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Studies on the formation of carnosine and anserine in pectoral muscle of the developing chick
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies on the Formation
of Carnosine
and Anserine
of the Developing
Chick’
I. ROSABELLE Department
444-453 (1967)
119,
McMANUS
AND
MARY
in Pectoral
Muscle
S. BENSON
oj Biochemistry and Nutrition, Graduate SchooE oj Public of Pittsburgh, Pittsburgh, Pennsylvania 15213
Health,
University
Received September 26, 1966 Changes in the concentrations of carnosine and anserine and the development of the p-alanyl peptide-synthesizing enzymes in pectoral muscle of the posthatched chick have been investigated. The concentration of the p-alanyl peptides in pectoral muscle of the developing chick increases markedly during 21 days after hatching. Carnosine increases from 0.25 pmole/gm fresh muscle at 24 hours to 9.7 pmoles at 21 days. Anserine, with a concentration of 0.4 pmole/gm fresh muscle at 24 hours, reaches a level of 17 pmoles at 21 days. n-Histidine-2-(ring)-% was utilized for the in vivo formation of carnosine and anserine in the chick. The rate of incorporation of “C into the carnosine in pectoral muscle of 2., G-, and 15-day-old chicks is indicative of a synthesis of peptide from precursor histidine which is influenced by the stage of development of the pectoral muscle. The activities of carnosine-N-methyltransferase and carnosine synthetase were investigated in the developing chick, and their activity patterns were found to differ. Carnosine N-methyltransferase reaches a maximum specific activity at 6 days and declines to about 55yo of maximum activity at 15 days. At 21 days, activity is 36% of peak levels. Carnosine synthetase shows a 50-fold increase in activity between 2 and 15 days, with some evidence for a decline in activity subsequent to 21 days. The development of carnosine N-methyltransferase is inhibited by puromycin and actinomycin D, consistent with a de novo synthesis of the enzyme.
At least two enzymes present in vertebrate skeletal muscle catalyze the formation of the p-alanyl peptides. Carnosine synthetase [Lhistidine:p-alanine ligase (AMP) ; E.G. 6.3.2.111 catalyzes the synthesis of carnosine and anserine as well as certain related analogues (l-4), and carnosine N-methyltransferase (X-adenosylmethionine : carnosine Nmethyltransferase; E.C. 2.1.1.12) mediates the synthesis of anserine from carnosine (3-5). Kalyankar and Meister (I), and Winnick and Winnick (3, 4) observed that the activity of carnosine synthetase was markedly higher in pectoral muscle from young chicks than in adult muscle. A qualitatively similar effect of age of pectoral 1 This investigation is supported by a grant-inaid from the Muscular Dystrophy Associations of America, Inc., New York, New York.
muscle tissue on enzyme activity was observed in studies on carnosine N-methyltransferase (5). In studies designed to investigate the generation of carnosine and anserine in leg skeletal muscle of chicks during embryonic development and into adult life, Parshin and Goryukhina (6), Skvortsova (7), and Severin and Fedorova (8) found that carnosine appeared at the days 12-14 of development and anserine at day 17, with subsequent postnatal increases in their concentrations in muscle. The latter workers remarked on the correlation between the appearance of the peptides and their increased concentration with the development of the mechanical functioning of the muscle. The present report is concerned with an investigation of the changes in the concen trations of the @alany peptides in chicak 444
CARNOSINE
AND ANSERINE
pectoral muscle during a 3-week period after hatching and a study of t,he incorporation of I>-histidine-2-14C into carnosine and anserine during that period. The development of the fi-alanyl peptide-synthesizing enzymes has been investigated with enzyme extracts prepared from developing chick pectoral muscle, and their act,ivity patterns have been compared and related to the accumulation of carnosine and anserine as a function of the age of t’he muscle. In addition, t’he effects of treatment with actinomycin D and puromytin dihydrochloride on the pattern of development of carnosine N-methyltransferase in young chicks have been studied. A preliminary report of some of these results has been published (9). MATERIALS
AND METHODS
L-histidine-2-(ring)-1°C and L-carnosine were obtained from California Corporation for Biochemical Research. p-Alanine-l-l% and adenine8.1% were plrrchased from the New England NIlclear Corporation. S-Adenosylmethioninemethyl-l% was isolated from yeast after incubation with L-methionine-methyl-l% according to the procedure of Stekol et al. (10). Actinomycin D was generously provided by Merck Sharp & Dohme, and puromycin dihydrochloride was purchased from the Sigma Chemical Company. White Rock chicks, aged 24 hours to 21 days, were maintained on a stock chick diet (Red Letter, All Mash, Jesse C. Stewart Company, Pittsburgh, Pennsylvania). Carnosine and anserine were isolated from chick pectoral muscle according to procedures described previously (11). The chicks were sacrificed by decapitation and the pectoral muscle was quickly removed, trimmed to remove bits of fat and other extraneous tissue, and frozen in liquid nitrogen if the muscle was not used immediately. Four to five gm of muscle was cut into small pieces and blended in a Waring Blendor with 10 volumes of 1% picric acid (12). The mixture was centrifuged in a PR-2 International centrifuge at 3000 rpm and the residue was re-extracted with 5 volumes of lyO picric acid. After removal of excess picrate by passing the supernatant mixture through a Dowex-l-Cl-l, 100-200 mesh column (11). the mixture was ready for isolation of the peptides by previously described chromatographic techniques (5, 11). The chromatographic separation involves selective displacement of the compounds from Dowex 50 by volatile organic bases (13, 14). The mixture is applied to Dowex SOW-H+, 49& 2m400 mesh ion-exchange columns,
IN THE CHICK
345
and the neutral, acidic, and aromatic amino acids are displaced by 0.2 M pyridine, followed by 0.2 M lutidine to displace the imidazolyl fraction. This fraction is rechromatographed on Dowex 50 saturated with 2,6-lutidine; 0.2 hr lntidine is used as developer in order to separate free histidine and 1-methylhistidine from the mixed carnosineanserine fraction. The peptide fraction is hydrolyzed in 6 N HCl in a sealed tube for 18 hours, and after removal of HCl by repeated in uucuo concentration, the hydrolyzate is chromatographed on a Dowex 50.a-picoline column which is developed with 0.1 M a-picoline. @-Alanine is obtained at the solvent front), and the histidine and l-methylhistidine residues representative of carnosine and anserine respectively are separated. These fractions are concentrated in V~CUOand the concentrations of the fractions are determined by ninhydrin analysis (15). In studies on the incorporation of histidine into carnosine and anserine in vivo as a function of the age of the chick, chicks were injected intraperiwith L-histidine-2-(ring)-1% in the toneally amount of 2 pC/lOO gm chick. After 4 hours, the chicks were sacrificed, and the peptides, as well as free hist,idine, were isolated from the nonprotein fraction of pectoral muscle as described above. Radioactive free histidine obtained by rechromatography on a column of Dowex 50 saturated with 2,6-lutidine was saved for determination of specific activity. The resulting histidine and I-met,hylhistidine residues representative of carnosine and anserine, respectively, as well as the fraction containing free histidine, were concentrated to a suitable volume, and an aliquot was plated, dried on an aluminum planchet’, and counted in a Geiger gas-flow counter. After counting to +2% accuracy, the plates were eluted with water, and histidine and l-methylhistidine were determined by t,he ninhydrin method (15). Result,s are expressed as the percentage of administered 1% incorporated into carnosine and anserine per gram of muscle, and cpm/pmole peptide (or histidine). Purity of all samples was ascertained by ascending paper chromat)ography of a suitable aliqllot using butanol:acetate acid:water, 4:1:5; and phenol-0.1 M phosphate buffer, pH 6.6, 7: 1 (16). For the enzyme studies, the chicks were sacrificed by decapitation and the pectoral muscle was quickly removed, weighed, and minced with scissors. A 1:5 suspension of muscle and ice-cold 0.25 M sucrose was prepared and homogenized for 1 minute in a cold Potter-Elvehjem homogenizer. The homogenate was centrifuged in the cold for 10 minutes at 7000g. The supernatant fraction was used as the source of carnosine N-met,hyltransferase and carnosine synthetase. The incubation medium and assay for carno-
446
McMANUS
AND
sine N-methyltransferase have been described previously (5). In these studies the isolation of I-methylhistidine after hydrolysis of isolated carnosine and anserine was omitted, and the incorporation of 1% from S-adenosylmethioninemethylJ% into anserine was determined by measuring 14C in the combined carnosine-anserine fraction. The conditions for the assay of carnosine synthetase were a modification of the conditions used by Kalyankar and Meister (1). The reaction mixture contained 0.3-0.5 ml of extract obtained after centrifugation of the crude homogenate at 7OOOg, 10 pmoles L-histidine, 7.5 rmoles MgC12, 20 rmoles ATP, 100 pmoles Tris buffer, pH 7.4, and 7 pmoles /3-alanine-l-l% (0.225 pC/pmole) in a final volume of 2.0 ml. The mixtures were incubated for 1 hour at 37”, and the reaction was stopped by addition of 1 volume of ethanol and heated for 1 minute in a boiling water bath. After centrifugation to remove precipitated protein, the mixture was chromatographed on Dowex 5OW-H+, 4%, 2@%400 mesh ion-exchange columns, 1 X 10 cm, to obtain the imidazolyl compounds by displacement chromatography as described above, followed by rechromatography on a Dowex 50-2,6-lutidine saturated column (0.8 X 9 cm) in which 0.2 M lutidine was used as eluting agent to separate histidine from carnosine. The carnosine fraction was concentrated in vacua to 5 ml and an aliquot was used for measurement of radioactive carnosine. The aliquot was plated and air-dried on an aluminum planchet, and counted as an infinitely thin sample in a Geiger gas-flow counter with a micromil window. Counting efficiency was 16%. Results of the assay of both of the enzymes are expressed as mrmoles product synthesized/mg protein. Assay conditions were chosen so that the rate of the reaction was proportional to enzyme concentration. Protein was determined by the method of Lowry et al. (16); bovine serum albumin was used as the standard protein. In studies on the effect of actinomycin D on carnosine N-methyltransferase activity, duplicate experiments were performed on 2- to 4-day-old chicks divided into three groups consisting of 2 control groups of 8 chicks each and one actinomytin-treated group of 32 chicks. A stock solution of 5 mg Actinomycin D/ml propylene glycol was diluted with sterile 0.9% NaCl and neutralized buffer, pH 7.5, to give a with 0.5 M phosphate final concentration of 100 @g/ml. Thirty-two 2day-old chicks, weighing between 40 and 50 gm, were injected intraperitoneally with 10 pg actinomycin D/50 gm body weight. The control chicks were sham injected with 0.9% NaCl and immediately sacrificed, and muscle extracts were prepared and assayed for carnosine N-methyltrans-
