of the rates of protein synthesis and degradation · protein turnoverrates in lemna ter....

Plant Physiol. (1972) 49, 40-46

Determination of the Rates of Protein Synthesis and Degradationin Lemna minor

Received for publication June 3, 1971

A. TREWAVAS1Biological Sciences, University of East Anglia, Norwich, England

ABSTRACT

Attempts to measure the rates of synthesis and degrada-tion of protein in plant tissues with isotopes are complicatedby the presence of at least two pools of amino acids, only oneof which contributes to the synthesis of protein. Direct meas-urement of the protein precursor pool is thus difficult. Thispaper shows that one solution to this problem is to assumethat the amino-acyl transfer RNA is the strict precursor ofprotein amino acid. By using labeled methionine, the varia-tion with time of the specific radioactivities of methioninebound to RNA and protein have been examined under twodifferent growth conditions in Lemna minor. From these datarates of flux of methionine into and out of protein may beeasily determined.A second method of determining the rates of protein synthe-

sis and degradation assumes that the specific radioactivity ofthe methionyl transfer RNA in label chase conditions is effec-tively zero. Evidence is presented to support this contention,and the rates determined by this method agree with those cal-culated by the first method.

The overriding difficulty in making definitive measurements ofthe rates of synthesis and degradation of protein in plants is thedetermination of the specific radioactivity of the precursor aminoacid. The simple assumption that extractable free amino acidrepresents the free precursor pool may be criticized on the evi-dence showing the presence in many tissues of active and inac-tive pools (7). One attempt which has been made to overcomethis difficulty is to swamp the precursor pool with exogenousamino acid of known specific radioactivity (3). There are obviousdisadvantages to this method (4).These difficulties can be overcome if it is recognized that the

immediate precursor of protein is not the free amino acid but theamino acid attached to transfer RNA. The difference in chemicalnature between the amino acid attached to RNA and that which isfree or bound to protein greatly simplifies the necessary purifica-tion and subsequent determination of specific radioactivities.As far as the author is aware, there is no evidence for the pres-

ence of active and inactive pools of amino acyl-tRNA. The de-termination of the rate constants of synthesis and degradation ofprotein is thus made considerably easier.One problem in measuring the level of amino acid attached to

tRNA is that the amount is likely to be extremely small, and

1 Present address: Department of Botany, University of Edinburgh,King's Buildings, Mayfield Road, Edinburgh, EH9, 3JH, Scotland.

therefore difficult to assay accurately. We have solved this prob-lem for methionine by using a radioactive method which involvegrowing Lemna to isotopic equilibrium on 35SO4.

Determination of the level of methionine is then readily madeby measurement of 35S-radioactivity in purified methionine.Specific radioactivities may subsequently be obtained by labelingthe tissue with tritiated methionine.

MATERIALS AND METHODSThe same strain of Lemna minor was used as previously de-

scribed (10). Growth conditions were identical except that incertain experiments 2 mM MgSO4 was replaced by 0.2 mMMgSO4. Our last reported list of nutrients accidentally omittedCoCl2 6H20, 0.1 piM, ZnSO4,7H20, 0.76 ,M, H2MOO4, 0.5 pM.Cultures were checked for contamination at each transfer (10),and nonsterile experiments were discarded. During the course ofthese investigations the cultures were examined by electron mi-croscopy and no contaminating or symbiotic microorganismswere observed. I am grateful to Dr. D. Wildon for performingthe electron microscopy.The following were obtained from the Radiochemical Centre,

Amersham: Na235SO4 (carrier free) and 3H-methyl-methionine(10 curies/mmole).

Isolation and Estimation of Protein. Samples of Lemna wereground in a mortar and pestle in a total of 3 ml of 10 mM tris,pH 7.0, and centrifuged at iOOOg for 5 min. The supernatant wasremoved and precipitated with an equal volume of 5%,7 (w/v)trichloroacetic acid at 0 C for 24 hr. After centrifugation theprecipitate was extracted twice with 80% (v/v) ethanol and threetimes with methanol at 60 C for 5 min. The precipitate was al-lowed to drain until partly dry. For every 50 mg initial freshweight of tissue, 1 ml 0.3 N NaOH was added, the mixture wasincubated at 37 C for 2 hr, and any remaining insoluble materialwas removed by centrifugation. Assays for total protein werecarried out on aliquots of this solution using the biuret method.The relationship between protein content and fresh weight waslinear over the range 50 to 500 mg fresh weight and an averagevalue of 12 i 0.67 mg protein/g fresh weight was obtainedfor 10 tissue batches in this weight range. The protein contentvaries slightly between different strains.

