In vitro amino acid incorporation into particle protein from maize seedlings

BIOCHIMICA ET BIOPI-IYSICAACTA

IN

VITRO

287

AMINO ACID INCORPORATION INTO PARTICLE PROTEIN FROM MAIZE SEEDLINGS RUSTY J. MANS AND G. DAVID NOVELLI

Biology Division7, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A .) *

(Received November 25th, 196o)

SUMMARY An amino acid incorporating system has been isolated from young maize seedlings. The addition of adenosine triphosphate, an adenosine triphosphate generator, guanosine triphosphate, MgC12, KC1, and a soluble component are required for the incorporation of Ei-14Clleucine into washed lO5 ooo x g centrifuged particles. The incorporation is rapid and ahnost exclusively into particle protein. Although the soluble component can be supplied by the pH 5 precipitate from rat liver, the maize lO5 ooo ",,t g supernatant is required for full activity of the particles.

INTRODUCTION Previous work in this laboratory directed toward the eventual cell free synthesis of specific enzymes involved in starch biosynthesis has resulted in the isolation of a cell-free amino acid incorporating system from fresh maize kernels 1. Recently we have observed that the embryo constitutes the richest source of incorporating activity in the fresh kernel 2. We have turned, therefore, to the developing seedling as a more readily available as well as a more homogeneous source of experimental material than the kernel. Since the particles isolated from seedlings are quite comparable to preparations obtained from the kernel, these studies are directly applicable to investigation of the mechanisms involved in the gene-induced changes in enzymes that regulate starch biosynthesis in maize endosperm 3. We have defined the maize seedling system as to the requirements for the isolation of active particles and for incorporation of [I-14Clleucine into particle protein. The maize system is markedly different from the amino acid incorporating system isolated from pea seedlings described by WEBSTER4 and by RAAKE6 as well as the microsomal fraction isolated from tobacco leaves 6. The maize particles require the addition of a soluble component for incorporation, whereas the pea system does not require a soluble component. The soluble component can be obtained trom the highspeed supernatant fraction of maize seedling homogenates or a pH 5 fraction precipitated from the high-speed supernatant of either maize or of rat-liver homogenates. Abbreviations: ATP, adenosine triphosphate; GTP, guanosine triphosphate; PEP, phosphoenolpyruvic acid; PC, phosphocreatine; Tris, tris (hydroxymethyl)aminomethane; TCA, trichloroacetic acid; RNAase, ribonuclease; s-RNA, soluble ribonucleic acid; EI)TA, ethylenediamine tetraacetic acid. * Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission. Biochim. Biophys. dcta, 5o (1961) 287-3oo

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Consistent with the ability of the rat liver p H 5 fraction to substitute for a similar fraction of the maize supernatant we observed that the general properties and requirements for incorporation in the maize system are essentially the same as those of the rat-liver microsomal system described by ZAMECKNIK AND I~ELLER~. However, we found an all-maize system incorporated lencine more efficiently than the mixed maize and rat-liver system. MATERIALS AND METHODS

Materials I)L-[i-14Clleucine was purchased from New England Nuclear at specific activities of 8. 7 or lO.75 ~C//xmole. The potassium salts of ATP and PC and the sodium salt of GTP were procured from Sigma Chemical Company. Crystalline pyruvic kinase and P E P as the silver-barium salt were purchased from C. F. Bohringer and Sons. P E P as the tricyclohexylammonium salt was purchased from California Corporation for Biochemical Research. The P E P was converted to the potassium salt on a Dowex50 column according to the procedure of HALL AND COHEN8 and assayed by tile method of SCHMIDT9. The amino acid mixture was prepared according to an amino acid analysis of yellow dent maize seed 1° and contained the following L-amino acid~ (amounts in mg): L-alanine, 9.2; L-arginine, 4.6; L-aspartic acid, 5; t:cysteine, 2. 7 ; L-glutamic acid, I I . 2 ; glycine, 5.9 ; L-histidine, 2.8; L-isoleucine, 3.7 ; L-lysine, 4.7 ; L-methionine, 4; L-phenylalanine, 4.4; L-proline, 6.1; L-serine, 4.7; L-threonine, 8.2; L-tryptophan, 0.5; L-tyrosine, 2.8; L-valine, 3.7; L-asparagine, 3.7; and L-glutamine, 3.7. The mixture was placed in IOO ml of distilled water and adjusted to p H 7-5 with KOH. L-amino acids were purchased from Mann Research Laboratories. RNAase was obtained from Worthington Biochemicals. The creatine-ATP transphosphorylase was kindly supplied bY A. BEST. We are indebted to Dr. D. SCHWARTZ for generously supplying us with the maize kernels used in this work.

Preparation of biological materials Dried kernels of waxy yellow maize were stripped from one ear, rinsed, and soaked overnight in tap water at room temperature. The kernels were rinsed again and then placed individually on a thoroughly wetted filter paper-lined tray, covered, and incubated in the dark at 22.5 ° for 48 h. Under these conditions, more than 95 '~'i, of the kernels germinated with quite uniform plumule development (5-IO m m long) and in the absence of mold contamination. The plumules were dissected from approx. 350 etiolated seedlings and ground in 65 ml of cold 0.45 M sucrose-o.o5 M Tris (pH 7.5) o.ooi M MgClz medium with a motor-driven conical glass homogenizer (Konte Glass). In order to obtain active particles it was essential that the preparation be kept at near-freezing temperatures throughout all manipulations. The resultant homogenate was centrifuged at 2oooo < g for 15 min and the supernatant removed from beneath the lipid layer and centrifuged at lO5 ooo x g for 9 ° min in a Spinco preparative centrifuge. The supernatant was decanted through cheesecloth and retained. The golden yellow pellets were combined and resuspended in 2-3 ml of distilled water to a protein concentration of 15-2o mg particle protein/ml. To prepare washed maize particles, the combined pellets were resuspended in 12 ml of distilled water with a teflon homogenizer and Biockim. Biopkys. ~4cta, 5o (196t) 287 3o0

