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Biochimica et Biophysica Acta, 349 (1974) 114--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 97983

EVIDENCE AGAINST THE INCORPORATION INTO PROTEIN OF AMINO ACIDS DIRECTLY FROM THE MEMBRANE TRANSPORT SYSTEM IN RAT HEART

JOHN MOWBRAY and KEITH S. LAST*

Department of Biochemistry, University College London, Gower Street, London, WCIE 6BT (Great Britain)

(Received November 29th, 1973)

Summary

The possibility suggested recently [Hider, R.C., Fern, E.B. and London, D.R. (1969} Biochem. J. 114, 171--178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121 ,817- -827 ; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427--433; and Adamson, L.F., Herington, A.C. and Bornstein, J. (1972) Biochim. Biophys. Acta 282, 352--365] that protein synthesis takes place using amino acids directly from the membrane transport system and not from an intracellular pool has been investigated in rat heart. The tissue was perfused first for 30 min with either [14C]glycine or [14C]leucine and then for a further 30 min with identical medium containing [3 H] glycine or [3 HI leucine, respectively. After an initial lag, [14 C] glycine was incorporated into protein at a linear rate up to 60 min. The [3 H] glycine was accumulated into tissue water and incorporated just as readily as the [14 C] glycine had been. The rate of total protein synthesis agrees with literature values only if intracellular and not extracellular specific activity values are used in the calculation. Some glycine was converted to serine or threonine. Leucine influx and efflux were very rapid in contrast to the rela- tively slow exchange reported for incubated tissues [Hider, R.C., Fern, E.B. and London, D.R. (1969} Biochem. J. 114, 171--178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817--827; van Venrooij, W.J., Poort , C., Kramer, M.F. and Jansen, M.T. (1972} Eur. J. Biochem. 30, 427-- 433] . The results are consistent with the existence of an intracellular precursor pool for glycine. Some possible reasons for the discrepancies between this and the other studies are discussed.

* P r e s e n t A d d r e s s : D e n t a l Department , University College Hospital Medical Schoo l , Universi ty Street , L o n d o n , WC1E 6 J J , Great Britain.

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Introduction

In vitro studies of amino acid accumulation and incorporation into the protein of incubated tissues have suggested strongly that the intracellular amino acid pool is inhomogeneous. That part of the total which exchanges rapidly with the extracellular environment has properties appropriate to the amino acid pool for protein synthesis [1--4] , while the much less freely exchanging part derives from intracellular proteolysis [5] . Hider et al. [6,7] examined the nature of the amino acid pool used in protein synthesis in incubated rat exten- sor digitorum longus muscle and appear to have demonstrated clearly that this is an extracellular and no t an intracellular pool. Further they suggested, on the basis that no intracellular amino acid crossed the plasma membrane at 2°C in their experiments, that this extracellular pool may be distinct from the medium pool. Support for the surprising suggestion that the amino acid precursor pool is extracellular has come recently from studies of amino acid incorporation in isolated pancreas slices [8] and in embryonic chick cartilage [9] .

Thus, the possibility exists that, with an abundant amino acid supply in plasma, protein synthesis takes place directly from the membrane transport system and the ability of cells to accumulate certain amino acids is an inciden- tal consequence of the existence of the transport system, or is a related but separate ability. It is interesting, in this context , that active accumulation is largely confined to non-essential amino acids [10] which could suggest that these are acquired for purposes other than protein synthesis for which both essential and non-essential amino acids are required.

