264 BIOCHIMICA ET BIOPHYSICA ACTA

T H E I N C O R P O R A T I O N OF [32P~ORTHOPHOSPHATE INTO

A S P EC IF IC L I P I D FRACTION AND INTO A D E N I N E N U C L E O T I D E S

OF M I T O C H O N D R I A D U R I N G O X I D A T I V E P H O S P H O R Y L A T I O N

T. E. CONOVER*, G. V. MARINETTI, R. F. WITTER** AND ELMER STOTZ

Department o~ Biochemistry, University o] Rochester School o~ Medicine and Dentistry, Rochester, N.Y. (U.S.A.)

(Received November 7th, 1959)

SUMMARY

A method for the extraction of mitochondriat lipid using acetone to precipitate the mitochondrial lipoprotein is reported. This procedure gave rapid destruction of enzymic activity and high phosphatide recoveries under mild conditions and was used to simultaneously follow the incorporation of [3*P]orthophosphate into the adenine nucleotides and the lipid components of mitochondria during oxidative phosphorylation.

At io ° the incorporation of E3*P]orthophosphate into ATP proceeded rapidly and linearly during the initial minutes of reaction, but leveled off after IO to 15 min. ADP behaved similarly but the specific activity remained about half that of the ATP.

The incorporation into the lipids at IO ° occurred almost completely into a very minor fraction; however, this incorporation was not detectable for the first IO min of reaction, after which it increased rapidly. The conclusion was made that unless this lipid fraction contains a minor but highly active component, it cannot be the precursor of the phosphate moiety of ATP and therefore cannot act as an intermediate in the phosphate esterification reactions during oxidative phosphorylation.

INTRODUCTION

The localization of high concentrations of lipids in mitochondria 1-5, and their associa- tion with the insoluble oxidative enzymes e, 7 and sub-mitochondrial particles capable of carrying out oxidative phosphorylation s-l°, have suggested an important role for these compounds in mitochondrial function. However, there is as yet no clear evidence to indicate whether such a role might be as a specific reactant in oxidative phosphoryl- ation or merely as a component in the structural organization.

Abbreviations: TCA, trichloroacetic acid; AMP, adenosine 5'-phosphate; ADP, ade~xosine diphosphate; ATP, adenosine triphosphate; DNP, 2,4-dinitrophenol,

* This work was taken from a thesis submitted by T. E. Conover in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Present address: Wenner-Grens Institute, Stockholm (Sweden).

**Present address: U.S. Public Health Service, Communicable Disease Center, Technical Development Laboratories, Savannah, Ga. (U.S.A.).

Biochim. Biophys. Acta, 41 (196o) 264-271

[32p]ATP AND LIPIDS DURING OXIDATIVE PHOSPHORYLATION 265

With the development of paper and column chromatographic methods for phosphatide analysis, a high degree of resolution of phospholipid preparations became possible. This increased resolution has revealed a number of minor constituents hitherto unobserved n, 12 and raises the question of a specific role for these phosphatides.

Studies with isolated in vitro systems3,13 have shown that unidentified acidic glycerolphosphatides, which are found nearly exclusively in mitochondria, rapidly incorporate [~2P]orthophosphate during oxidative phosphorylation. During such incubation the common phosphatides showed very little incorporation of radio- activity. In this study the incorporation of Ea2P]orthophosphate into the individual phosphatide components of mitochondria was determined during oxidative phos- phorylation and compared with the incorporation into ATP. These studies indicate that the phosphatides do not incorporate [82P]orthophosphate at a rate fast enough to suggest their direct participation as intermediates in oxidative phosphorylation.

EXPERIMENTAL

Methods and reagents

Mitochondria were prepared by the method of DOUNCE et al. 14 modified by the omission of the citric acid. Paper chromatographic analysis of the phosphatides was carried out by procedures previously described 15. Paper chromatographic analysis of the adenine nucleotides was carried out by modifications of the procedures of EGGLESTON AND HEMS TM a n d KREBS AND HEMS 17. The methods for lipid detection have been described previously11, TM. Autoradiograms were prepared on Kodak no- screen X-ray film and required 3 to 4 weeks' exposure. The radioactivity was deter- mined on the paper spots which were cut from the chromatograms. Phosphorus determinations were made by a modification of the method of CHEN et al. TM or by the m e t h o d of HARRIS AND POPAT 2°.