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recentrifuged at lO5OOO × g for 9 ° min. The washed pellet then was resuspended in distilled water at a protein concentration of 15 to 2o mg/ml. The rat liver p H 5 fraction was prepared as described by HOAGLAND et al. 11 and stored in o.5 ml aliquots at - - I O °, and is referred to throughout this paper as rat liver p H 5. The fraction relerred to as the maize p H 5 was obtained by addition of I M acetic acid to the high-speed supernatant of the maize homogenate to bring it to pH 5.I. The precipitate was centrifuged off at 2oooo × g, washed onc~-n o.25 M sucrose, and resuspended in o.o5 M Tris buffer (pH 7.5)-

Assav of incorporation The composition of each reaction mixture is tabulated for each experiment. All the reagents were adjusted to pH 7.5 prior to their addition. Unless stated otherwise, incubations were routinely carried out in IO ml Erlenmeyer flasks on a Dubonoff shaker at 37 ° under an atmosphere of nitrogen-carbon dioxide (95:5) for 30 min. The reaction was stopped by the addition of I ml of IO °/o TCA-o.I M DL-[12C~leucine and chilling in an ice-bath for IO rain. The TCA-precipitated material was separated from the soluble components by centrifugation and washed b y a modification of the procedure of SIEKEVITZ~2. The precipitates were washed twice with 5 ~o TCA and then extracted with 5 ~/o TCA for 15 rain at 9 o°. The precipitates were washed once again with 5 o/(~TCA and then extracted with ether-alcohol (I : I, v/v) for 30 min at 37 °. Finally, tile precipitates were washed with ether, air-dried, and then dissolved in 0.5 ml of 80 ~)"oformic acid. A zero time control, prepared by the addition of io ~o TCA-o.I M DL-E12C]leucine to a complete reaction mixture at zero time, was included in each experiment. This control serves as a measure of the efficiency of the washing procedure in removing nonincorporated materials. All reported values are corrected for this error which was less than 5 counts/min/mg total protein. The incorporation of radioactive leucine was determined b y counting a 0.25 ml aliquot of the formic acid-dissolved protein in a Packard Tri-Carb scintillation spectrometer as described previouslyL An additional aliquot was used to determine the protein concentration b y the method of LOWRY et al. 13. RESULTS AND

DISCUSSION

Preparation of particles Initially, homogenates were prepared from maize seedlings in the same sucrosephosphate medium used for the isolation of active particles from fresh maize kernels 1. As shown in Table I, very low levels of leucine incorporation were obtained when plumules of maize seedlings were homogenized in 0.45 M sucrose-o.o5 M phosphate buffer. I t is known that microsomal particles isolated from pea seedlings 14, as well as from bacterial and mammalian sources, undergo reversible dissociation into smaller fragments and/or irreversible changes as a function of: (a) magnesium or phosphate concentration, (b) pH, and (c) the ionic strength of the suspending medium. Correspondingly, the sedimentation characteristics and biological activity of maize particles were expected to be influenced by these factors. Indeed, Table I shows that the addition of MgC12 to the sucrose-phosphate medium, the omission of the phosphate buffer or the substitution of Tris buffer for the phosphate resulted in a substantial increase in incorporating activity. Furthermore, when the sucrose-Tris medium was supple-

Biochim. Biophys. Acta, 50 (1961) 287-300

290

R. J. MANS, G. D. NOVELLI TABLE I I~SFFECT

OF

VARIOUS

AI)D1TIONS

INCORPORATING

ACTIVITY

TO

GRINDING

OF

MAIZE

MEDIUM

ON

PARTICLES

The r e a c t i o n m i x t u r e c o n t a i n e d 2 5 / t m o l c s p h o s p h a t e buffer (pH 7.5), 0.5/~mole ATI', o. t 5 / / m o l e GTP, 5 p m o l e s PC, 5 ° p g p h o s p h o c r e a t i n ATP t r a n s f e r a s e , 5/ ~ mol e s MgCI~, o.o45 p m o l e DI.II-14C] leucine (3.7" IO5 c o u n t s / m i n ) , a n d 2.2 m g m a i z e p a r t i c l e p r o t e i n in a t o t a l v o l u m e of 0.5 ml. I n c u b a t i o n s were c a r r i e d o u t as d e s c r i b e d in t e x t . One b a t c h of s e e d l i n g s w a s d i v i d e d i n t o s e ve ra l p a r t s a n d ~he p l u m u l e s f r o m each p a r t were h o m o g e n i z e d in t h e m e d i a listed. Specific a c t i v i t y is r e p o r t e d as c o u n t s / r a i n / r a g p a r t i c l e p r o t e i n . Leucine incorporation counts~rain/rag

Additions lo homogeni=in~ medium

o.45 M sucrose o.45 M sucrose + o.o 5 M p h o s p h a t e o.45 M sucrose 4- o.o 5 M p h o s p h a t e + o.ooI 3~/MgCI~ 0.45 M sucrose + 0.05 ~V/Tris buffer o.45 M sucrose + o.o 5 M T r i s buffer + o.ooi M MgC12 0.45 M sucrose + o.ooI ,,~,IMgC12 0.45 M sucrose + o.ooI M E D T A D i s t i l l e d 1-t20 i n s t e a d of sucrose

i

buffer (pH 7.5) buffer (pH 7-5)

o6 -8

73 lO8

(pH 7.5) (pH 7.5}

15,S 88 ~7

77

mented with MgC12, the particles isolated were more than 5 times as active as the material prepared in the sucrose-phosphate medium. Preparation of homogenates in distilled HiO or in sucrose medium plus either MgC12 or EDTA resulted in the isolation of particles with less activity than those prepared in sucrose alone. The sucroseTris-MgC12 mixture consistently yielded preparations of higher specific activity than any other combination tested and, therefore, was employed routinely as homogenizing medium. In early experiments we noted that particles prepared from either very young or relatively old seedlings were most active. In addition there was considerable day-to-day variability in the activity of particle preparations. Therefore, the effect of age of seedling on particle activity was investigated. Particles were prepared from seeds that were germinated for varying times at a constant temperature of 22.5 °. The data plotted in Fig. I show that very young seedlings yielded the most active particles, and that the activity fell abruptly with age followed by a return of incor400-

