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Effect of insulin on the incorporation of [13C]leucine into rat caudofemoralis protein
~I0
BIOCHIMICA ET BIOPHYSICA ACTA
~BA 25774 E F F E C T OF I N S U L I N ON T H E INCORPORATION OF [14C]LEUCINE INTO RAT CAUDOFEMORALIS P R O T E I N S. G O L D S T E I N * AND W.
J,
iREDDY
Baker Research Laboratory of the Diabetes Foundation, Department of 2efedicine, New England Deaconess Hospital and Peter Bent Brigham Hospital, Departments of Medicine and Biochemist
SUMMARY
This study sought first to develop an in vitro preparation representative of peripheral skeletal muscle and then to study the effects of insulin on the incorporation of labelled amino acid into various protein fractions thereof. Rat caudofemoralis muscle proteins were separated on the basis of their solubility in solutions of increasing ionic strength and pH. The specific radioactivity of various protein fractions was found to be inversely proportional to the ionic and alkaline strength of the extracting solution. Insulin stimulated incorporation of [i4C]leucine into each fraction with approximately equal magnitude. Likewise, the distribution (ratiot of counts in several protein fractions Muted from a DEAE-cellulose column was not materially affected b y insulin. Our results fit the hypothesis that insulin stimulates the incorporation of [l~C]leucine into skeletal muscle protein b y increasing its entry into ceils and not by stimulation of synthesis of one or more specific proteins.
INTRODUCTION
Current hypotheses ascribe the enhancing effects of insulin on protein synthesis to a stimulation at two loci: At the cell membrane via stimulation of amino acid transport 1-a and at a site distal to amino acid penetration of the cell membrane ~, 5 Studies on the isolated perfused rat heart have further substantiated the membrane transport phenomenon G,~ and more recent work on ribosomal preparations of rat heart has adduced evidence for the effect of insulin at tile ribosomal level of translation 8 or perhaps the nuclear level of RNA translocation to cytoplasm ~. It m a y be hazardous to extrapolate data obtained in highly specialized and nearly continuously active muscle like heart and diaphragm to the rest of the only intermittently active skeletal muscle, the latter comprising over 40 % of our body weight. The purpose of the present study was to develop an i~ vitro preparation more representative of peripheral skeletal muscle, to re-examine earlier work describing the effects of insulin on protein synthesis, and zo obtain information regarding tile * P r e s e n t address: D e p a r t m e n t of Bacteriology and [ m m u n o ! o g y , H a r v a r d Niedical School, Boston, Mass. o 2 I I 5 , U.S.A.
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fraction into which labelled amino acids are incorporated. This report describes experiments concerned with the incorporation of tracer amino acid into various protein fractions of rat caudofemoralis muscle. METHODS
Male C-D strain (Charles River Breeding Lab.) rats weighing I5O-2OO g were employed in all experiments. Rats were fed Purina chow and water ad libitum following arrival from the supplier. They were housed 8 to a cage till the day prior to experiment in an animal room of constant temperature and humidity with automatically alternated i2-h intervals of light and darkness. The animals were then transferred to separate cages and fasted for 16 h prior to sacrifice b y cervical section and exsanguination. The hind limbs were skinned and the superficial layer of muscles was divided and retracted thus exposing the origins and insertions of the caudofemoralis which was excised intact. The muscles from each side were alternated between test and control samples to obviate any bias that might be introduced b y laterality. Krebs-Ringer phosphate buffer 1° was used throughout, modified b y reduction of the Ca 2+ concentration to one-half. Uniformly l~C-labelled leucine was obtained from New England Nuclear Corporation at specific activity of 223/,C//~mole and used at a final concentration of 0.2 /~C/ml. Glucagon-free insulin was supplied at specific activity of 23 units/rag and used at a final concentration of o.I unit/ml. The buffer mixture was gassed with IOO % O~ for IO rain prior to distribution of 5 ml into each of the 25-ml erlenmeyer flasks fitted with serum sleeve-type rubber stoppers. Following weighing, the tissues, averaging 125 rag, were added to the flasks, transferred to a metabolic shaker, and incubated at 37 -- 0.5 ° for 60 rain. Agitation at 42 cycles/rain with an amplitude of 3.5 inches was carried on throughout the hour. For the first io rain of the incubation, flasks were charged with IOO % 0 2. After I h, metabolic activity was interrupted b y immersing all flasks in ice water. Incorporation of label into protein is negligible at o ° (GOLDSTEINAND REDDY, unpublished observations). To prepare a protein extract approximating o ionic strength, tissue was removed from the incubating medium (ionic strength = o.18), drained and placed in a 3o-ml Virtis homogenizing flask containing IO ml distilled water, and homogenized. Homogenization to fine particulate matter at high speeds on the Virtis "23" homogenizer, using No. I6-1o7 sharp-edged Virtis stainless steel blades, required about I - 2 rain. Homogenizing flasks were then emptied into conical bottom centrifuge tubes and centrifuged at 16oo × g for 20 rain. An aliquot of the homogenate supernatant was worked up for liquid scintillation counting. Protein was precipitated with 2 vol. of 20 % trichloroacetic acid, treated for 15 rain with 2 vol. of 5 % boiling trichloroacetic acid to extract nucleic acids, washed consecutively with 2 vol. of ethanol-ether ( I : I ) and ether to extract lJpids, then dried under negative pressure in a desiccator. Two ml hyamine (I M in methanol, Packard Instrument Co.) were added for overnight hydrolysis in 60 ° waterbath. To this hydrolysate were added 15 ml of toluene-2,5-diphenyloxazole-i,4 bis-2(4methyl-5-phenyloxazolyl)benzene fluor solution. The mixture was then transferred to counting vials and counted in a liquid scintillation counter. Quenching, which was always less than 5 %, was corrected b y the internal standard method. Another aliquot of the supernatant was treated identically except for omission of boiling 5 % Biochim. Biophys. ,4ct~, 141 (1967) 3Io-318
