the proteins of green leaves · stored in a deep freeze to minimize enzymatic changes in the...

THE PROTEINS OF GREEN LEAVES

V. A CYTOPLASMIC NUCLEOPROTEIN FROM SPINACH AND TOBACCO LEAVES*

BY LUTHER EGGMAN, S. J. SINGER,? AND SAM G. WILDMANS

(From the Kerckhoff Laboratories of Biology and the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, California)

(Received for publication, June 26, 1953)

In a previous publication of this series (l), evidence was presented to show that as much as 50 per cent of the total soluble protein contained in the leaves of several plant species consists of a protein component that is apparently homogeneous by sedimentation and electrophoretic criteria. This component, designated as Fraction I protein, is easily identified in the analytical ultracentrifuge because its sedimentation constant of about 19 Svedberg units (19 S) is much greater than that of any of the other soluble protein components of normal cytoplasm. The present commu- nication is concerned with the isolat.ion and further chemical and physicochemical characterization of the Fraction I protein component. By means of simple centrifugal procedures it is possible to isolate Fraction I protein in large yield and in a state of about 95 per cent purity as judged by analysis in the analytical ultracentrifuge. Chemical characterization of this material demonstrates that it is a nucleoprotein.

Materials and Methods

Plant Material-Two varieties of Nicotiana tabacum, Turkish and Mary- land Mammoth, were grown in the Earhart air-conditioned greenhouse under conditions previously found to be optimal for growth. Spinach (Xpinacia sp.) was secured from a local market. Only well expanded leaves free of pathological symptoms were used, and the midribs and petioles were removed prior to maceration of the tissue and extraction of the proteins.

Preparation and IiSTactionation of Leaf Protoplasm-The met,hod of preparation and fractionation was essentially the same as that described previously (1, 2). The leaf tissue was disrupted in a cold Eppenbach colloid mill, 2 parts by weight of leaves to 1 part of 0.5 M KOH-maleate buffer,

* This work was supported in part by grants from the United States Public Health Service, The National Foundation for Infantile Paralysis, Inc., and the Atomic Energy Commission.

t Present address, Chemistry Department, Yale University, New Haven, Con- necticut.

$ Present address, Botany Division, University of California at Los Angeles.

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970 PROTEINS OF GREEN LEAVES. V

pH 7.0, as a dispersion medium being used. This procedure yields a cell- free protoplasmic extract of pH 6.7 to 6.8, a pH which prevents denatura- tion of the cytoplasmic proteins which might otherwise arise from the mix- ing of the acid vacuolar contents of the cell with the cytoplasm.

The particulate matter was removed from the protoplasmic extract by centrifugation for 1 hour at 25,000 r.p.m. in the No. 30 rotor of a Spinco model L preparative centrifuge and the resulting supernatant solution was used for further analysis. Unless this centrifugation could be performed immediately after grinding, the protoplasmic extract was quick-frozen and stored in a deep freeze to minimize enzymatic changes in the protein.

Estimation of Protein Content-2 volumes of ice-cold 1.0 N trichloroacetic acid (TCA) were added to aliquots of the protein solution estimated to contain 10 to 30 mg. of protein and the mixture was held for 18 to 24 hours in an ice bath. The precipitated protein was removed by centrifugation at 2”, washed twice with 0.5 N TCA, and then dried to constant weight at 105”.

Estimation of Trichloroacetic Acid-Precipitable Phosphorus (TCA-P)- Determination of the amount of phosphorus precipitated with protein was carried out on aliquots precipitated with TCA as for protein estimation, except that the precipitate was centrifuged and washed after standing only 3 to 6 hours. The shorter precipitation time is necessary because it was found that even at 0” detectable amounts of TCA-P were removed from the protein if the precipitate was allowed to remain in contact with TCA for 8 hours, and that as much as 10 to 15 per cent was removed in 24 hours. The washed precipitates (or suitable aliquots of the protein solution if total P was being determined) were digested with 60 per cent perchloric acid and the inorganic phosphorus released was determined according to the method of Allen (3).

Ultracentrifugal and Electrophoretic Analysis-The equipment and procedures used were the same as those described previously (1). The pH of the buffers was determined with a glass electrode at 22-26”. Equilib- rium dialysis for both types of analyses was performed at 2’. Electrophore- sis experiments were conducted at 1.8” and ultracentrifugal analyses at 20-25’.