Hydrolysis of Labeled Protein. One-ml aliquots of the alkalineextract were removed and precipitated with 1 ml of 5% trichloro-acetic acid for 24 hr at 0 C. The precipitate was washed once with5% trichloroacetic acid. Excess acid was removed, the precipi-tate was dissolved in 0.2 ml 90% formic acid, and the dissolvedprotein was transferred to an ampule, together with 0.2 ml of 6 NHCl. Hydrolysis was carried out in vacuo (6) at 110 C for 16 hr,and the HCI was removed as described by Moore and Stein (6).The amino acids were dissolved in water and chromatographedone-dimensionally on Whatman No. 3MM using butanol-aceticacid-water (12:3:5 v/v). Methionine was located by the use ofmarkers and eluted chromatographically in about 0.2 ml of wa-

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PROTEIN TURNOVER RATES IN LEMNA

ter. Radioactivity was determined by mixing the eluate with adioxane-cellosolve (5:1) scintillator and counting in a scintilla-tion counter.

Determination of the Specific Radioactivity of Methionine inProtein. Lemna was grown for at least eight generations on su-crose-mineral salts containing 0.1 or 1 p.c of 3'S04/mL. Isolationof the methionine from protein and determination of its 35S-radioactivity enabled the accurate measurement of the level of

exogenous methionine -* endogenous synti

also be electrophoresed on glass fiber paper. Under these condi-tions the nucleic acids move about twice as fast as the most highlycharged pectins (1), indicating elimination of these substancesduring the nucleic acid purification.Methods of Determining Flux Constants of Amino Acids through

Protein. Using methionine as an example, current theory indi-cates the following sequence of reactions for the movement ofamino acids into and out of protein.

I Vtacid-soluble methionine (A) - methionyl-tRNA(T)

pool

other reactionsI V.

Vd \ acid-insol ublemethionine (P)

this amino acid to be made. Methionine represented about 40%of the total acid-insoluble sulfur and 0.6 to 0.7 umoles methi-onine/g fresh weight were normally recoverable. There was nodetectable loss of added radioactive methionine during the hy-drolysis procedure.For the purposes of obtaining a specific radioactivity, the 35S-

Lemna were transferred for 1 hr to 1 p.c/ml 3H-methyl-methi-onine. Isolation of the protein methionine from these samplesand determination of the 3H/35S ratio gave a direct measurementof the specific radioactivity.

Determination of the Specific Radioactivity of the Methionyl-tRNA. Plants were grown to equilibrium on 35SO4 and labeledwith 3H-methyl-methionine as described above. Nucleic acidswere isolated by homogenizing the tissue in equal volumes of0.1 M sodium acetate-10 mm disodium EDTA-1 mm methionine,pH 5.0 (AEM2 buffer 1) and phenol mixture (80%- phenol-10%m-cresol-0.01 % 8-hydroxyquinoline) (2). After 15 min stirringat room temperature, the mixture was centrifuged, the aqueouslayer was removed and precipitated with two volumes of ethanolat -15 C. The pellet was redissolved in AEM buffer and repre-cipitated twice more. The pellet was redissolved in about 10 piof AEM buffer and electrophoresed on cellulose acetate strips(Oxoid Ltd.) for 90 min at 0 C at 10 v/cm. The buffer compart-ments contained AEM buffer without the methionine. The mo-bility of the nucleic acids was followed using an ultraviolet light,and the nucleic acid band was moved about 6 cm during the run.The band was cut out and eluted with 0.5 ml AEM buffer and

