Fig. 3B) and

5

mM

Mg2+ (Fig. 3D), these profiles indicated polysome degradation because of the increased proportion of small polysomes. Similar but less pronounced degradation was also apparent in the free polysomes isolated in buffers that contained 1 mm (Fig. 3A) and 5 mm Mg2+ (Fig. 3C). An analysis of the profiles in Figure 3 is presented in Table I. The ratio of the large polysomes to the total polysomes has previously been used as an indication of the relative degradation of polysomes (6, 11). The proportion of large free polysomes isolated in buffers containing 1, 5, and 50 mm Mg2+ was 37, 59, and 80%, respectively. The membrane-bound polysomes were similarly affected, and concentrations of 1, 5, and 50 mm Mg2+ yielded 45, 67, and 94%, respectively, large polysomes. The Mg2+ concentration also affected total recovery of ribosomal material. Increasing the Mg2+ concentration from 1 or 5 mm to 50 mm increased recovery of free polysomes by 8%

Mg2+ 1 mM 5 mM 50 mM

Free Polysomes An, Units

Membrane-bound Polysomes Aim Units

28.7 28.8 31.2

21.1 32.7 36.4

Total

Amno Units

49.9 61.5

67.6

(Table II). The Mg2+ concentration had an even greater effect on membrane-bound polysome recovery. Increasing the Mg2+ concentration from 1 to 5 mm improved recovery by 54%, and 50 mM Mg2+ further increased recovery by 18%. Less total A260 material was recovered from tissue homogenized in buffers containing 1 and 5 iM Mg2+ because small polysomes were incompletely pelleted. This result is in agreement with a previous report of sucrose pads discriminating against small size classes of polysomes (14).


ZEIN SYNTHESIS

743

..A

%,, t ir Ir

I

.

X..

w Cl.

141. I -

.i

--

P,

FIG. 4. Electron micrographs of negatively stained initially membrane-bound polyribosomes from a fractionated sucrose gradient. A; sample of

the monosomes; B to E: samples from the region of the gradient containing polysomes bearing more than 9 ribosomes. A, C, D, E: x 110,000; B: x 62,000.

LARKINS, BRACKER, AND TSAI

744

Electron Microscopy of Polyribosomes. A gradient of membrane-bound polysomes prepared from kernels ground in buffer A was fractionated into monosomes, polysomes containing 2 to 9 ribosomes, and polysomes containing more than 9 ribosomes. Figure 4 shows an electron micrograph of negatively stained samples of monosomes (Fig. 4A) and the polysomes containing more than 9 ribosomes (Fig. 4, B to E). The monosome preparation contained a homogeneous population of well separated single ribosomes and a few small polysomes. Samples from the region of the gradient containing 2 to 9 ribosomes per messenger contained primarily small polysomes (data not shown). The sample from the lower portion of the gradient (Fig. 4B) revealed many large polyribosomes, some containing as many as 25 ribosomes (Fig. 4, C to E). The polysomes often appeared to be in long helical configurations, which is consistent with previous observations of polysome structure (7). Large helical polysomes were also present in negatively stained preparations of total free polysomes (data not shown). These preparations contained a large proportion of monosomes consistent with their polysome profiles. RNase treatment of both the free and initially membrane-bound polysomes prior to negative staining produced single ribosomes on the grids. In Vitro Protein Synthesis. To determine if zein was synthesized predominantly by one class of polysomes, the free and membrane-bound polysomes were incubated in an in vitro protein-synthesizing system derived from wheat germ (16). A comparison of total hot acid-insoluble protein and hot ethanolsoluble protein synthesized in vitro is shown in Table III. The membrane-bound polysomes incorporated more than twice as much _4C-leucine into trichloroacetic acid-insoluble protein as did free polysomes. Approximately 50% of the acid-insoluble '4C-leucine incorporated by the membrane-bound polysomes was soluble in 70% hot ethanol, whereas only 10% of the labeled protein produced by the free polysomes was ethanolsoluble. The similarity between the lysine and leucine composition of the ethanol-soluble protein synthesized in vitro and that of native zein has been reported (12). The hot trichloroacetic acid-insoluble and hot ethanol-soluble protein synthesized in vitro were also analyzed by SDS-polyacrylamide gel electrophoresis. The hot trichloroacetic acid-insoluble protein synthesized by the membrane-bound polysomes produced one major radioactive peak with the same mobility as the two major, close migrating zein bands (Fig. SA). This peak was also the major component of the ethanol-soluble radioactive protein (Fig. SA). A radioactive peak of similar mobility was also present in the hot ethanol-soluble protein produced by the free polysomes (Fig. SB), although it represented a smaller proportion of the labeled product. Ethanol-soluble protein of a lower mol wt than the major zein bands was present in both gels. This protein may consist of incomplete polypeptide chains not removed during dialysis of the samples. DISCUSSION Polysome degradation may occur prior to or during isolation. Undegraded free and membrane-bound polysomes from maize