BENSON
ferase activity. These are designated as the “2-day-old control group.” After 24 hours, the treated chicks were reinjected with actinomycin D, the second control group was sham injected, and both groups were then sacrificed after an additional 24 hours. These are designated as the “4-day-old treated group” and the “4-day-old control group,” Carnosine Nrespectively. methyltransferase activity was measured in pectoral muscle extracts prepared from the treated and control chicks. The dosage of actinomycin D was chosen so that less than half of the actinomycin D-treated chicks died during the experimental period. In order to evaluate the effect of this level of actinomycin D administered over 48 hours on hepatic and pectoral muscle RNA, duplicate experiments were performed in which a similar series of chicks were injected over a 4%hour period with two doses of 10 pg actinomycin D/50 gm body weight. Control chicks were sham injected with 0.9% NaCl. Six hours prior to sacrifice these chicks were injected intraperitoneally with 1 pC adenine8-14C (5 &/pmole) per 50 gm body weight. Pectoral muscle and liver were quickly removed, quick frozen in a dry-ice-acetone mixture and stored at -20” until ready to be used. Partially thawed tissue was homogenized in 5 volumes of ice-cold 0.4 M perchloric acid in a Sorvall Omni-mixer for 1 minute at 30-second intervals. The homogenate was centrifuged for 30 minutes at SOOg, the supernatant portion was reserved for isolation of soluble adenosine 5’-phosphate, and the residue was washed two times with 0.2 M perchloric acid, followed by two washes with 50/, trichloroacetic acid. Ribonucleic acid in the washed residue was hydrolyzed by incubating with 5 volumes 0.5 N KOH for 21 hours at 37” with occasional shaking (17). Excess potassium perchlorate was removed after adjustment of the hydrolysate to pH 4, and the resulting supernatant was then adjusted to neutrality and chromatographed on a Dowex l-formate column using stepwise elntion with 0.1 M, followed by 0.25 M, formic acid which elutes adenosine 2’-phosphate and adenosine 3’-phosphate (18). Adenosine 5’-phosphate was isolated from the soluble fractions of the tissue extracts on a similar Dowex 1-formate column after removal of potassium perchlorate and adjustment to neutrality. The adenosine phosphates were concentrated almost to dryness, dissolved in a small volume of 0.01 N HCl, and checked for purity by determining the 250 mr/260 rnp and 280 mN/260mp ratios with a Zeiss spectrophotometer. Concentrations were determined from the absorbance at 260 rnp. Aliquots of 1 ml were transferred to scintillation counting vials and made to 20 ml with a modified Bray’s Cab-o-sil mixture, and the mixture was
CARNOSINE AND ANSERINE counted in a Packard 4000 liquid scintillation comlter. Results are expressed as cpm/Gmole ndenosine 2’-, adenosine 3’., or adenosine 5’.phosphate. In cases where the rat,ios indicated the presence of a possible impurity, sliquots of t,he samples were concentrated to dryness, dissolved in 5y0 ammonium hydroxide, and subjected to thin-layer chromatography on cellulose-silica gel plates which were developed with isopropanol-ammonium hydroxide-water, 120:70:3.5, as described by Gebicki and Freed (19). The adenosine phosphate area was outlined under an ultraviolet lamp, scraped into a small centrifrlge tube, and eluted to rewith 1 ml of 0.01 N HCl. After centrifuging move the cellulose-silica gel mixture, the absorbance at 260 rnp was measured and the sample was then counted as described above. In studies on the effect of puromycin on carnosine N-methyltransferase activity, 4-day-old chicks were divided into three groups consisting of two control groups of 17 chicks and a treated group of 12 chicks. The treat,ed group of 4-day-old chicks was injected intraperitoneally with a total dose of 40 mg puromycin dihydrochloride in 1.39y0 NaHCO per 100 gm body weight administered in 5 doses over a period of 2 days. The control groups were injected with 0.9% XaCl, and half were immediately sacrificed and muscle extracts were prepared and assayed for carnosine X-methpltransferase activity. This constituted the 4-day-old cont,rol group. After 48 hours, the remaining chicks of the control group, and the puromycintreated B-day-old chicks, were sacrificed and enzyme activity was determined in pectoral mns-
cle extracts. Nine of the original 12 treated chicks survived the experimental period. are designated as the “6-day-old “6-day-old treated” groups.
These groups control” and
RESULTS
Concentrations of the P-alanyl peptides in developing muscle. Figure 1 shows the changes in the concentration of carnosine and anserine in the pectoral muscle of I-, 6-, 13-, and 21-day-old chicks. The results are expressed as micromoles carnosine and anserine per gram fresh muscle, and represent average values obtained from analyses of the muscle from 5 chicks. The peptides are clearly demonstrable one day after hatching, but the amounts are low when contrasted to the levels observed in the older muscle. When levels of 0.25 pmole carnosine and 0.4 pmole anserine per gram of fresh muscle at 24 hours are compared, a significant increase is ob-
FIG.
pectoral
IN THE
CHICK
1. Concentrations of @-alany peptides muscle of the developing chick.
447
in
served in both peptides in muscle from B-day-old chicks. Carnosine shows a smaller increment, reaching 0.64 bmole, while anserine is increased to 2.75 bmoles. Eetween 6 and 21 days, a rapid increase in both peptides is observed, and the final analyses at 21 days show levels of 9.7 pmoles carnosine and 17 pmoles anserine. These figures may be compared with those reported by Davey (20) for adult chick pectoral muscle. He found 12.3 pmoles carnosine and 43.5 pmoles anserine per gram adult pectoral muscle. The pattern of a faster rate of increase of anserine than carnosine observed in our experiments is in agreement with the figures reported by Vul’fson (21) on duck pectoral muscle. Employing paper chromatographic techniques for isolation and measurement, he found that anserine increased much more rapidly than did carnosine with a ratio of anserine to carnosine of 6.5 at 15 days. The results reported here are based on the wet weight of muscle, which fails to take into account the variations in liquid content of the muscles at the several phases of growth. Dickerson (22) has determined the water concentration of pectoral muscle in the newly hatched chick and found 845 gm/kg, which decreased to 771 gm at 2.5 weeks. However, a comparison of the peptide patterns of duck pectoral muscle (21) based on wet and dry
448
McMANUS
AND
BENSON
FIG. 2. Rate of incorporation of L-histidine-2-(ring)-W into L-earnosine in 2-, 6-, and 15-day-old chicks. Chicks were injected with 2 PC/Z pmoles/100 gm body weight L-histidine2-(ring)-W and carnosine was isolated from pectoral muscle at the indicated times. Open circles represent results from a-day-old chicks; closed circles, 6-day-old chicks; halfclosed circles, l&day-old chicks. -
weight of tissue shows that the two patterns are essentially similar, and suggests that no serious error in interpretation may be attributed to this variable. A more serious limitation encountered in relating the changes in these constituents to the weight of the muscle tissue lies in the knowledge that this period constitutes a time of rapid growth and increase in cell mass. The results shown here may be interpreted only in terms of a pattern of change with relation to the whole tissue (23). Incorporation of L-hi&dine-% (ring) -14C into carnosine in the developing chick. The rapid accumulation of the peptides in the muscle during the early phases of chick growth stimulated interest in an investigation of the in viva utilization of histidine-214C for the synthesis of carnosine. Histidine is readily incorporated into both carnosine and anserine in the chick (24) and it is well established that skeletal muscle is a major site of synthesis of the fl-alanyl-peptides (l-5, 25). Therefore, it appeared reasonable to compare the in vivo utilization of Lhistidine-2-(ring)-r4C for the synthesis of carnosine over a short-time period as a function of the age of the chick. Thirty 2-day-old chicks were injected intramuscularly with 2 PC L-histidine-2-(ring)-14C (2 ,umoles) per 100 gm body weight and sacrificed in groups of 6 after 15, 30, 60, 120, and 240 minutes. A total of fifteen B-day-old and
fifteen 15-day-old chicks were injected with similar doses of labelled histidine and sacrificed in groups of 3 at the indicated time intervals. Radioactive histidine and carnosine were isolated and their specific activities were determined as described in the Methods. Figure 2 shows incorporation of radioactive histidine into carnosine over a $-hour period in muscle from 2-, 6-, and 15-day-old chicks. The points shown represent averages from two separate determinations of specific activity. These results, together with deterof the specific activities of mination histidineJ4C isolated from the muscle at these time periods, can be used to estimate the magnitude of synthesis of carnosine. Calculation of the fractional rate of synthesis is based on the Zilversmit treatment (26) of a situation where a single injection of labelled precursor follows a first-order regression and where the product does not contribute to the precursor. These conditions are fulfilled within the first hour after injection of labelled histidine, and data obtained during this period have been used in estimates of the rates of synthesis. Therefore, using knowledge of the specific activities of precursor and product over a onehour period and with the further assumption here that the rate of growth over the chosen interval is small compared with the rate. of metabolic activity, it is estimated that the fractional rate of synthesis of carnosine in
CARNOSINE
I 0
4
6
I DAYS
12
I
16
AND
ANSERINE
I
20
FIG. 3. Effect of age on carnosine N-methyltransferase and carnosine synthetase activity in chick pectoral muscle. Closed circles represent results for carnosine N-methyltransferase activity; half-closed circles, carnosine synthetase.