Preparation of lipid extracts

Preliminary studies on the preparation of lipid extracts from TCA precipitates of mitochondria revealed that a considerable loss of both lipid and lipid phosphorus was incurred. Paper chromatographic analysis of such extracts demonstrated some degradation of phosphatide and the appearance of a lipid material whose chromato- graphic behavior was very similar to lysophosphatides.

From the results of these experiments it was felt that a non-acidic precipitant would cause less lipid degradation, particularly of a possible labile phosphate inter- mediate. Acetone was chosen on the basis of its use as a phosphatide precipitant and its high solubility in water. Phosphatides show maximum insolubility in acetone when the acetone is anhydrous. Such conditions would not exist during the mitochondria precipitation. It was felt, however, that the phosphatides of mitochondria might also be relatively insoluble in acetone-water mixtures containing acetone in concentra- tions of 50 O~jo or less. Various concentrations of acetone were therefore tested at o ° and 45 ° by pipetting a suspension of mitochondria into the appropriate volume of acetone. It was found that concentrations between 4 ° and 50 % acetone gave nearly complete precipitation of mitochondrial protein. The effect of temperature did not appear to be appreciable on the extent of protein precipitation, but lower tempera- tures (o ° versus 45 °) retarded considerably the rate of protein flocculation.

Biochim. Biophys. Acta, 41 (I96o) 264-271

266 T. E. CONOVER, G. V. MARINETTI, R. F. WITTER, E. STOTZ

Lipid extracts were prepared from washed precipitates of mitochondria by heating at 60 ° for 4 min with chloroform-methanol ( I : I ) . This extract was dried under nitrogen in vacuo and the residue re-extracted with warm chloroform-petro- leum ether-methanol (5 : 5 : I). With mitochondria precipitated by 4 ° or 50 % acetone at 45 ° and washed once with 4 ° % acetone, the recovery of lipid phosphorus was 80-90 % when compared with the recovery obtained by the direct extraction of KC1- washed mitochondria (Table I). The lipid (dry wt.) usually varied between 6.0 and 6.5 mg/ml of mitochondrial preparation. Extraction of the combined acetone supernatant fluids and wash solutions with chloroform-petroleum ether (I : I) yielded less than IO % of the lipid phosphorus and about I5-2o % of the lipid (dry wt).

T A B L E I

T H E R E C O V E R Y O F L I P I D F R O M T R I C H L O R O A C E T I C A C I D A N D

k C E T O N E P R E C I P I T A T E S O F R A T - L I V E R M I T O C H O N D R I A

Expt. Precipitant* Precipitation temperature

I 5 % TCA o °

Cont ro l * * o °

I I 4 ° % a c e t o n e o °

5 ° % a c e t o n e o ° 4 ° % a c e t o n e 45 ° 50 % a c e t o n e 45 ° Cont ro l** 45°

Lipid phosphorus

mg/ml % mitochondria recovery

0.36 64 0.57 i o o

0.30 72 0.34 8i 0.37 88 0.35 83 0.42 ioo

* P r o t e i n loss w a s d e t e r m i n e d b y o b s e r v i n g the p r e c i p i t a t e f o r m e d on a d d i t i o n of one ha l f v o l u m e of 2o % TCA to t h e s u p e r n a t a n t f luid a t 5 o°.

** L ip id P r e c o v e r y f r o m t h e d i r e c t e x t r a c t i o n of KC1 w a s h e d m i t o c h o n d r i a se rved as t he con t ro l .

Fig. I shows a chromatographic comparison of the phosphatides obtained by direct extraction of mitochondria, the extraction of an acetone precipitate, and extraction of the combined acetone supernatant fluid and wash solution. No qualita- tive differences were detected chromatographically between the two methods. Chromatography of the lipid extracted from the combined acetone supernatant fluid and wash solution demonstrated small amounts of lecithin, phosphatidyl ethanol- amine, and inositol phosphatide and only trace amounts of the other phosphatides. I t is noteworthy that in a preparation of mitochondria incubated with [szP]phosphate, the combined acetone supernatant solution and wash solution contained a labeled material which behaved on chromatograms like a lysophosphatide. This was the only labelled phosphatide material detected on autoradiograms in the extracts of these solutions. Moreover, the phosphatide was not detectable in the extracts of the acetone precipitates of mitochondria. The method for lipid extraction may therefore lead to losses of certain lipids, in particular water soluble lipids like the lysophos- phatides.