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s. GOLDSTE!N, W. J REDDY
trichloroacetic acid. The cleaned and dried pellet was dissolved in o.I ~i NaOH and quantitaled according to LowRy et al. I~ using bovine albumin as standards. To prepare an extract of approximately o.o5 and o.Io ionic strength, I5 and 5 ml, respectively of distilled water were added directly to the tissue and medium in the original incubating flask. The entire contents were then transferred to the Virtis flasks for homogenization. All processing following homogenization was performed as described above. For o.2o ionic strength extracts, 5 ml of o.22 M KC1 were added ±o each flask, followed by transfer and homogenization, etc. Where direct o.I M NaOH extracts of whole tissue were processed, 5 ml of o.2 N NaOIt were added to the incubating flasks, again followed by transfer, homogenization and overnight extraction at o-4 °. To obtain o.I M NaOH extracts of residual protein remaining after extraction with the o.2o ionic strength solution, the o.2o ionic strength supernatant was decanted and the residue washed with distilled water. Then o.i M N a 0 H was added to the residue and extraction at o-4 ° was carried on overnight with mixing at intervals to facilitate solubilization. Following centrifugation, the supernatant fluid was processed as above. A similar overnight treatment was carried out using I.o M NaOH on the residue re~ rosining after o.I M NaOH extraction. When total tissue protein was processed directly, I vol. of 2o % trichloroaeetic acid was added to each flask followed b y nucleic acid and lipid extraction and drying. Protein was then dissolved overnight in I.O M NaOH and aliquots removed for counting and LOWRY determination. A modification of the FDNB N-terminal analysis method of SANCER12 was carried out on pooled aliquots. Over 98 % of the amino groups were found to be protected from reacting with FDNB, indicating that incorporation into interior peptide linkage had occurred. Hydrolysis of other protein aliqnots followed by isolation of [z~C leucine, using descending paper chromatography with butanol-acetic acid-water (5 : I : 4) as developing solvents, showed that over 99 % of the incorporated radioactivity had a mobility identical to teucine standards. DEAE-cellulose 0. 9 mequiv/g dry weight was washed with distilled water several times to remove fines and finally with 2 fresh changes of starting buffer (o.o3 M KCt, o.o2 M Tris-HC1, pH 7.6). A column of 3o cm ~ em diameter/total vot. 23.6 mI) was packed with the cleaned DEAE-celluiose by gravity and then flushed through with several volumes of starting buffer It was then ready for application of the sample. Material to be applied to DEAE-cellulose columns was prepared as follows. Tissues were obtained as described above from several anima!s and pooled into 2 groups, one for the test and one for the control incubation. Incubation was carmed out as described above with 2 modifications. A mixture of x8 amino acids in concentrations corresponding to intracellular levels found in rat thigh musclO 3 was employed in the incubating medium. Secondly, the labelled leucine used in the insulin, containing flask was uniformly ~4C-labelled L-leucine at I ~C/ml (final specific activity, 3.2 ~C//~mole) whereas the labelled leucine employed in the control flask was L[4,5-aH]leucine at Io ~C/m! (final specific activity, 32 ~Cl~rnole). After z h of incubation at 37 °, metabolic activity was interrupted by chilling in ice water, followed by pooling the contents of both flasks. A KC1 solution was used ro adjust the ionic strength to o.2o. Tissues were then finely minced with scissors, homogenized till particulate and centrifuged. The decanted supernatant was then dialyzed agains~ starting buffer for approximately 24 h. During this period the buffer was renewed Biochim. Biophys. Acta, x41 (:I967) 3io-318
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several times until registered counts failed to exceed background levels. The precipitate formed in the dialysis bag, due presumably to both reduction of ionic strength and change in ionic environment plus some inevitable denaturation of protein, was carefully removed and discarded. An aliquot of this dialyzed solution was applied to the DEAE-cellulose column followed by gradient elution from starting buffer to o.2 M KC1-Tris solution succeeded b y a gradient from o.2 M KC1-Tris to o.5 M KC1Tris solution. Fractions of approximately 5 ml were collected with a turntable shift every IO min. Both the tubes in the turntable and the jacketed column were constantly refrigerated with circulating glycerated water at o-4 °. Aliquots from each fraction were monitored for protein by the LowRY method and chlorides using the Buchler Company apparatus. After removal of these aliquots, I ml of a I mg/ml albumin solution was added to each tube to serve as carrier protein. An aliquot was then removed and radioactivity measured as described above except for the substitution of NCS* solubilizer for hyamine to facilitate double isotope counting. Results were then expressed as the 14C/8H ratio. A sample of the solution originally applied to the column was similarly worked up for counts to ascertain the starting I~C/aH ratio. Ratios reported are derived only from those tubes where both 14C and ~H counts were 2.5 times background or greater. RESULTS
The stimulation by insulin of uniformly 14C-labelled leucine incorporation into various fractions of rat caudofemoralis protein is shown in Table I. Low ionic strengths were expected to solubilize fractional amounts of the enzymatic components while excluding most of the structural and contractile proteins 14,is. As the ionic strength of the extracting solution is increased for the homogenization of different muscle batches, protein yields can be seen to increase, whereas the specific radioactivity of TABLE I THE STIMULATION BY INSULIN OF [14C]LEUCINE INCORPORATION INTO EXTRACTS OF PROTEIN FROM CAUUOFEMORALIS MUSCLE
Approximate Average Counts~rain ionic strength protein yield per mg protein* of extract (% wet wt.) Insulin Control
Counts~rain per total protein Insulin
Control
-->o 0.05 o.Io 0.20
951 17o 4 1816 1958
684 13o2 1384 I5o 7
1. 5 3.0 4.0 5.5
634i64"* 568~58"* 454~52"* 3 5 6 ~ 4 3 ***
456~44 434±41 346ii3 274±26
% Difference due to insulin
+39 +31 +31 +3o
* zk S.E. ** I n s u l i n effect P < 0 . 0 I . *** I n s u l i n effect .P ~ o.o2.
