THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 253. No. 4, Issue of February 25, PP. 1030-1040. 1978

Printed m U.S A

Measurement of the Rate of Protein Synthesis and Compartmentation of Heart Phenylalanine*

(Received for publication, August 22, 1977)

EDWARD E. MCKEE, JOSEPH Y. CHEUNG, D. EUGENE RANNELS, AND HOWARD E. MORGAN

From the Department of Physiology, The Milton S. Hershey Medical Center, The Pennsyluania State University, Hershey, Pennsylvania 17033

Calculation of rates of protein synthesis, based upon incorporation of [14C]phenylalanine into protein, depended upon use of the specific activity of phenylalanyl-tRNA. At a perfusate phenylalanine concentration of 0.01 mM, the spe- cific activity of phenylalanyl-tRNA was 65 and 155% of extracellular and intracellular specific activities, respec- tively. At this concentration, the rate of protein synthesis was overestimated if calculated using the intracellular spe- cific activity, but underestimated if the extracellular spe- cific activity was employed. Thus, neither the extracellular nor total intracellular pool of phenylalanine served as the sole precursor for protein synthesis.

When the concentration of perfusate phenylalanine was increased from 0.01 to 3.6 mM, the concentration of intracel- lular phenylalanine increased linearly. At a perfusate phen- ylalanine concentration of 0.4 mM, specific activities of extracellular, intracellular, and tRNA-bound phenylalanine were the same, and the calculated rate of protein synthesis was 106 nmol of phenylalanine incorporated/g of heart/h. The same rate was obtained using the specific activity of V2lphenylalanyLtRNA, when perfusate phenylalanine was 0.01 mM. Thus, protein synthesis did not depend on extracel- lular phenylalanine concentration over this range. Simi- larly, raising perfusate phenylalanine from 0.01 to 3.6 mM had no effect on the incorporation of [14Clhistidine.

Addition of insulin to the perfusate did not modify the relationship between the specific activities of extracellular, intracellular, and tRNA-bound phenylalanine at perfusate phenylalanine levels of either 0.01 or 0.4 mM, but increased the rate of protein synthesis approximately 100%. Specific activities of heart phenylalanine and phenylalanyl-tRNA equilibrated with perfusate phenylalanine (0.01 mM) within 3 to 5 min. Addition of insulin resulted in even faster equilibration.

Two models of amino acid compartmentation involving (a) aminoacylation of tRNA from both the extracellular and intracellular compartments or (b) aminoacylation of tRNA from a compartmented intracellular pool were con- sistent with the steady state and transient data. Inclusion of an intracellular pool of nonradioactive phenylalanine was not required to fit the data. Preferential aminoacyla-

* This work was supported by National Institutes of Health Grants HL-20388, HL-18258, and HL-07223. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “Wuertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

tion of tRNA using amino acids arising from protein degra- dation was not supported by the finding that the specific activities of intracellular and tRNA-bound phenylalanine were the same at a perfusate phenylalanine concentration of 0.4 rnM.

Measurement of the rate of protein synthesis requires that the specific activity of an amino acid serving as the immediate precursor for peptide-bond formation be known (for review, see Rannels et al., 1977). At least a partial solution is to use the specific activity of a tRNA-bound amino acid as represent- ative of the immediate precursor (Henshaw et al., 1971; Khairallah and Mortimore, 1976; Vidrich et al., 1977; Martin et al., 1977).

In several recent studies, the specific activity of an amino- acyl-tRNA was found to be higher than the specific activity of the free amino acids in the total intracellular pool (Khairallah and Mortimore, 1976; Vidrich et al., 1977; Martin et al., 1977). One explanation for this finding is compartmentation of the intracellular pool (Mortimore et al., 1972; Khairallah and Mortimore, 1976). Early experiments supporting compartmen- tation demonstrated that specific activities of [“‘Cllysine in nuclear, mitochondrial, microsomal, and cytoplasmic fractions of liver cells equilibrated with plasma specific activity at different rates (Portugal et aZ., 1970). An alternative explana- tion of the higher specific activity of tRNA bound as compared to intracellular amino acids is aminoacylation of tRNA from both the intracellular and extracellular compartments (Air- hart et al., 1974; Hod and Hershko, 1976; Vidrich et al., 1977).

In the present experiments, compartmentation of phenylal- anine in the rat heart was evaluated by comparison of the rates of equilibration of interstitial, intracellular and tRNA- bound phenylalanine with [Wlphenylalanine in the perfu- sate, and by comparison of these specific activities in the steady state. These values were used to define conditions under which the rate of protein synthesis could be measured accurately.

EXPERIMENTAL PROCEDURES

Heart Perfusion -Hearts were perfused by a modified Langendorff technique (Neely et al., 1967) using Krebs-Henseleit bicarbonate buffer gassed with 95% 0,:5% CO, and containing glucose (15 mM) and amino acids as indicated. Aortic perfusion pressure was main- tained at 60 mm Hg. During the first 10 to 40 min of perfusion, the buffer passed through the heart a single time. In some experiments,

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Measurement of Rate of Protein Synthesis in Heart 1031

recirculation of a measured volume of buffer containing “C-amino- acid, other nonradioactive amino acids and glucose followed. The first 20 ml of radioactive buffer was washed through the heart to reduce dilution of specific activity of “C-amino-acid. Recirculation was continued for 30 to 180 min. In other experiments, the radioac- tive buffer was not recirculated but passed through the heart only once. At the end of perfusion, hearts were cut from the cannulae into beakers of 0.15 M NaCl (2’1, opened, blotted on filter paper, and frozen with a Wollenberger clamp cooled in liquid nitrogen (Wollen- berger et al., 19601. In some experiments, hearts were frozen while still attached to the perfusion cannulae.