the 2-day-old chicks is less than 0.001 per hour, as compared with 0.005 per hour and 0.2 per hour in 6- and 15-day-old chicks, respectively. The numerous assumptions implicit in such a calculation (Zilversmit, 1960) make these values useful only as a qualitative indication of an increasing rate of synthesis of the ,&alanylpeptides in the developing chick, but the pattern of incorporation of labelled histidine as a function of age encouraged investigation of the behavior of the enzymes involved in the synthesis of the peptides. Development of carnosine synthetase and carnosine N-methyltransferase in the chick. Carnosine synthetase represents the major known route leading to synthesis of carnosine and, with 1-methylhistidine as substrate, it is able to catalyze the synthesis of anserine (l-4). Carnosine N-methyltransferase catalyzes the methylation of carnosine by Sadenosylmethionine and, in common with carnosine synthetase, is found prominently in pectoral muscle (3-5). Previous studies have shown carnosine N-methyltransferase to be present in the soluble protein fraction as a constituent of the muscle sarcoplasm (9, and soluble extracts were used as a
IN THE
449
CHICK
source of the enzyme in the present studies. Figure 3 shows the pattern of changes in the activity of the methyl t,ransferase and carnosine synthetase as a function of age of the chick. Results are expressed here as a percentage of maximal activity. It is seen that maximal activity of carnosine N-methyltransferase is reached between 4 and 6 days. In these experiments, in which 70009 extracts were the source of enzyme, 12 mpmoles anserine synthesized/hour/mg protein was the maximal absolute specific activity attained. This activity is sustained until at least day S, but by day 13, the activity has decreased to 62% of the maximal level. A progressive decline in enzyme activity is observed and reaches 55 % of maximal levels by day 15 and 36 % by day 21. Thus, by day 21 the activity is approaching levels seen in the 2-day-old chick in which the activity is only 25 % of the maximum. A different pattern of activity is observed with carnosine synthetase. The enzyme catalyzes the overall reaction (l-4) : P-Alanine
+ L-IIistidine
L-Carnosine
+ AMP
+ ATP 4 + PP
involving the intermediary formation of enzyme bound p-alanyladenylate (1) followed by peptide bond formation between the a-amino of n-histidine and the a-carboxyl of the p-alanyl derivative. A marked increase in activity is observed between 2 and 15 days, and if results are expressed using the activity measured at 15 days as 100 %, the Ievel at 2 days is only 2 % of the maximum, which is a 50.fold increase in activity. In absolute terms maximal activity is equal to 8.04 mFmoles carnosine synthesized/hour/mg protein. In contrast to the pattern obtained for carnosine N-methyltransferase, carnosine synthetase is still increasing rapidly at 15 days. Considerable variation has been noted in t#he pattern of activity subsequent to this time, and the results obtained using 21-day-old chicks do not as yet allow a final conclusion regarding the later phases of the activity pattern. In some instances, the specific activity remains constant, whereas in other cases, a drop in activity occurs with levels generally decreased to one-half of maximal activity. It is
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McMANUS
AND
clear, however, from inspection of the results presented here that the pattern of the activity of the two enzymes is not parallel. Carnosine N-methyltransferase peaks considerably earlier than does the synthetase and declines in activity at a time when the synthetase is approaching peak activity. It should be pointed out that considerable variation may be observed in different series of experiments, but the general pattern of activity is sustained. Table I illustrates some of the variations seen in absolute levels of carnosine N-methyltransferase specific activity together with mean specific activity, and also shows the changes in soluble muscle protein concentration found in 2- to 21-dayold chicks. Enzyme activity is expressed as mclmoles anserine synthesized/hour/mg protein, and each result represents activity of an enzyme preparation obtained by pooling pectoral muscle tissue from two chicks. It is apparent that the level of enzyme as measured by its activity is not proportional to the concentration of soluble protein. Enzyme activity shows a $-fold increase in preparations, but protein increases approximately 40% above the level in 2-day-old muscle. Despite an increase in concentration of soluble protein, a decrease in enzyme activity is noted in the 13- and 21-day-old chicks. Whether this decrease represents an actual decline in the proportion of enzyme protein or is due to the presence of an inhibitor or a competition for substrate cannot be stated with present information. However, a similar decrease in activity is observed using welldialyzed enzyme preparations, and the deTABLE I ACTIVITY OF CARNOSINE N-METHYLTRANSFERASE IN PECTORAL MUSCLE OF DEVELOPING CHICK
Ate of chicks (dd 2 4 8 13 15 21
Protein EOIIC. (mg/ml extract) 4.6 5.3 5.8 6.6 7.6 8.5
(4.5-4.7)4 (4.9-6.0) (4.8-6.4) (6.4-6.8) (6.8-8.3) (8.4-8.7)
Anserine synthesized (mpmoles/hour/mg protein) 2.80 7.44 11.90 5.87 4.94 2.85
(1.64-4.58) (5.88-10.00) (10.90-12.20) (6.07-7.50) (4.15-6.48) (1.67-3.80)
(1Figures in parentheses indicate the range values obtained from 4 separate experiments.
of
BENSON
TABLE II EFFECTS OF ACTINOMYCIN AND PUROMYCIN ON THE DEVELOPMENT OF CARNOSINE N-METHYLTRANSFERASE IN YOUNG CHICKS NO. tllizkSXllpleS (dvs) 2 4 4 6 6
6 12 12 11 9
a Average theses.