* T r a d e m a r k of t h e N u c l e a r Chicago C o r p o r a t i o n for a p r e p a r a t i o n of t o l u e n e - s o l u b l e q u a t e r n a r y a m m o n i u m b a s e in t h e mol. wt. r a n g e of 250-600. The use of t h i s m a t e r i a l ha s been d o c u m e n t e d in t h e f o l l o w i n g a r t i c l e : " I m p r o v e d S o l u b i l i z a t i o n P r o c e d u r e s for L i q u i d S c i n t i l l a t i o n C o u n t i n g of B i o l o g i c a l M a t e r i a l s " b y D. L. HANSI;N AND E. T. BusI:I i n Analyt. Biochem., 18 (1967) 320-3 32 .
Biochim. Biophys. Acta, 141 (1967) 31o-318
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S. G O L D S T E I N ,
xeV. ~. t~EDDY
both control and insulin-treated proteins is seen to decrease, The simultaneous increase in protein yield and decrease in specific radioactivity with increasing ionic strength is presumably due to the progressive solubilization of contractile protein containing less [14C]1eucine. This dilutional effect of relatively poorly labelled proteii~ 400
320
-
-
240.c_
o
IL
cL 1 6 0 -
o_
.g
80
-
o
L)
O0.20 I <
0.1M N a O H "
1.0iv NaOH ~
0.1M NaOH ~
i.0M NaQH *
F i g . i . E f f e c t of i n s u l i n o n t h e specific a c t i v i t y of m u s c l e p r o t e i n o b t a i n e d b y v a r y i n g t h e e x t r a c t i o n p r o c e d u r e . T i s s u e s w e r e i n c u b a t e d a n d a n a l y z e d as d e s c r i b e d u n d e r -~XTHODS. * C o l u m n s so m a r k e d i n d i c a t e a s i g n i f i c a n t effect of i n s u l i n P % o.o2 5, 240
J J 160
J
J
g ,,-7-1
&o 0.8
.o
0,6
I
u 02 I 1
I 5
I 10
~ 15
I 20
1 25 Tube No.
[ 30
i 35
I 40
i 45
5(3
Fig. 2. P a i r e d s a m p l e s of c a n d o f e m o r a l i s m u s c l e w e r e p o o l e d a n d i n c u b a t e d i n 2 flasks, t h e c o n t r 0 i f l a s k i n t h e p r e s e n c e of [sI-I]leucine, t h e i n s u l i n - c o n t a i n i n g f l a s k i n t h e p r e s e n c e of [i~C]leucine. F o l l o w i n g i n c u b a t i o n , t i s s u e s w e r e c o m b i n e d , e x t r a c t e d a t o.2o i o n i c s t r e n g t h a n d p r e p a r e d for D E A E - c e l l u l o s e c h r o m a t o g r a p h y . F o r t h i s a n d s u b s e q u e n x s t e p s , see t e x v .
Biochi~n. Biophys. dora, z4z (I967) 3 I o - 3 1 8
INSULIN AND PROTEIN SYNTHESIS
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is clearly shown in the total count columns. There is, however, no significant alteration in the insulin stimulation taken as percentage above baseline. Identical results were obtained in the presence of 5 mM glucose in the incubating medium. Fig. i reveals further data derived after extracting tissues in various ways following incubation. In the left section of the graph are presented the results of 3 fractions of protein derived from the same tissue. As expected, the highest speciflic radioactivity is found in the o.2o ionic strength extract with the o.I M NaOH extract showing a decrease and the I.O M NaOH extract showing a further decrease. In all three fractions, a significant effect of insulin to stimulate [~4CJleucine incorporation is demonstrated. The right section of Fig. I shows data making it unlikely that insulin is merely modifying the extractability of various proteins by simple physical alteration of their solubility properties. When, following incubation, tissues were directly extracted in o. I M NaOH or b y adding 2o % trichloroacetic acid directly to incubation flasks and then worked up for total protein, the resultant protein specific radioactivities were seen to approximate what might be expected were the component fractions on the left side of the diagram pooled. Again, an insulin effect is seen in these proteins worked up directly. Fig. 2 reveals data examining in further detail the distribution of counts in proteins soluble at very low ionic strength. Small deviations are found to occur in the ~C/~H ratio of fractionated proteins eluted from the column compared to the ratio of the starting mixture. Cationic proteins emerging with the void volume and collected as tile forerun were found to have a ratio identical to the starting mixture. A significant increase in the ratio of some fractions would be expected were insulin to be stimulating the synthesis of a specific protein. DISCUSSION
Rat diaphragm has been used extensively as a i~ vitro system to study the effects of insulin on protein synthesis since 1952 when KRAHL1G,1~ and SINEX, MacMULLEN AND HASTINGSls found that insulin stimulated the incorporation of labelled amino acids into its protein. Several objections have been raised to the use of intact or cut diaphragm as a valid representative of skeletal 19-29 muscle. Preliminary dissections and experiments comparing various anatomically designated thigh muscles ~° indicated that the caudofemoralis was well suited for the following reasons. It is accessible to rapid and intact excision via its readily identifiable ligamentous origins from the posterior aspects of the sacral and caudal vertebrae to its tendinous insertion into the femur. Its long thin-bellied dimensions which produce long parallel muscle fibers facilitate metabolic exchanges with synthetic media, thus approximating a quasi-physiological capillary-muscle fiber relationship al. Finally, caudofemoralis in pilot experiments incorporated radioactive amino acid more actively than other muscles examined. Caudofemoralis has been used previously in a study of muscle phosphorylase ~2 and glycogen metabolism 33. The present data do not delineate the specific step or steps in protein synthesis influenced by insulin. The present experiments have been carried out on the premise that insulin, employed in a I-h incubation of muscle in vitro, should stimulate [I~C]leucine incorporation into proteins of rapid turnover or short half-life. In rat muscle, these proteins are mainly enzymatic in nature and are easily extractable in dilute Biochim. Biophys. Acta, 141 (1967) 31o-318