Determination of “C-amino-acid Incorporation and Space, and Amino Acid Concentration and Specific Activity- Incorporation of “C-amino-acid into protein and ‘*C-amino-acid space were deter- mined as described earlier (Morgan et al., 1971a). Concentrations of amino acids were estimated by ion exchange chromatography using Beckman amino acid analyzers (Spackman et al., 1958; Morgan et al., 1971a). Since, in previous experiments (Morgan et al., 1971a1, radioactivity added as [‘“Clphenylalanine remained only in this compound during 3 h of perfusion, specific activity of this amino acid was calculated by dividing the radioactivity of phenylalanine- containing solutions (disintegrations per min/ml) by the phenylala- nine concentration (micromoles/ml).

Aminoacyl-tRNA Extraction and Purification - Aminoacyl-tRNA was extracted and purified by a method modified from Rogg et al. (1969), and Davey and Manchester (1969). Frozen hearts were pulverized in a percussion mortar at the temperature of liquid nitrogen. The heart powder was homogenized in a Waring blendor in a solution containing (3 volumes) 0.4 M sucrose, 0.1 M KCI, 6 rnM MgCl,, 30 rnM NaCl, 1 mM sodium acetate (pH 4.51, and 0.4 mg/ml of bentonite and (7 volumes) redistilled water-saturated phenol. Bentonite was sized and deionized as described by Wallyn et al. (1974). The homogenate was diluted by addition of 4 volumes of a solution containing 0.15 M NaCI, 1 rnM EDTA, and 0.4 mgtml of bentonite and shaken at 4” for 30 min. After centrifugation (15,000 x g, 10 min), the aqueous phase was removed and re-extracted with 0.5 volume of phenol by shaking for 15 min at 4”. After a second centrifugation, nucleic acids were precipitated from the aqueous phase by adding 2.5 volumes of absolute ethanol t-20”) containing potassium acetate (12 g/liter) and by allowing the mixture to stand overnight at -20”. The precipitate was removed by centrifugation and dissolved in a solution containing 50 mhl NaCl, 10 mM MgCl,, 1 mM Na*EDTA, 10 mM sodium acetate (pH 4.51, and 0.4 mg/ml of bentonite. Sodium chloride (5 M) was added to give a final concentra- tion of 1.25 M and the mixture was allowed to stand at 2” for 2 h. This mixture was centrifuged (10,000 x g) and the supernatant containing aminoacylated-tRNA was retained. The precipitate, con- taining high molecular weight RNA, was washed with a solution containing 1.25 M NaCl, 10 rn~ MgCl,, and 1 rnM Na,EDTA. The supernatants were combined and the tRNA was precipitated by addition of 2.5 volumes of absolute ethanol C-20”). After standing overnight, the precipitate was collected and washed (2”) with a solution ofethanol:HiO (2.51). The washed precipitate was dissolved in 50 rnM sodium carbonate (pH 10.0) and incubat,ed for 1.5 h at 37”. The deacylated tRNA was precipitated with ethanol (2.5 volumes). The supernatant was taken to dryness either under nitrogen or in an evacuated centrifuge (Savant Instruments). Samples were stored dry at -20” until analyzed.

In separate experiments, [i4Clphenylalanine did not exchange with nonradioactive phenylalanyl-tRNA during the isolation of aminoacyl-tRNA. When radioactive phenylalanine was added to nonradioactive powder prepared from perfused and unperfused hearts, no radioactivity was detected in aminoacyl-tRNA. Recovery of added [*~Clphenylalanyl-tRNA from nonradioactive heart powder was about 60%.

Dns’-Cl Analysis of Phenylalanine Specific Activity- The Dns-Cl method used in these studies was modified from Peterson et al. (1973) and Brie1 and Neuhoff (1972). Dried amino acid residues, resulting from deacylation of tRNA, were dissolved in 0.2 ml of distilled water. The samples contained about 100 pmol of sodium carbonate (pH 101 that arose from the previous deacylation step. Dns-Cl was added in 0.2 ml of acetone (2 pmol/ml of Dns-Cl, Sigma Chemical Co.; 50 to 100 &i/ml of [G-3HlDns-Cl, Amersham). This solution was incubated for 30 min (30”) in the dark. Acetone (3.6 ml) was added to precipitate the salt and Dns-OH. Following centrifu-

I The abbreviation used is: Dns or dansyl, 5-dimethylamino- naphthalene-1-sulfonyl.

gation in a table top centrifuge, the supernatants were decanted and evaporated to dryness either under N, or in an evacuated centrifuge. The dried residues were dissolved in 20 ,ul of 50% pyridine and applied to polyamide sheets. Samples of perfusate were treated similarly except that 10 ~1 of 2.5 M Na,CO, (pH 10) were added to 0.1 ml of perfusate followed by 0.1 ml of the [G- “H]Dns-Cl solution.

Chromatography of the Dns-derivatives was carried out in two dimensions on micropolyamide sheets (Schleicher and Schuell) ac- cording to the method of Woods and Wang (19671, as modified by Hartley (19701 and Peterson et al. (1973). The chromatograms were developed in the first dimension in water:formic acid (100:3) and, after thorough drying, in the second direction in benzene:acetic acid (9O:lOl. After drying, the chromatograms were developed again in the second dimension in ethyl acetate:methanol:acetic acid (2O:l:l). Dns-phenylalanine was well-separated from other Dns-derivatives by this method and was identified by use of standards. Upon completion of the chromatography, Dns-phenylalanine was identi- tied by its fluorescence in ultraviolet light and marked with a soft lead pencil. The entire area was cut out of the sheet and attached to a bent al-gauge needle on a 3-ml syringe. Dns-phenylalanine was eluted from the sheet with 2 ml of water-saturated ethyl acetate into scintillation vials. Recovery of radioactivity due to :‘H and ‘Y! from the sheet was about 80% whether eluted immediately or 24 h later. Radioactivity of the eluate was measured in 10 ml of scintilla- tion fluid (New England Nuclear, Formula 947) using a scintillation spectrometer (Beckman, model 350). Efficiency of counting was assessed using internal standards. Specific acitity of [G-3HlDns-Cl was determined using [“Clphenylalanine of known specific activity. Subsequently, the [G-3H]Dns-Cl was used to measure the specific activity of P4Clphenylalanine in tRNA and perfusate. The calcula- tions were as follows: (a) specific activity of [G-“HlDns-Cl, dpml nmol = specific activity of [“Clphenylalanine standard, dpml nmol . (radioactivity due to :‘H/radioactivity due to “C); (b) specific activity of phenylalanine samples, dpmlnmol = specific activity of [G-3H]Dns-Cl, dpmlnmol . (radioactivity due to ‘C/radioactivity due to 3H).