On the other hand, Morgan et al. [11] studying protein synthesis in a perfused rat heart preparation have interpreted some of their results to show that an intracellular amino acid pool is used in protein synthesis. Unfortunately their conclusions are open to serious question since they depend on: (1) the claim that glycine incorporation to protein shows a time-lag and this the authors interpret as being due to the increasing specific activity of the (intracel- lular) precursor pool. Their data, however, do not prove the existence of such a lag since their time-course is constructed from only two time points (at 10 and 30 min) with no marker for lost material to set the zero point. Even if a lag exists, this argument should be treated with caution since Hider et al. [6] also report a lag in glycine incorporation while presenting data to show that the precursor pool is extracellular. (2) on continued 14C incorporation after all ,4 C-labelled amino acids had been removed from perfusate. In this case the only source of amino acids for protein synthesis was the previously accumu- lated intracellular pool, which is available to the transport system. As a neces- sary preliminary to a tracer s tudy involving amino acid incorporation into protein in perfused rat heart we have re-examined the nature of the precursor pool in this tissue. The results re-affirm the role of an intracellular pool as the source of amino acids in protein synthesis and it is suggested that the indica- tions to the contrary may be artifacts.

Materials and Methods

Tuck strain male white rats weighting 220--270 g were deprived of food

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overnight. Hearts were removed under ether anaesthesia and perfused as de- scribed by Mowbray and Ottaway [12] . The medium contained in addition to 5.5 mM D-glucose and 40 mM raffinose the following L-amino acids in pM: aspartate (13), threonine (212), serine (235), glutamine (495), glutamate (112), proline (112), citrulline (56), glycine {400), alanine (299), valine (169), methionine (39), isoleucine (101), leucine (150), tyrosine (86), phenylalanine (66), ornithine (79), lysine (328), histidine {64), arginine (75), t ryp tophan (60), aspargine (38), taurine (194), cysteine (305) which resembles the com- position of rat plasma [13--15] .

Changing the perfusion medium after 30 min of perfusion was achieved by transferring the heart and eannula assembly to a second apparatus where either the new perfusate was not reeirculated for 5 min or washout perfusion with medium as detailed above but containing no radioactive tracers was continued for 7 rain before the assembly was retransferred to a recirculating system con- taining the new medium. Mixing of an injected nicotinamide marker is com- plete in under 2 min in this perfusion apparatus 116].

After the perfusion period, the heart was clamped with liquid nitrogen- cooled Wollenberger tongs [171 and disintegrated in 3 ml 10% trichloroacetic acid at 0°C with an Ultra-Turrax homogeniser (Janke and Kunkel, Staufen i.b., W. Germany) and the homogeniser washed with another 3 ml trichloroacetic acid. The trichloroacetic acid protein mixture was incubated at 80°C for 10 min. The precipitated protein was weighed and washed three times with 10% trichloroacetic acid containing unlabelled glycine (leucine) before being dried to constant weight. The dried protein was extracted three times with ether and then dissolved in either 1.5 ml formic acid or in 2 ml 40% KOH + 0.5 ml methyl cellusolve.

Raffinose was estimated by the method of Davies and Gander [18] . Radiochemicals obtained from the Radiochemical Centre, Amersham were added to give the following initial specific activities in perfusate: [U 2 4C], [2 -~ It] glycine, 2.47 • 10 s dpm-pmole ' ; L_[I_I 4 C] leucine, 4.9 • l 0 s dpm'pmole-~ ; L-[4,5 -3 H2 ]leucine, 6.7 • l 0 s dpm.pmole -1 Liquid scintilla- tion quench correct ion was by external standardisation (Packard 2420 or Beck- man LS-200 spectrometers) and checked with internal standards. The counting error was never greater than 2%. Automatic amino acid analysis was performed using a Technicon Analyser with the stream split to collect 50% of the eff luent in about fifty 4-ml fractions. 0.1 pmole norleucine was used as standard.

Results and Discussion

Glycine incorporation A series of hearts were perfused for 30 min with medium containing glu-

cose, a complete amino acid mixture and tracer [14C]glycine" At 30 min, medium was replaced by identical medium except that the tracer was now [3 It] glycine and the perfusate was allowed to recirculate after 5 min. Hearts were freeze-clamped at intervals during this regime.