precipitated by the addition of 0.05 ml of 1 cetyltrimethyl-ammonium bromide at 0 C for 5 min. The pellet was washedwith 70%70 ethanol containing 0.1 M sodium acetate, pH 5.0, dis-solved in 0.1 ml 0.2 M ammonium carbonate, pH 9.0, and in-cubated at 37 C for 1 hr to strip the amino acids from the tRNA.Two volumes of ethanol were added, and the RNA was removedby centrifugation. One hundred micrograms of methionine wasadded to the clear supernatant and this was applied to a sheet ofWhatman No. 3MM and developed with butanol-acetic acid-water (12:3:5 v/v). The methionine was located using markersand eluted and counted in dioxane-based scintillation mixture.In general 200 to 400 pmoles of methionine were isolated per mgnucleic acid. The unstable nature of this complex makes it un-likely that these figures represent in vivo levels of methionyl-tRNA. Overnight storage of the purified nucleic acid at -15 Cappeared to result in some loss of the methionyl-tRNA, and it islikely, therefore, that losses occur during the extraction procedure.The inclusion of EDTA in the electrophoresis buffer was found

to be essential to prevent an otherwise irreversible sticking of thenucleic acid to the origin. Using AEM buffer, the RNA could

2 Abbreviation: AEM: sodium acetate-disodium EDTA-methioninebuffer.

where Vt = absolute rate of synthesis of methionine into methi-onyl-tRNA; V8 = absolute rate of synthesis of methionine intoprotein from methionyl-tRNA; and Vd = absolute rate of degra-dation of methionine from protein.

Proteins may be heterogenous with respect to their rates ofsynthesis and degradation in plants. V8 and Vd will representrespectively average rates of synthesis and average rates of lossof methionine from protein.

If Lemna is placed on labeled methionine, then the cellularpools will become labeled. Putting St = specific radioactivity ofmethionine attached to RNA; Sp = specific radioactivity of acid-insoluble methionine; P = total acid-insoluble methionine orprotein methionine; then the relationship between these threeparameters and V8,,the rate of synthesis, is given by the equationfirst described in Reiner (8), namely:

dSp = (St -Sp)V

dt Sp (1)

In a growing system the quantity of acid-insoluble methioninewill vary, and its rate of accumulation or loss will clearly be thedifference between the rate of synthesis and rate of degradation.Thus

dP1-- = V. - Vd (2)dt

In a growing culture of Lemna, V8, the rate of synthesis ofacid-insoluble methionine of the culture and P, the total acid-insoluble methionine of the culture, will vary. If a steady state ofgrowth is to be maintained, then both V8 and P will be directlydependent on the size of the culture and the ratio V8/P should beconstant and may be designated K8, the rate constant of synthe-sis of protein. Thus

V.y= K8, or V. = K.P(3

The evidence available (e.g., 8) suggests that the degradationof protein is a simple first order process. In this case then

Vd = Kd-P (4)where Kd = the rate constant of degradation of methionine fromprotein. Combining equations 2, 3, and 4 we obtain

dP= (K., - Kd) -P()

Therefore integrating and taking logs gives

log P = (K8-Kd)t + C (6)

Plant Physiol. Vol. 49, 1972 41

,

(3)

(5)

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Plant Physiol. Vol. 49, 1972

and a graph of log P against time should yield a straight line (seeFig. 3) whose slope = K8 - Kd . If either K8 or Kd can be deter-mined independently, the use of equation 6 will enable K. or Kdto be determined by difference. Two methods are described in thispaper for independent determination of K8 .

METHOD 1

Combining equations 1 and 3 gives

dt = (St - Sp)KS (7)dt

If conditions are set such that both methionyl-tRNA and acid-insoluble methionine are labeled, then K, may be determineddirectly from equation 7, providing the following information isknown: the specific radioactivity of methionine attached to tRNAat time t, and a curve showing the variation of the specific radio-activity of methionine in protein with time. From the latter re-sult both Sp, the specific radioactivity of methionine in proteinat time t, and the slope (dSp) /dt at time t can be determined.

METHOD 2

In this method conditions are set such that, after labelinggrowth is allowed to take place for a considerable period fromunlabeled sources. The assumption is then made for which evi-dence will be found later in this paper, that the specific radioac-tivity of methionine attached to tRNA, (St), may be consideredeffectively zero. In this case equation (7) may be simplified to

dStdt = -S (8)

Integrating and taking logs of both sides of the equation yields

log Sp = -Kst + C8 (9)

A graph of log of the radiospecific activity of acid-insoluble me-thionine with time should yield a straight line (see Fig. 3) whoseslope is -K,, the rate constant of synthesis.