Validation of Dns-CI Meth.od -This method requires that Dns- phenylalanine be separated from all other radioactive compounds, and that extreme care be used to avoid contamination of the sample. When only [G-3H]Dns-Cl and sodium carbonate were allowed to react, no radioactivity was present in the area of Dns-phenylalanine. When samples containing complete mixtures of amino acids, except phenylalanine, in concentrations 0.1 to 2 times the normal plasma levels were reacted with [G-3HlDns-Cl, radioactivity appeared in the area of Dns-phenylalanine in proportion to the specific activity of the [G-3HlDns-Cl. The amount of 3H in this area of the chromato- gram did not exceed 10% of the 3H present in this area when the specific activity of [‘*C]phenylalanine was measured. This blank was subtracted before calculating the [‘Qphenylalanine specific activity.

Specific activities of [“Clphenylalanine obtained by the Dns-Cl method were compared with those obtained using the amino acid analyzer. As seen in Table I, excellent agreement was found between these methods both for perfusate phenylalanine and phenylalanyl- tRNA. The dansyl chloride method was 20 to 100 times more sensitive than the amino acid analyzer.

RESULTS

Relationship between Perfusate and Intracellular Concen- trations and Specific Activities of Phenylalanine - As seen in Table II, heart phenylalanine content increased progressively as perfusate phenylalanine was raised. When intracellular concentrations were calculated from these measurements, a linear relationship between perfusate and intracellular con- centrations was apparent up to a perfusate concentration of 3.6 mM (Fig. 1). In other experiments, this relationship appeared to be established by 30 min of perfusion. Extrapola- tion to zero perfusate phenylalanine indicated that an intra- cellular level of 0.017 mM was maintained, presumably through proteolysis. Similarly, in hearts that were perfused for 3 h with buffer containing no phenylalanine but normal plasma levels of other amino acids, intracellular phenylala- nine was 0.016 f 0.0003 mM. This value was approximately

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1032 Measurement of Rate of Protein Synthesis in Heart

15% of the intracellular phenylalanine concentration that was present in hearts perfused with normal plasma levels of the amino acid (0.08 mM), but was 50% of the intracellular phenylalanine concentration at a perfusate level of 0.01 mM.

Estimation of the specific activity of intracellular phenylal- anine indicated that the ratio of intracellular to perfusate specific activity rose from 0.38 at a perfusate phenylalanine level of 0.01 rn~ to unity at 0.4 mM phenylalanine and above

TABLE I

Comparison of specific activities obtained using [G-Widens-C1 with specific activities obtained using amino acid analyzer

A preliminary perfusion of 10 min with buffer containing phenyl- alanine at the concentration indicated, 15 rn~ glucose, and normal plasma levels of other amino acids was followed by recirculation of the same buffer containing YClphenylalanine for either 30 (0.01 mM) or 90 (0.40 mM) min. These amino acid levels were as follows (in millimolar concentrations): aspartic acid, 0.014; asparagine, 0.68; glutamic acid, 0.12; glutamine, 0.65; serine, 0.25; threonine, 0.20; proline, 0.11; glycine, 0.34; alanine, 0.33; valine, 0.19; isoleucine, 0.11; leucine, 0.17; tyrosine, 0.069; lysine, 0.38; histidine, 0.052; arginine, 0.12; methionine, 0.054; cystine, 0.10; and tryptophan, 0.10. The normal plasma level of phenylalanine was 0.08 mM. The figures in parentheses indicated the number of buffer samples or pools of hearts (60 each) that were analyzed. At the final stage of preparation, each sample of phenylalanine deacylated from tRNA was divided so that one-tenth was used for determination of specific activity by the Dns-Cl method, while the remainder was used for the amino acid analyzer. Values represent the mean ? S.E.

Specific activity of: Perfilsate phmvld. Perfusate phenylalanine Phenvlalanvl-tRNA, I____.j _-.

anine Amino acid Dns-Cl Amino acid analvzer analvzer Dns-Cl

I ” ~~ T7lM dpmlpmol dpmlpmol

0.01 106 r 2 (12) 108 k 2 (12) 72.3 f 5.7 (3) 70.4 -t 5.8 (3) 0.4 62.0 r 0.9 (5) 60.7 k 2.3 (5) 21.3 20.0 f 1.0 16)

I 2 3 4 z ,025 .050 .075 .:oo

PERFUSATE PHENYLALANINE, mM

FIG. 1. Relationship between perfusate and intracellular concen- trations of phenylalanine in the perfused heart. Data from hearts perfused for 180 min were taken from Table II. The following assumptions were involved in calculation of intracellular phenylal- anine concentrations. (a) Phenylalanine concentrations in the intra- vascular and interstitial compartments were considered equal to those in the perfusate. (b) The volume of the interstitial and intravascular compartments was taken to be the sorbitol space (0.35 ml/g). Total water was 0.80 ml/g (Morgan et al., 1971a; Cheung ef al., 1978). Intracellular phenylalanine was calculated using the following formula:

intracellular phenylalanine = (mM)

heart phenvlalanine (umolle) iperf&.ate phenylalanine (pmol/ml) . sorbitol space, (ml/g)

Total water (ml/g) - sorbitol space (ml/g)

The line relating intracellular and perfusate phenylalanine concen- trations in Panel A includes the entire range of perfusate phenylal- anine concentration. In Panel 8, only the four lower perfusate levels are plotted. In this instance, the line was fitted by the method of least squares (y = 0.9& + 0.017). Eachpoint represents 6 to 10 observations. The standard error is not shown if it falls within the symbol.