Treatment None None Actinomycin None Puromycin
values;
ranges
Enzyme activitya (~moles anserine/ hour/mg protein) 1.4 7.5 2.7 12.3 5.2
(0.7-2.1) (5.9-9.2) (1.6-3.8) (7.7-16.4) (3.1-6.2)
are shown
in paren-
crease is not reversed by increasing the concentrations of S-adenosylmethionine and carnosine in the incubation mixture. That the increase in the activity of carnosine N-methyltransferase in the first 7 days after hatching does in fact represent a de novo synthesis of the enzyme rather than an activation not directly attributable to a net synthesis of enzyme protein (27) is indicated by the results of two studies on the effects of injection of young chicks with actinomycin and puromycin. As described in Methods, 2-day-old chicks were injected with a total of 40 pg actinomycin/lOO gm over a period of 48 hours, control and treated groups were sacrificed, and pectoral muscle extracts were assayed for carnosine Nmethyltransferase. In the puromycin experiment, 4-day-old chicks were injected with a total of 40 mg puromycin/lOO gm body weight administered in 5 doses over a 48hour period. Table II shows the changes in the activity of the enzyme expressed as mpmoles anserine synthesized/hour/mg protein in 2-, 4-, and g-day-old chicks and the effects of actinomycin and puromycin on the activities. It is apparent that in these experiments the specific activity of the enzyme in the normal 4-day-old control chicks is approximately 5 times higher than that of the 2-day-old chicks. Treatment with actinomycin interfered with this increase in enzyme activity and essentially prevented the normal rise in activity seen between 2 and 4 days. The 4-day-old actinomycin-treated chicks have only 36 % of the activity seen in the untreated 4-day-old chicks. A similar alteration in activity is seen in the puromy-
CARNOSINE
AND
ANSERINE
tin-treated chicks, and it is evident that puromycin has effectively interfered with the normal increase in carnosine N-methyltransferase activity. In an effort to evaluate the effect of actinomycin un the synthesis of muscle and hepatic RNA under the conditions employed in these experiments, chicks mere treated with actinomycin as described above, and control and treated chicks were injected intraperitoneally with adenine-8-14C 6 hours prior to sacrifice. Soluble adenosine 5’-phosphate was isolated, and adenosine 2’- and adenosine 3’-phosphates were obtained from RNA hydrolyzates as described in Methods. Table III shows the specific activities of these compounds isolated from the livers of control chicks and from two treated groups. One treated group (group II) received 20 pg actinomycin/lOO gm over 24 hours, and the other group (group III) received a total of 40 pg actinomycin/IOO gm over a 4%hour period. The results are expressed as cpm/pmole product, and it is seen that the specific activities of the adenosine 2’- and 3’-phosphates are generally in close agreement, in keeping with an origin from RNA. No difference is seen in the specific activity of RNA from the control group and the 24-hour treated group, but RNA specific activity in the 4%hour treated group is decreased to approximately 15 70 of control levels. However, a consideration of the specific activit.ies of adenosine 5’-phosphate, which may be presumed to be in isotopic equilibrium with ATP in the cell, suggests that the decrease in the specific activity of RNA is not due directly to an effect on the synthesis of RNA (28). The specific activity of adenosine 5’-phosphate was also depressed in the 4%hour actinomycin-treated chicks so that the ratio of specific activites of adenosine 2’- and 3’-phosphates to adenosine 5’-phosphate is unchanged. Since the ratio provides an indicator of the relative incorporation rate of ATP into RNA, it is difficult to ascribe the depressed specific activity of RNA to a specific effect of actinomycin on the synthesis of RNA. A similar result was obtained in a duplicate separate experiment. There was no significant alteration in the incorporation of adenine-W4C into muscle RNA. The depressed incorporation of
IN
THE
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CHICK
TABLE III EFFECT OF ACTINOMYCIN ON INCORPOR.~TION OF ADENINE-8-14C INTO HEPATIC RNA AND ADENINE NUCLEOTIDES
-
-
-
ispecific
I-
Treatment
Group
activity
(cpm/fimole
Ratio !‘-(3’). AMP/ iI’-AMP
!I;
2‘-AMF
-AMP
5’.AMP
Control Sham injected with 0.9% NaCl
2500 4294 6772 5421 6719 4084
3571 4107 6164 5748 6093 4189
15,352 14,890 26,592 11,395 19,774 18,642
0.16 0.29 0.25 0.48 0.34 0.22
II
20 pg actinomycin/lOO gm body wt. over 24 hours
7077 6777 4625 2750 3766
5000 7416 5059 3085 3354
42,331 39,955 21,676 22,919 36,963
0.17 0.17 0.21 0.12 0.10
III
40 pg actinomycin/lOO gm body wt. over 48 hours
700 268 160 1542 206 133 515
I
i’
-
l-
I-
-
-
1000 436 245 1427 237 139 589
1636 962 725 3682 1538 1185 2763 L
O.i3
-
0.28 0.22 0.42 0.13 0.11 0.19
adenine-8J4C into the s ible adenine nucleotide fraction isolated from the liver may indicate a change in the permeability of the liver cell as a result of a relatively long exposure to sublethal levels of actinomycin, or alternatively, it may reflect an imbalanced catabolic activity (29) prior to injection of adenine-8J4C which results in dilution of the administered labeled adenine. Spector et al. (30) failed to observe a decrease in the incorporation of adenine-SJ4C into the soluble adenine nucleotides from calf lens after a 24-hour exposure to actinomycin, but no data were provided on the effects of longer periods. It is of interest that Olson (31) failed to note a significant change in the synthesis of hepatic RNA in young chicks after administration of 160 pg actinomycin per 100 gm body weight. DISCUSSION
That the first 3 weeks after hatching constitute a period of intensive change in the pectoral muscle is attested to by extensive data (22, 23, 32-34). The pectoral muscles
452
McMANUS
AND
increase in weight almost tenfold during the first 17 days, proportionally much more than the total weight of the chick, which increases from about 35 gm to over 100 gm in the same period. Sarcoplasmic protein nitrogen increases from 4.0 mg/gm to 6.6 mg/gm (22, 34). These latter figures are in agreement with our data in which protein contained in a 7000s supernatant portion was equivalent to 28 mg (4.48 mg N) per gram fresh muscle in 2-day-old muscle as compared with 43 mg protein (6.9 mg N) at 21 days. Nonprotein nitrogen exhibits a dramatic rise in concentration just prior to hatching (23), which then remains essentially constant when the amounts are based on the individual muscle cell. A sharp increase in actomyosin (Csapo et al., 1951), and labile phosphate compounds (35), are observed during this same period, attesting to an increase in components of the muscle cell which are vital to its structure and function at a rather late stage in development. The results reported here concerning the changes in levels of the P-alanyl peptides indicate a similar pattern of accumulation. It is of interest to speculate on the relative contribution of carnosine synthetase and carnosine N-methyltransferase to the synthesis of anserine in chick pectoral muscle. Anserine and carnosine occur in adult chick pectoral muscle in a ratio of about 3 to 1 (20), while the ratio at 21 days is approximately 1.8. In the period subsequent to 13 days, the enzyme activity patterns suggest that the contribution of carnosine synthetase to the formation of anserine in the later phases of growth is of greater significance than the synthesis mediated by carnosine N-methyltransferase. However, the immediate source of n-1-methylhistidine needed as a substrate for the synthetase is unclear. Attempts to demonstrate an in vitro synthesis of I-methylhistidine in this system via methyl group transfer to histidine have been unsuccessful (5), The inhibition of carnosine N-methyltransferase activity by both puromycin and actinomycin suggests that the normal early increase in activity is a consequence of a de novo synthesis of the enzyme, although no real evidence exists here for a primary effect
BENSON
of actinomycin on RNA synthesis. However, it is perhaps significant that the adenine% incorporation studies were carried out 24 hours subsequent to actinomycin administration and Schwartz et al. (36) observed that the incorporation of orotate-7-14C into rat liver RNA returned essentially to normal levels 16 hours after administration of 100 pg actinomycin/lOO gm. After administration of actinomycin to adrenalectomized rats at levels similar to those employed in these experiments, Rosen et al. (37) found that the activities of liver tyrosine-a-ketoglutarate transaminase and serine dehydrase actually increased. Moog (38) observed an increase in alkaline phosphatase activity in mouse duodenum and a decrease in this activity in mouse kidney after two injections of actinomycin at levels of 40 pg actinomycin/lOO gm over a a-day period. The levels of actinomytin used in their experiments produced marked toxic effects, including weight loss, fluid in the peritoneal cavity, hemorrhagic areas in tissues (37), and reduction in spleen weight (38). Some of these changes, particularly fluid in the peritoneal cavity and hemorrhagic areas, were observed in the chicks used in our experiments, but no attempt tias made here to evaluate the possible influence of this toxicity on the change in activity of carnosine N-methyltransferase. ACKNOWLEDGMENT The authors wish to acknowledge the able assistance of Miss Dorothy Anderson in this investigation. REFERENCES 1.
G. D., AND MEISTER, A., J. Biol. Chem. 234, 3210 (1959). 2. STENESH, J. J., .~ND WINNICK, T., Biochem. J. ‘77, 575 (1960). 3. WINNICK, R. E., AND WINNICK, T., Biochim. Biophys. Acta 31, 47 (1959). 4. WINNICK, T., AND WINNICK, R. E., Nature 183, 1466 (1959). 5. MCMANUS, I. R., J. Biol. Chem. 237, 1207 (1962). T. A., 6. PARSHIN, A. N., AND GORYUKHINA, Dokl. Akad. Nauk 73, 531 (1950). 7. ~KVORTSOV~, R. I., Biokemika 13, 594 (19%). 8. SEVERIN, S. E., AND FEDOROVA, V. N., Dokl. Akad. Nauk 83, 443 (1952). KALYANKAR,
CARNOSINE
AND
ANSERINE
9. MCMANUS, I. R., S&h Intern. Congr. Biothem. 6, 400 (1964). 10. STEKOL, J. A., ANDERSON. E. I., .IND WEISS, S., J. Biol. Chem. 233, 425 (1958). 11. MCMANUS, 1. R., J. Biol. Chem. 236, 1398 (1960). 12. HI\MILTON, P. B., AND TAI~R, R. R., J. Biol. Chem. 168, 375 (1945). 13. BUCHN.ZNSN, D. L., J. BioZ. Chem. 229, 211 (1957). 14. BUCHANAN, 1). L., Anal. Chem. 31, 833 (1959). 15. MOORE, S., SND STEIN, W. H., J. Biol. Chem. 211, 907 (1954). 16. COWGILL, R. W., AND FREEBURG, B., Arch. Biochem. Biophys. 71, 466 (1957). S. J., J. 17. SCHMIDT, G., A~~~ TH.~NNHAUSER, Biol. Chem. 161, 83 (1945). 18. COHN, W. E., in “The Nucleic Acids” (Chargaff and Davidson, eds.), Vol. 1, pp. 211-241. Academic Press, New York (1955). 19. GEBICKI, J. M., AND FREED, S., Anal. Biochem. 14, 253 (1960). 20. DAVEY, C. L., Arch Biochem. Biophys. 89, 303 (1960). P. L., Biokemika 23, 300 (1958). 21. VUL’FSON, 22. DICKERSON, J. W.T., Biochem. J. 76, 33 (1960). 23. LESLIE, I., ,IND DAVIDSON, J.W. T., Biochim. Biophys. Beta 7, 413 (1951).