316
S. GOLDSTEIN, W. j. REDDXY
ionic soiutions14,1~, 3~. This holds especially for the glycolytic enzymes which are easily extractable and abundant in skeletal muscle 35. Moreover, these are the same enzymes one might expect to be susceptible to an insulin effect. The data in Table and Fig. I show that there is an inverse realtionship between ionic strength and specific radioactivity of the extracted protein. Extracting with solutions of progressively increasing ionic strength and eventually with dilute alkali progressively solubilizes the more viscous contractile proteins. These proteins reported to have ha!f-lives ranging from 4o to I20 days would not be expected to show- either high baseline incorporation or an insulin effect in a I-h incubation i ~ vitro. The connective tissue proteins, such as elas{in and collagen whose half-lives m a y indeed approach the life of the animal36, a7 would least likely be expected to show an insulin effect. Nevertheless, a substantial incorporation of [l~C]leucine into peptide linkage and an insulin stimulation of this process in all the reported protein fractions have been observed. FuI th.ermore, the percentage stimulation is about the same in all fractions. I t is possible t h a t insulin is influencing messenger RNA production, translocation, or activation in mediating its effect. However, an effect on messenger RNA production de ~ovo seems improbable with the observation that incorporation of labelled amino acid into protein proceeds undiminished in the presence of actinomycin concentrations sufficient to b!ock messenger RNA synthesis as. An insulin effect at the level of translocation or activation seems unlikely in that it would not be expected to influence equally messengers corresponding to all of the fractions. I t m a y be argued that insulin is simultaneously promoting the synthesis of some proteins while retarding the synthesis of others within each fraction. Our crude fractionaiion would then cancel these subtle changes wrought b y insulin. The data in Fig. 2 do no*_ support such a hypothesis. An insulin effect to alter the ratio of a specific fraction was not observed. The simultaneous promoting-retarding ef~ee~ resulting in cancellation appears remote, and more credible is a non-specific insulin effec~ on all prozeins. The data can be reconciled with an insulin effect mainly, if not entirety, on transport. In this way, insulin could accelerate the presentation of rate-limiting precursor amino acids to the synthetic machinery and enhance protein synthesis perhaps via mass action or perhaps b y stabilizing the polysome. The difference in synthetic potential between diabetic and normal muscle polysomes reported by W o o l s may merely be secondary to the increased influx of amino acid brought about b y adequate insulin levels. The recent report a9 of enhanced protein synthesis in ribosomes prepared after hearts from diabetic donors were perfused with insulin in substrate-free m e d m m could also be explained on the basis of augmented retention of effluent amino acids consequent to the stimulating effect of insulin transport. It is implicitly assumed in the discussion above and that of others that the [l~C]leucine in the medium equilibrates with the [~2C]leucine in the intrace!iular pool equally in the presence or absence of insulin. In other words, it is assumed that during the incubation, nascent proteins are drawing upon a ]~4C]leueine pool of identical specific radioactivity in both contro]s and insulin-treated samples. [t is probable that insulin in stimulating transport of labelled amino acids including [l~C]teucine is merely providing the peptide-forming machinery, here operating az constanz velocity. with a precursor pool of higher specific radioactivity than controls. This pe~, se would result in the formation of proteins of higher specific radioactivity and the as:ensible insulin effect. This phenomenon is still consistent with a ~ranspor* locus of insulin Biochim. Biophys. Acts, x4I (I967) 3~o-318
INSULIN AND PROTEIN SYNTHESIS
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action and could well account for the insulin effect evident in the present observations and those of others. Experiments concerned with controlling and measuring specific radioactivity of intracellular 14C-labelled amino acids in this system are now in progress. ACKNOWLEDGEMENTS
We acknowledge the generosity of Dr. MARY A. ROOT of the Eli Lilly Company for her assistance in providing us with insulin, Lot P J 4609 and the assistance of Dr. DAVID P. LAULER and Mr. J. H. M0WBRAY who provided the chloride analysis and Mr. EDWARD BOYLE and Miss MARYANNE BRIENZ0 for their technical assistance. This research was supported by a grant from National Institutes of Health AM 09262. Dr. S. GOLDSTEINwas a postdoctoral fellow of the Diabetes Training Grant National Institutes of Health AM 05077.