After an initial lag [ ~ 4 C] glycine was accumulated at a steady rate into the raffinose-impenetrable space and moreover there was no appreciable loss of this ' 4C radioactivity during the subsequent wash and incubation with [ 3H ] glycine

117

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8

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Fig. 1. Glyc ine a c c u m u l a t i o n and in c or p or a t ion into hearts . (a) The a c c u m u l a t i o n o f [ 14C] g lyc ine ( e ) and [ 3 H ] g l y c i n e (m) radioact iv i ty into the ra f f inose - impenetrab le space o f hearts . (b) The inco rpo ra t io n o f these tracers into the h o t t r i ch loroace t i c ac id- insoluble fract ion o f the same hearts . Each po in t is the m e a n of 5 hearts and the error bars represent S.E. The 3H tracer perfusate was n o t ree irculated b e t w e e n 3 0 and 3 5 rain.

(Fig. la). Virtually all the soluble 14C radioactivity could be recovered as glycine (see below). Glycine uptake in the second part of the experiment (3 H tracer) was not significantly different from that observed in the initial 30 min of perfusion (Fig. la). The prior accumulation of [' 4 C] glycine, therefore, does not appear to have impaired the tissue's ability to take up external amino acid, and presumably, therefore, access of external amino acid to the transport sys- tem is undiminished.

The incorporation of tracer into the hot trichloroacetic acid-insoluble fraction is shown in Fig. lb . After an initial lag [14 C] glycine was incorporated throughout the 60-min of perfusion: most significantly incorporation con- tinued at a linear rate during the second 30-min of perfusion when virtually all extracellular tracer glycine was 3H-labelled. These results, which contrast directly with those obtained with incubated rat skeletal muscle [6] when all 14 C incorporation ceased in the 3 H medium, argue strongly that the source of glycine for protein synthesis in perfused rat heart is an intracellular and not an extracellular pool. If the amino acids were incorporated directly from the transport system, both perfusate and previously accumulated amino acid could be precursors. The observed rate of incorporation in the 30--50-min period {Fig. lb ) is much greater for [' 4 C] glycine than for [3 H] glycine. Thus, if the transport system pool and not the intracellular pool were the precursor, this would imply that the transport system is very much more accessible to intracel-

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lular amino acid. There seems, a priori, no reason to suppose this unless there were a large concentrat ion gradient across the plasma membrane. Making the simplifying assumption that intracellular glycine is evenly distributed in a volume of 0.506 ml intracellular water per g wet heart [16] , the distribution ratio for glycine ranged from 1.6 to 2.6 in these experiments. Thus the results suggest a preference for an intracellular amino acid rather than one on the transport system. The incorporation of [3 H] glycine mirrors the initial 30-min [14 C] glycine time-course demonstrat ing that there was no barrier to the use of medium amino acid in the final 30-min period.

Conversion of glycine to other amino acids To check the possibility that there was significant conversion of glycine

carbon (or tritium) into other amino acids, 3-ml samples of extract from hearts perfused for the same time were pooled and dried down in a nitrogen stream. They were then applied with 0.1 pmole norleucine standard to an amino acid analyser with a split effluent stream to allow collection of samples for radio- assay. The only fraction apart from that containing glycine in which there was greater than background radioactivity was the serine fraction (strictly, the unresolved serine/threonine fraction). 4% of the total intracellular activity was found in serine after 30 min and 7.5% of the total after 60 min perfusion in the presence of [14 C] glycine. Although this relatively small amount of conversion does not alter the argument for an intracellular precursor pool it is possibly large enough to render glycine unreliable as a tracer for kinetic measurements of protein synthesis under differing physiological or experimental conditions.