IN
THE
453
CHICK
W. S., AND WINNICH, T., Biochim. Biophys. Acta 16, 48 (1954). 25. RAZINA, L. G., Bull. Exptl. Biol. Med. 43, 606 (1957). 26. ZILVERSMIT, D. B., Bm. J. Med. 29,832 (1960). 24. HARMS,
27. GREENGARD,
J. Biol.
28. 29.
30. 31. 32. 33.
34. 35.
36.
37. 38.
O.,
SMITH,
M.
A.,
.~ND Acs,
G.,
Chem. 238, 1548 (1963). REICH, IX., FHAIL‘BLIN, R. M., SHATKIN, J., .~ND T.\TuM, E. L., Science 134, 556,(1961). CHANTRENNE, H., Biochim. Biophys. Acta 96, 351 (1965). SPECTOR, A., .~ND KINOSHITA, J. II., Biochim. Biophys. Acta 96, 5Gl (1965). OLSON, R. E., Science 146, 926 (1964). HERRMANN, H., /Inn. l\r. Y. dead. Sci. 66, 99 (1952). MOOG, F., in “Embryonic Nutrit,ion” (D. Rudnick, ed.), pp. 87-102. Univ. of Chicago Press (1956). ROBINSON, D. S., Biochem. J. 62, 621 (1952). HERRMANN, H., .IND Cox, W. M., Am. J. Physiol. 166, 711 (1951). SCHWARTZ, H. S., SODERGREN, J. E., GAROF.ILO, M., .\ND STERNBERG, S. S., Cancer Res. 26, 307 (1965). ROSEN, F., RAINA, P. N., MILHOLLBND, R. J., .~ND NICHOL, C. A., Science 146, 661 (19G4). MOOG, F., Science 144, 414 (~1964).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies on the Formation
of Carnosine
and Anserine
of the Developing
Chick’
I. ROSABELLE Department
444-453 (1967)
119,
McMANUS
AND
MARY
in Pectoral
Muscle
S. BENSON
oj Biochemistry and Nutrition, Graduate SchooE oj Public of Pittsburgh, Pittsburgh, Pennsylvania 15213
Health,
University
Received September 26, 1966 Changes in the concentrations of carnosine and anserine and the development of the p-alanyl peptide-synthesizing enzymes in pectoral muscle of the posthatched chick have been investigated. The concentration of the p-alanyl peptides in pectoral muscle of the developing chick increases markedly during 21 days after hatching. Carnosine increases from 0.25 pmole/gm fresh muscle at 24 hours to 9.7 pmoles at 21 days. Anserine, with a concentration of 0.4 pmole/gm fresh muscle at 24 hours, reaches a level of 17 pmoles at 21 days. n-Histidine-2-(ring)-% was utilized for the in vivo formation of carnosine and anserine in the chick. The rate of incorporation of “C into the carnosine in pectoral muscle of 2., G-, and 15-day-old chicks is indicative of a synthesis of peptide from precursor histidine which is influenced by the stage of development of the pectoral muscle. The activities of carnosine-N-methyltransferase and carnosine synthetase were investigated in the developing chick, and their activity patterns were found to differ. Carnosine N-methyltransferase reaches a maximum specific activity at 6 days and declines to about 55yo of maximum activity at 15 days. At 21 days, activity is 36% of peak levels. Carnosine synthetase shows a 50-fold increase in activity between 2 and 15 days, with some evidence for a decline in activity subsequent to 21 days. The development of carnosine N-methyltransferase is inhibited by puromycin and actinomycin D, consistent with a de novo synthesis of the enzyme.
At least two enzymes present in vertebrate skeletal muscle catalyze the formation of the p-alanyl peptides. Carnosine synthetase [Lhistidine:p-alanine ligase (AMP) ; E.G. 6.3.2.111 catalyzes the synthesis of carnosine and anserine as well as certain related analogues (l-4), and carnosine N-methyltransferase (X-adenosylmethionine : carnosine Nmethyltransferase; E.C. 2.1.1.12) mediates the synthesis of anserine from carnosine (3-5). Kalyankar and Meister (I), and Winnick and Winnick (3, 4) observed that the activity of carnosine synthetase was markedly higher in pectoral muscle from young chicks than in adult muscle. A qualitatively similar effect of age of pectoral 1 This investigation is supported by a grant-inaid from the Muscular Dystrophy Associations of America, Inc., New York, New York.
muscle tissue on enzyme activity was observed in studies on carnosine N-methyltransferase (5). In studies designed to investigate the generation of carnosine and anserine in leg skeletal muscle of chicks during embryonic development and into adult life, Parshin and Goryukhina (6), Skvortsova (7), and Severin and Fedorova (8) found that carnosine appeared at the days 12-14 of development and anserine at day 17, with subsequent postnatal increases in their concentrations in muscle. The latter workers remarked on the correlation between the appearance of the peptides and their increased concentration with the development of the mechanical functioning of the muscle. The present report is concerned with an investigation of the changes in the concen trations of the @alany peptides in chicak 444
CARNOSINE
AND ANSERINE
pectoral muscle during a 3-week period after hatching and a study of t,he incorporation of I>-histidine-2-14C into carnosine and anserine during that period. The development of the fi-alanyl peptide-synthesizing enzymes has been investigated with enzyme extracts prepared from developing chick pectoral muscle, and their act,ivity patterns have been compared and related to the accumulation of carnosine and anserine as a function of the age of t’he muscle. In addition, t’he effects of treatment with actinomycin D and puromytin dihydrochloride on the pattern of development of carnosine N-methyltransferase in young chicks have been studied. A preliminary report of some of these results has been published (9). MATERIALS
AND METHODS
L-histidine-2-(ring)-1°C and L-carnosine were obtained from California Corporation for Biochemical Research. p-Alanine-l-l% and adenine8.1% were plrrchased from the New England NIlclear Corporation. S-Adenosylmethioninemethyl-l% was isolated from yeast after incubation with L-methionine-methyl-l% according to the procedure of Stekol et al. (10). Actinomycin D was generously provided by Merck Sharp & Dohme, and puromycin dihydrochloride was purchased from the Sigma Chemical Company. White Rock chicks, aged 24 hours to 21 days, were maintained on a stock chick diet (Red Letter, All Mash, Jesse C. Stewart Company, Pittsburgh, Pennsylvania). Carnosine and anserine were isolated from chick pectoral muscle according to procedures described previously (11). The chicks were sacrificed by decapitation and the pectoral muscle was quickly removed, trimmed to remove bits of fat and other extraneous tissue, and frozen in liquid nitrogen if the muscle was not used immediately. Four to five gm of muscle was cut into small pieces and blended in a Waring Blendor with 10 volumes of 1% picric acid (12). The mixture was centrifuged in a PR-2 International centrifuge at 3000 rpm and the residue was re-extracted with 5 volumes of lyO picric acid. After removal of excess picrate by passing the supernatant mixture through a Dowex-l-Cl-l, 100-200 mesh column (11). the mixture was ready for isolation of the peptides by previously described chromatographic techniques (5, 11). The chromatographic separation involves selective displacement of the compounds from Dowex 50 by volatile organic bases (13, 14). The mixture is applied to Dowex SOW-H+, 49& 2m400 mesh ion-exchange columns,
IN THE CHICK
345
and the neutral, acidic, and aromatic amino acids are displaced by 0.2 M pyridine, followed by 0.2 M lutidine to displace the imidazolyl fraction. This fraction is rechromatographed on Dowex 50 saturated with 2,6-lutidine; 0.2 hr lntidine is used as developer in order to separate free histidine and 1-methylhistidine from the mixed carnosineanserine fraction. The peptide fraction is hydrolyzed in 6 N HCl in a sealed tube for 18 hours, and after removal of HCl by repeated in uucuo concentration, the hydrolyzate is chromatographed on a Dowex 50.a-picoline column which is developed with 0.1 M a-picoline. @-Alanine is obtained at the solvent front), and the histidine and l-methylhistidine residues representative of carnosine and anserine respectively are separated. These fractions are concentrated in V~CUOand the concentrations of the fractions are determined by ninhydrin analysis (15). In studies on the incorporation of histidine into carnosine and anserine in vivo as a function of the age of the chick, chicks were injected intraperiwith L-histidine-2-(ring)-1% in the toneally amount of 2 pC/lOO gm chick. After 4 hours, the chicks were sacrificed, and the peptides, as well as free hist,idine, were isolated from the nonprotein fraction of pectoral muscle as described above. Radioactive free histidine obtained by rechromatography on a column of Dowex 50 saturated with 2,6-lutidine was saved for determination of specific activity. The resulting histidine and I-met,hylhistidine residues representative of carnosine and anserine, respectively, as well as the fraction containing free histidine, were concentrated to a suitable volume, and an aliquot was plated, dried on an aluminum planchet’, and counted in a Geiger gas-flow counter. After counting to +2% accuracy, the plates were eluted with water, and histidine and l-methylhistidine were determined by t,he ninhydrin method (15). Result,s are expressed as the percentage of administered 1% incorporated into carnosine and anserine per gram of muscle, and cpm/pmole peptide (or histidine). Purity of all samples was ascertained by ascending paper chromat)ography of a suitable aliqllot using butanol:acetate acid:water, 4:1:5; and phenol-0.1 M phosphate buffer, pH 6.6, 7: 1 (16). For the enzyme studies, the chicks were sacrificed by decapitation and the pectoral muscle was quickly removed, weighed, and minced with scissors. A 1:5 suspension of muscle and ice-cold 0.25 M sucrose was prepared and homogenized for 1 minute in a cold Potter-Elvehjem homogenizer. The homogenate was centrifuged in the cold for 10 minutes at 7000g. The supernatant fraction was used as the source of carnosine N-met,hyltransferase and carnosine synthetase. The incubation medium and assay for carno-