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 i6 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32
J. J. CASTLES AND I. G. WOOL, Bioehem. dr., 91 (1964) I I C . D. M. KIPNIS AND M. W. NOALL, Biochim. Biophys. Acta, 28 (1958) 226. D. M. 1KIPNIS AND J. E. PARRISH, Federation Proc., 24 (1965) lO51. K. L. MANCHESTER AND M. ]~. I~RAHL, J. Biol. Chem., 234 (1959) 2938. I. G. WOOL AND M. E. •RAHL, Nature, 183 (1959) 1399. R. SCHARFF AND I. G. WOOL, Biochem. J., 97 (1965) 257. R. SCHARFF AND I. G. WOOL, Biochem. J., 97 (1965) 272. I. G. WOOL, Federation Proe., 24 (1965) lO6O. I. G. WOOL, in P. KARLSON, Mechanisms of Hormone Action, Academic Press, N e w York, 1965, p. 98. P. P. COHEN, in W. W. UMBREIT, R. H. BIIRRIS AND J. F. STAUFFER, Manometric Techniques, Burgess, Minneapolis, Minn., 1957, p. 148. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. R. R. PORTER, in L. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 4, Academic Press, N e w York, 1957, p. 221. E. N. SASSENRATH AND D. M. GREENBERG, Cancer Res., 14 (1954) 563. D. S. ROBINSOI;, Biochem. dr., 52 (1952) 621. A. SZENT-GY6RGI, in G. H. BOURN~, The Structure and Function of Muscle, Academic Press, N e w York, 196o, p. i. M. E. KRAHL, Science, 116 (1952) 524 . M. E. KRAHL, dr. Biol. Chem., 200 (1953) 99F. M. SINEX, J. MACMULLEN AND A. B. HASTINGS, dr. Biol. Chem., 198 (1952) 615. E. BAR AND M. C. BLANCHAER, Am. dr. Physiol., 209 (1965) 905. C. H. BEATTY, R. D. PETERSON AND R. M. BOCEK, Am. J. Physiol., 204 (1963) 939M. C. BLANCHAER, A~n. dr. Physiol., 206 (1964) lO15. K. HAMA, dr. Biophys. Biochem. Cytol., 7 (196o) 575. D. L. ODOR, Am. J. Anat., 95 (1954) 433D. L. ODOR, dr. Biophys. Biochem. Cytol., 2 (1956) lO5, Suppl. R. D. PETERSON, C. H. BEATTY AND R. M. BOCEK, Am. J. Physiol., 200 (1961) 182. D. M. tKIPNIS AND C. F. CORI, jr. Biol. Chem., 224 (1957) 681. D. M. KIPNIS, Ann. N. Y. Acad. Sei., 82 (1959) 354. P. J. RANDLE AND G. H. SMITH, Biochem. dr., 7 ° (1958) 5Ol. P. J. RANDLE AND G. H. SMITH, in F. G. YOUNG, W. A. BROOM AND F. W. WOLFF, The Mechanism of Action of Insulin, Blackwell, Oxford, 196o, p. 65. E. C. GREENE, Anatomy of the Rat, Hafner, Philadelphia, Pa., 1935; Trans. Am. Phil. Soe., 27 (1935). W. BLOOM AND D. W. FAWCET~, A Textbook of Histology, 8th ed., Saunders, London, 1962, p. 192. W. H. DANFORTI-IAND J. B. LYON, .J. Biol. Chem., 239 (1961) 4047 .
Biochim. Biophys. 2tcta, 14i (1967) 31o-318
3!8 33 34 35 36 37 38 39
s. GOLDSTEIN,
W. ~, R E D D Y
R. M. BOCEK, R. D. PETERSON ANn C. H. BEATTY, Am. f . Physiol, 2 i o (I966) ~r~ol /E. C. BATS-SMITH,J. SOt;. Chem. fn& (London) Trans., 53 (I934) T 3 5 I . D. PETTE AND T. B0CHER, Z. Physiol. Chem., 331 (z963) I8o. A. NEUBERGZR, J. C. P~RRONE AND H. G. B. SLACK, Biochem. J., 49 (I95 I) I99. W. VAN B. ROBERTSON, J. Biol. Chem., I97 (I952) 495. I). EBOUA-BONIS, A. M. CHAMBAUT, P. VOLFIN AND H. CLAUSER, Nature, I99 (I963) zi83. W . S. STIREWALT AND I. G. W'OOL, Physiologist, 9 (I966) 297.
Biochim. Biophys. Aeta, z4x (I967) 310-318
BIOCHIMICA ET BIOPHYSICA ACTA
~BA 25774 E F F E C T OF I N S U L I N ON T H E INCORPORATION OF [14C]LEUCINE INTO RAT CAUDOFEMORALIS P R O T E I N S. G O L D S T E I N * AND W.
J,
iREDDY
Baker Research Laboratory of the Diabetes Foundation, Department of 2efedicine, New England Deaconess Hospital and Peter Bent Brigham Hospital, Departments of Medicine and Biochemist
SUMMARY
This study sought first to develop an in vitro preparation representative of peripheral skeletal muscle and then to study the effects of insulin on the incorporation of labelled amino acid into various protein fractions thereof. Rat caudofemoralis muscle proteins were separated on the basis of their solubility in solutions of increasing ionic strength and pH. The specific radioactivity of various protein fractions was found to be inversely proportional to the ionic and alkaline strength of the extracting solution. Insulin stimulated incorporation of [i4C]leucine into each fraction with approximately equal magnitude. Likewise, the distribution (ratiot of counts in several protein fractions Muted from a DEAE-cellulose column was not materially affected b y insulin. Our results fit the hypothesis that insulin stimulates the incorporation of [l~C]leucine into skeletal muscle protein b y increasing its entry into ceils and not by stimulation of synthesis of one or more specific proteins.