>-a_ ~_ ~ 500-

>J

v_ o_

/ /

o ~ 200. u_ m c) o

ca N .~ 1 0 0

°~o__o__O_~-°

v

o

2

a

,b

~

GERMINATION TIME (&lys)

Fig. I. T h e effect of age of s e e d l i n g on l e u c i n e - i n c o r p o r a t i n g a c t i v i t y of m a i z e particles. The r e a c t i o n m i x t u r e s ar e t h e s a m e as t h o s e d e s c r i b e d in T a b l e I I . E a c h p o i n t r e p r e s e n t s t h e a c t i v i t y of a h o m o g e n a t e p r e p a r e d f r o m a p p r o x i m a t e l y 35 ° s e e d l i n g s as d e s c r i b e d in METHODS.

Biochim. Biophys. _4eta., 50 (I96I) 287 3oo

AMINO ACID INCORPORATION

291

porating activity in particles prepared from older seedlings. Since it is difficult to obtain sufficient material from very young seedlings we do not know when activity is initially present, nor the age at which m a x i m u m activity is present. Germination of seeds at higher temperatures permits isolation of particles from seedlings younger than 2 days and furthermore, the intervening period prior to the isolation of active particles from older seedlings is shortened considerably. In a series of experiments conducted at different temperatures the most active particles were obtained from plumules which measure 5-1o m m in length, indicating that m a t u r i t y of the seedling is critical to the isolation of the leucine-incorporating system. This result is in marked contrast to the finding of RAAKEs that there is no effect of age of pea seedlings on the incorporating activity of isolated microsomal particles. The reasons for the different age effect in the two plant tissues is not known, but m a y he related to differences in pool size of amino acids at various stages of maturity 15. I t is known that some parts of a developing seedling are metabolically more active than others TMand further, that the mobilization of nitrogen from storage proteins varies within different areas of the seedlinglL If we interpret microsomal amino acid incorporation as a measure of protein biosynthesis we can expect that certain areas of the seedling will yield particles of greater incorporating activity than others. Therefore particles were prepared from 3 major regions of the seedling. We were particularly interested in the plumule since its excision from the germinated kernel is a relatively easy and rapid process compared to the tedious procedure of removing the entire seedling from the endosperm. Each seedling was removed from its adhering endosperm ; approx. 2/3 of the seedlings were then divided into a plumule, a scutellar, and a root fraction. Each fraction as well as the remaining intact seedlings were ground with o.45 M sucrose and sand in a cold mortar. The various homogenates were centrifuged and tile high-speed pellets were assayed (see METHODS). The data in Table I I indicate that the scutellum was richest in total amount of particle protein and also in leucine-incorporating activity of the fractions examined. Twice as much particle protein was isolated from the plumule than the root; however, the ability of the particle material from the plumule or the root to incorporate leucine was only 1/3 that of the scutellum. The scutellum plays a major role in mobilizing the storage materials of the endosperm for utilization b y the growing embryo TMand thus m a y be TABLE II ABILITY

OF

PARTICLES

FROM DIFFERENT REGIONS OF MAIZE INCORPORATE [I-14C] LEUCINE

SEEDLINGS

TO

E a c h r e a c t i o n flask contained. 50 / , m o l e s p h o s p h a t e buffer (pH 7.5), i t~mole A T P , o. 3 /imole G T P , io # m o l e s PC, I o o # g p h o s p h o c r e a t i n A T P t r a n s f e r a s e , I o / t m o l e s MgCI~, 0 . 0 9 / , m o l e DL-II-14C~leucine (7.4" l°5 c o u n t s / m i n ) , 1.8 m g r a t liver p H 5, a n d 1. 5 m g maize particle p r o t e i n in a t o t a l v o l u m e of i ml. Specific a c t i v i t y is reported as c o u n t s / m i n / m g particle protein. Leucinc incorporation Region

E n t i r e seedling Plumule Scutellum P r i m a r y root

Total protein mg

42 14 19 6.6

Particles alone counts/min]mg

86 45 123 37

Particles + rat liver pH 5 countslmin/mg

124 144 18o 60

Biochim. Biophys. Acta, 50 (1961) 287-300

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R. J. MANS, G. D. NOVELLI

expected to exhibit relatively high incorporating activity. The low incorporating activity of the particles reported in Table I I as compared with the activities in Table I and Fig. I is due to less effective grinding and the use of sucrose medium.

Requirementsfor incorporation RABSON AND NOVELLI1 had demonstrated that particles prepared from fresh maize kernels can be stimulated to incorporate leucine by the addition of rat liver pH 5. We therefore considered the possibility that differences in activity among regions of the seedling m a y not be an exclusive property of the particle protein but rather the lack of a soluble component. Since our early a t t e m p t s to isolate an active p H 5 fraction from maize seedling supernatant were consistently unsuccessful, we resorted to rat liver p H 5 as a source of soluble material. In the last column of Table I I are presented data obtained b y assaying the different particle preparations in the presence of rat liver p H 5. It is seen that the addition of rat liver pH 5 results in a 3-fold stimulation of incorporation b y plumule particles. Preparations from the other 2 fractions as well as the intact seedlings also were stimulated by the rat liver preparation but not to the same extent as the particles isolated from plumules. Since particles isolated from the plumule region exhibited a marked response to soluble materials, and because of the relative ease of removing the plumule from the seedling, all subsequent experiments were conducted with these particles. Since we had observed that unwashed particles could be stimulated by rat liver p H 5, it was desirable to study the effect of rat liver pH 5 on washed particles in order to determine if other requirements of the system could be revealed. Therefore, the effect of rat liver pH 5 on washed and unwashed particles was compared. The data in Table I I I show that washing particles removed 80 o/,,oof their activity. This residual activity probably was caused by supernatant material still adhering to the particles after washing, since more extensive washing further reduced the residual activity. TABLE Ill SOLUBLE