Intracellular glycine specific activity and protein synthesis rate The overall intracellular glycine specific radioactivity at each sample time

is given in Table I, and Fig. 2 shows the time-courses of the amounts of glycine incorporated into protein estimated from these specific activities and the incor- porated radioactivity data (Fig. 1). After a pronounced lag, the incorporation of glycine (14 C data) is a linear function of time. The error due to [14 C] serine incorporation in this period up to 60 min will be small (around 5%) and has

T A B L E I

I N T R A C E L L U L A R G L Y C I N E S P E C I F I C R A D I O A C T I V I T Y

3 - m l samples o f e x t r a c t w e r e p o o l e d and appl ied to an a u t o m a t i c a m i n o acid analyser (see t e x t ) . R a f f i n o s e w a s used as extrace l lu lar marker . The spec i f ic rad ioact iv i ty o f the per fusate was 2 4 6 . 5 d p m ' n m o l e -1 . [ 14C] G l y c i n e - c o n t a i n i n g m e d i u m was rep laced by [ 3H] g lyc ine -conta in ing m e d i u m at 3 0 r a i n .

T i m e Spec i f i c ac t iv i ty ( d p m / n m o l e ) ( m i n )

[ 14C] G l y c i n e [ 3H] G l y c i n e

10 16 - - 20 51 - - 3 0 91 - - 4 0 8 5 14 50 78 36 60 83 73

119

200

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Fig. 2. The amount of glycine incorporated into heart protein. The values (o [14C]glycine; • [3H]gly- cine) are derived from the incorporated radioactivity (Fig. 1) and the overall specific activity of the intracellular glycine obtained from amino acid analysis (Table I). 4-&, the time-course using the 14C data

and the extracellular specific activity.

been ignored. The rate of incorporation of glycine between 20 and 60 min from the intraceUular carbon specific activity data is 226 nmoles/h per g wet tissue. Using the data of Morgan et al. [11] for the glycine content of rat heart myosin and a value of 180 mg protein per g wet tissue [12] gives a value for total protein glycine of 59.2 pmoles/g wet tissue. Thus the incorporation rate from Fig. 2 represents a synthesis rate of 9% of total protein per day which is very like the value of 10--11% found for hearts perfused for 1 h in the absence of insulin by Sender and Garlick [19]. The same value estimated from the 50--60-min 3 H data is 7% per day. The value calculated using the perfusate [l 4C]glycine specific activity {246.5 dpm/nmole) yields a value of 2.5% per day. Thus these data agree with that of Sender and Garlick [19] only if the intracellular amino acid pool is the source of amino acids for protein. Since Sender and Garlick [19] obtained their data under conditions where the pre- cursor (tyrosine) specific activity was not very different inside and outside the cell, their estimate is largely independent of the source of the precursor amino acid.

The linear nature of the incorporation after the lag would suggest (see Mortimore et al. [5] ) that there is not great inhomogeneity in intracellular glycine. If there is a pool of glycine which exchanges with medium glycine more slowly than that used in protein synthesis then this pool must most probably represent a small fraction of the total pool.

The incorporation of tracer leucine An amino acid whose transport is very different from that of glycine [10]

and which appeared also to be incorporated directly from the extracellular medium into skeletal muscle [6,8] is leucine. Since it is possible that essential amino acids like leucine might be treated differently in protein synthesis from glycine and indeed originate in the extracellular pool we repeated the incor- poration experiment using [14 C]- and [3 H]-labelled leucine. The regime was exactly as for the glycine experiment except that between the ' 4C perfusion and the 3H perfusion there was a 7-min washout with unlabelled amino acid perfusate. The results are presented in Fig. 3. Each point is the result from only one heart.

120

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Fig. 3. Leu c i n e a c c u m u l a t i o n and i n c o r p o r a t i o n in to hea r t s . (a) The a c c u m u l a t i o n of L-[ 14 C] leuc ine ($) a n d L - [ 3 H ] leucine (m) r a d i o a c t i v i t y in to the r a f f i n o s e - i m p e n e t r a b l e space of hear t s . (b) The i n c o r p o r a t i o n

of these t r ace r s in to the h o t t r i c h l o r o a c e t i c ac id- inso luble f r a c t i o n of t he same hear t s . Each p o i n t repre-

sen t s on ly 1 hea r t . The re was a 7-rain w a s h o u t p e r f u s i o n at 30 m i n (see t ex t ) .