For both methods 1 and 2, Kd is determined from the differencebetween the measured value of K, and the slope of the line log Pagainst time.

RESULTS

DETERMINATION OF PROTEIN TURNOVER RATES USING METHOD 1

High Growth Rate Conditions. Figure 1 shows an experimentin which Lemna, growing on sucrose-mineral salts containing"5SO4 has been placed on 3H-methyl-methionine. Figure IA showsthe rate of uptake of radioactive methionine from the mediuminto the tissue using an arbitrary initial value of 10. Over 80%c ofthe labeled methionine in the medium has been taken up into thetissue by 4 to 4.5 hr. Measurements of specific radioactivitieshave not therefore been made past this time period. Figure 1, Band C shows the variation with time of the specific radioactivitiesof the methionyl-tRNA and acid-insoluble methionine duringthe 4.5-hr period. The specific radioactivity of the methionyl-tRNA shows a peak about 0.5 hr after commencement of label-ing and then settles to a plateau value of 5.5 after 0.75 hr. Thispattern of labeling is not dissimilar to that noted by Hoaglandet al. (2). The specific radioactivity of the acid-insoluble methi-onine during this period shows a steady, almost linear increasewith time. In some experiments there have been indications of alag period of 5 to 10 min in length before this steady rate oflabeling is observed. Because of the fluctuations in labeling inearly time periods, calculations of the rate constants of synthesishave been made at 1, 2, 3, and 4 hr.

For these calculations we have used equation 7, a value of 5.5for the specific radioactivity of the methionyl-tRNA and tangentsto the curve and specific radioactivities of protein methionine inFigure IC at 1, 2, 3, and 4 hr. Calculated values of K, the rateconstant of synthesis of protein methionine, are 0.02, 0.0199,0.0198, and 0.0199 hr-'. This gives an average of value 0.0199 hr-or 0.477 days-'. To determine Kd, the rate constant of degrada-tion of protein methionine, the data in Figure 1D has been used.This shows the rate of accumulation of acid-insoluble methioninein an identical culture to that used for the experiment shown inFigure 1, A, B, and C. Equation 6 shows that the slope of thisline in Figure ID, which is 0.39 days-', is equal to the differencebetween the rate constant of synthesis of protein and the rateconstant of degradation. Thus from KS and Figure 1D a value of0.087 days-' may be calculated for the rate constant of degrada-tion of methionine from protein.An alternative method of estimating K, from the data in Figure

1, B and C, is suggested by the fact that over most of the experi-mental period examined the specific radioactivity of the methi-onyl-tRNA is at a constant value of 5.5. Equation 7 in this casecan be integrated, as shown by Russel (9), to give the equationSp = St(l - e- t)K The line which has been drawn in FigureIC is the equation Sp = 5.5 (1 - e-0.0') and this represents anexcellent fit with the data which are the points. Thus this methodgives an independent assessment of 0.02 hr-' for K, .Low Growth Rate Conditions. To investigate the possibility

that different growth conditions for Lemna may alter the patternof labeling of methionyl-tRNA and acid-insoluble methionine,Lemna has been grown on sucrose-mineral salts containing 0.2mm magnesium sulfate instead of the normal 2 mm. Growth onthis medium is exponential but slower. The rate constant forfresh weight increase for the experiment shown in Figure 2 inwhich this altered medium was used was 0.18 days-', instead ofthe more typical 0.37 days-' for the experiment shown in Figure 1.