TABLE II Effect of perfusate phenylalanine concentration on specific activity and content of heart phenylalanine and incorporation of

[‘4C]phenylalanine and [%‘]histidine into heart protein.

A preliminary perfusion of 10 min with buffer containing phenyl- perfusate that was recirculated was varied to minimize the fall in alanine at the concentrations indicated, 15 mM glucose, and normal perfusate specific activity. When 0.01, 0.02, and 0.04 rnM phenylala- plasma levels of other amino acids was followed by recirculation of nine were present, 170 ml of perfusate were recirculated; 0.08 mM, the same buffer containing [YJphenylalanine or [‘Qhistidine 75 ml; and 0.4 mM and greater, 25 ml. The figures in parentheses (916,000 dpm/wmol) for 180 min. In the 0.01 mM phenylalanine indicate the number of pools of hearts that were analyzed. These experiments, YC]histidine was purified on an amino acid analyzer pools contained 1 to 6 hearts. Values represent the mean -c SE. to avoid contamination with [14Clphenylalanine. The volume of

Parameter 0.0094(8) 0.017(7)

Phenylalanine concentration in original perfusate, rn~

0.040(7) 0.079(10) 0.358(6) 1.15(6) 3.43(6)

Perfusate phenylala- 0.011 ? 0.0001 0.020 2 0.0003 0.037 ? 0.001 0.084 -+ 0.003 0.385 f 0.018 1.10 * 0.02 3.36 k 0.03 nine imr.0

Heart phenylalanine 0.018 f 0.001 0.023 5 0.001 0.036 5 0.001 0.075 2 0.006 0.304 2 0.014 0.857 ? 0.007 2.56 2 0.02 (kmollg)

Specific activity of per- fusate phenylala- nine, 10-3.dpm/ pm01

Original perfusate 330 369 361 370 369 380 364 180-min perfusion 247 + 5 316 r 4 356 + 6 344 + 6 332 c 4 364 2 6 368 r 8

Specific activity of heart 122 * 8 194 ?z 5 252 f 10 293 2 7 333 2 12 369 2 5 372 ‘- 1 phenylalanine (10-3.dpm/pmol)

Phenylalanine incorpo- 268 k 5 350 rt_ 12 380 2 12 449 2 9 572 rt_ 14 614 -c_ 19 634 k 11 ration (dpmlmg protein)

Histidine incorporation 364 f 15 384 k 16 377 t 15 357 2 27 359 k 26 345 2 12 (dDmima Drotein)

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Measurement of Rate of Protein Synthesis in Heart 1033

(Fig. 2). Lower specific activities of the intracellular pool

reflected the balance between the rates of entry of amino acid from the extracellular space and protein degradation.

Calculation of Apparent Rates of Protein Synthesis-The effect of perfusate phenylalanine concentration on protein synthesis was investigated by measuring incorporation of [‘“Clhistidine into heart protein over a range of perfusate phenylalanine concentrations (0.01 to 3.6 mM). As seen in Table II, [“Clhistidine incorporation was unaffected. In ear- lier studies (Rannels et al., 1975), the rate of protein degrada-

tion was found to be unchanged by raising perfusate phenyl- alanine from 0.01 to 0.08 mM.

Although studies of [“Clhistidine incorporation indicated that protein synthesis did not increase as perfusate phenylal- anine was raised, incorporation of [“‘Clphenylalanine into heart protein more than doubled (Table II). Apparent rates of protein synthesis were calculated by dividing the radioactivity in protein by the average specific activity of either perfusate or intracellular phenylalanine (Fig. 3). This rate was not constant for either compartment of phenylalanine, suggesting that neither the extracellular nor total intracellular pool of phenylalanine represented the sole precursor pool serving protein synthesis.

Effect of Perfusate Phenylalanine Concentration on Specific Activity of Phenylalanyl-tRNA and Rate of Protein Synthe- sis - As seen in Table III, the rate of protein synthesis calcu- lated using the specific activity of phenylalanyl-tRNA was the same at 0.01 and 0.40 mM perfusate phenylalanine in hearts perfused for 30 or 90 min. These results support the conclusion that the rate of protein synthesis was not affected over this range of perfusate phenylalanine. As reported above, rates of protein synthesis calculated using the specific activi- ties of intracellular or perfusate phenylalanine were not the same at these concentrations.

$ I.b-l,A *IA 0 .02 .04 .06 .08 .I0 .38 40 1.10 1.12 336 538

PERFUSATE PHENYLALANINE, mM

FIG. 2. Effect of perfusate phenylalanine concentration on the equilibration of intracellular and perfusate specific activities. Data were taken from Table II. In addition to the assumptions involved in calculating intracellular phenylalanine concentrations (Fig. l), calculation of intracellular specific activity involved the assumption that phenylalanine present in the intravascular and interstitial spaces had the same specific activity as that in the perfusate. Intracellular specific activity was given by the following formula:

intracellular specific = activity, dpm/pmol (heart phenylalanine, pmol/g)

(heart specific activity, dpm/pmol)) - (perfusate specific activity, dpm/Fmol).