446
McMANUS
AND
sine N-methyltransferase have been described previously (5). In these studies the isolation of I-methylhistidine after hydrolysis of isolated carnosine and anserine was omitted, and the incorporation of 1% from S-adenosylmethioninemethylJ% into anserine was determined by measuring 14C in the combined carnosine-anserine fraction. The conditions for the assay of carnosine synthetase were a modification of the conditions used by Kalyankar and Meister (1). The reaction mixture contained 0.3-0.5 ml of extract obtained after centrifugation of the crude homogenate at 7OOOg, 10 pmoles L-histidine, 7.5 rmoles MgC12, 20 rmoles ATP, 100 pmoles Tris buffer, pH 7.4, and 7 pmoles /3-alanine-l-l% (0.225 pC/pmole) in a final volume of 2.0 ml. The mixtures were incubated for 1 hour at 37”, and the reaction was stopped by addition of 1 volume of ethanol and heated for 1 minute in a boiling water bath. After centrifugation to remove precipitated protein, the mixture was chromatographed on Dowex 5OW-H+, 4%, 2@%400 mesh ion-exchange columns, 1 X 10 cm, to obtain the imidazolyl compounds by displacement chromatography as described above, followed by rechromatography on a Dowex 50-2,6-lutidine saturated column (0.8 X 9 cm) in which 0.2 M lutidine was used as eluting agent to separate histidine from carnosine. The carnosine fraction was concentrated in vacua to 5 ml and an aliquot was used for measurement of radioactive carnosine. The aliquot was plated and air-dried on an aluminum planchet, and counted as an infinitely thin sample in a Geiger gas-flow counter with a micromil window. Counting efficiency was 16%. Results of the assay of both of the enzymes are expressed as mrmoles product synthesized/mg protein. Assay conditions were chosen so that the rate of the reaction was proportional to enzyme concentration. Protein was determined by the method of Lowry et al. (16); bovine serum albumin was used as the standard protein. In studies on the effect of actinomycin D on carnosine N-methyltransferase activity, duplicate experiments were performed on 2- to 4-day-old chicks divided into three groups consisting of 2 control groups of 8 chicks each and one actinomytin-treated group of 32 chicks. A stock solution of 5 mg Actinomycin D/ml propylene glycol was diluted with sterile 0.9% NaCl and neutralized buffer, pH 7.5, to give a with 0.5 M phosphate final concentration of 100 @g/ml. Thirty-two 2day-old chicks, weighing between 40 and 50 gm, were injected intraperitoneally with 10 pg actinomycin D/50 gm body weight. The control chicks were sham injected with 0.9% NaCl and immediately sacrificed, and muscle extracts were prepared and assayed for carnosine N-methyltrans-
BENSON
ferase activity. These are designated as the “2-day-old control group.” After 24 hours, the treated chicks were reinjected with actinomycin D, the second control group was sham injected, and both groups were then sacrificed after an additional 24 hours. These are designated as the “4-day-old treated group” and the “4-day-old control group,” Carnosine Nrespectively. methyltransferase activity was measured in pectoral muscle extracts prepared from the treated and control chicks. The dosage of actinomycin D was chosen so that less than half of the actinomycin D-treated chicks died during the experimental period. In order to evaluate the effect of this level of actinomycin D administered over 48 hours on hepatic and pectoral muscle RNA, duplicate experiments were performed in which a similar series of chicks were injected over a 4%hour period with two doses of 10 pg actinomycin D/50 gm body weight. Control chicks were sham injected with 0.9% NaCl. Six hours prior to sacrifice these chicks were injected intraperitoneally with 1 pC adenine8-14C (5 &/pmole) per 50 gm body weight. Pectoral muscle and liver were quickly removed, quick frozen in a dry-ice-acetone mixture and stored at -20” until ready to be used. Partially thawed tissue was homogenized in 5 volumes of ice-cold 0.4 M perchloric acid in a Sorvall Omni-mixer for 1 minute at 30-second intervals. The homogenate was centrifuged for 30 minutes at SOOg, the supernatant portion was reserved for isolation of soluble adenosine 5’-phosphate, and the residue was washed two times with 0.2 M perchloric acid, followed by two washes with 50/, trichloroacetic acid. Ribonucleic acid in the washed residue was hydrolyzed by incubating with 5 volumes 0.5 N KOH for 21 hours at 37” with occasional shaking (17). Excess potassium perchlorate was removed after adjustment of the hydrolysate to pH 4, and the resulting supernatant was then adjusted to neutrality and chromatographed on a Dowex l-formate column using stepwise elntion with 0.1 M, followed by 0.25 M, formic acid which elutes adenosine 2’-phosphate and adenosine 3’-phosphate (18). Adenosine 5’-phosphate was isolated from the soluble fractions of the tissue extracts on a similar Dowex 1-formate column after removal of potassium perchlorate and adjustment to neutrality. The adenosine phosphates were concentrated almost to dryness, dissolved in a small volume of 0.01 N HCl, and checked for purity by determining the 250 mr/260 rnp and 280 mN/260mp ratios with a Zeiss spectrophotometer. Concentrations were determined from the absorbance at 260 rnp. Aliquots of 1 ml were transferred to scintillation counting vials and made to 20 ml with a modified Bray’s Cab-o-sil mixture, and the mixture was
CARNOSINE AND ANSERINE counted in a Packard 4000 liquid scintillation comlter. Results are expressed as cpm/Gmole ndenosine 2’-, adenosine 3’., or adenosine 5’.phosphate. In cases where the rat,ios indicated the presence of a possible impurity, sliquots of t,he samples were concentrated to dryness, dissolved in 5y0 ammonium hydroxide, and subjected to thin-layer chromatography on cellulose-silica gel plates which were developed with isopropanol-ammonium hydroxide-water, 120:70:3.5, as described by Gebicki and Freed (19). The adenosine phosphate area was outlined under an ultraviolet lamp, scraped into a small centrifrlge tube, and eluted to rewith 1 ml of 0.01 N HCl. After centrifuging move the cellulose-silica gel mixture, the absorbance at 260 rnp was measured and the sample was then counted as described above. In studies on the effect of puromycin on carnosine N-methyltransferase activity, 4-day-old chicks were divided into three groups consisting of two control groups of 17 chicks and a treated group of 12 chicks. The treat,ed group of 4-day-old chicks was injected intraperitoneally with a total dose of 40 mg puromycin dihydrochloride in 1.39y0 NaHCO per 100 gm body weight administered in 5 doses over a period of 2 days. The control groups were injected with 0.9% XaCl, and half were immediately sacrificed and muscle extracts were prepared and assayed for carnosine X-methpltransferase activity. This constituted the 4-day-old cont,rol group. After 48 hours, the remaining chicks of the control group, and the puromycintreated B-day-old chicks, were sacrificed and enzyme activity was determined in pectoral mns-
cle extracts. Nine of the original 12 treated chicks survived the experimental period. are designated as the “6-day-old “6-day-old treated” groups.