REQUIREMENT

FOR

L p 2 U C I N E 1 N C O R P O R A ' r I O N B Y ~,VASIIED P A R T I C L E S

TILe reaction m i x t u r e s used were prepared as described in "Fable I with the addition (where indicated) of 0.9 mg of r a t liver p H 5. The a m o u n t of particle protein added was between 1.8 and i 9 mg/o.5 rill reaction m i x t u r e . Specific a c t i v i t y is expressed as c o u n t s / m i n / m g particle protein. Particle prepara¢ion

Unwashed \¥ashed

Particles alone cot nts/min, mg

Particles plus rat liver p H 5 counts~rain/rag

Stimulation by rat liver pH .5 %

.-81 36

345 128

23 255

Preparing the particles from a dilute homogenate was much more effective in reducing residual activity than increasing the washing volume. Addition of the rat liver p H 5 stimulated incorporation into both the washed and the unwashed particles. The stimulation was IO times greater into washed compared to unwashed particles. In spite of the large stimulatory effect of rat liver pH 5 on washed particles the soluble fraction did not restore the full activity to that of the original unwashed particles. Further, the unwashed particles were stimulated by addition of rat liver p H 5- I t seems likely, therefore, that a maize soluble component adhering to the unwashed particles t3iochim.

Biophys.

Acta,

5 ° (1961) 287 300

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AMINO ACID INCORPORATION

and not contained in rat liver pH 5 is required for full activity. Subsequent data will be presented in this paper to further substantiate this idea. Additional requirements for the incorporating system are shown in Table IV. The complete system incorporated 364 counts/min/mg particle protein, which is almost 3 times the incorporation obtained with washed particles in the experiment presented in Table III. The increased incorporation resulted from slight alterations in the composition of the reaction mixture, i.e., Tris was substituted for phosphate buffer, the MgCI~ level was lowered to 2.5/,moles, and o.I/xmole of KC1 was included. The critical levels of both magnesium and potassium seem to be characteristic of incorporating systems in general. The requirements for and levels of ATP, an ATP generator, and soluble material are essentially those observed for the fresh maize kernel system described by RABSON AND NOVELLI1. Deletion of GTP caused no decrease in leucine incorporation. However, in other experiments the addition of GTP to the reaction mixture appeared to stimulate leucine incorporation and, therefore, was routinely included in the reaction mixture. In the absence of maize particles, rat liver pH 5 did not incorporate leucine. TABLE IV REQUIREMENTS FOR LEUCINE INCORPORATION 1N THE PRESENCE OF RAT LIVER p H 5 T h e c o m p l e t e r e a c t i o n m i x t u r e c o n t a i n e d 2 5 / , m o l e s Tris a t p H 7.5, 0.5/~mole A T P , o.15/~mole G T P , 2 . 5 / , m o l e s MgCI~, 4.75/~moles P E P , i o / ~ g p y r u v i c kinase, o.i /tmole KC1, 0 . 0 5 7 5 / , m o l e DL-El-laClleucine (3.7" lO5 c o u n t s / m i n ) , 0. 4 m g r a t liver p H 5, a n d 1.6 m g w a s h e d maize particle p r o t e i n in a t o t a l v o l u m e of 0. 5 ml. Specific a c t i v i t y is p r e s e n t e d as c o u n t s / m i n / m g particle p r o t e i n in all i n s t a n c e s e x c e p t where particles were o m i t t e d ; in t h i s i n s t a n c e t h e specific a c t i v i t y is p r e s e n t e d as c o u n t s / m i n / m g t o t a l protein. Incorporation counts/min/m~

System Complete Complete Conlplete Complete Complete Complete Complete

minus minus minus minus minus minus

ATP MgCI 2 PEP and pyruvic kinase GTP r a t liver p H 5 maize particles

364 66 59 i 14 378 io o

TABLE V EFFECT

OF

SOME

INHIBITORS

ON

~I-14CJLEUCINE INCORPORATION BY MAIZE PARTICLES

T h e r e a c t i o n m i x t u r e w a s p r e p a r e d as described in T a b l e I V r a t liver p H 5 a n d 2 m g of w a s h e d maize particle p r o t e i n T h e specific a c t i v i t y is r e p o r t e d as c o u n t s / m i n / m g particle indicate a m o u n t s of c o m p o u n d

Incorporation counts/min/mg

System Complete Complete plus Complete plus Complete plus C o m p l e t e plus Complete plus (5/*moles)

w i t h t h e e x c e p t i o n s t h a t 0.48 m g of were a d d e d to each reaction flask. protein. T h e figures in p a r e n t h e s i s added.