kernels have been isolated during all stages of development when the ears were initially frozen in liquid N2. A temperature of -20 C appears to be adequate for subsequent storage of frozen kernels. Rapid freezing appears to be a prerequisite for the preparation of undegraded polysomes from frozen kernels. Polysomes prepared from fresh kernels allowed to become slightly dehydrated showed evidence of degradation particularly in the free polysome preparation (data not shown). This suggests that the degraded polysomes isolated from excised kernels (Fig. 1, A and B) may have been the result of increased RNase activity due to water stress (10) prior to homogenizing in buffer A. The effectiveness of the high pH, high ionic strength tris-HCl buffer (6) may be partially due to the high Mg2+ concentration (23). High concentrations of Mg2+ inhibited RNase activity in rat liver (9) and hen oviduct (23), and we obtained similar results in maize kernels (data not shown). Morton et al. (18) reported improved polysome isolation with buffers containing 50 mm Mg2+. However the large size of the membrane-bound polysomes isolated in buffers of high Mg2+ concentration might reflect stabilization of the membrane-polysome complex (15). Since polysome degradation is the most sensitive method to assay for messenger ribonuclease activity (5), we incubated pea polysomes in maize extract. The results presented in Figure 2 clearly demonstrate that endogenous RNase has very low activity during polysome isolation. The electron micrographs confirm that our procedures permitted the isolation of large polysomes and that the methods for isolating polysomes and preparing grids of negatively stained samples did not cause aggregation into polysomes. The absence of polysome structures in RNase-treated or monosome samples demonstrates that aggregation of monosomes did not occur. Samples from the region of the gradient containing small polysomes support the conclusion that small polysomes did not aggregate into large polysomes. The electron micrographs further show that the purified preparations of initially membranebound polysomes were free from membrane contamination. The unique size classes of polysomes observed in gradients of membrane-bound polysomes do not appear to be artifacts. These distinct peaks were absent from the profiles of the free polysomes prepared by the same isolation procedure (Fig. 3E). Both classes of polysomes had A260/A280 ratios which indicated a high degree of purification (22), and both were susceptible to RNase activity. We recently found that these large membranebound polysomes are not present at earlier stages of kernel development or in the opaque-2 mutants (manuscript in preparation). Both the free and membrane-bound polysomes isolated in buffers of high Mg2+ concentration are functional in in vitro protein synthesis. The membrane-bound polysomes synthesized one main radioactive protein similar to purified zein in ethanol solubility. The lysine and leucine composition of this protein was also similar to that of native zein (12). Furthermore, this protein corresponded to the two major zein bands with mol wt of 19,000 and 21,800 daltons (K. H. Lee et al., in preparation). A similar protein was also produced by the free polysomes, although it represented a smaller proportion of the protein synthesized in

III.

Comparison of Hot Trichloroacetic Acid-insoluble and Hot Ethanol-soluble Protein Synthesized By Free and Membrane-bound Polyribosomes and extraction Procedures for in vitro protein synthesis procedures are described under "Materials and Methods." The radioactive counts were the averages of triplicate assays and the counts/min are expressed per total reaction mixture. TCA: trichloroacetic acid; EtOH: ethanol. Table

Free Polysomes TCA-insoluble

Expt. 1 Expt. 2

EtOH-soluble

Membrane-bound Polysomes EtOH-soluble/TCA-insoluble

TCA-insoluble

EtOH-soluble

EtOH-soluble/TCA-insoluble

cpm

cpm

ratio

cpm

cpm

ratio

10,430 18,062

1,120 1,615

0.11

27,724

12,424

0.45

0.09

35,358

18,810

0.53

745

ZEIN SYNTHESIS


o

Specific classes of polysomes have also been found in reticulocytes (8), thyroid (24), and legume nodules (D.P.S. Verma, personal communication) all of which synthesize large quantities of one particular protein. The only way to unequivocally demonstrate that these large membrane-bound polysomes are indeed large polysomes is to isolate the mRNA from them and show that it has a correspondingly large mol wt. Such experiments are in progress. We have not determined which of the polysome size classes synthesize the two major zein proteins. Preliminary results indicated that both the small (2- to 9-mers) and large (>9-mers) membrane-bound polysomes synthesize ethanol-soluble protein. Considering the size of the large polysomes and the small mol wt (about 20,000 daltons) of the zein proteins, it is possible that the mRNA may be redundant or code for both proteins.

,

Acknowledgements- We

wish to thank P.