These groups control” and
RESULTS
Concentrations of the P-alanyl peptides in developing muscle. Figure 1 shows the changes in the concentration of carnosine and anserine in the pectoral muscle of I-, 6-, 13-, and 21-day-old chicks. The results are expressed as micromoles carnosine and anserine per gram fresh muscle, and represent average values obtained from analyses of the muscle from 5 chicks. The peptides are clearly demonstrable one day after hatching, but the amounts are low when contrasted to the levels observed in the older muscle. When levels of 0.25 pmole carnosine and 0.4 pmole anserine per gram of fresh muscle at 24 hours are compared, a significant increase is ob-
FIG.
pectoral
IN THE
CHICK
1. Concentrations of @-alany peptides muscle of the developing chick.
447
in
served in both peptides in muscle from B-day-old chicks. Carnosine shows a smaller increment, reaching 0.64 bmole, while anserine is increased to 2.75 bmoles. Eetween 6 and 21 days, a rapid increase in both peptides is observed, and the final analyses at 21 days show levels of 9.7 pmoles carnosine and 17 pmoles anserine. These figures may be compared with those reported by Davey (20) for adult chick pectoral muscle. He found 12.3 pmoles carnosine and 43.5 pmoles anserine per gram adult pectoral muscle. The pattern of a faster rate of increase of anserine than carnosine observed in our experiments is in agreement with the figures reported by Vul’fson (21) on duck pectoral muscle. Employing paper chromatographic techniques for isolation and measurement, he found that anserine increased much more rapidly than did carnosine with a ratio of anserine to carnosine of 6.5 at 15 days. The results reported here are based on the wet weight of muscle, which fails to take into account the variations in liquid content of the muscles at the several phases of growth. Dickerson (22) has determined the water concentration of pectoral muscle in the newly hatched chick and found 845 gm/kg, which decreased to 771 gm at 2.5 weeks. However, a comparison of the peptide patterns of duck pectoral muscle (21) based on wet and dry
448
McMANUS
AND
BENSON
FIG. 2. Rate of incorporation of L-histidine-2-(ring)-W into L-earnosine in 2-, 6-, and 15-day-old chicks. Chicks were injected with 2 PC/Z pmoles/100 gm body weight L-histidine2-(ring)-W and carnosine was isolated from pectoral muscle at the indicated times. Open circles represent results from a-day-old chicks; closed circles, 6-day-old chicks; halfclosed circles, l&day-old chicks. -
weight of tissue shows that the two patterns are essentially similar, and suggests that no serious error in interpretation may be attributed to this variable. A more serious limitation encountered in relating the changes in these constituents to the weight of the muscle tissue lies in the knowledge that this period constitutes a time of rapid growth and increase in cell mass. The results shown here may be interpreted only in terms of a pattern of change with relation to the whole tissue (23). Incorporation of L-hi&dine-% (ring) -14C into carnosine in the developing chick. The rapid accumulation of the peptides in the muscle during the early phases of chick growth stimulated interest in an investigation of the in viva utilization of histidine-214C for the synthesis of carnosine. Histidine is readily incorporated into both carnosine and anserine in the chick (24) and it is well established that skeletal muscle is a major site of synthesis of the fl-alanyl-peptides (l-5, 25). Therefore, it appeared reasonable to compare the in vivo utilization of Lhistidine-2-(ring)-r4C for the synthesis of carnosine over a short-time period as a function of the age of the chick. Thirty 2-day-old chicks were injected intramuscularly with 2 PC L-histidine-2-(ring)-14C (2 ,umoles) per 100 gm body weight and sacrificed in groups of 6 after 15, 30, 60, 120, and 240 minutes. A total of fifteen B-day-old and
fifteen 15-day-old chicks were injected with similar doses of labelled histidine and sacrificed in groups of 3 at the indicated time intervals. Radioactive histidine and carnosine were isolated and their specific activities were determined as described in the Methods. Figure 2 shows incorporation of radioactive histidine into carnosine over a $-hour period in muscle from 2-, 6-, and 15-day-old chicks. The points shown represent averages from two separate determinations of specific activity. These results, together with deterof the specific activities of mination histidineJ4C isolated from the muscle at these time periods, can be used to estimate the magnitude of synthesis of carnosine. Calculation of the fractional rate of synthesis is based on the Zilversmit treatment (26) of a situation where a single injection of labelled precursor follows a first-order regression and where the product does not contribute to the precursor. These conditions are fulfilled within the first hour after injection of labelled histidine, and data obtained during this period have been used in estimates of the rates of synthesis. Therefore, using knowledge of the specific activities of precursor and product over a onehour period and with the further assumption here that the rate of growth over the chosen interval is small compared with the rate. of metabolic activity, it is estimated that the fractional rate of synthesis of carnosine in
CARNOSINE
I 0
4
6
I DAYS
12
I
16
AND
ANSERINE
I
20
FIG. 3. Effect of age on carnosine N-methyltransferase and carnosine synthetase activity in chick pectoral muscle. Closed circles represent results for carnosine N-methyltransferase activity; half-closed circles, carnosine synthetase.
the 2-day-old chicks is less than 0.001 per hour, as compared with 0.005 per hour and 0.2 per hour in 6- and 15-day-old chicks, respectively. The numerous assumptions implicit in such a calculation (Zilversmit, 1960) make these values useful only as a qualitative indication of an increasing rate of synthesis of the ,&alanylpeptides in the developing chick, but the pattern of incorporation of labelled histidine as a function of age encouraged investigation of the behavior of the enzymes involved in the synthesis of the peptides. Development of carnosine synthetase and carnosine N-methyltransferase in the chick. Carnosine synthetase represents the major known route leading to synthesis of carnosine and, with 1-methylhistidine as substrate, it is able to catalyze the synthesis of anserine (l-4). Carnosine N-methyltransferase catalyzes the methylation of carnosine by Sadenosylmethionine and, in common with carnosine synthetase, is found prominently in pectoral muscle (3-5). Previous studies have shown carnosine N-methyltransferase to be present in the soluble protein fraction as a constituent of the muscle sarcoplasm (9, and soluble extracts were used as a
IN THE
449
CHICK
source of the enzyme in the present studies. Figure 3 shows the pattern of changes in the activity of the methyl t,ransferase and carnosine synthetase as a function of age of the chick. Results are expressed here as a percentage of maximal activity. It is seen that maximal activity of carnosine N-methyltransferase is reached between 4 and 6 days. In these experiments, in which 70009 extracts were the source of enzyme, 12 mpmoles anserine synthesized/hour/mg protein was the maximal absolute specific activity attained. This activity is sustained until at least day S, but by day 13, the activity has decreased to 62% of the maximal level. A progressive decline in enzyme activity is observed and reaches 55 % of maximal levels by day 15 and 36 % by day 21. Thus, by day 21 the activity is approaching levels seen in the 2-day-old chick in which the activity is only 25 % of the maximum. A different pattern of activity is observed with carnosine synthetase. The enzyme catalyzes the overall reaction (l-4) : P-Alanine
+ L-IIistidine
L-Carnosine
+ AMP
+ ATP 4 + PP
involving the intermediary formation of enzyme bound p-alanyladenylate (1) followed by peptide bond formation between the a-amino of n-histidine and the a-carboxyl of the p-alanyl derivative. A marked increase in activity is observed between 2 and 15 days, and if results are expressed using the activity measured at 15 days as 100 %, the Ievel at 2 days is only 2 % of the maximum, which is a 50.fold increase in activity. In absolute terms maximal activity is equal to 8.04 mFmoles carnosine synthesized/hour/mg protein. In contrast to the pattern obtained for carnosine N-methyltransferase, carnosine synthetase is still increasing rapidly at 15 days. Considerable variation has been noted in t#he pattern of activity subsequent to this time, and the results obtained using 21-day-old chicks do not as yet allow a final conclusion regarding the later phases of the activity pattern. In some instances, the specific activity remains constant, whereas in other cases, a drop in activity occurs with levels generally decreased to one-half of maximal activity. It is
450
McMANUS
AND
clear, however, from inspection of the results presented here that the pattern of the activity of the two enzymes is not parallel. Carnosine N-methyltransferase peaks considerably earlier than does the synthetase and declines in activity at a time when the synthetase is approaching peak activity. It should be pointed out that considerable variation may be observed in different series of experiments, but the general pattern of activity is sustained. Table I illustrates some of the variations seen in absolute levels of carnosine N-methyltransferase specific activity together with mean specific activity, and also shows the changes in soluble muscle protein concentration found in 2- to 21-dayold chicks. Enzyme activity is expressed as mclmoles anserine synthesized/hour/mg protein, and each result represents activity of an enzyme preparation obtained by pooling pectoral muscle tissue from two chicks. It is apparent that the level of enzyme as measured by its activity is not proportional to the concentration of soluble protein. Enzyme activity shows a $-fold increase in preparations, but protein increases approximately 40% above the level in 2-day-old muscle. Despite an increase in concentration of soluble protein, a decrease in enzyme activity is noted in the 13- and 21-day-old chicks. Whether this decrease represents an actual decline in the proportion of enzyme protein or is due to the presence of an inhibitor or a competition for substrate cannot be stated with present information. However, a similar decrease in activity is observed using welldialyzed enzyme preparations, and the deTABLE I ACTIVITY OF CARNOSINE N-METHYLTRANSFERASE IN PECTORAL MUSCLE OF DEVELOPING CHICK
Ate of chicks (dd 2 4 8 13 15 21
Protein EOIIC. (mg/ml extract) 4.6 5.3 5.8 6.6 7.6 8.5
(4.5-4.7)4 (4.9-6.0) (4.8-6.4) (6.4-6.8) (6.8-8.3) (8.4-8.7)
Anserine synthesized (mpmoles/hour/mg protein) 2.80 7.44 11.90 5.87 4.94 2.85
(1.64-4.58) (5.88-10.00) (10.90-12.20) (6.07-7.50) (4.15-6.48) (1.67-3.80)
(1Figures in parentheses indicate the range values obtained from 4 separate experiments.
of
BENSON
TABLE II EFFECTS OF ACTINOMYCIN AND PUROMYCIN ON THE DEVELOPMENT OF CARNOSINE N-METHYLTRANSFERASE IN YOUNG CHICKS NO. tllizkSXllpleS (dvs) 2 4 4 6 6
6 12 12 11 9
a Average theses.