R N A a s e (ioo/tg) R N A a s e (2oo #g) e h l o r a m p h e n i c o l (50/*g) ehloramphenicol (too/,g) p-fluorophenylalanine

228 43 16 18o 83 17o

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R . J . MANS, G. D. NOVELLI

General properties of the incorporating system The analogy between the maize incorporating system and systems isolated from animal, bacterial, and other plant materials is manifested further by the sensitivity of the maize system to some known inhibitors ol protein synthesis. The date in Table V show that addition of 200/xg RNAase to the complete reaction mixture inhibited 95 "~, of the leucine incorporation. Chloramphenicol also inhibited incorporation; however, 600. f

Z 13/ >_13_

400-

_ ~ 400 >..,J F_o

~o~

O~

~_ LJ 300"

(_) t -

sd

/

o-t~

7oo

<

o ~ 200. 5o

0 oo~°

/ F-~oo_ ?


cq -.. c_

/

~

o/

o

us

2'0 40 INCUBATION TIME (min)

60

Fig. 2. Kinetics of leucine incorporation in the presence of maize particles and rat liver p H 5The reaction m i x t u r e s are as described in Table I V w i t h the exception t h a t 1-7 mg w a s h e d maize particle p r o t e i n was added to each flask and i n c u b a t i o n s were carried out for the t i m e s indicated.

o

o.~5

o.~3

o.654

O.'SB

RAT LIVER pH 5 ADDED(rag)

Fig. 3. Leucine incorporation in the presence (ff maize particles and increasing a m o u n t s of r a t liver p H 5- The reaction m i x t u r e s are as described in Table I V with the exception of the protein components. A total of 1.24 mg washed particle protein plus increasing a m o u n t s of r a t liver p H 5 were added as indicated.

IOO fig were required to give 75 (}~, inhibition. The degree of inhibition obtained at the concentration of inhibitors used is comparable to that seen in both the mammalian and the plant systems. Therefore, that amount of incorporation that is energydependent is interpreted as a measure of the protein-synthesizing capacity of the system. Leucine incorporation is reduced by only 25 0~, in the presence of p-fluorophenylalanine. However, only one concentration of the amino acid analogue was tested, and furthermore, it is not known if the analogue is incorporated into maize protein. Analogous to the rat-liver microsomal system 7, the maize incorporating system is also sensitive to air. The reaction flasks were routinely shaken under an atmosphere of nitrogen-carbon dioxide (95:5) (see METHODS). However, when the incubations were carried out under air, a I5-2o(}'o reduction in incorporation is observed. We have not been able to stimulate the aerobic incorporation rate by inclusion of reduced glutathione and at present have no explanation for the deleterious effect of aerobiosis. Fig. 2 shows the time course of the reaction. It is seen that a rapid rate of incorporation occurs in the first IO min of incubation, again analogous to the rat system. If the incubation is continued the rate of incorporation slows markedly. Raising the initial concentration of ATP, MgC12, or the ATP generator, failed to increase the Biochim. Biophys. Acta, 5o (1961) 287---3oo

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AMINO ACID INCORPORATION

specific activity of the particle protein after 30 min incubation, indicating that these reagents are probably not rate limiting. We next considered the possibility of saturation of the particles with leucine as an explanation for the time course observed. In order to test this possibility the optimal levels of the components of the incorporating system were determined. The optimum concentrations of ATP, MgC12, and P E P were ascertained and the system was then titrated with rat liver p H 5. The data plotted in Fig. 3 show that after addition of 0.65 mg rat liver p H 5 the particles apparently are saturated with respect to soluble material An interpretation of the kinetic data in terms of particle saturation requires that essentially all of the radioactivity incorporated is associated with the particle material. The data presented in Table VI were obtained by reseparating the particle protein from the soluble material at the end of the incubation period. The data are reported as total counts of radioactivity incorporated into a particle protein fraction. TABLE VI TOTAL [I-14CJLEUCINE INCORPORATED BY MAIZE PARTICLES The complete reaction m i x t u r e contained: 125/*moles Tris buffer at p H 7.5, 2-5/*moles ATP, 0.75/*mole GTP, 12. 5 / , m o l e s MgC12, 25/*moles P E P , 5 ° / * g p y r u v i c kinase, o. 5/*mole KCI, 5.75/*moles DL-[I-14C~leucine (1.8.1o 7 counts/min), 2 mg rat liver p H 5 and 9-9 mg washed maize particle protein in a t o t a l volume of 2. 5 ml. To a second flask containing the same reaction m i x t u r e was added 0. 5 ml of the amino acid m i x t u r e described u n d e r METHODS. B o t h flasks were incubated at 37 ° w i t h shaking u n d e r n i t r o g e n - c a r b o n dioxide (95:5). Aliquots were removed and assayed as described in the text. Total radio~tivity in protein counted Addition to complete system

None A m i n o acids

Incubation time rain

Total protein counts/rain

5 45 5 45

1746 3472 1754 3796

Soluble protein counts~rain

io8 24o 211 526

Particle protein counts~rain

Radioactivityin particleprotein %

1218 218o 145o 3208

92 94 88 88

Total protein refers to the material obtained by precipitating the reaction mixture with TCA. The soluble and particle proteins were prepared by centrifuging the reaction mixture after incubation and then precipitating the fractions with TCA. Therefore, the soluble and the particle counts/rain should sum to give the total protein counts/ min. Discrepancies are ascribed to the failure to recover all the protein in the recentrifuged samples. The data obtained in the absence of added amino acids (Table VI) show that more than 9 ° % of the counts incorporated after 5 and 45 min incubation are bound to the particle material. There seems to be no significant release of labeled protein into the soluble fraction throughout the incubation period; however, only about 7 ° % of the total protein was recovered in the recentrifuged material. WEBSTER4 has shown that the addition of a mixture of 18 amino acids and a preparation of s-RNA transforms the pea incorporating system into one that carries out the net synthesis of soluble protein. The data presented in Table VI demonstrate that the addition of a mixture of amino acids to the incorporating system has little effect on the reaction. The total incorporation was slightly higher in the presence of amino B i o c h i m . B i o p h y s . A c t a , 5o (1961) 287-30o

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R.j.