Figure 2A shows the variation with time of the specific radio-activity of the methionyl-tRNA and acid-insoluble methioninewhen Lemna was placed on 3H-methyl-methionine under theselow growth rate conditions. Comparing with Figure 1, certaindifferences can be seen. The methionyl-tRNA no longer exhibitsan early peak in labeling, but instead simple saturation kineticsare observed. The pattern of incorporation of the label into theacid-insoluble methionine, on the other hand, is similar to thatshown in Figure IC. Using equation 7, tangents and points at 1,2, 3, and 4 hr to the acid-insoluble methionine curve and the1 to 4 hr values of the methionyl-tRNA, the rate constant ofsynthesis, Ks, can be calculated to be 0.015, 0.014, 0.127, and0.0118 giving an average value of 0.0133 hr-' or 0.32 days-' forthe first 4 hr. Figure 2B shows the increase in acid-insolublemethionine in a duplicate culture and the slope of this line is0.19 days-'. The rate constant of degradation may therefore becalculated by use of equation 6 to be 0.13 days-'. One effect,therefore, of growing Lenmna on medium with a 10-fold reductionin the magnesium sulfate is a reduction in the rate constant ofsynthesis from 0.48 to 0.32 and an increase in the rate constantof degradation from about 0.09 to 0.13 days-'.

Further information can be obtained from Figure 2A. The linedrawn through the points of the methionyl-tRNA data is theequation, specific radioactivity = 7.4 (1 - e-3t). This can beexplained in the following manner. When Lenina is placed onlabeled methionine, the precursor pool of methionine rapidlyassumes a constant specific radioactivity. The relationship be-tween this precursor pool and methionyl-tRNA is given by theequation (dSt,'dt) = (Sa - St)Kt where Sa, is the specific radio-activity of the precursor pool of methionine, and Kt is the rateconstant of synthesis of methionyl-tRNA from methionine.If Sa is constant then the equation may be integrated, as shown byRussel (9), to yield S, = Sa(l - e 'f'). Thus, the methionyl-

42 TREWAVAS




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A hours B hours

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D1 2 3 4 5 6 7

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C hours

FIG. 1. Variation of the uptake of methionine, the specific radioactivity of methionyl-tRNA and acid-insoluble methionine with time aftertransfer of fronds to 3H-methyl-methionine. Cultures of Lemna were grown for at least eight generations on sucrose-mineral salts containing1 ,uc of 35SO4/ml. At time zero 100 Mc of 3H-methyl-methionine was added to the culture, samples were taken for the succeeding 4 to 4.5 hr, andthe specific radioactivity of the methionine (cpm per pmole methionine) attached to RNA and acid-insoluble methionine was determined fromthe 3H/35S ratio of the purified methionine. A: The rate of uptake of methionine from the medium with an arbitrary initial value of 10. B: Thespecific radioactivity of the methionyl-tRNA. C: The specific radioactivity of the acid-insoluble methionine. Data from two experiments are shownin the graph. The line which has been drawn is the theoretical curve specific radioactivity = 5.5 (1 e-00t). D: The long term accumulation ofacid-insoluble methionine in Lemna.

RNA data in Figure 2A suggest that a rate constant of synthesisof methionyl-tRNA from methionine may be 3 hours-l.

DETERMINATION OF PROTEIN TURNOVER USING METHOD 2

In this method Lemna is labeled with 3H-methyl-methionine,and conditions are then set so that growth takes place from un-labeled sources for a considerable portion of the experimentalperiod. The assumption behind this method is that the specificradioactivity of the precursor during experimental analysis maybe assumed to be at or near zero. Evidence for this contention ispresented in the section below and in the following paper.

In the experiment shown in Figure 3 Lemna grown on sucrose-mineral salts was labeled for 1 hr with 3H-methyl-methionine,the fronds were distributed among seven flasks and allowed togrow in the absence of labeled methionine. One flask was removedeach day, and the total acid-insoluble methionine and the specificradioactivity of the acid-insoluble methionine was determined.The rate constant for frond number increase was 0.35 days-'.The results in Figure 3 support the predictions of equations 6 and9, that the variations of log,0 P and logi0 Sp with time are linear.The slopes of the lines are 0.48 days'l for the specific radioac-tivity, and 0.36 days-' for the total acid-insoluble methionine;thus the calculated rate constant for degradation in this experi-

ment is 0.12 days'l. Determination of the rate constant of degra-dation by this method has been made from seven different ex-periments using 3H-methyl-methionine as a precursor and themean 41 standard error was 0.103 + 0.022. Using the equationhalf-life = log. 2/rate constant an average half-life of 6.8 daysmay be determined.The average value for the rate constant of degradation, 0.103,

determined by method 2 is in excellent agreement with the deter-mined rate constant of degradation obtained from the data inFigure 1, 0.087.