EXPERIMENTAL

Partial PuriJication of Fraction I Protein by (NH&SO~ Precipitation- As previously noted (4), about 70 to 80 per cent of the total protein in cytoplasmic protein solutions can be precipitated by 0.4 saturated (NH&SO4 and completely redissolved in a neutral buffer. However, examination of the salt-precipitated fraction in the analytical ultracentrifuge indicated that only about 70 per cent of the redissolved protein consisted of the 19 S


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L. EGGMAN, S. J. SINGER, AND S. G. WILDMAN 971

component, and repeated reprecipitation with (NH&S04 failed to increase the proportion of this component appreciably. Consequently, a physical method for the isolation of Fraction I protein was developed.

PuriJication of Fraction I Protein by Preparative Ultracentrifugation- Since Fraction I protein constitutes a large part of the total cytoplasmic proteins and possesses a sedimentation constant considerably greater than the other proteins of cytoplasm, it is possible to achieve partial purification of the protein by high speed centrifugation. The following procedure has resulted in Fraction I protein preparations of approximately 95 per cent purity from tobacco, spinach, and pea leaves, as judged by their behavior in the analytical ultracentrifuge.

Approximately 1 per cent solutions of cytoplasmic proteins are centrifuged at 40,000 r.p.m. for 45 minutes in the No. 40 rotor of a Spinco preparative centrifuge. The rotor is precooled to 0” and the centrifuge refrigerator set to maintain the temperature of the rotor chamber at about -20”. The small pellet which forms during this first centrifugation, con- stituting less than 5 per cent of the total cytoplasmic proteins and often containing traces of green matter, is discarded and the supernatant solution centrifuged for 2.5 hours at 40,000 r.p.m. A pellet containing 20 to 35 per cent of the total cytoplasmic proteins is deposited during this second centrifugation. The supernatant solution is discarded and the pellet dissolved in 1 to 2 ml. of 0.1 p KOH-maleate buffer, pH 7.0, at 0”. The pellet will dissolve in about 4 hours if the centrifuge tube is inclined to about a 20” angle and gently agitated on a mechanical shaker. The slight amount of material which fails to redissolve is removed by a low speed centrifugation and discarded. This completes the first fractionation cycle.

The redissolved protein is again centrifuged for 2 hours at 40,000 r.p.m. The supernatant solution is discarded and the pellet dissolved in 0.1 p buffer as before. The small amount of protein which fails to redissolve is removed by a low speed centrifugation and discaided. The resulting clear, amber to brown colored, non-opalescent Fraction I protein solution contains about 70 to 80 per cent of the protein which dissolved after the first cycle of centrifugation.

As shown in Fig. 1, two centrifugal treatments are sufficient to pcepare Fraction I protein solutions containing only about 5 per cent of low molecular weight components (Fig. 1, B) from spinach leaf cytoplasm which initially contained approximately 60 to 70 per cent low molecular weight material (Fig. 1, A). Further purification of Fraction I protein by centrifugation has not been successful because a large proportion of a pellet sedimented three times fails to redissolve.

The process of purifying Fraction I protein from tobacco leaf cytoplasm has been followed in detail by electrophoretic and by ultracentrifugal


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analysis as shown in Figs. 2 and 3. Fig. 2, A is an electrophoretic scanning pattern of tobacco cytoplasmic proteins in 0.1 P NaCl-Na cacodylate buffer, pH 7.20. Under these conditions there is little separation of the components known from ultracentrifugal analyses to be present in cytoplasm. The electrophoretic pattern of twice sedimented Fraction I protein solutions, however, shows a much sharper and more symmetrical peak than that obtained with the unfractionated prot,eins. The calculated mobility of the principal component of cytoplasm is -5.25 X 1O-5 and of purified Fraction I protein solutions is -5.15 X 10M5 cm.2 volt-l sec.-I.