(perfusate phenylalanine, pmol/ml. (sorbitol space, ml/g)

(heart phenylalanine, pmollg) - (perfusate phenylalanine, pmol/ml). (sorbitol space, ml/g)

r

z I c 0 ,022 .04 .06 .08 / -1.12.s .I0 .38 tlo I.10

PERFUSATE PHENYLPILANINE. mM

FIG. 3. Effect of perfusate phenylalanine concentration on appar- ent rates of protein synthesis. Rates were calculated by division of phenylalanine incorporation at 180 min (Table II) by the average specific activity of perfusate (e-0) or intracellular (0- - -0) phenylalanine. This value was converted to an hourly rate and expressed per g of heart (0.16 g of protein/g of heart, Morgan et al., 1971a). The horizontal dashed line indicates the average rate of protein synthesis as estimated at 0.38, 1.10, and 3.36 rnM perfusate phenylalanine.

TABLE III

Effect ofperfusate phenylalanine on specific activity of phenylalanyl- tRNA and rate of protein synthesis in control hearts

Hearts were perfused for a preliminary period of 10 min with buffer containing phenylalanine at the concentration indicated, 15 rnM glucose, and normal plasma levels of other amino acids. Subse- quently, the same buffer containing [‘4Clphenylalanine was recir- culated for 30 or 90 min. An initial perfusate sample was taken after 5 min of recirculation. Perfusate, heart, and phenylalanyl- tRNA specific activities were determined as described under “Exper- imental Procedures.” Intracellular phenylalanine specific activity was calculated as described in Fig. 2. Apparent rates of protein synthesis were calculated using the mean specific activity of perfu- sate, intracellular and tRNA-bound phenylalanine, as described in Fig. 3. Initial specific activities of intracellular phenylalanine and phenylalanyl-tRNA were taken to be 38 and 65% of the initial perfusate specific activity, respectively. Each value represents the mean ? standard error of five or six determinations. N.D., not done.

Specific activity of perfusate phenylalanine (lo-’ dpml nmoll

Specific activity of intracel- lular phenylalanine

(lo-‘! dpmlnmoll

Specific activity of phenyl- alanyl-tRNA

(lo-’ dpm/nmoll

Phenylalanine incorporation (lo-’ dpmimg protein)

Protein synthesis (nmol/g heart/h)

Perfusate specific acivity

Intracellular specific activ- ity

tRNA specific activity

Parameter Minutes of

Perfusate phenylalanine

perfusion 0.01 nml 0.4 InM

5 30 90

30

90

30

90

30 90

30 90 30 90 30 90

30-90

1300 f 20 570 f 5 1080 f 16 N.D. 598 2 14 568 + 5

410 k 16 N.D.

227 2 5 569 A 6

701 -t 20 N.D.

388 f 18 556 f 9

306 f 11 N.D. 579 2 23 550 5 19

82 + 3 N.D. 65 f 3 105 f 2

231 f 8 N.D. 184 f 7 105 k 2 127 f 5 N.D. 101 2 4 108 f 4

80 k 7 N.D.

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1034 Measurement of Rate of Protein Synthesis in Heart

It should be noted that the rates reported in Table III are higher than those found in Fig. 3. The rates in Table III were measured over 30 or 90 min of perfusion, while those in Fig. 3 were measured over 3 h. Control hearts have a higher rate of protein synthesis during the first 30 min of perfusion clue to the presence of endogenous substrates and insulin (Morgan et al., 1971a). If the rate of protein synthesis obtained using 0.01 mM perfusate phenylalanine was calculated for the period between 30 and 90 min, good agreement with the value in Fig. 3 was obtained (Table III, last line).

Concentration and Specific Activity of Intracellular Phen- ylalanine, Specific Activity of Phenylalanyl-tRNA, and Rate of Protein Synthesis in Hearts Perfused with Buffer Contain- ing Insulin - Provision of insulin maintained ribosomal aggre- gation and a linear rate of phenylalanine incorporation during 3 h of perfusion (Morgan et al., 1971a, 1971b). When exposed to insulin (Table IV), heart and intracellular levels of phenyl- alanine were lower than in control tissues. This reduction reflected a shift in the balance between rates of protein synthesis and degradation to favor the synthetic pathway (Rannels et al., 1975).

As was found in control hearts (Table ID, incorporation of [‘Qhistidine into protein was not reduced by lowering perfu- sate phenylalanine from 0.347 to 0.0084 mM in insulin-treated hearts. The rates of histidine incorporation were 902 2 44 and 1012 t 36 dpmlmg at these concentrations, respectively.

In the presence of insulin (Table V), the relationship be- tween the specific activities of phenylalanyl-tRNA and intra- cellular and extracellular phenylalanine was similar to that in control hearts (Table III). The rates of protein synthesis at 0.4 mM perfusate phenylalanine, calculated using specific activities of perfusate, intracellular, or tRNA-bound phenyl- alanine, were 60 to 100% greater than in control hearts, depending upon period of measurement. The rates of protein synthesis, calculated using the specific activities of phenyla- lanyl-tRNA, were the same at perfusate phenylalanine con- centrations of 0.01 and 0.4 mM.

In other experiments, the specific activity of phenylalanyl- tRNA was measured in hearts perfused with buffer containing normal plasma level of amino acids, including phenylalanine (0.08 mM). The specific activity of phenylalanyl-tRNA was 80.3 2 3.8 and 90.0 2 3.1% of perfusate specific activity in control and insulin-treated hearts, respectively. Three pools of six hearts each were included in each group.

TABLE IV

Effect of insulin on heart, and intracellular concentrations of phenylalanine

A preliminary perfusion of 10 min with buffer containing 15 mM glucose, 25 milliunits/ml of insulin, where indicated, and normal plasma levels of amino acids except phenylalanine was followed by recirculation of the same buffer for 180 min. Intracellular concentra- tions of phenylalanine were calculated as described in Fig. 1. Values represent the mean + SE. of four to six determinations on pools of one to six hearts.