Incorporation of [~2P]orthophosphate into the phosphatides of mitochondria When mitochondria were incubated for 20 min with [32P]orthophosphate in the

Bioch im. B i o p h y s . Acta , 41 (i96o) 264-271

[a2P]ATP AND LIPIDS DURING OXIDATIVE PHOSPHORYLATION 267

presence of appropriate substrates, a lipid extract was obtained which was radio- active. Chromatography and subsequent autoradiography of the extracted lipids revealed that the radioactivity was almost exclusively in 3 or 4 spots (Fig. I-D). In agreement with earlier work a, 13 extremely little or no activity was detected in the common phosphatide components such as lecithin, phosphatidyl serine, phosphatidyl ethanolamine, or sphingomyelin.

Fig. I. Paper chromatograms and autoradiogram of total f'~ lipid extracts of rat liver mitochondria and acetone precipi- g A rates of mitochondria. A, the lipids were prepared by 8 direct extraction of mitochondria. B, the lipids were pre- t~ pared by extraction of acetone precipitates of mitochondria. 7 C, the lipids were prepared by extraction of the acetone supernatant fluid and wash solution. D, tracing of an 6---#:=~ autoradiogram of a2p labeled mitochondrial lipids. The 5( ,_J lipids in this case were prepared by direct extraction of the mitochondria. Chromatography was carried out on 4 ( ~ silicic acid impregnated paper as described previously 15. 3 0 In A, B, and C the lipid spots were detected by staining with Rhodamine 6 G. In D the spots were detected by 2 C) exposure of X-ray film. The identification of spots is as 1 C) follows15: spots i and 2, unidentified; spot 3, inositol phosphatide; spot 4, sphingomyelin; spot 5, lecithin; spot 6, phosphatidyl serine; spot 7, phosphatidylethanol- amine; A

spots 8 and 9, polyglycerolphosphatides.

r~

J @

@o C ) O °° | 0 © @

A ,~ 0 v B c D

One of the spots observed on the autoradiograms was located near the origin and corresponded to none of the lipid spots detected with the dye, Rhodamine G. These origin components are believed to be traces of inorganic phosphate, ATP, and other water soluble phosphates.

A second faint elongated spot (RF value of about 0.20-0.30) is believed to be due to traces of lysophosphatide. I t was found in lipid extracts prepared directly on mitochondria. However, with acetone precipitation it was not found in the extracts of the mitochondrial precipitate, but rather occurred in extracts of the acetone supernatant fluid and washes.

The major portion of the radioactivity was found in spots 8 and 9 (Fig. i). These components are believed to be polyglycerolphosphatides a. The autoradiograms sometimes revealed a double spot, but often the radioactivity was concentrated along the leading edge of spot No. 9, suggesting a material of rather low concentration but with a high specific activity. In the following rate studies the total values for spots No. 8 and No. 9 were used so as to minimize variation in specific activity due to this unequal distribution.

The incorporation of [3~P]orthophosphate into this fast moving phosphatide component was very sensitive to uncoupling of oxidative phosphorylation by lO -4 M DNP. KCN in concentrations of IO -a M, inhibited both respiration and [a2P]ortho - phosphate incorporation into ATP, but gave a less pronounced inhibitory effect on the incorporation into this lipid than did DNP.

Biochim. Biophys. Acta, 41 (196o) 264-271

268 T. E. CONOVER, G. V. M A R I N E T T I , R. F. W I T T E R , E. STOTZ

Rate of incorporation o/[3zPJorthophosphate into the phosphatides and adenine nucleotides of mitochondria

The acetone precipitation procedure was applied to the problem of determining the initial rate of [32Plorthophosphate incorporation into the fast-moving fraction of mitochondrial phosphatide. In these experiments the reaction mixtures were first prepared containing the mitochondria and all constituents except orthophosphate. The mixtures were attached to the manometers and equilibrated for 5 rain at IO °. The phosphate buffer containing the Ia2P~phosphorus was then added (this time was called zero time) and the reaction was followed manometrically by measuring the oxygen consumption. At the end of various time intervals the flasks were removed and an aliquot of 3.1 ml was removed and rapidly pipetted into 3.o ml of acetone at a temperature of 45 ° . Simultaneous with the addition of the aliquot to the acetone, o.5 ml of cold 2o % TCA was added to the remainder of the reaction mixture. Both solutions were immediately transferred to ice and kept cold. The lipid extracts were prepared from the acetone precipitates in the manner described. The TCA supernatant