Ultracentrifugal patterns of these same solutions are presented in Fig. 3, A and C, respectively. Fig. 3, B is the pattern of the supernatant

FIG. 1. Analytical ultracentrifuge patterns of a typical spinach whole cytoplasm (A) and the Fraction I protein preparation made from it (B). The arrows indicate the 19 S, Fraction I protein component. The direction of sedimentation is from left t,o right. The time in seconds after reaching the rotor speed of 50,220 r.p.m. is indicated in the upper right corner of each diagram. A, 0.3 M KOH-maleate buffer, pH 6.9; 13.0 mg. of protein per ml. B, KOH-maleate buffer, pH 7.5, 0.1 p; 4.0 mg. of protein per ml.

solution remaining after the first 2.5 hours of centrifugation to remove Fraction I protein as a pellet. The depletion of the 19 S component relative to the trailing, lower molecular weight components is evident. The degree of purification of the 19 S component with respect to low molecular weight material accomplished in the Fraction I protein preparat,ion illustrated by the pattern in Fig. 3, C is about the same as that achieved with spinach (Fig. 1, B). With tobacco leaves there is, however, a very notice- able increase in the amount of components heavier than Fraction I protein and, in this instance, one component not apparent in whole cytoplasm has appeared. Evidence that the components heavier than Fraction I prot,ein are aggregates of the 19 S component which are formed during centrifugation will be considered next.

Components of Larger Sedimentation Constant Than Fraction I Protein- In a previous survey of the soluble cytoplasmic proteins contained in the leaves of eight species of plants, a small percentage of a component sedi-


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menting faster than Fraction I protein was found in four species (tomato,

Turkish tobacco, pea, and gherkin) (1). In no case was more than one component which sedimented more rapidly than Fraction I protein found in whole cytoplasm; yet, in every instance, twice sedimented Fraction I protein preparations obtained from any species have contained at least

1300

Fm. 2 FIG. 3

FIG. 2. Electrophoretic diagrams of tobacco cytoplasmic proteins (A) and Frac- tion I protein (B) isolated by high speed centrifugation. Proteins equilibrated against NaCl-Na cacodylate buffer, pH 7.20, 0.1 p. Protein concentration is 5.50 and 4.85 mg. per ml. in A and B, respectively. Only descending boundaries are shown. The time of migration in minutes is given to the right of each diagram; the arrows indicate the starting boundary.

FIG. 3. A typical series of analytical ultracentrifuge patterns illustrating the course of fractionation of tobacco cytoplasmic proteins by high speed centrifugation. Rotor speed 50,220 r.p.m. A, whole cytoplasm before centrifugation; NaCl-Na cacodylate buffer, pH 7.1, 0.1 p; 5.0 mg. of protein per ml. B, supernatant solution remaining after first centrifugation cycle; same buffer as in A; 5.6 mg. of protein per ml. C, twice sedimented Fraction I protein solution; KOH-maleate buffer, pH 7.5, 0.1 p; 5.0 mg. of protein per ml. A and C used for electrophoretic analysis (Fig. 2).

one component which sedimented more rapidly than 19 S. All Fraction I protein preparations made from cytoplasms of Turkish and Maryland Mammoth tobacco, which have been studied most extensively, have contained at least two components which sediment more rapidly than does Fraction I protein itself. Sedimentation diagrams of several typical preparations are shown in Fig. 4, A and B. More rarely, preparations which contain three or more components heavier than the Fraction I protein are observed. The ultracentrifugal analysis of one such preparation is illus-


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trated by the diagrams reproduced in Fig. 4, C. Since these faster sedimenting components were not present in the original cytoplasmic preparations, we believe that the following arguments support the conclusion that they are aggregates of the 19 S component which arise from centrifugal packing of protein molecules.

During the initial survey of cytoplasmic proteins cited above (l), no component with a sedimentation constant greater than 19 S was ever observed in spinach cytoplasm. This observation has been confirmed in the present work even when spinach cytoplasm was examined in the ultracentrifuge at concentrations as high as 1.5 per cent total protein. How- ever, as may be seen in Fig. 1 and in Table I, there is a small amount of a 26 S component contained in Fraction I protein solutions prepared from spinach. That this 26 S material did not appear as the simple result of centrifugal enrichment of a small amount of the same material, which was initially present in the whole cytoplasm in a concentration too low to be detected in the ultracentrifugal analysis, is evident from the following consideration.

As little as 0.1 mg. per ml. of the 26 S component can be detected by ultracentrifugal analysis of Fraction I protein preparations. Failure to find the 26 S component in spinach whole cytoplasm solutions at a total protein concentration of 15 mg. per ml. suggests that the 26 S component constitutes less than 1 part in 150 parts of total protein in such preparations. Therefore, the ratio of 19 S protein to 26 S protein must be greater than 75: 1 in cytoplasmic protein solutions containing about 50 per cent Fraction I protein. From the ratios of the sedimentation constants and the relative compositions, calculation reveals that simple enrichment of the 26 S protein during two cycles of centrifugation of such a mixture should result in a preparation containing not more than 1 part of 26 S protein to 59 parts of 19 S protein. However, measurement of areas shows the presence of approximately 1 part of 26 S protein to 10 parts of 19 S protein in twice sedimented Fraction I protein preparations. Thus, the amount of 26 S component present in such twice sedimented preparations is too great to have resulted merely from centrifugal enrichment and must therefore have been formed at the expense of the 19 S component during the isolation procedure.