Kinetics of Methionyl-tRNA during Label-Chase Conditions.

One requirement for the validity of method 2 given above is thatthe specific radioactivity of the precursor, methionyl-tRNA, beeffectively zero during the experimental period. The agreementbetween methods 1 and 2 for the rate constant of degradationsuggests that this should be the case. Attempts to confirm thisdirectly have been made by examining the kinetics of the specificradioactivity of methionyl-tRNA and acid-insoluble methionineunder label-chase conditions.

In the experiment in Figure 4A, Lemna grown on sucrose-mineral salts was labeled for 1 hr with 3H-methyl-methioninebefore transfer to sucrose-mineral salts in the presence or absenceof 10 JM methionine. The specific radioactivity (cpm per pmolemethionine) of the methionine attached to RNA and the acid-

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FIG. 2. Variation of the specific radioactivity of methionyl-tRIand acid-insoluble methionine with time after transfer of fronds groon low magnesium sulfate to 3H-methyl-methionine. Cultures of Lenwere grown for at least eight generations on sucrose-mineral saltswhich the magnesium sulfate was 0.2 mM instead of 2 mm and ctaining 1 uc of 35SO4/ml. At time zero, 100 ,c of 3H-methyl-methionwas added to the culture, samples were taken for the succeedinghr, and the specific radioactivity (cpm per pmole methionine) ofmethionine attached to RNA and acid-insoluble methionine v

determined from the 3H/35S ratio of the purified methionine. A:Methionyl-tRNA. The curve drawn through the points is the equatispecific radioactivity = 7.4 (1 -e-t). 0: Acid-insoluble methioniB: The long term accumulation of acid-insoluble methionine in Lengrown on low magnesium sulfate.

insoluble methionine was determined on samples incubated.up to 2.5 hr and at 16 hr.Removal of the 3H-methyl-methionine results in a rapid dr

in the specific radioactivity of the methionyl-tRNA, and thismore pronounced if the transfer is onto 10 pM methionine. Trapid drop is followed by a much slower decay which continifor at least 16 hr to specific radioactivities which are abiK00 of the initial specific radioactivity. The 16-hr values <at the limit of detectability of the method used for measurispecific radioactivities and are not above the level of contamiition normally encountered in this type of experiment. Thus,could be argued that they are effectively zero. Incorporationmethionine into protein is strongly reduced after 0.5 hr in Icase of Lemna incubated on 10 ,u.M methionine and after 1.5in the case of Lemna incubated on sucrose-mineral salts onThis disparity seems to be related to the rate at which the specradioactivity of the methionyl-tRNA decays under the twocubation conditions.The data in Figure 4B have been taken from the experime

shown in Figure 4A and show the variation with time ofpmoles 35S-methionine attached to RNA during the label chconditions. During incubation on 10 uM methionine there is vconsiderable loss of 35S-methionine from the RNA, while in

no absence of unlabeled methionine the 35S-methionine remainsM constant or increases slightly. As described in "Materials and: Methods," the specific radioactivity of the methionyl-tRNA has- been determined from the ratio of 3H/35S in isolated methionine.

In the methionine decay curve the elimination of 35S-methioninefrom the tRNA (and its possible replacement by unlabeled

9 methionine from the medium) means that the ratio 3H/35S maya . no longer represent a genuine specific radioactivity. These data

indicate that the true specific radioactivity of the methionyl-tRNA in the 10 ,AM methionine decay curve must be considerablylower than the quoted values.The data shown in Figure 4 were measured on Lemna grown on

sucrose-mineral salts.In the experiments shown in Figure 5, Lemna, grown on low

magnesium sulfate which reduces growth rates by half, were la-beled for 1 hr with 3H-methyl-methionine before transfer ontounlabeled medium in the presence or absence of 10 ,M methi-onine. Figure 5A shows the decay of methionyl-tRNA and com-parison with Figure 4A indicates that the decline in the specificradioactivity is very much slower than normally grown Lemna.Inclusion of 10 p.M methionine in the chase medium makes verylittle difference to the rate of decay of the specific radioactivity of

?6 the methionyl-tRNA, but the elimination of 35S-methionine fromthe RNA, shown previously in Figure 4B, was again observed.Figure SB shows that incorporation of methionine into proteincontinues under label chase conditions, but begins to plateauafter 2 hr. This continued high rate of incorporation is probablyrelated to the slow rate of decay of the methionyl-tRNA shown

8 in Figure 5A.