Treatment None None Actinomycin None Puromycin
values;
ranges
Enzyme activitya (~moles anserine/ hour/mg protein) 1.4 7.5 2.7 12.3 5.2
(0.7-2.1) (5.9-9.2) (1.6-3.8) (7.7-16.4) (3.1-6.2)
are shown
in paren-
crease is not reversed by increasing the concentrations of S-adenosylmethionine and carnosine in the incubation mixture. That the increase in the activity of carnosine N-methyltransferase in the first 7 days after hatching does in fact represent a de novo synthesis of the enzyme rather than an activation not directly attributable to a net synthesis of enzyme protein (27) is indicated by the results of two studies on the effects of injection of young chicks with actinomycin and puromycin. As described in Methods, 2-day-old chicks were injected with a total of 40 pg actinomycin/lOO gm over a period of 48 hours, control and treated groups were sacrificed, and pectoral muscle extracts were assayed for carnosine Nmethyltransferase. In the puromycin experiment, 4-day-old chicks were injected with a total of 40 mg puromycin/lOO gm body weight administered in 5 doses over a 48hour period. Table II shows the changes in the activity of the enzyme expressed as mpmoles anserine synthesized/hour/mg protein in 2-, 4-, and g-day-old chicks and the effects of actinomycin and puromycin on the activities. It is apparent that in these experiments the specific activity of the enzyme in the normal 4-day-old control chicks is approximately 5 times higher than that of the 2-day-old chicks. Treatment with actinomycin interfered with this increase in enzyme activity and essentially prevented the normal rise in activity seen between 2 and 4 days. The 4-day-old actinomycin-treated chicks have only 36 % of the activity seen in the untreated 4-day-old chicks. A similar alteration in activity is seen in the puromy-
CARNOSINE
AND
ANSERINE
tin-treated chicks, and it is evident that puromycin has effectively interfered with the normal increase in carnosine N-methyltransferase activity. In an effort to evaluate the effect of actinomycin un the synthesis of muscle and hepatic RNA under the conditions employed in these experiments, chicks mere treated with actinomycin as described above, and control and treated chicks were injected intraperitoneally with adenine-8-14C 6 hours prior to sacrifice. Soluble adenosine 5’-phosphate was isolated, and adenosine 2’- and adenosine 3’-phosphates were obtained from RNA hydrolyzates as described in Methods. Table III shows the specific activities of these compounds isolated from the livers of control chicks and from two treated groups. One treated group (group II) received 20 pg actinomycin/lOO gm over 24 hours, and the other group (group III) received a total of 40 pg actinomycin/IOO gm over a 4%hour period. The results are expressed as cpm/pmole product, and it is seen that the specific activities of the adenosine 2’- and 3’-phosphates are generally in close agreement, in keeping with an origin from RNA. No difference is seen in the specific activity of RNA from the control group and the 24-hour treated group, but RNA specific activity in the 4%hour treated group is decreased to approximately 15 70 of control levels. However, a consideration of the specific activit.ies of adenosine 5’-phosphate, which may be presumed to be in isotopic equilibrium with ATP in the cell, suggests that the decrease in the specific activity of RNA is not due directly to an effect on the synthesis of RNA (28). The specific activity of adenosine 5’-phosphate was also depressed in the 4%hour actinomycin-treated chicks so that the ratio of specific activites of adenosine 2’- and 3’-phosphates to adenosine 5’-phosphate is unchanged. Since the ratio provides an indicator of the relative incorporation rate of ATP into RNA, it is difficult to ascribe the depressed specific activity of RNA to a specific effect of actinomycin on the synthesis of RNA. A similar result was obtained in a duplicate separate experiment. There was no significant alteration in the incorporation of adenine-W4C into muscle RNA. The depressed incorporation of
IN
THE
451
CHICK
TABLE III EFFECT OF ACTINOMYCIN ON INCORPOR.~TION OF ADENINE-8-14C INTO HEPATIC RNA AND ADENINE NUCLEOTIDES
-
-
-
ispecific
I-
Treatment
Group
activity
(cpm/fimole
Ratio !‘-(3’). AMP/ iI’-AMP
!I;
2‘-AMF
-AMP
5’.AMP
Control Sham injected with 0.9% NaCl
2500 4294 6772 5421 6719 4084
3571 4107 6164 5748 6093 4189
15,352 14,890 26,592 11,395 19,774 18,642
0.16 0.29 0.25 0.48 0.34 0.22
II
20 pg actinomycin/lOO gm body wt. over 24 hours
7077 6777 4625 2750 3766
5000 7416 5059 3085 3354
42,331 39,955 21,676 22,919 36,963
0.17 0.17 0.21 0.12 0.10
III
40 pg actinomycin/lOO gm body wt. over 48 hours
700 268 160 1542 206 133 515
I
i’
-
l-
I-
-
-
1000 436 245 1427 237 139 589
1636 962 725 3682 1538 1185 2763 L
O.i3
-
0.28 0.22 0.42 0.13 0.11 0.19
adenine-8J4C into the s ible adenine nucleotide fraction isolated from the liver may indicate a change in the permeability of the liver cell as a result of a relatively long exposure to sublethal levels of actinomycin, or alternatively, it may reflect an imbalanced catabolic activity (29) prior to injection of adenine-8J4C which results in dilution of the administered labeled adenine. Spector et al. (30) failed to observe a decrease in the incorporation of adenine-SJ4C into the soluble adenine nucleotides from calf lens after a 24-hour exposure to actinomycin, but no data were provided on the effects of longer periods. It is of interest that Olson (31) failed to note a significant change in the synthesis of hepatic RNA in young chicks after administration of 160 pg actinomycin per 100 gm body weight. DISCUSSION
That the first 3 weeks after hatching constitute a period of intensive change in the pectoral muscle is attested to by extensive data (22, 23, 32-34). The pectoral muscles
452
McMANUS
AND
increase in weight almost tenfold during the first 17 days, proportionally much more than the total weight of the chick, which increases from about 35 gm to over 100 gm in the same period. Sarcoplasmic protein nitrogen increases from 4.0 mg/gm to 6.6 mg/gm (22, 34). These latter figures are in agreement with our data in which protein contained in a 7000s supernatant portion was equivalent to 28 mg (4.48 mg N) per gram fresh muscle in 2-day-old muscle as compared with 43 mg protein (6.9 mg N) at 21 days. Nonprotein nitrogen exhibits a dramatic rise in concentration just prior to hatching (23), which then remains essentially constant when the amounts are based on the individual muscle cell. A sharp increase in actomyosin (Csapo et al., 1951), and labile phosphate compounds (35), are observed during this same period, attesting to an increase in components of the muscle cell which are vital to its structure and function at a rather late stage in development. The results reported here concerning the changes in levels of the P-alanyl peptides indicate a similar pattern of accumulation. It is of interest to speculate on the relative contribution of carnosine synthetase and carnosine N-methyltransferase to the synthesis of anserine in chick pectoral muscle. Anserine and carnosine occur in adult chick pectoral muscle in a ratio of about 3 to 1 (20), while the ratio at 21 days is approximately 1.8. In the period subsequent to 13 days, the enzyme activity patterns suggest that the contribution of carnosine synthetase to the formation of anserine in the later phases of growth is of greater significance than the synthesis mediated by carnosine N-methyltransferase. However, the immediate source of n-1-methylhistidine needed as a substrate for the synthetase is unclear. Attempts to demonstrate an in vitro synthesis of I-methylhistidine in this system via methyl group transfer to histidine have been unsuccessful (5), The inhibition of carnosine N-methyltransferase activity by both puromycin and actinomycin suggests that the normal early increase in activity is a consequence of a de novo synthesis of the enzyme, although no real evidence exists here for a primary effect
BENSON
of actinomycin on RNA synthesis. However, it is perhaps significant that the adenine% incorporation studies were carried out 24 hours subsequent to actinomycin administration and Schwartz et al. (36) observed that the incorporation of orotate-7-14C into rat liver RNA returned essentially to normal levels 16 hours after administration of 100 pg actinomycin/lOO gm. After administration of actinomycin to adrenalectomized rats at levels similar to those employed in these experiments, Rosen et al. (37) found that the activities of liver tyrosine-a-ketoglutarate transaminase and serine dehydrase actually increased. Moog (38) observed an increase in alkaline phosphatase activity in mouse duodenum and a decrease in this activity in mouse kidney after two injections of actinomycin at levels of 40 pg actinomycin/lOO gm over a a-day period. The levels of actinomytin used in their experiments produced marked toxic effects, including weight loss, fluid in the peritoneal cavity, hemorrhagic areas in tissues (37), and reduction in spleen weight (38). Some of these changes, particularly fluid in the peritoneal cavity and hemorrhagic areas, were observed in the chicks used in our experiments, but no attempt tias made here to evaluate the possible influence of this toxicity on the change in activity of carnosine N-methyltransferase. ACKNOWLEDGMENT The authors wish to acknowledge the able assistance of Miss Dorothy Anderson in this investigation. REFERENCES 1.
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CARNOSINE
AND
ANSERINE
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