An extreme and unusual case of such aggregation during centrifugal preparation of tobacco Fraction I protein is illustrated by the diagrams in Fig. 5. The ultracentrifugal pattern of the original cytoplasm is shown in Fig. 5, A. This preparation is typical of the many that we have examined and, characteristically, contains only a small amount of the 26 S component.

The protein solution resulting after once sedimenting and redissolving the 19 S component can also be considered typical. As shown in Fig. 5,


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B, the 19 S component constitutes the major portion of the total protein in this sample as expected, although low molecular weight material still accounts for about 15 to 20 per cent of the total protein at this stage of

600

Ll l-----T

LLI A

1450

;L, /----7q 1 1500 16001

FIG. 4 C

FIG. 5

FIG. 4. Ultracentrifuge patterns illustrating the sedimentation behavior of several typical Fraction I protein preparations from Turkish (A and B) and Maryland Mammoth (C) tobacco leaves. Rotor speed 50,220 r.p.m. Solvent, KOH-maleate buffer, pH 7.0,O.l p. The 19 S component is the largest in each diagram. In A and B, two faster moving aggregates of Fraction I protein are evident, and in C there are three.

FIG. 5. Ultracentrifuge patterns showing the sedimentation behavior of a Mary- land Mammoth tobacco whole cytoplasm preparation (A) together with once sedimented (B) and twice sedimented (C) Fraction I protein preparations derived from it. The patterns illustrate the formation of aggregates during the preparative procedure. In the whole cytoplasmic proteins and once sedimented Fraction I protein preparation, only a small amount of one component moving faster than 19 S is evident; in the twice sedimented preparation, at least five components moving faster than 19 S are resolved. Numbers by the peaks indicate the 19 S component (No. 1) and higher particle weight aggregates (Nos. 2 to 6) of the 19 S units. Rotor speed 50,220 r.p.m. A, 0.3 M KOH-maleate buffer, pH 6.9; 8.6 mg. of protein per ml. B, KOH-maleate buffer, pH 7.0, 0.1 p; 12.5 mg. of protein per ml. 6, KOH-maleate buffer, pH 7.5, 0.1 p; 21.6 mg. of protein per ml.


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976 PROTEINS OF GREEN LEBVES. V

purification. In accord with the results obtained with spinach, the amount of 26 S component has also been greatly increased, but it is noteworthy that no resolution of components sedimenting more rapidly than 26 S is apparent. In marked contrast, the Fraction I protein solution (Fig. 5, C) of this unique preparation after being twice sedimented contained not only the 19 S and 26 S components which were present in the once sedimented preparation, but also four other readily distinguishable components which sediment more rapidly than either of these. Thus, the analysis of this Fraction I preparation seems to establish clearly that t,he high molecular weight components are artifacts formed by aggregation of the 19 S component during the centrifugal preparation, that the aggregation occurs largely during the second sedimentation, and that for analytical purposes such aggregates should be treated as Fraction I protein.

The corrected sedimentation constants of the components represented by Peaks 1,2, and 3 in Fig. 5, C are 18.0,25.7, and 30.7, respectively. The sedimentation constants of the other three components could not be determined accurately from the material at hand. However, if the three components characterized by sedimentation constants 18 to 19 S, 26 to 28 S, and 31 to 35 S (Table I) are considered, the values of these constants are consistent with the hypothesis that these components are related as mon- omer, dimer, and trimer, respectively. Furthermore, the latter two components, although exhibiting more variation in sedimentation constant from preparation to preparation than does Fraction I protein, nevertheless show but little more dependence of sedimentation constant upon total protein concentration than does Fraction I protein itself. This behavior is also consistent, because dimers and trimers of a relatively spherical molecule such as Fraction I protein, as will be shown below, are also relatively symmetrical molecules and should show little dependence of their sedimentation constants on concentration.