T A B L E I I

T H E I N C O R P O R A T I O N OF [ 3 2 p ] O R T H O P H O S P H A T E INTO A D E N I N E N U C L E O T I D E S A N D

P H O S P H A T I D E S B Y R A T - L I V E R M I T O C H O N D R I A

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 t h e fo l lowing : 2 . 1 o - 3 M D L - f l - h y d r o x y b u t y r a t e , 5.O. lO -2 M I ~ 2 P ] o r t h o p h o s p h a t e ( p H 7.4), 3.3" lO-3 M A D P , 1.7- lO-a M A T P , 6.7" lO -3 T r i s bu f f e r ( p H 7.4). 6.7" lO -2 M MgC12, 5" IO-8 M KC1, I • i o 2 M K F , a n d 1.2 m l of m i t o c h o n d r i a . [ 3 2 p ] o r t h o p h o s p h a t e c o n t a i n e d a p p r o x i m a t e l y 500 # C / m l of [22Plphospl lorus T o t a l v o l u m e was 4.65 ml . T e m p e r a t u r e

XVaS IO °.

Specific activity counts/min/l,g P Time

Flask (rain) Inorganic P A M P A DP A T P Phosphatide spots no. 8 and 9

I o 43.4" lO2 0.2 • lO 2 0 .02 . lO 3 0 .25 . lO 3 o 2 5 43 .6 . i o 3 0. 3 • lO 2 t .7 ° . t o 3 4 .11 . lO 3 o 3 IO 4o.7 • i o 3 0 . 8 . 1 o 2 3 .59 ' Ios 7.34" IO3 0.88 4 20 39 .4 ' 1o2 t .8. lO 2 6.34" l o 2 lO.73. I o 3 2.28 5 3 ° 37-7" Io3 1.9 ' 1o 2 7.09 • t o8 12 .25 ' Io 3 2.94 6 5 ° 30.8. lO 3 1. 9 • 1o 2 7.84. i o 3 14.39. lO 3 9.13

Phosphotlde Nucleotlde count $/min/~ 9 cou ntslrnM/#Lg

15 15 • 10 ~' I" A

' ~ 1 0 1 0 . 1 0 z'

i,#," _...-.."'i t i * I 10 2 0 3 0 4 0 5 0

T i m e ( r a i n )

Fig. 2. I n i t i a l r a t e of i n c o r p o r a t i o n of r a d i o a c t i v e o r t h o p h o s p h a t e illtO t h e f a s t m o v i n g l ip id f r a c t i o n a n d in to a d e n i n e n u c l e o t i d e of r a t l i v e r m i t o c h o n d r i a . T h e r e a c t i o n m i x t u r e is t h e s a m e as t h a t g i v e n i n T a b l e I I . C u r v e A c o r r e s p o n d s to A T P ; C u r v e B c o r r e s p o n d s to A D P ; a n d C u r v e C

c o r r e s p o n d s t o t h e f a s t m o v i n g l ip id f r a c t i o n (spots 8 a n d 9 s h o w n in Fig . i ) .

Biochim. Biophys. Acta, 41 (196o) 264--27I

[aap]ATP AND LIPIDS DURING OXIDATIVE PHOSPHORYLATION 26 9

fluids were prepared for chromatography of the adenine nucleotides and orthophos- phate by washing with ethyl ether in order to remove the TCA.

The results of a typical experiment are given in Table II and Fig. 2. The specific activity of the inorganic phosphate given in Table II showed a slow decline during the course of the experiment. This was assumed to be due to the exchange of labeled orthophosphate in the medium with unlabeled pools of phosphate in the mitochon- dria. The ATP showed an initial rise in specific activity for the first IO or 20 min and then leveled off to a considerably slower rate of increase. At the end of 50 min, the total ATP phosphorus had attained a specific activity which was only slightly less than half that of the inorganic phosphate. The ADP behaved in a very similar manner, but the rate of orthophosphate incorporation was little more than half the rate into ATP. As might be expected, the AMP showed a small incorporation of radioactivity.

In contrast to the adenine nucleotides, the combined phosphatide spots No. 8 and No. 9 showed no appreciable incorporation of radioactive phosphorus during the first 5 or IO min. After IO min radioactivity appeared in this fraction and rapidly increased with longer time intervals. Within 50 minutes there was no sign of leveling off.