By contrast the incorporation of tracer leucine shows no lag comparable to glycine (Fig. 3b) and after switching to [3H]leucine-containing medium, [, 4 C] leucine incorporation ceases. The influx of leucine is linear and rapid and the exchange with the perfusate pool is great enough for there to be no detect- able intracellular [1 4C]leucin e after the wash period and 10 min perfusion with tri t ium tracer. Also there appeared to be no difference between intracel- lular and extracellular specific activities at 10 min. Because of this rapid trans- fer of leucine it is no t possible to tell from this experiment whether the amino acid precursor was intra- or extracellular. The fractional protein synthesis rate represented by these data is 11% per day which is very close to the glycine value.

This rapid exchange of leucine is very different from the situation re- ported for skeletal muscle [6,7] or pancreas fragments [8], and the possible significance of this is discussed below.

General discussion

The data presented here demonstrate that glycine is incorporated into rat heart protein from an intracellular pool. This conclusion contrasts sharply with those of other workers who have postulated the existence of an extracellular or membrane precursor amino acid pool [6--9]. It seems possible to suggest some reasons for this discrepancy.

These other studies were carried out with incubated tissues or tissue frag- ments, which immediately raises the possibility of uneven amino acid or nutr ient distribution. Van Venrooij et al. [8] investigated the penetration of

121

labelled substrate into their pancreas fragments and showed that, provided the volume was 1 mm 3 or less, then cells throughout the tissue became labelled. However they also found that a considerable proport ion of the cells incor- porated little or no radioactive amino acid. This implies that either there are cells synthesising protein to which labelled amino acid is denied or that some cells are not synthesising protein but nevertheless may have access to amino acid. In addition, these authors demonstrated that an extracellular marker reached saturation level within 10 min and they presumed that equilibration of extracellular and medium amino acid was equally rapid. However, their data show that the total tissue water showed an increase in accumulation of amino acid over the 10--30-min period which was 50% greater than the accumulation shown by the intracellular amino acid pool. This additional slow penetration must have been to extracellular space, which therefore appears to equilibrate slowly with medium amino acid.

Thus, results showing tissue retention of previously accumulated tracer do not automatically mean that that tracer is available to cells active in protein synthesis. In particular it is noticeable that both glycine and leucine are re- tained in the incubated muscle [6] while leucine is lost rapidly from heart. Since glycine appears to be actively accumulated by cells, whereas leucine is not and is subject to rapid exchange diffusion [10] , the results with heart agree more closely with those expected of an A system and an L system than do the skeletal muscle data. If this leucine data is questioned, the glycine data of Hider et al. [6] by itself is not convincing, and the lag in the incorporation as well as the reduced [3 H] glycine incorporation following [14 C] glycine accumulation would leave open the question of an intracellular pool.

The other premise upon which the studies [7--9] favouring an extracel- lular precursor pool rests is that there is no appreciable efflux of intracellular amino acid at 0--2°C in the experimental times used, while all the extracellular amino acid is lost. The demonstrat ion of an extracellular pool exchanging slowly at 37°C (see above) would not support a rapid loss from this pool at 0°C. Moreover amino acid transport does take place at 1°C [22] and some transport processes can show very appreciable rates at 0°C [23] . So the possi- bility exists that transport out of the cells at low temperature is faster than efflux from some extracellular space.

Thus we believe that there are good reasons to question the data support- ing the existence of a distinct extracellular or membrane transport system amino acid pool with priority in supplying t-RNA amino acids. Our findings with a tissue where amino acid was supplied by the normal arterial system and in which none of the cells appears deprived of nutrient [12] , confirm the traditional role of the intracellular amino acid pool as the precursor in protein synthesis. Correspondingly, protein synthesis rates must properly be computed from incorporation data using intracellular and not extracellular [8] specific activity values.

Acknowledgements

We are grateful to Mr M.I. Khan who carried out the automatic amino acid analysis and to Dr V.M. Pain for helpful discussion.

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