Evidence presented below indicates that Fraction I protein is a nucleoprotein of the ribose type. Aggregation of nucleoproteins is now a well established fact, particularly with virus proteins, since the dimerization of tobacco mosaic virus (TMV), prepared by centrifugation, is readily demonstrated upon ultracentrifugal analysis (5). Even more convincing is the finding (6) that, when two electrophoretically distinguishable strains of TMV are compacted by centrifugation into a pellet and the latter redissolved and analyzed by electrophoresis, an appreciable amount of a third component of intermediate mobility appears. Although Fraction I protein is much smaller than TMV, it does not seem unreasonable to expect this nucleoprotein also to aggregate under similar conditions of centrifugal preparation, and it is possible that further examination of the behavior of other nucleoproteins during centrifugation may reveal aggregation to be a rather common property of nucleoproteins.


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L. EGGMAN, S. J. SINGER, AND S. G. WILDMAN

Chemical Characterization

977

Phosphorus Content of Fraction I Protein-Various preparations of twice sedimented Fraction I protein obtained from tobacco and spinach leaves have all contained organically bound phosphorus which is precipitated with the protein by TCA. The amount of organic phosphorus has varied from 0.15 to 1.5 per cent. Although the precise explanation of this variation must await further experimentation, one source of variation appears to reside in the fact that the cytoplasmic extracts themselves differ in

TABLE I Sedimentation Constants of Fraction I Protein in Several Whole Cytoplasm and

Fraction I Preparations

Species and preparation

-4. Spinach, Fraction ItI

B. Turkish tobacco, Fraction It (Sam- ple E-6)$

C. Turkish tobacco (Sample E-S) Whole cytoplasmj Whole cytoplasm supernatant af-

ter 1st centrifugation cycle$ Fraction ItI

D. Turkish tobacco (Sample E-11) Whole cytoplasm§ Whole cytoplasm supernatant af-

ter 1st centrifugation cycles Fraction Its

E. Maryland Mammoth tobacco, Frac- tion It (Sample E-20)$

Concentra- tion

mg. per ml.

4.0 2.0 1.0 0.5

21.6

18.1 18.6 17.9 18.4 18.0 25.7 30.7

5.0 18.8 5.6 18.7

5.1 18.7 1.3 18.6

5.0 5.5

19.1 18.9

9.85 4.92 9.90 4.95 2.47 1.23 3-15

19.5 27.4 33.9 19.1 27.9 34.8 19.2 28.4 36.8 19.1 28.0 19.0 28.7 19.0

F. Various spinach and tobacco whole cytoplasmsll

1s.9g

SompIonent

.%.*0 x 10’3 *

corn onen, HI

26.0 26.4

ComKoInent

* Values corrected to the density and viscosity of water at 20”. t Prepared by two high speed centrifugation cycles as described in the text. 3 KOH-maleate buffer, pH 7.0, 0.1 p. $ KOH-maleate buffer, pH 7.5, 0.1 p. 11 Na cacodylate-cacodylic acid-NaCl buffer, pH 6.9,0.1 p, or KOH-maleate buffer

pH 7.0, 0.1 /L. 7 Average of twenty independent experiments.


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TCA-P content. These differences are, in turn, a reflection of the physiological age and nutritional status of the plant at the time of harvest. There is much evidence to indicate that essentially all of the TCA-P of cytoplasm is associated with the 19 S component since, in many experiments, approximately the same percentage of TCA-P and of 19 S protein is removed as a pellet by centrifugation. However, since the amount, of TCA-P in cytoplasm has varied over a 5-fold range, in contrast to the amount of 19 S protein which has varied surprisingly little between different samples, it seems probable that the amount of TCA-P per unit of 19 S protein in the original cytoplasm as well as in the final Fraction I protein preparations is also somewhat variable.

Nature of Organic Phosphorus Associated with Fraction I Protein-Solu- tions of Fraction I protein show strong absorption in the region of 260 mp. When a TCA precipitate of purified Fraction I protein or of proteins precipitated from cytoplasm is heated with either 1 N HCI or HC104 for 5 minutes at 90°, all of the phosphorus which precipitated with the protein is released into solution. This acid solution also absorbs strongly at 260 mp. Continued heating of the 1 N acid extract releases approximately one-half of the phosphorus as inorganic phosphorus in 60 minutes and, concurrently, free ribose appears in the solution.