DISCUSSION

A discussion of the rate of incorporation of E32Plorthophosphate in the fast-moving phosphatide fraction of mitochondria must consider the incorporation into the nucleotides as well, particularly ATP, since this is the reference compound to which the incorporation into the lipids is compared if the lipid is an intermediate in oxidative phosphorylation.

The incorporation of E32p]orthophosphate into ATP followed an apparently linear course during the first I0 min. After this time the rate of incorporation di- minished markedly due to the decline of orthophosphate and phosphate-acceptor concentrations. ADP labeling followed a very similar pattern at a somewhat slower rate. The incorporation of [*2Plphosphorus into ADP is believed to be due to adenylate kinase activity. Adenylate kinase has been found in mitochondria by BARKULIS AND LEHNINGER 21 and KIELLEY AND KIELLEY 22. At zero time the concentrations of AMP, ADP, and ATP were approximately equal. Since only ADP and a small amount of ATP were added, a transfer of phosphate groups between the nucleotides is believed to have occurred during the 5-min equilibration period before the addition of radio- active orthophosphate. This further indicates the presence of an active adenylate kinase. Throughout the first part of the experiment the ADP levels remained approxi- mately constant. The increase in the ATP concentration appeared to be accompanied by a concomitant decrease in AMP concentration. These results may be explained by the simultaneous occurrence of ATP synthesis through oxidative phosphorylation and ADP resynthesis by the adenylate kinase reaction.

In contrast to the rapid rise in nucleotide activity in the first I0 min., the lipids showed a lag period during which no incorporation of radioactivity was measurable. This lipid fraction was found to contain several components and it was observed that the radioactivity may be in a minor component of high activity. The major portion of this fraction consists of polyglycerolphosphatide 3. However, an intermediate in the phosphate esterification reactions of oxidative phosphorylation would, in all likelihood, rapidly obtain a specific activity which approached that of the ortho-

Biochirn. Biophys. Acta, 41 (196o) 264-271

27 ° T . E . CONOVER, G. V. MARINETTI, R. F. WITTER, E. STOTZ

phosphate and might be expected to occur in approximately the same concentrations as the cytochromes. CHANCE 23 has calculated io -6 M as the approximate concentration of the cytochromes in a three fold diluted heart muscle preparation. It is probably approximately the same or somewhat higher in the isolated mitochondria preparations which were used in these experiments. On this basis a compound at the concentration of IO -6 M in the mitochondria preparation with the specific activity of the ortho- phosphate should produce, even if present as a minor component, a minimal specific activity in the total fast-moving phosphatide of about 5 counts/min/~g phosphorus. This specific activity was not reached in the lipid fraction until after 30 min. During this time, however, ATP was being rapidly labeled.

In addition, criteria for the establishment of a precursor-product relation between two components have been proposed by ZILVERSMIT et al. 2~ for the case where a steady- state condition exists. During the time that the specific activity of both compounds is increasing, the specific activity of the precursor will always be greater than that of the product. Furthermore, the specific activity of the product will continue to increase until it equals the decreasing specific activity of the precursor. It would appear that neither criteria is met in the case of the fast-moving fraction and the ATP. It is considered therefore highly unlikely that an intermediate in oxidative phosphorylation could be present in the fast-moving phosphatide fraction even as a minor component. Unless the major part of the active phosphatide was lost in the acetone precipitation procedure, it would appear that phosphatides cannot function as intermediates in phosphate esterification prior to transfer to the nucleotides.

Earlier results on the rate of [~2P~orthophosphate incorporation into the phos- phatides 25 obtained by centrifugal isolation of the mitochondria, followed by washing with KC1 and direct extraction of the mitochondria pellet, showed a high incorporation after short incubation periods. In light of the experiments reported here it is assumed that the incorporation of orthophosphate into the phosphatide continued during the centrifugation and washing procedures in these experiments, after the supernatant fluid had been removed.

Although the active lipid fraction of mitochondria does not appear to participate directly in oxidative phosphorylation, its high turnover relative to the other phos- phatides must be considered to have special significance. I t is possible that this fraction may be an intermediate in the biosynthesis of the glycerolphosphatides a.

ACKNOWLEDGEMENTS

This work was supported in part by a grant, H2o63, from the National Heart Institute, National Institutes of Health, U.S. Public Health Service and by the Life Insurance Medical Research Fund.