MANS, G. D. NOVELLI

acids and recovery of the recentrifuged material was enhanced; however, 88'I{~ of the radioactivity remained associated with the particle material. Furthermore, no significant increase in total protein concentration was detected after incubation of the reaction mixture. Thus, even in the presence of amino acids all the incorporated leucine remained associated with the particle. This experimental result demonstrates the validity of the presentation of incorporation data in terms of counts/min/mg particle protein. The conclusion that maize particles become saturated with respect to leucine incorporation is indicated by (a) the finding that essentially all the radioactivity incorporated remains with the particle protein throughout the incubation period, (b) the nature cf the time course of incorporation, and (c) the saturation of the system with rat liver pH 5 in the presence of excess cofaetor requirements. A reasonable explanation of these observations is to assume that each particle possesses a limited number of leucine sites which are rapidly filled in the presence of excess rat liver pH 5. If this assumption is correct than addition of more particles to the system in the presence of excess rat liver pH 5 should result in particles of equal specific activity independent of particle concentration. This idea was tested. Increasing amounts of washed maize particles were added to identical reaction mixtures in the presence of o.43 mg rat liver p H 5. When the data are plotted in terms of the total counts recovered versus the amount of particle protein added, it is readily apparent (curve A, Fig. 4) that the amount of leucine incorporated into the TCA-precipitated protein is a linear function of the amount of maize particle protein added to the reaction mixture. At the end of 3o min incubation the specific activity of the particle protein recovered from each flask was essentially the same (curve B, Fig. 4)- This is the result predicted on the assumption of a limited number of leucine sites per maize particle. 2500-

A/

E 2oooo

-Boo

z

>-~_ L~ >~ 7__ ° -600 ~

_o ~500-

g oo ~O00z o 500-

/°

-4oo

-2oo

~ E

o

Fig. 4" I,eucine incorporation in the presence ~f rat liver p H 5 and increasing a m o u n t s of maize particle protein. The reaction m i x t u r e s are as described in Table I V w i t h the exception of the protein components. A total of o.43 m g r a t liver p H 5 plus increasing a m o u n t s of washed maize particle protein were added as indicated. Curve A is a plot of the total a c t i v i t y and curve 13 is a plot of the specific a c t i v i t y of the particle protein.

0.66 1,32 t82 2.64 313 MAIZE P/~RTICLEPROTEIN ADDED(rng)

Investigation of the maize soluble

As discussed in an earlier section, tile data in Table I I I indicated that in the mixed maize-rat liver system a component of the maize soluble might be lacking in rat liver pH 5. This could be a soluble enzyme component responsible for stripping protein from the particle as reported by BISHOP et al. 19 for the hemoglobin system. Therefore, the properties of the maize soluble fraction were reinvestigated. By precipitating the 105000 × g supernatant at p H 5.1 with acetic acid, instead of at p H 5.2 as is done with rat liver 11, and centrifuging at 20 ooo × g for 20 rain we Biochim. Biophys. ,4cta, 50 (1961) 287 3 °0

297

AMINO ACID INCORPORATION

were able to isolate a maize p H 5 fraction which was as active as rat liver p H 5 on a protein basis. When the maize supernatant was adjusted to p H 5, the precipitate had no stimulatory activity, indicating the critical nature of the isoelectric precipitation. We tested the requirements for leucine incorporation in the presence of an active maize p H 5 fraction. The results are presented in Table VII. In the same reaction mixture used to assay leucine incorporation with rat liver pH 5, the all-maize system incorporated 195 counts/min/mg protein. This incorporation represents slightly less than half of the activity seen in the presence of rat liver p H 5 (cf. Table IV). TABLE VII REQUIREMENTS

FOR

LEUCINE

INCORPORATION

IN

THE

PRESENCE

OF M A I Z E

pH

5

FRAC'I1ON

T h e c o m p l e t e m i x t u r e w a s t h e s a m e as t h a t described in Table IV w i t h t h e e x c e p t i o n s t h a t 1.8 m g of m a i z e p H 5 w a s s u b s t i t u t e d for t h e r a t liver p H 5 a n d 1.3 m g of w a s h e d maize particle p r o t e i n was used. Specific a c t i v i t y is r e p o r t e d as c o u n t s / m i n / m g particle protein. System

Complete Complete Conlplete Complete Complete Complete

minus minus minus minus minus

Incorporation countumin/mg

ATP MgC12 P E P a n d p y r u v i c kinase GTP maize p H 5 fraction

195 46 7 93 85 48

Note that the particles minus the maize pH 5 fraction incorporated 48 counts/min/mg protein. This activity indicates that a significant amount of supernatant material remained with the particles. Stimulation b y the addition of ATP, an ATP generator, MgC12, and GTP was demonstrated in this system as it was in the mixed system, i.e., maize particles and rat liver p H 5. However, the GTP stimulation was pronounced in the all-maize system in contrast to the erratic results obtained in the mixed system. Interpretation of this latter observation requires further investigation of the role of GTP as well as the nature ot the intermediate components involved in the incorporation. The requirement for magnesium is absolute in the all-maize system and appears to be more critical than in the mixed system. The maize p H 5 preparations generally are less active than comparable preparations from rat liver. Furthermore, the maize preparations are labile to storage at - - i o °. I t is conceivable that the major proportion of the components necessary to stimulate leucine incorporation into maize particles was not precipitated b y adjusting the supernatant to p H 5.1. We therefore tested the activity of the whole maize supernatant. The data plotted in Fig. 5 were obtained by incubating the maize particles in the presence of maize high-speed supernatant. Aliquots were removed at the times indicated and then recentrifuged in order to separate the soluble and the particle proteins. Curve A represents the labeling of the soluble protein, and Curve B represents the labeling of the particle protein with [i-14Clleucine. From curve B of Fig. 5, it can be seen that 9 o % of the radioactivity remains associated with the particle protein throughout the course of the incubation period. The same proportion of bound radioactivity was found with the mixed system (see Table VI). It also can be seen that the time course for leucine incorporation is essentially the same as that obtained in the mixed system. When one compares this experiment (conducted with Biochim. Biophys. Acta, 50 (1961) 287-300