INTRODUCTION
Current hypotheses ascribe the enhancing effects of insulin on protein synthesis to a stimulation at two loci: At the cell membrane via stimulation of amino acid transport 1-a and at a site distal to amino acid penetration of the cell membrane ~, 5 Studies on the isolated perfused rat heart have further substantiated the membrane transport phenomenon G,~ and more recent work on ribosomal preparations of rat heart has adduced evidence for the effect of insulin at tile ribosomal level of translation 8 or perhaps the nuclear level of RNA translocation to cytoplasm ~. It m a y be hazardous to extrapolate data obtained in highly specialized and nearly continuously active muscle like heart and diaphragm to the rest of the only intermittently active skeletal muscle, the latter comprising over 40 % of our body weight. The purpose of the present study was to develop an i~ vitro preparation more representative of peripheral skeletal muscle, to re-examine earlier work describing the effects of insulin on protein synthesis, and zo obtain information regarding tile * P r e s e n t address: D e p a r t m e n t of Bacteriology and [ m m u n o ! o g y , H a r v a r d Niedical School, Boston, Mass. o 2 I I 5 , U.S.A.
Biochim. Biophys. Acta, z4z
i967) 3IO-318
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fraction into which labelled amino acids are incorporated. This report describes experiments concerned with the incorporation of tracer amino acid into various protein fractions of rat caudofemoralis muscle. METHODS
Male C-D strain (Charles River Breeding Lab.) rats weighing I5O-2OO g were employed in all experiments. Rats were fed Purina chow and water ad libitum following arrival from the supplier. They were housed 8 to a cage till the day prior to experiment in an animal room of constant temperature and humidity with automatically alternated i2-h intervals of light and darkness. The animals were then transferred to separate cages and fasted for 16 h prior to sacrifice b y cervical section and exsanguination. The hind limbs were skinned and the superficial layer of muscles was divided and retracted thus exposing the origins and insertions of the caudofemoralis which was excised intact. The muscles from each side were alternated between test and control samples to obviate any bias that might be introduced b y laterality. Krebs-Ringer phosphate buffer 1° was used throughout, modified b y reduction of the Ca 2+ concentration to one-half. Uniformly l~C-labelled leucine was obtained from New England Nuclear Corporation at specific activity of 223/,C//~mole and used at a final concentration of 0.2 /~C/ml. Glucagon-free insulin was supplied at specific activity of 23 units/rag and used at a final concentration of o.I unit/ml. The buffer mixture was gassed with IOO % O~ for IO rain prior to distribution of 5 ml into each of the 25-ml erlenmeyer flasks fitted with serum sleeve-type rubber stoppers. Following weighing, the tissues, averaging 125 rag, were added to the flasks, transferred to a metabolic shaker, and incubated at 37 -- 0.5 ° for 60 rain. Agitation at 42 cycles/rain with an amplitude of 3.5 inches was carried on throughout the hour. For the first io rain of the incubation, flasks were charged with IOO % 0 2. After I h, metabolic activity was interrupted b y immersing all flasks in ice water. Incorporation of label into protein is negligible at o ° (GOLDSTEINAND REDDY, unpublished observations). To prepare a protein extract approximating o ionic strength, tissue was removed from the incubating medium (ionic strength = o.18), drained and placed in a 3o-ml Virtis homogenizing flask containing IO ml distilled water, and homogenized. Homogenization to fine particulate matter at high speeds on the Virtis "23" homogenizer, using No. I6-1o7 sharp-edged Virtis stainless steel blades, required about I - 2 rain. Homogenizing flasks were then emptied into conical bottom centrifuge tubes and centrifuged at 16oo × g for 20 rain. An aliquot of the homogenate supernatant was worked up for liquid scintillation counting. Protein was precipitated with 2 vol. of 20 % trichloroacetic acid, treated for 15 rain with 2 vol. of 5 % boiling trichloroacetic acid to extract nucleic acids, washed consecutively with 2 vol. of ethanol-ether ( I : I ) and ether to extract lJpids, then dried under negative pressure in a desiccator. Two ml hyamine (I M in methanol, Packard Instrument Co.) were added for overnight hydrolysis in 60 ° waterbath. To this hydrolysate were added 15 ml of toluene-2,5-diphenyloxazole-i,4 bis-2(4methyl-5-phenyloxazolyl)benzene fluor solution. The mixture was then transferred to counting vials and counted in a liquid scintillation counter. Quenching, which was always less than 5 %, was corrected b y the internal standard method. Another aliquot of the supernatant was treated identically except for omission of boiling 5 % Biochim. Biophys. ,4ct~, 141 (1967) 3Io-318
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s. GOLDSTE!N, W. J REDDY
trichloroacetic acid. The cleaned and dried pellet was dissolved in o.I ~i NaOH and quantitaled according to LowRy et al. I~ using bovine albumin as standards. To prepare an extract of approximately o.o5 and o.Io ionic strength, I5 and 5 ml, respectively of distilled water were added directly to the tissue and medium in the original incubating flask. The entire contents were then transferred to the Virtis flasks for homogenization. All processing following homogenization was performed as described above. For o.2o ionic strength extracts, 5 ml of o.22 M KC1 were added ±o each flask, followed by transfer and homogenization, etc. Where direct o.I M NaOH extracts of whole tissue were processed, 5 ml of o.2 N NaOIt were added to the incubating flasks, again followed by transfer, homogenization and overnight extraction at o-4 °. To obtain o.I M NaOH extracts of residual protein remaining after extraction with the o.2o ionic strength solution, the