NA DISCUSSIONiwnnna This paper describes two methods which may be used to deter-iin mine the rate constants of synthesis and of degradation of protein

on- in Lemna mninor. Measurement of the rate constants by these twoin methods on cultures of Lemna grown on sucrose-mineral saltsthe gave values in excellent agreement and suggested a rate constant,vas of degradation of about 0.1 days-' under the labeling conditionsA: used. As Koch (5) has pointed out, the labeling conditions usedon,ine.nna

for

rop, ishisues)utareingna-, itofthehrily.II.ificin-

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0

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FIG. 3. Long term variation of the specific radioactivity and thetotal acid-insoluble methionine with time. Lemna was grown for atleast eight generations on sucrose-mineral salts containing 0.1 Mc of35S04/ml. At time zero, the culture was labeled with 100 Ac of 3H-methyl-methionine for 1 hr. The fronds of the culture were then dis-persed onto seven flasks containing sucrose-mineral salts and 0.1 Mc of3'5S04/ml. A flask was removed each day, and the total acid-insolublemethionine and the specific radioactivity were determined from the3H/35S ratio of the isolated purified methionine. Slopes were determinedby the method of least squares and the rate constant from the slopesby multiplying by log, 10; 0: log,o specific radioactivity (cpm pernmole methionine); A: log1o total acid-insoluble methionine (nmolesmethionine).

44 TREWAVAS




in this paper would tend to emphasize the short lived componentsin a mixed population. The apparent half-life of protein in thistissue of about 7 days suggests that the shortest lived proteins infast growing Lemna may have half-lives of days rather than hours.This would also be the conclusion from the data shown in Figure4, where the establishment of label-chase conditions did not re-sult in detectable loss of label from the protein for at least 2.5 hr.The type of technique used for measuring protein turnover, and

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FIG. 4. Variation of the specific radioactivity of methionyl-tRNAand acid-insoluble methionine with time after transfer of cultureslabeled with 3H-methyl-methionine to unlabeled media. Cultures ofLemnia were grown for at least eight generations on sucrose-mineralsalts containing 1 Mc of 35SO4/ml. One hundred microcuries of 3H-methyl-methionine were added for 1 hr before transfer of the cultureat time zero to sucrose-mineral salts containing 1 Muc of '5SO4/ml inthe presence or absence of 10 Mm unlabeled methionine. Samples weretaken for a further 2.5 hr and at 16 hr, and the specific activity (cpmper pmole methionine) of the methionyl-tRNA and acid-insolublemethionine were determined. A: A, E: methionyl-tRNA; A, U:

acid-insoluble methionine; o, *: 10 AM methionine in the chasemedium; A\, A: no methionine in the chase medium. B: Variation of35S-methionine attached to RNA with time. Data were calculated fromthe methionyl-tRNA decay curves shown in Figure 4A and representthe total 5S-labeled methionine attached to RNA. Total RNA wasmeasured on the pellet remaining after pH 9.0 extraction and ethanolprecipitation. *: 10 Mm methionine in the chase medium; A: no me-thionine in the chase medium.

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0.o

B hours

FIG. 5. Variation of the specific radioactivity of the methionyl-tRNAand acid-insoluble methionine after transfer of fronds labeled with'H-methyl methionine and grown on low magnesium sulfate to un-labeled media. Batches of Lemna were grown for at least eight genera-tions on a sucrose-mineral salts medium containing 0.2 mm magnesiumsulfate instead of 2 nm~, and also containing 1 A.C Of 3'SO4,/Ml. Onehundred microcuries of 3H-methyl-methionine was added for 1 hrbefore transfer of the fronds to a sucrose-mineral salts medium con-taining the low magnesium sulfate, 1 AC of 35SO4/'ml and the presenceor absence of 10 Am methionine. Samples were taken for the succeeding3 hr and the specific radioactivity (cpm per pmole methionine) of themethionyl-tRNA and the acid-insoluble methionine were determined.A: 0, A: no unlabeled methionine in chase medium; A: 10umM me-thionine in the chase medium; B: 0: no unlabeled methionine in thechase medium; A: 10 Am methionine in the chase medium.

shown in Figure 1 , is similar to that described by Koch (5) as a"continuous labeling procedure." One feature of this technique isthat in a population of proteins of mixed stability the apparentrate constants are dependent on the period of exposure to theisotope. In the rather short period of time for which the experi-ment was conducted, there was no evidence for metabolic heter-ogeneity in protein turnover, the rate constants for each of thehourly periods being identical.