Gutay,

R. Allen, and Mrs. C.-Y.

Cheng

for

technical assistance. LITERATURE CITED

(I~~~~~~~~~~~~~ 511

BREEN, M. D., E. I. WHITEHEAD, AND D. G. KENEFICK. 1972. Requirements for extraction of polyribosomes from barley leaves. Plant Physiol. 49: 733-739. 2. BRUNNGRABER, E. F. 1962. A simplified procedure for the preparation of soluble RNA from rat liver. Biochem. Biophys. Res. Commun. 8: 1-3. 3. DALBY, A. 1974. Rapid method for determination of zein content of whole maize seed or isolated endosperm. Cereal Chem. 51: 586-592. 4. DALBY, A. AND C. Y. TSAI. 1974. Zein accumulation in phenotypically modified lines of opaque-2 maize. Cereal Chem. 51: 821-825. 5. DAVIES, E. AND B. A. LARKINS. 1974. Polysome degradation as a sensitive assay for endolytic messenger ribonuclease activity. Anal. Biochem. 61: 155-164. 6. DAVIES, E., B. A. LARKINS, AND R. H. KNIGHT. 1972. Polyribosomes from peas. An for their isolation in the absence of ribonuclease inhibitors. Plant ~~~~~~~~~~~~~~3.0 improved50:method 581-584. Physiol. 7. FLICenGER, CH. J. 1972. Ribosomal aggregates in Ameoba exposed to the protein synthesis inhibitor emetine. Exp. Cell. Res. 74: 541-546. 8. GOODMAN, H. M. AND A. RICH. 1963. Mechanism of polyribosome action during-protein synthesis. Nature 199: 318-322. 9. GRIBNAU, A. A. M., J. G. G. SCHOENMAKERS, M. VAN KRAAiAmwp, M. HILAK, AND H. 1.

BLOEMENDAL. 1970. Further studies on the ribonuclease inhibitor and other properties. Biochim. Biophys. Acta 224: 55-62.

4

~~~0

o 0

co~~~~~~~

from rat liver:

stability

10. HSiAo, T. C. 1970. Rapid changes in levels of polyribosomes in Zea mays in response to water stress. Plant Physiol. 46: 281-285. 11. JACKSON, A. 0. AND B. A. LARKINS. 1976. Influence of ionic strength, pH, and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57: 510. 12. LARKINS, B. A. AND A. DALBY. 1975. In vitro synthesis of zein-like protein by maize polyribosomes. Biochem. Biophys. Res. Commun. 66: 1042-1047. 13. LARwNS, B. A. AND E. DAVIES. 1973. Polyribosomes from peas. III. Stimulation of

polysome degradation by exogenous and endogenous calcium.

Plant

Physiol.

52: 655-659.

14. LARnNs, B. A. AND E. DAVIES. 1975. Polyribosomes from peas. V. An attempt to characterize the total free and membrane-bound polysomal population. Plant Physiol. 55:

749-756. 15.

AND D. BOULTER. 1973. Protection of polyribosomes by attachment to by diethylpycarbonate during their extraction from Vicia faba. Phyto12: 39-41.

LONSDALE, D. M.

membranes and

chemistry ORIGIN

)

uRONT

(±)

dodecyl sulfate polyacrylamide gel electrophoresis of protein synthesized by free and membrane-bound polyribosomes. A: hot acid-insoluble ( -0-0 - ) and hot ethanol-soluble protein (- -) synthesized by the membrane-bound polynbosomes. B: hot acid-insoluble (*-) and hot ethanol-soluble (-- -) protein ) were synthesized by the free polyribosomes. The zein standards ( FIG.

5.

Sodium

'4C-leucine

labeled

stained with Coomassie blue and scanned at 600 nm.

vitro. We do not know if the ethanol-soluble

by

the free

artifact of

protein produced

polysomes is a reflection of the in vivo the polysome isolation procedure.

condition or an

polysurprising, since zein accounts for as much as 50% of the cereal protein (19). Zein is synthesized primarily between 16 and 28 days after pollination and is approximately 30% complete by 22 days after pollination (4). The isolation of distinct size classes of membrane-bound

somes from maize kernels is not

16. MARCus, A. 1974. The wheat embryo cell-free system. Methods Enzymol. 30: 749-761. 17. MERTz, E. T., L. S. BATES, AND 0. E. NELSON. 1964. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 145: 279-280. 18. MORTON, B., N. CHIMEZIE, E. C. HENSHAW, C. A. HIRSCH, AND H. H. HiATr. 1975. The isolation of large polyribosomes in high yield from unfractionated tissue homogenates. Biochim. Biophys. Acta 395: 28-40. 19. MosSE, J. 1966. Alcohol-soluble proteins of cereal grains. Fed. Proc. 25: 1663-1669. 20. MURPHY, J. J. AND A. DALBY. 1971. Changes in the protein fractions of developing normal and opaque-2 endosperm. Cereal Chem. 48: 336-349. 21. NELSON, 0. E., E. T. MERTz, AND L. S. BATES. 1965. Second mutant gene affecting the amino acid pattern of maize endosperm proteins. Science 150: 1469-1470. 22. NOLL, H. 1969. Polysomes: Analysis of Structure and Function. In: P. N. Campbell and J. R. Sargent, eds., Techniques in Protein Biosynthesis. Academic Press, New York. pp. 101-179. 23. PALMrrER, R. D. 1974. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonudeic acid. Biochemistry 13: 3606-3615. 24. VASSART, G. AND J. E. DumoNT. 1973. Identification of polysomes synthesizing thyroglobulin. Eur. J. Biochem. 32: 322-330. 25. WEEIs, D. P. AND A. MARCUS. 1969. Polyribosome isolation in the presence of diethylpycarbonate. Plant Physiol. 44: 1291-1294. 26. Wow., M. J., U. KHOO, AND H. L. SECKINGER. 1967. Subcellular structure of endosperm proteins in high-lysine and normal corn. Science 157: 556-557.