R E F E R E N C E S

1 N. KRETCHMER AND C. P. BARNUM, Arch. Biochem. Biophys., 31 (I95I) 141. 2 C. LEVlNE AND E. CHARGAFF, Exptl. Cell Research, 3 (1952) 154. 3 G. V. MARINBTTI, J. ERBLAND, M. ALBRECHT AND E. STOTZ, Biochim. Biophys. A cta, 26 (I958) 13 O. 4, M. J. SPIRO AND J. M. McKmBIN, J. Biol. Chem., 219 (1956) 643.

M. A. SWANSON, Biochim. Biophys. Acta, 2o (1956) 85. 6 G. V. 1V[ARINETTI, J. KOCHEN, J. ERBLAND AND E. STOTZ, J. Biol. Chem., 229 (1957) lO27. 7 G. V. 1V[ARINETTI, D. J. SCARAMUZZINO AND E. STOTZ, J. Biol. Chem., 224 (1957) 819.

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[32PlATP AND LIPIDS DURING OXIDATIVE PHOSPHORYLATION 271

8 L. G. ABOOD AND L. ALEXANDER, I . Biol. Chem., 227 (1957) 717 . 9 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 219 (1956) 489.

10 D. E. GREEN, Harvey Lectures, 22 (1957) 177. n G. V. MARINETTI AND E. STOTZ, Biochim. Biophys. 21cta, 21 (1956) 168. 12 G. V. MARINETTI, R. F. WITTER AND E. STOTZ, J. Biol. Chem., 226 (1957) 475. 13 G.V. MARINETTI, J. ERBLAND, M. ALBRECHT AND E. STOTZ, Biochim. Biophys. 2t cta, 25 (1957) 585 . 14 A. L. DOUNCE, R. F. WITTER, K. J. MONTY, S. PATE AND IV[. A. COTTONE, J. Biophys. Biochem.

Cytol., i (1955) 139. is G. V. MARINETTI, J. ERBLAND AND J. KOCHEN, Federation Proc., 16 (1957) 837. 16 L. V. EGGLESTON AND R. HEMS, Biochem. J., 52 (1952) 156. 1~ H. A. KREBS AND R. HEMS, Biochim. Biophys. Acta, 12 (1952) 172. 18 G. ROUSER, G. V. MARINETTI, R. F. WITTER, J. F. BERRY AND E. STOTZ, J . Biol. Chem., 223

(1956 ) 485 • 19 p. S. CHEN, JR., T. Y. TORIBARA AND H. WARNER, 21hal. Chem., 28 (1956) 1756. 20 W. D. HARRIS AND P. POPAT, J. Am. Oil Chemists' Soc., 31 (1954) 124. 21 S. S. BARKULIS AND A. L. LEHNINGER, J. Biol. Chem., 19o (1951) 339. 22 W. W. KIELLEY AND R. K. KIELLEY, J. Biol. Chem., 191 (1951) 485 . 23 B. CHANCE in W. D. MCELROY AND B. GLASS, The Mechanism o/ Enzyme Action, The Johns

Hopkins Press, Baltimore, 1954. 24 D. B. ZILVERSMIT, C. ENTENMAN AND M. C. FISHLER, I" Gen. Physiol., 26 (1943) 325. 35 T. E. CONOVER AND R. F. WITTER, Federation Proc., 17 (1958) 205.

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I N T E S T I N A L T R A N S P O R T OF AMINO ACIDS S T U D I E D I N VITRO

W I T H L- [131I] M O N O I O D O T Y R O S I N E

D A N I E L NATHANS*, D O N A L D F. T A P L E Y AND J O A N E. ROSS

Department o/Medicine, College o/Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A.)

(Received October 23rd, 1959)

S U M M A R Y

The transport of L-E131I]monoiodotyrosine by everted sacs of small intestine is dependent on active accumulation of this amino acid in the gut wall, the terminal ileum being the most active segment. Its transport is stimulated by glucose, which depresses the effiux of monoiodotyrosine from the mucosal surface of the intestine. Certain amino acids and potassium ion competitively inhibit monoiodotyrosine transport, a-amino acids of L-configuration with a free amino group and a long non-polar side-chain are the most effective inhibitors.

INTRODUCTION

Intestinal absorption of most amino acids occurs by way of an active transport mechanism a-3. Using the gut sac technique, WISEMAN a has shown that certain amino acids are transferred from the mucosal to the serosal side of the small intestine against

* Present address: Rockefeller Institute, New York, N.Y.

Biochim. Biophys. Acta, 41 (196o) 271-282