Parameter Control hearts Insulin-treated hearts

Perfusate phenylalanine (rnM)

Original 0.0094 0.0084 180 min of perfusion 0.011 + 0.0001 0.0083 2 0.0002

Heart phenylalanine (pmol/ 0.018 2 0.001 0.0079 k 0.0002” g)

Intracellular phenylalanine 0.031 2 0.003 0.0112 ? 0.006” (~mol/ml)

a p < 0.05 uers~s control hearts.

Time Course of Equilibration and Incorporation of I’C]Phenylalanine in Control and Insulin-treated Hearts - Accurate estimates of protein synthesis over short periods of perfusion depended upon a rapid approach of the specific activity of phenylalanyl-tRNA to the steady state value. In order to validate the use of the specific activity of this precursor pool as the basis for calculating rates of protein synthesis, the time course of equilibration of specific activity of heart phenylalanine (intracellular and extracellular) and phenylalanyl-tRNA and of incorporation of [14Clphenylalanine into protein was determined. As seen in Fig. 4, the [‘~Clphenylalanine space in both control and insulin-treated hearts increased rapidly and reached a plateau in 4 to 8 min. However, if perfusion was continued for 30 min, a value 15% greater was achieved. Despite this increase in [“C]- phenylalanine space, the specific activity of phenylalanyl- tRNA after 5 min of perfusion was 64 k 3% of the specific activity of per&sate phenylalanine (seven pools of six hearts each). This value was not significantly different from the extent of equilibration observed after 30 and 90 min of perfu- sion (Table III). The rate of incorporation of [14Clphenylalanine into protein also was the same after 5 and 30 min of perfusion (data not shown). These findings suggested that this slow phase of [‘4C1phenylalanine equilibration involved a pool unrelated to protein synthesis, such as a slow equilibrating compartment in the extracellular space (Morgan et al., 1961). Since the specific activity of phenylalanyl-tRNA was equili- brated in 5 min, the plateau value for [i4Clphenylalanine distribution at 4 to 8 min (Fig. 4) was used for calculation of the time course of equilibration of phenylalanine in insulin-

TABLE V

Effect of perfusate phenylalanine on specific actiuity of phenylalanyl- tRNA and rate of protein synthesis in insulin-treated hearts

Hearts were perfused as in Table III, except for addition of insulin (25 milliunits/ml) to the perfusate. Calculations were the same as those described in Table III. The initial specific activities of intracellular and tRNA-bound phenylalanine were taken to be 38 and 70% of perfusate phenylalanine, respectively. Each value rep- resents the mean * S.E. of five or six determinations. N.D. = not done.

Parameter Minutes of Perfusate phenylalanine

perfusion 0.01 rnM 0.4 rnM

Specific activity of perfusate phenylalanine (lo-’ dpm/ nmol)

Specific activity of intracel- lular phenylalanine (lo-* dpm/nmol)

Specific activity of phenyl- alanyl-tRNA (lo-’ dpm/ nmol)

Phenylalanine incorporation (IO-’ dpm/mg protein)

Protein synthesis (nmol/g heart/h)

Perfusate specific activity

Intracellular specific activ- ity

t-RNA specific activity

5 30 90

30 90

30 90

30 90

30 90 30 90 30 90

1300 2 20 440 f 6 1131 + 20 N.D.

807 f 20 438 f 5

430 k 8 N.D. 3072 8 437 -c 7

790 f 18 N.D. 523 f 14 441 f 11

447 i 7 N.D. 1071 f 32 661 f 41

118 f 4 N.D. 108 +- 4 162 ? 8 311 2 11 N.D. 286 f 12 162 f 8 168 f 6 N.D. 160 k 6 160 ? 8

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Measurement of Rate of Protein Synthesis in Heart 1035

% IL0 fj 09

$ 0.8

uo7 z 5 06

4 5 0.5

$04 w 2 0.3

CENTRoL N 9

” 0

0 P

-------------------- ---_____ & 11 e

l k

INSULIN

/ 5 6 7 8 Tc I 2 3 4

MINUTES

lOOF+

80- !;

: 60- i t 6,

E i 40- \* 5 s ‘?:

! i

4 ‘\ ‘,)

20- .\

2 .’ : \ . ‘.\

7 ‘.\

z

PHE-tRNA ‘.‘\ \

In \

2 3 4 s -0 I MIN

FIG. 5. Time course of equilibration of heart phenylalanine and phenylalanyl-tRNA and incorporation of radioactivity into protein of control hearts. Hearts were perfused as described in Fig. 4. Data for heart phenylalanine and incorporation of [L4C1phenylalanine into protein are shown as mean f SE. of 6-8 determinations. If a standard error is not shown, it did not extend beyond the data point. Data for equilibration of phenylalanyl-tRNA are shown as individual determinations. The dashed and dotted lines represent the theoretical time courses of equilibration predicted from mathe- matical analysis of two models, (11 the two-site activation model (- - -) or (2) one of the solutions of the compartmented intracellular pool model (. . .). These models and their solutions are presented in the miniprint. The solutions of equations describing equilibration of heart phenylalanine were:

‘+tt) + P41V + q6(t) “Odel2: l- _ m m = 0.033e-19t + 0.30e-6.7t

9* + 94 + qs

+ 0.6&3.59t

The solutions of equations describing equilibration of phenyla- lanyl-tRNA were:

treated hearts. In control hearts, the average value obtained at 7 and 8 min was employed.