o.2o ionic strength supernatant was decanted and the residue washed with distilled water. Then o.i M N a 0 H was added to the residue and extraction at o-4 ° was carried on overnight with mixing at intervals to facilitate solubilization. Following centrifugation, the supernatant fluid was processed as above. A similar overnight treatment was carried out using I.o M NaOH on the residue re~ rosining after o.I M NaOH extraction. When total tissue protein was processed directly, I vol. of 2o % trichloroaeetic acid was added to each flask followed b y nucleic acid and lipid extraction and drying. Protein was then dissolved overnight in I.O M NaOH and aliquots removed for counting and LOWRY determination. A modification of the FDNB N-terminal analysis method of SANCER12 was carried out on pooled aliquots. Over 98 % of the amino groups were found to be protected from reacting with FDNB, indicating that incorporation into interior peptide linkage had occurred. Hydrolysis of other protein aliqnots followed by isolation of [z~C leucine, using descending paper chromatography with butanol-acetic acid-water (5 : I : 4) as developing solvents, showed that over 99 % of the incorporated radioactivity had a mobility identical to teucine standards. DEAE-cellulose 0. 9 mequiv/g dry weight was washed with distilled water several times to remove fines and finally with 2 fresh changes of starting buffer (o.o3 M KCt, o.o2 M Tris-HC1, pH 7.6). A column of 3o cm ~ em diameter/total vot. 23.6 mI) was packed with the cleaned DEAE-celluiose by gravity and then flushed through with several volumes of starting buffer It was then ready for application of the sample. Material to be applied to DEAE-cellulose columns was prepared as follows. Tissues were obtained as described above from several anima!s and pooled into 2 groups, one for the test and one for the control incubation. Incubation was carmed out as described above with 2 modifications. A mixture of x8 amino acids in concentrations corresponding to intracellular levels found in rat thigh musclO 3 was employed in the incubating medium. Secondly, the labelled leucine used in the insulin, containing flask was uniformly ~4C-labelled L-leucine at I ~C/ml (final specific activity, 3.2 ~C//~mole) whereas the labelled leucine employed in the control flask was L[4,5-aH]leucine at Io ~C/m! (final specific activity, 32 ~Cl~rnole). After z h of incubation at 37 °, metabolic activity was interrupted by chilling in ice water, followed by pooling the contents of both flasks. A KC1 solution was used ro adjust the ionic strength to o.2o. Tissues were then finely minced with scissors, homogenized till particulate and centrifuged. The decanted supernatant was then dialyzed agains~ starting buffer for approximately 24 h. During this period the buffer was renewed Biochim. Biophys. Acta, x41 (:I967) 3io-318
INSULIN AND PROTEIN SYNTHESIS
313
several times until registered counts failed to exceed background levels. The precipitate formed in the dialysis bag, due presumably to both reduction of ionic strength and change in ionic environment plus some inevitable denaturation of protein, was carefully removed and discarded. An aliquot of this dialyzed solution was applied to the DEAE-cellulose column followed by gradient elution from starting buffer to o.2 M KC1-Tris solution succeeded b y a gradient from o.2 M KC1-Tris to o.5 M KC1Tris solution. Fractions of approximately 5 ml were collected with a turntable shift every IO min. Both the tubes in the turntable and the jacketed column were constantly refrigerated with circulating glycerated water at o-4 °. Aliquots from each fraction were monitored for protein by the LowRY method and chlorides using the Buchler Company apparatus. After removal of these aliquots, I ml of a I mg/ml albumin solution was added to each tube to serve as carrier protein. An aliquot was then removed and radioactivity measured as described above except for the substitution of NCS* solubilizer for hyamine to facilitate double isotope counting. Results were then expressed as the 14C/8H ratio. A sample of the solution originally applied to the column was similarly worked up for counts to ascertain the starting I~C/aH ratio. Ratios reported are derived only from those tubes where both 14C and ~H counts were 2.5 times background or greater. RESULTS
The stimulation by insulin of uniformly 14C-labelled leucine incorporation into various fractions of rat caudofemoralis protein is shown in Table I. Low ionic strengths were expected to solubilize fractional amounts of the enzymatic components while excluding most of the structural and contractile proteins 14,is. As the ionic strength of the extracting solution is increased for the homogenization of different muscle batches, protein yields can be seen to increase, whereas the specific radioactivity of TABLE I THE STIMULATION BY INSULIN OF [14C]LEUCINE INCORPORATION INTO EXTRACTS OF PROTEIN FROM CAUUOFEMORALIS MUSCLE
Approximate Average Counts~rain ionic strength protein yield per mg protein* of extract (% wet wt.) Insulin Control
Counts~rain per total protein Insulin
Control
-->o 0.05 o.Io 0.20
951 17o 4 1816 1958
684 13o2 1384 I5o 7
1. 5 3.0 4.0 5.5
634i64"* 568~58"* 454~52"* 3 5 6 ~ 4 3 ***
456~44 434±41 346ii3 274±26
% Difference due to insulin
+39 +31 +31 +3o
* zk S.E. ** I n s u l i n effect P < 0 . 0 I . *** I n s u l i n effect .P ~ o.o2.
* T r a d e m a r k of t h e N u c l e a r Chicago C o r p o r a t i o n for a p r e p a r a t i o n of t o l u e n e - s o l u b l e q u a t e r n a r y a m m o n i u m b a s e in t h e mol. wt. r a n g e of 250-600. The use of t h i s m a t e r i a l ha s been d o c u m e n t e d in t h e f o l l o w i n g a r t i c l e : " I m p r o v e d S o l u b i l i z a t i o n P r o c e d u r e s for L i q u i d S c i n t i l l a t i o n C o u n t i n g of B i o l o g i c a l M a t e r i a l s " b y D. L. HANSI;N AND E. T. BusI:I i n Analyt. Biochem., 18 (1967) 320-3 32 .