298

R . J . MANS, C. D. NOVELLI

an all-maize system) with the experiment of Fig. 2 (conducted with the mixed system), the most striking difference is that the particles in the all-maize system become saturated with maize supernatant at 3 times the specific activity of the particles in the mixed system (cf. Figs. 5 and 2). The steady rise in the specific activity of the soluble protein for the first 30 rain incubation, curve A, Fig. 5, is probably caused by contamination of the reseparated soluble fraction with labeled particle protein, since the radioactivity in the soluble protein represents a constant percentage of the total incorporation. Therefore, in the absence of a net increase in total protein, the appearance of radioactivity in the soluble protein cannot be interpreted as a "stTipping" Off of newly synthesized protein from the particles. The erratic results obtained in earlier experiments with the maize p H 5 are explained by the fact that the major portion of the stimulating activity remains soluble at p H 5.1. The data presented in Table V I I I were obtained by preparing a pH 5 fraction from maize and then adjusting the nnprecipitated material to pH 7.5. As shown in Table V I I I , essentially all the activity originally present in the high-speed supernatant is recovered in the supernatant after removal of material precipitated at p H 5.1, analogous to the findings of GRossI AND MOLDAVE2° and SATAKE el al. 2'

...I000 I 8oot

/

ij a0400~ /

0

2}) 4() INCUI3ATIONTIME(min)

60

Fig. 5. K i n e t i c s of leucine i n c o r p o r a t i o n in t h e presence of maize p a r t i c l e s a n d m a i z e supern a t a n t . T h e r e a c t i o n m i x t u r e c o n t a i n e d 125 /,moles Tris buffer (pH 7.5), 5 /,moles ATP, 1. 5 ffmoles G T P , 25 ffmoles MgC12, 47.5/*moles P E P , ioo fig p y r u v i c kinase, 0..57.5 ffmole DL~I-14C] leucine (3.7" 1°6 counts/rain), i ffmole KCI, 4 m g maize high speed s u p e r n a t a n t p r o t e i n a n d 16 m g w a s h e d maize particle p r o t e i n in a t o t a l v o l u m e of 5 ml. i - m l aliquots were r e m o v e d at t h e t i m e s i n d i c a t e d a n d a n a l y z e d as described in t h e t e x t . C u r v e A is t h e specific a c t i v i t y of t h e soluble protein a n d c u r v e B is t h e specific a c t i v i t y of t h e particle protein.

with mammalian preparations. The addition of twice as much lO5 ooo >~ g supernatant or p H 5 supernatant resulted in no increase in specific activity. This finding indicates that the supernatant was present in excess. On the other hand, increasing amounts of maize p H 5 precipitate resulted in slight increases in specific activity of the particle protein. When increasing amounts of maize particles were added to a reaction mixture in the presence of excess maize supernatant, again it was found that the particles appear saturated with respect to leucine incorporated in 30 rain. Total counts incorporated and milligrams of particle protein added is plotted in curve A of Fig. 6. Curve B, a straight line, indicates that the specific activity of the particles is relatively constant at 760 counts/min/mg and is independent of particle concentration. This result is to be compared with the data obtained with rat liver pH 5 plotted in Fig. 4, in which case the maize particles appeared saturated at 47 ° counts/min/mg particle protein. Furthermore, the data in Table I X demonstrate that addition of maize high-speed supernatant to washed particles results in the complete restoration of the activity measured in unwashed particles. The addition of maize supernatant to unwashed particles causes no appreciable increase in activity. These results are in Biochim. ]3iophys. _4eta, 5o (I96t) 2 8 7 3o,~

AMINO ACID INCORPORATION

299

c o n t r a s t to the d a t a o b t a i n e d with the mixed system (see Table I I I ) . As suggested previously, the maize s u p e r n a t a n t m a y c o n t a i n a factor or factors required for m a x i m u m incorporation t h a t m a y n o t be present in rat liver p H 5, a n d the factor does not seem to be a " s t r i p p i n g e n z y m e " . Variation in specific a c t i v i t y of particle protein is e n c o u n t e r e d in d a y - t o - d a y preparations. For example, in experiments not shown here, specific activities as TABLE VIII FRACTIONATION

OF

MAIZE

HIGH-SPEED

SUPERNATANT

The complete reaction mixture was prepared as described in Table IV with the omission of rat liver pH 5. The soluble components indicated in this table were added to the complete reaction mixture in the amounts shown. Flasks were incubated as described in METHODSfor IO min. Specific activities are reported as counts/min/mg particle protein. lncorpomtion counts/min/mg

System

Complete Complete plus maize IO5,OOOx g supernatant (225/ig) Complete plus maize pH 5 precipitate (36o/*g) Complete plus maize pH 5 supernatant (16o/~g)

4I 945 20o 920

J l °°°

?~ 600-

.L

o

Fig. 6. Leucine incorporation in the presence of maize supernatant and increasing amounts of maize particle protein. The reaction mixtures are as described in Table IV with the exception of the protein components. A total of o.54 mg maize high speed supernatant plus increasing amounts of washed maize particle protein were added as indicated. Curve A is a plot of the total activity and curve B is a plot of the specific activity of the particle protein.

•

2o-"-----L

c~4co

o'

40%~

o u-u.