Specific activities of heart phenylalanine and phenylalanyl- tRNA reached greater than 90% of equilibrium in 4 min in control hearts (Fig. 5). A faster rate of equilibration was observed in the presence of insulin, probably as a result of a decrease in intracellular phenylalanine concentration (Fig. 6). Both control and insulin-treated hearts demonstrated a

FIG. 4. Time course of the accumu- lation of [“Clphenylalanine in control and insulin-treated hearts. Hearts were perfused for 30 min with buffer containing 15 rnM glucose, 0.01 mM phenylalanine, and normal plasma levels of other amino acids. Insulin (25 milliunits/ml) was added as indicated. After this period, perfusion was contin- ued for the time indicated with the same buffer containing [‘Qphenylala- nine. This buffer passed through the heart a single time and was discarded. Phenylalanine spaces were calculated as described earlier (Morgan et al., 1971al. Values represent the mean + S.E. of four to six determinations at each time point. When standard errors are not shown, they did not extend beyond the symbol.

I

MIN

Model 2: l -g ! = 0.023e-19t - 0.120e-6.7t + 1.01e-0.69t q3

+ 0.164e-l.17t

The solutions of equations describing incorporation of radioactiv- ity into protein were:

Model 1: q51t1 = d i53 q3(t)dt 1.17 2.32 x - = [I 105t

105e-o~64~ - 104e-1.17t 1 t

1.76 103e-lot x + 3.36 x 3.63 x

0

Model 2: q5ct> = As3 6’

y3(t1 dt = 1.17 C

2.31 x dt + 2.73

x lo*e-lgt - 6.78 x 3.38 103e-6.'t 105e-0.69t + x

+ 3.22 x 104e-1.17t I t 0 lag in achieving the steady state rate of [14Clphenylalanine incorporation. This lag was related to the time required for phenylalanyl-tRNA to reach a steady state specific activity. As expected, the rate of [Wlphenylalanine incorporation achieved was about 100% greater in insulin-treated hearts (Fig. 6).

Models of amino acid compartmentation that would fit both the steady state and transient data in regard to phenylalanine

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Measurement of Rate of Protein Synthesis in Heart 1036

f I I 110 ‘- z ‘0 I 2 3 4

I 1 “0 20 40 60 60

7 W 2

PHE-tRNA

MIN

FIG. 6. Time course of equilibration of heart phenylalanine and phenylalanyl-tRNA and incorporation of radioactivity into protein of insulin-treated hearts. Hearts were perfused as in Fig. (,-except that insulin (25 mu/ml) was added to all buffers. Data are plotted as described in Fig. 5. The dashed and dotted lines represent the theoretical time courses from mathematical analyses of two models: (1) the two-site activation model (- - -1 or (2) one of the solutions of the compartmented intracellular pool model (. . . .l, as presented in the miniprint. The solutions of equations describing equilibration of heart phenylalanine were:

q2(t) + q4it1

“dell: l- _ m

= 0.386e-10t + 0.614e-0.8’t

42 + 94

9*(t) + q4(t1 + c+,(t) MOdel2: l- m m = 0.040e-=t + 0.447e-9t

92 + q4 + q;

+ 0.512e-0.94t

The solutions of equations describing equilibration of phenylalanyl- tRNA were:

MO&l 1: 1 _ gl = -13.196e-10t + 0.580e-0.8't + 0.616e-2.42t

93

concentration and specific activities were considered. As dis- cussed in the miniprint,” the two-site activation model (Air- hart et al., 1974; Hod and Hershko, 1976; Vidrich et al., 1977) and the compartmented intracellular pool model (Mortimore et al., 1972; Rhairallah and Mortimore, 1976) were tested. Steady state data obtained with 0.01 mM perfusate phenylala- nine were used to predict the rate of equilibration of the specific activities of heart phenylalanine and phenylalanyl- tRNA and the rate of [14C]phenylalanine incorporation. Both models appeared to provide a good fit to the experimental observations (Figs. 5 and 6). It was not necessary to postulate the existence of an intracellular pool of nonradioactive phen- ylalanine as was described for valine in liver (Mortimore et al., 1972). The size of the phenylalanyl-tRNA pool that pro- vided the best fit was 1.2 nmol/g of heart. This value is approximately l/20 of the total tRNA content (Davey and Manchester, 1969). The mathematical derivation and analysis of these models are presented in the miniprint.

DISCUSSION

Valid estimates of the rate of protein synthesis, based on incorporation of radioactive amino acids, require that the

* Portions of this paper (including Figs. 7 to 10 and Table VI) are presented in miniprint following the references. Full size photoco- pies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 77M- 1366, cite authors, and include a check or money order for $1.80 per set of photocopies.

; ‘i-/( ,PROT;l

Y 01 2345

+ 0.851e-24*t

The solutions of equations describing incorporation of radioactivity into protein were:

Model 1: q5(t) = b3

d

q3(tj dt = 2.42 2.52 x 105t

C

- 5.19 x 103e-lot + 1.78 x 105.-o~8't + 6.81

1 t

x lo4e-*.42t

0

6.42 x 102e-21t - 1.46 x 104e-8t + 1.5 x 105e-o.94t

1 t

+ 8.84 x lo4e-*.42t

0

specific activity of the pool of amino acids serving as direct precursor for the synthetic pathway be determined. The most direct approach to this problem would be to measure the specific activity of an appropriate amino acid in peptidyl- tRNA. In practice, however, the methods available for sepa- ration and purification of nascent polypeptides are difficult (Slabaugh and Morris, 1970). Nascent chains make up a very small portion of the mass of polysomes, and contamination with other peptides is hard to avoid. Furthermore, polysomes are recovered in poor yield from heart homogenates (Morgan et al., 1971b), and the period of time required for their isolation could lead to modification of the nascent chain specific activity.