This may not be the case for the experiments shown in Figure2. The Lemna in these experiments were growing more slowly,on a magnesium sulfate reduced medium, and the average rateconstant of degradation of protein was higher than the culturesshown in Figure 1. The average apparent rate constant of syn-thesis of protein for the first 2 hr of labeling was 0.0145 hr-iand for the next 2 hr, 0.0122 hr-1. On this basis the possible rateconstants of degradation for these two time periods would be0.16 days-' and 0.103 days-'. These data suggest that the in-crease in the rate of degradation of protein resulting from growing





Lemna on reduced magnesium sulfate may be accompanied bythe production of metabolic heterogeneity in protein turnover.The average value for the turnover of methionine from protein

was a half-life of about 7 days. The rate of turnover of rRNA inthis tissue is an approximately similar value between 5 and 8days. The similarity between these two values may be fortuituous.Although it was concluded (10) that the turnover of rRNA wasprobably a reflection of the turnover of the ribosome itself, thecontribution of ribosomal protein to total protein turnover willbe rather small. Thus, there is for example 250 to 300 p,g rRNA/gfresh weight. If the ribosomes are composed of equal quantitiesof RNA and protein, then the level of ribosomal protein is 250to 300 ,ug against a total level of cellular protein of at least 12mg/g fresh weight. The turnover of ribosomal protein will nottherefore contribute extensively to total protein turnover.

Acknowledgments-This work was performed under receipt of a grant fromthe Agricultural Research Council to Professor D. D. Davies. I am grateful toProfessor Davies for useful discussion at the beginning of this project. I am

also grateful to Dr. J. Ingle and Dr. C. Leaver for criitical reading of thismanuscript.

LITERATURE CITED

1. BARETT, A. J. AND D. H. NXORTHCOTE. 1965. Apple fruit pectic substances.Biochem. J. 94: 617-627.

2. HOAGLAND, M. B., M. L. STEPHENSON, J. F. SCOTT, L. I. HECHT, AND

P. C. ZAMECNIK. 1938. A soluble ribonucleic acid intermediate in proteinsynthesis. J. Biol. Chem. 231: 241-257.

3. HOLLEMAN, J. H. AN-D J. L. KEY. 1967. Inactiv e and protein precursor poolsof amino acids in the soybean hypocotyl. Plant Physiol. 42: 29-36.

4. Joy, K. W. AN-D B. F. FOLKES. 1965. The uptake of amino acids and theirincorporation into the proteins of excised barley embryos. J. Exp. Bot.16: 646-666.

5. KOCH, A. L. 1962. The ev-aluation of the rates of biological processes fromtracer kinetic data. J. Theor. Biol. 3: 283-303.

6. MIOORE, S. AND W. H. STEIN. 1963. Chromatographic determination ofamino acids by the use of automatic recording equipment. In: S. P.Colowick and N. 0. Kaplan, eds., Methods in Enzymology. ol. V-I.Academic Press, New York, pp. 819-831.

7. O.Ks, A. AND R. G. S. BIDWELL. 1970. Compartmentation of intermediarymetabolism. Annu. Rev. Plant Physiol. 21: 43-66.

8. REINER, J. M. 1953. The study of metabolic turnover rates using isotopictracers. Arch. Biochem. Biophys. 46: 53-79.

9. RuSSELL, J. A. 1958. The use of isotopic tracers in estimating rates ofmetabolic reactions. Perspect. Biol. Med. 1: 138-173.

10. TREWAVAS, A. J. 1970. The turnover of nucleic acids in Lemna 7ninor. PlantPhysiol. 45: 742-751.

46 TREWAVAS



of the rates of protein synthesis and degradation · protein turnoverrates in lemna ter....

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