That the phosphorus which precipitates with the protein in the presence of TCA is in the form of ribonucleic acid was determined by chromato- graphic methods. The TCA-P was released from the precipitated protein by treatment with 1 N HCl at 90” for 5 minutes, the protein removed by centrifugation, and the supernatant solution further hydrolyzed in the 1 N acid for 1 hour at 100” in a sealed tube. The hydrolysis products were chromatographed on filter paper according to the method of Smith and Markham (7) and identified as guanine, adenine, cytidylic acid, and uridylic acid by cochromatography with similar hydrolysates of highly purified yeast nucleic acid and with authentic samples of the individual compounds. The hydrolysate also gave a positive orcinol reaction; the presence of ribose was established by cochromatography with aut,hentic ribose in two different solvent systems.

Quantitative studies of hydrolysis products separated by chromatography demonstrated that all of the TCA-P in Fraction I protein preparations and in whole cytoplasm solutions may be accounted for as components of ribonucleic acid. For these studies, samples were prepared as for the qualitative studies reported above, the volume of 1 N acid used in removal of the TCA-P from the protein being such that the concentration of P in the hydrolysate was 0.1 to 0.3 y, equivalent to 1 to 3 y of nucleotides, per ~1. After chromatography of the hydrolysate, the developed chromatograms were dried at room temperature, the spots located, and


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the marked spots and suitable blanks for each replicate were cut out and eluted in 4.00 ml. of 1.0 N HCl for 15 to 20 hours at 25”. The optical density of the eluates was determined at the maximal absorption of each component with a Beckman model DU spectrophotometer. From the optical density, the free base or nucleotide concentration was calculated from the extinction coefficients of Markham an,d Smith (8). In general, duplicate aliquots of the solution to be analyzed, containing 50 to 120 y

TABLE II

Analysis of Nucleic Acid Associated with Fraction I Protein Prepared from Maryland Mammoth Tobacco Whole Cytoplasm by Two Fractionation Cycles

Each sample contained 3.3 PM of TCA-P before hydrolysis.

component

Guanine.................................... Adenine. ., .._...__.____.__. Cytidylic acid. Uridylic “

Preparation I*

PM lrx

0.97 1.22 0.72 0.83 0.69 0.84 0.61 0.73

Preparation II*

Total.................................... 2.99 I 3.62 Average total. _........................ 3.3

Ratio of components in nucleic acid, based on above analyses

Guanine ~.................................... Adenine Cytidylic acid

1.35

Uridylic acid . ’ . . . 1.13

1.47

1.15

Total purines Total pyrimidines. 1.30 I

1.31

* Average of six replicates.

of TCA-P, were precipitated and hydrolyzed. Two chromatograms, each with three 20 to 40 ~1. aliquots of the hydrolysate and three blanks, were prepared from each hydrolysate.

In a typical experiment, hydrolysates of 1 N HCl extracts of aliquots of Fraction I protein solution containing 3.3 PM of TCA-P were analyzed as described above. As shown by the data in Table II, the average sum of the nucleotides in each hydrolysate was equivalent to 3.3 j!M per aliquot. Thus, 1 mole of nucleot.ide was present for each mole of phosphorus in Fraction I. This indicates that the TCA-P associated with Fraction I protein is in the form of ribonucleotide polymers (ribonucleic acid) and, hence, that Fraction I protein is a nucleoprotein.


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980 PROTEINS OF GREEN LE$VES. V

Physicochemical Characterization of Fraction I Protein

Several solutions of Fraction I protein prepared by the two cycle centrifugation method from spinach and tobacco cytoplasms have been examined electrophoretically and in the analytical ultracentrifuge.

Electrophoretically, the preparations, regardless of the number of aggregates present, possess but a single component, migrating as a sharp rather symmetrical peak with a mobility of -4.0 to -5.5 X lo-” (*m.2 sec.-* volt-l in 0.1 p Na cacodylate buffer at p1-I 7.20 (Fig. 2, B).

The sedimentation constants of the components in Fraction I protein solutions prepared from spinach and from tobacco cytoplasms are essentially the same, as is shown in Table I. Furthermore, the sedimentation constants are practically independent of concentration and are not affected by the presence of higher molecular weight aggregates, since the sedimentation rate is the same in highly purified preparations as in whole cytoplasm. Consequently, the low degree of spreading of the 19 S peak during sedimentation is not to be attributed to the boundary sharpening which occurs with substances which exhibit a large dependence of sedimentation constant upon concentration. It is, rather, indicat#ive of the homogeneity of the protein with respect to molecular weight.