Alternatively, specific activity of the precursor pool could be determined by measurement of specific activity of an amino acid within a newly induced protein. This approach was used successfully in liver by Loftfield and Harris (1956) to show that the tissue pool of amino acids served as precursor for the synthetic pathway. Fifteen years later, Righetti et al. (1971) examined the specific activities of three amino acids in the newly synthesized ferritin of HeLa cells. Their results indicated that lysine, leucine, and phenylalanine derived from proteolysis mixed with the intracellular amino acid pool to varying extents before being reincorporated into protein. Studies of this type have not been carried out in the heart since rapid induction of specific proteins has not been de- scribed. This approach requires complete purification of a protein synthesized from a negligible level prior to the experi-

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Measurement of Rate of Protein Synthesis in Heart 1037

ment. Determination of the precursor pool specific activity, based upon isolation of newly synthesized protein, may not provide a reliable index of the average precursor specific activity. In liver, the ratio of the specific activities of serine/ glycine in ferritin was higher than in albumin, and both ratios were different than the ratio found in total liver protein (Fern and Garlick, 1976).

A third approach to the determination of precursor pool specific activity is measurement of this parameter in a tRNA- bound amino acid. This approach to measurement of synthesis of whole heart protein assumes that the average specific activity of aminoacyl-tRNA is equal to that of peptidyl-tRNA. In liver, the studies of Fern and Garlick (1976) and Ilan and Singer (1975) suggest that both of these pools may be compart- mented. In the latter study, the specific activity of leucine in peptidyl-tRNA of free polysomes was 2-fold higher than in membrane-bound polysomes. In present studies, compartmen- tation of aminoacyl-tRNA did not distort the measurement of synthesis of whole heart protein at a perfusate phenylalanine concentration of 0.4 mM since the specific activity of phenyl- alanine in tRNA was equal to the specific activity of perfusate phenylalanine. The same rate of protein synthesis was calcu- lated when the specific activity of phenylalanyl-tRNA was 65% of perfusate phenylalanine (0.01 mM), suggesting that compartmentation did not introduce errors in this calculation at either phenylalanine level.

Practical problems complicate measurement of aminoacyl- tRNA specific activity. Aminoacyl-tRNA is present in low amounts in heart (Davey and Manchester, 1969). In spite of this, purification must be rigorous. In addition, the tRNA pool turns over with a tliZ of several seconds, a situation that demands rapid methods for freezing and homogenization. If these technical problems are overcome, the specific activity of aminoacyl-tRNA may be used in calculating accurate rates of synthesis of whole heart protein.

It is more difficult to establish that the specific activities of either extracellular or intracellular amino acids provide a reliable index of the specific activity of the precursor pool serving protein synthesis (for review, see Ranneis et al., 1977). The complexities of using specific activities of free amino acids to estimate synthetic rates were illustrated by Mortimore et al. (1972) and are shown in Fig. 3. In the present studies, the apparent rate of protein synthesis ob- tained from these calculations varied with the extracellular concentration of phenylalanine. However, the rate of protein synthesis was constant over this range, when calculated using phenylalanyl-tRNA specific activities (Tables III and V), or based on incorporation of [“Clhistidine (Table ID. These studies indicate that accurate rates of protein synthesis in heart muscle can be calculated from the specific activity of free amino acids only under well defined conditions. The least complicated approach to this calculation is to use high perfu- sate concentrations of phenylalanine; a situation in which the specific activities of perfusate, intracellular, and tRNA-bound phenylalanine are the same.

Data presented in this paper confirm earlier studies report- ing that insulin accelerated protein synthesis in heart (Mor- gan et al., 1971b). The major effect of the hormone in heart was to stimulate steps involved in peptide chain initiation. In addition, the hormone affects net protein turnover by inhibit- ing protein degradation (Rannels et al., 19751, as was reflected in lower intracellular levels of phenylalanine (Table IV).

Analysis of amino acid compartmentation indicates that both a two-site activation model (Airhart et al., 1974; Hod

and Hershko, 1976; Vidrich et al., 1977) and a compartmented intracellular pool model (Mortimore et al., 1972; Khairallah and Mortimore, 1976) provide a good fit to the experimental observations. This is not surprising, since both models are operationally the same. The two-site activation model allows mixing of extracellular and intracellular amino acids at the level of aminoacyl-tRNA. The compartmented intracellular pool model allows the mixing to occur as free amino acids with subsequent acylation to tRNA.

Either of these models appears feasible based on present knowledge of cell structure and enzyme localization. Subcel- Iular organelles, including mitochondria and lysosomes, are known to contain free amino acids that may be restricted from reaching isotopic equilibrium with the cytosol specific activity. Complex aggregates containing lipids were found in association with tRNA and tRNA synthetases (Bandyopa- dhyay and Deutscher, 1971, 1973; Tscherne et al., 1973). Whether this reflects association with cellular membranes is not clear. Phenylalanyl-tRNA synthetases have been found in three forms: complex aggregates (Roberts and Coleman, 1972), bound to ribosomes (Irvin and Hardesty, 1972; Roberts and Coleman, 1972; Tanaka et al., 1976; Ussery et al., 1977), and in postribosomal supernatants (Hampel and Enger, 1973; Tanaka et al., 1976). Ussery et al. (1977) reported that phen- ylalanyl-tRNA synthetases did not occur in complex aggre- gates in a number of eukaryotic cells but were bound to ribosomes in variable, but generally high proportions. Up to 90% of phenylalanyl-tRNA synthetase activity was recovered bound to ribosomes from rabbit reticulocytes (Irvin and Har- desty, 1972). Ultimately, the ability to distinguish between the two models discussed above will depend on knowledge of the localization of phenylalanyl-tRNA synthetase in the heart.

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Measurement of Rate of Protein Synthesis in Heart 1039

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1040 Measurement of Rate of Protein Synthesis in Heart

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E E McKee, J Y Cheung, D E Rannels and H E Morganphenylalanine.

Measurement of the rate of protein synthesis and compartmentation of heart

1978, 253:1030-1040.J. Biol. Chem. 

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