/o

S

g

/° 0

2co~ E

0.62 t.24 t86 2.48 MAIZE PARTICLE PROTEIN ADOEO(mg)

high as 1600 c o u n t s / m i n / m g particle protein have been o b t a i n e d in the all-maize system. I n view of the lability of the maize soluble c o m p o n e n t , fresh p r e p a r a t i o n s are used for each experiment. This lability m a y explain the differences seen in final specific activity. On the other hand, v a r i a t i o n in the relative competence of the washed maize particles from different homogenates also is observed. I n c o r p o r a t i o n b y washed particles alone ranges from 25 to Io0 c o u n t s / r a i n / r a g particle protein a m o n g different preparations. An e x p l a n a t i o n is not a p p a r e n t for the s a t u r a t i o n of the maize particles with leucine in the mixed system at a lower level t h a n the s a t u r a t i o n of the particles with leucine in the all-maize system. The all-maize system differs from the pea systems described b y RAAKEa a n d WEBSTER4,22 in 3 m a j o r aspects: (a) The maize system requires a soluble fraction for full a c t i v i t y whereas the pea i n c o r p o r a t i n g systems do not. WEBSTER22 has reported, however, t h a t washing pea seedling particles in o.oi M KC1 removes incorp o r a t i n g a c t i v i t y which can be restored b y the a d d i t i o n of pea p H 5 protein. (b) The pea systems are s t i m u l a t e d b y the addition of a m i x t u r e of a m i n o acids, whereas the maize system shows little or no effect upon the addition of amino acids. Differences Biochim. Biophys. Acta, 50 (1961) 287-300

300

R. J. MANS, G. D. NOVELLI TABLE I X RESTORATION

OF

ACTIVITY

OF

WASHED

PARTICLES

BY

MAIZE

SUPERNATANT

The complete reaction mixture was the same as t h a t described in Table IV witll the exceptions t h a t 1.6 mg of particle protein was used and the rat liver pH 5 was omitted. Where indicated, o.25 mg of maize supernatant protein was added. Specific activity is expressed as counts/min/nlg particle protein. Particle preparation Unwashed Washed

Particlesa~onr I'articlesplus c.ltnts/min/mg maize supernatanl COIO~[S~ni~t,'tn~

540 139

51~o 552

in t h e e n d o g e n o u s l e v e l of free a m i n o a c i d s p r e s e n t in t h e t w o p l a n t m a t e r i a l s 17 or a r i s i n g d u r i n g i n c u b a t i o n of t h e p a r t i c l e s m a y a c c o u n t for t h i s r e s u l t . S h o r t - t e r m d i a l y s i s ot t h e m a i z e h i g h - s p e e d s u p e r n a t a n t r e s u l t e d in a m o d e r a t e i n c r e a s e in i n c o r p o r a t i o n w h i c h is p r o b a b l y d u e t o t h e r e m o v a l of n o n r a d i o a c t i v e leucine. (c) N e t p r o t e i n s y n t h e s i s h a s b e e n d e m o n s t r a t e d in t h e p e a s y s t e m 4 w h e r e a s o n l y a m i n o a c i d i n c o r p o r a t i o n h a s b e e n d e m o n s t r a t e d in t h e m a i z e s y s t e m . A t t e m p t s t o d e m o n s t r a t e n e t s y n t h e s i s of p r o t e i n in t h e m a i z e s y s t e m b y t h e a d d i t i o n of a m i x t u r e of a m i n o a c i d s a n d s - R N A h a v e n o t b e e n s u c c e s s f u l as y e t . S u b t l e d i f f e r e n c e s in t h e level of s o l u b l e c o m p o n e n t s i n c l u d e d in t h e i n c u b a t i o n m i x t u r e , as well as d i f f e r e n c e s in t h e n a t u r e of t h e s o l u b l e a n d p a r t i c l e p r e p a r a t i o n s , m a y b e r e s p o n s i b l e for t h e d i f f e r e n c e in c a p a b i l i t y b e t w e e n t h e m a i z e a n d t h e p e a s y s t e m s .

REFERENCES 1 R. RABSON AND G. D. NOVELLI, Proc. Natl. Acad. Sci. U.S., 46 1196o) 484 . 2 R. J. MANS AND G. D. NOVELLI, unpublished data. 3 D. SCHWARTZ, Science, 129 11959) 1287. 4 a . C. WEBSTER,Arch. Biochem. Biophys., 85 11959) 159. I. D. RAAKE, Biochim. Biophys. Acta, 34 11959) I. M. L. STEPHENSON, K. V. THIMANN AND 17. C. ZAMECNIK,Arch. Bioehem. Biophys., 65 (1956) 194. P. C. ZAMECNIKAND E. B. KELLER, J. Biol. Chem., 209 11954) 337. s L. M. HALL AND 17. 17. COHEN, J. Biol. Chem., 229 11957) 345. 9 G. SCHMIDT in S. 17. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vnl. II, Academic Press, New York, 1957, p. 227. 10 M. WOLFE AND L. FOWDEN, Cereal Chem., 34 11957) 286. 11 M. B. HOAGLAND, E. B. KELLER AND P. C. ZAMECNIK,J. Biol. Chem., 218 11956) 34512 17. SIEKEVITZ,J. Biol. Chem., 195 11952) 549la O. H. LOWRY, N. J. ROSEBROUGH,A. g. FAAR AND R. J. RANDALL,J. Biol. Chem., 193 (1951 ) 265. 14 17. O. P. Ts'o, J. BONNER AND J. V1NOGRAD,Biochim. Biophys. Acta, 3° (1958) 57 o. 15 A. C. CHIBNALL, Protein Metabolism in the Plant, Yale University Press, New Haven, 1939. 16T. A. KIESSELBACH, University o/ Nebraska, College o/ Agriculture, Agricultural Experiment Station, Research Bulletin, 61, 1949. 17 j . M. LAWRENCE, K. M. DAY AND J. E. STEPHENSON, Plant Physiol., 34 11959) 668. 18 j . S. EDELMAN, I. SHIBKO AND A. J. KEYS, J. Exptl. Botany, io 11959) 178. 19 j . BISHOP, E. ALLEN, J. LEAHY, A. MORRIS AND R. SCHWEET,Federation Proc., 19 1196o) 346. 20 L. G. GROSSI AND K. MOLDAVE, Biochim. Biophys. Acta, 35 11959) 275. 21 M. SATAKE, K. MASE, Y. TAKALTASHIAND K. OGATA, Biochim. Biophys. Acta, 41 1196o) 366. 22 G. C. WEBSTER, J. Biol. Chem., 229 11957) 535Biochim. Biophys. Acta, 51) (1901) 287 30o