The molecular weight of the protein was determined by use of a modified form of Svedberg’s equation (Equation 1 (9)) relating t,he molar frictional coefficient, f, the sedimentation constant, s, and partial specific volume, P, of the protein, and the density of the solvent to the anhydrous molecular weight of the sedimenting molecule. All of these quantities except the molar frictional coefficient were determined experimentally.

The partial specific volume of the nucleoprotein XT--as determined in a pycnometer at 25.00” from the densit’ies of a dialyzed preparation of Frac- tion I protein from Maryland Mammoth tobacco (Sample E-20, Table I) containing 4.95 mg. of TCA-precipitable material per ml. and of the buffer solution against which it was dialyzed. From these data, the partial specific volume was found to be 0.6g1 (9).

This value is low for the partial specific volume of a protein, but it is in the range expected for a nucleoprotein because of the relat.ively high density of nucleic acid. The particular preparat.ion used for this determination contained 11.2 per cent nucleirn acid, computed either from TCA-I’ analysis or by direct nucleotide analysis.

Normally, f is evaluated from the diffusion coefficient (D), obtained from independent diffusion measurements, by means of the Einstein rela- tion, f = RT/D. Diffusion measurements of Fraction I protein solutions are at present impractical because of the presence of aggregates of Frac- tion I protein which seriously complicate determination of a diffusion constant and because of the instability of the preparations over the long


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periods of time required for the experiments. However, since it has been shown (10) that concentration dependence of s increases with increasing asymmetry of the sedimenting molecule, the lack of concentration dependence of s for Fraction I protein suggests that the molecule is nearly spherical. For unhydrated spherical molecules, f = fo, where f~ is the molar frictional coefficient of an unhydrated equivalent spherical molecule and may be evaluated from Stokes law (9). Hydration increases the ef- fective particle size and hence the value of f; thus f/j0 is always greater than unity.

The hydration of Fraction I protein is unknown, but, if it is assumed that the molecule is hydrated to the extent of 40 per cent by weight, the ratio f/j0 has the value 1.15 (11). With this value for flfo, and the experimental values i7 = 0.69 and s = 19.0 S, the calculated molecular weight is 375,000. This value should be regarded as a good first approxi- mation to the true molecular weight of a preparation containing approximately 11 per cent nucleic acid and refinements of this figure will depend upon the preparation of stable solutions containing no aggregates of the Fraction I protein.

DISCUSSION

In addition to the previous identification of Fraction I protein in the soluble, cytoplasmic protein extracts of a variety of leaves (l), a component of 19 S has now been isolated from spinach, pea, and tobacco leaves. As far as can be ascertained at present, there is a striking physicochemical similarity in the preparations of this protein derived from all three species. Variations in the nucleic acid content and sedimentation constant are no greater among preparations made from three genera of plants than among different preparations made from the same species. Detection of generic and species differences evidently will depend upon more refined techniques, such as detailed nucleic acid and amino acid analyses, rather t,han upon the gross chemical and physicochemical analyses of the type which we have utilized.

The quest)ion arises as to how much reliance may be placed on the homogeneity of Fraction I protein when the nucleic acid content of the isolated protein is variable. As indicated earlier, there is good reason to believe that all of the TCA-P, and hence nucleic acid, found in t.he soluble protein fraction of leaves is associated with the 19 S component, but that the variable amount of TCA-P in the soluble proteins is a reflection of the nutritional and physiological age status of the plant, and that these conditions will in turn determine the amount of TCA-P to be found in Frac- tion I protein when it is isolated. In addition to these factors, another source of possible variation in TCA-P content arises from the intense phos-


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phatase, protease, and ribonuclease activity found in the cytoplasmic proteins of leaves. Such protein preparations are capable of bringing about the rapid degradation of yeast nucleic acid to ribosides, inorganic P, and a residue which resists further enzymatic hydrolysis. Hence, it is reasonable to suspect that these enzymes are also responsible for some degradation of the nucleic acid associated with Fraction I protein during the time required for its preparation. Although it has always been our practice to work rapidly and under cold conditions, nevertheless it is possible to demonstrate losses in the TCA-P contained initially in the soluble proteins even when the material is maintained at 0” for only a few hours. The fact that this loss is accelerated at higher temperatures suggests that the losses may be attributed to enzymatic action.

An important matter for future experimentation concerns the question of whether the amount of nucleic acid per molecule of 19 S component is indeed variable in amount, or whether some molecules contain a constant amount of nucleic acid whereas others contain none whatsoever. The latter situation is not without parallel, since Markham and Smith (12) have found two fractions of protein in purified turnip yellow mosaic virus which are indistinguishable by chemical and serological means, except that one fraction contains nucleic acid and is infective, while the other fraction is devoid of nucleic acid and is non-infective.

When the nucleic acid content of Fraction I protein from young leaves in the process of expansion and rapid protein synthesis is compared to that obtained from older leaves in which the proteins are in a relatively steady state condition (13), the former is much richer in nucleic acid than Fraction I protein obtained from older leaves. However, analysis of the two preparations reveals no change in the nucleotide composition of the nucleic acid, and no significant change in the sedimentation constant, suggesting that there is no change in the protein moiety of Fraction I protein. It seems possible, therefore, that a limit may be placed on the amount of nucleic acid that a leaf may synthesize, in contrast to the less restricted synthesis of the protein, with the consequence that there is not enough uncombined nucleic acid to provide for those protein molecules formed during the later stages of synthesis. Also plausible is the possi- bility that all Fraction I protein molecules initially contain the same amount of nucleic acid, but that one function of the protein is to supply nucleic acid to other materials of the cell during the rapid synthesis of protoplasm which occurs when a leaf grows by expansion.

We believe that the real significance of this work lies in the ability to recognize and study by simple and straightforward analysis the behavior of a specific nucleoprotein which constitutes a large proportion of the total soluble protein fraction of leaves. Thus, it is possible now to study protein synthesis under conditions completely divorced from increased cellular


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divisions. Although not often recognized by biochemists, the leaf is a remarkable organ for the study of protein synthesis by virtue of the fact that an expanding leaf grows by a process of cell elongat,ion rather than cell division. As will be shown in subsequent publications, an expanding leaf synthesizes protein at a rapid rate, and, after expansion is complete, a steady state condition prevails for a relatively long period during which protein synthesis is well balanced against protein catabolism. Yet, de- tachment of the leaf at any time during the steady state condition will upset this balance in favor of rapid protein catabolism. Consequently, a challenging opportunity exists for the study of the metabolism of Fraction I protein under changing conditions which can be controlled largely by the investigator.

SUMMARY

1. Previous studies have revealed the presence of a major protein component comprising as much as 50 per cent of the total soluble proteins in cytoplasmic extracts of the leaves of several species of plants. A simple high speed centrifugal method has been developed for the preparation of this protein in good yield and in a high degree of purity.

2. The protein is shown to be a nucleoprotein containing ribonucleic acid. Physicochemical characterization of t,his nucleoprotein indicates that it is relatively homogeneous with respect to molecular weight and electrophoretic mobility, and that the molecules of this protein are very nearly spherical and have a molecular weight of the order of 375,000. They appear to form dimers, trimers, and larger aggregates under suitable conditions.

3. The ribonucleic acid content of preparations of this nucleoprotein varies somewhat. Possible factors involved in this variation and their significance are discussed.

BIBLIOGRAPHY

1. Singer, S. J., Eggman, L., Campbell, J. M., and Wildman, S. G., J. Biol. Chem., 197, 233 (1952).

2. Wildman, S. G., Cheo, C. C., and Bonner, J., J. Biol. Chem., 180,985 (1949). 3. Allen, R. J. L., Biochem. J., 34, 858 (1940). 4. Wildman, S. G., and Jagendorf, A., Ann. Rev. Plant Physiol., 3,131 (1952). 5. Lauffcr, M. A., J. Phys. Chem., 44, 1137 (1940). 6. Singer, S. J., Bald, J., Wildman, S., and Owen, R., Science, 114, 463 (1951). 7. Smith, J. D., and Markham, R., Biochem. J., 46,509 (1950). 8. Markham, R., and Smith, J. D., Biochem. J., 49,401 (1951). 9. Svedberg, T., and Pedersen, K. O., The ultracentrifuge, Oxford (1940).

10. Singer, S. J., J. Chem. Phys., 15, 341 (1947). 11. Oncley, J. L., Ann. New York Acad. SC., 41, 121 (1941). 12. Markham, R., and Smith, K. M., Parasitology, 39, 330 (1949). 13. Eggmnn, L., Thesis, California Institute of Technology (1953).


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WildmanLuther Eggman, S. J. Singer and Sam G.

LEAVESFROM SPINACH AND TOBACCO

A CYTOPLASMIC NUCLEOPROTEIN THE PROTEINS OF GREEN LEAVES: V.

1953, 205:969-983.J. Biol. Chem.

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