Biochim. Biophys. Acta, 141 (1967) 31o-318
3Z4
S. G O L D S T E I N ,
xeV. ~. t~EDDY
both control and insulin-treated proteins is seen to decrease, The simultaneous increase in protein yield and decrease in specific radioactivity with increasing ionic strength is presumably due to the progressive solubilization of contractile protein containing less [14C]1eucine. This dilutional effect of relatively poorly labelled proteii~ 400
320
-
-
240.c_
o
IL
cL 1 6 0 -
o_
.g
80
-
o
L)
O0.20 I <
0.1M N a O H "
1.0iv NaOH ~
0.1M NaOH ~
i.0M NaQH *
F i g . i . E f f e c t of i n s u l i n o n t h e specific a c t i v i t y of m u s c l e p r o t e i n o b t a i n e d b y v a r y i n g t h e e x t r a c t i o n p r o c e d u r e . T i s s u e s w e r e i n c u b a t e d a n d a n a l y z e d as d e s c r i b e d u n d e r -~XTHODS. * C o l u m n s so m a r k e d i n d i c a t e a s i g n i f i c a n t effect of i n s u l i n P % o.o2 5, 240
J J 160
J
J
g ,,-7-1
&o 0.8
.o
0,6
I
u 02 I 1
I 5
I 10
~ 15
I 20
1 25 Tube No.
[ 30
i 35
I 40
i 45
5(3
Fig. 2. P a i r e d s a m p l e s of c a n d o f e m o r a l i s m u s c l e w e r e p o o l e d a n d i n c u b a t e d i n 2 flasks, t h e c o n t r 0 i f l a s k i n t h e p r e s e n c e of [sI-I]leucine, t h e i n s u l i n - c o n t a i n i n g f l a s k i n t h e p r e s e n c e of [i~C]leucine. F o l l o w i n g i n c u b a t i o n , t i s s u e s w e r e c o m b i n e d , e x t r a c t e d a t o.2o i o n i c s t r e n g t h a n d p r e p a r e d for D E A E - c e l l u l o s e c h r o m a t o g r a p h y . F o r t h i s a n d s u b s e q u e n x s t e p s , see t e x v .
Biochi~n. Biophys. dora, z4z (I967) 3 I o - 3 1 8
INSULIN AND PROTEIN SYNTHESIS
315
is clearly shown in the total count columns. There is, however, no significant alteration in the insulin stimulation taken as percentage above baseline. Identical results were obtained in the presence of 5 mM glucose in the incubating medium. Fig. i reveals further data derived after extracting tissues in various ways following incubation. In the left section of the graph are presented the results of 3 fractions of protein derived from the same tissue. As expected, the highest speciflic radioactivity is found in the o.2o ionic strength extract with the o.I M NaOH extract showing a decrease and the I.O M NaOH extract showing a further decrease. In all three fractions, a significant effect of insulin to stimulate [~4CJleucine incorporation is demonstrated. The right section of Fig. I shows data making it unlikely that insulin is merely modifying the extractability of various proteins by simple physical alteration of their solubility properties. When, following incubation, tissues were directly extracted in o. I M NaOH or b y adding 2o % trichloroacetic acid directly to incubation flasks and then worked up for total protein, the resultant protein specific radioactivities were seen to approximate what might be expected were the component fractions on the left side of the diagram pooled. Again, an insulin effect is seen in these proteins worked up directly. Fig. 2 reveals data examining in further detail the distribution of counts in proteins soluble at very low ionic strength. Small deviations are found to occur in the ~C/~H ratio of fractionated proteins eluted from the column compared to the ratio of the starting mixture. Cationic proteins emerging with the void volume and collected as tile forerun were found to have a ratio identical to the starting mixture. A significant increase in the ratio of some fractions would be expected were insulin to be stimulating the synthesis of a specific protein. DISCUSSION
Rat diaphragm has been used extensively as a i~ vitro system to study the effects of insulin on protein synthesis since 1952 when KRAHL1G,1~ and SINEX, MacMULLEN AND HASTINGSls found that insulin stimulated the incorporation of labelled amino acids into its protein. Several objections have been raised to the use of intact or cut diaphragm as a valid representative of skeletal 19-29 muscle. Preliminary dissections and experiments comparing various anatomically designated thigh muscles ~° indicated that the caudofemoralis was well suited for the following reasons. It is accessible to rapid and intact excision via its readily identifiable ligamentous origins from the posterior aspects of the sacral and caudal vertebrae to its tendinous insertion into the femur. Its long thin-bellied dimensions which produce long parallel muscle fibers facilitate metabolic exchanges with synthetic media, thus approximating a quasi-physiological capillary-muscle fiber relationship al. Finally, caudofemoralis in pilot experiments incorporated radioactive amino acid more actively than other muscles examined. Caudofemoralis has been used previously in a study of muscle phosphorylase ~2 and glycogen metabolism 33. The present data do not delineate the specific step or steps in protein synthesis influenced by insulin. The present experiments have been carried out on the premise that insulin, employed in a I-h incubation of muscle in vitro, should stimulate [I~C]leucine incorporation into proteins of rapid turnover or short half-life. In rat muscle, these proteins are mainly enzymatic in nature and are easily extractable in dilute Biochim. Biophys. Acta, 141 (1967) 31o-318
316
S. GOLDSTEIN, W. j. REDDXY
ionic soiutions14,1~, 3~. This holds especially for the glycolytic enzymes which are easily extractable and abundant in skeletal muscle 35. Moreover, these are the same enzymes one might expect to be susceptible to an insulin effect. The data in Table and Fig. I show that there is an inverse realtionship between ionic strength and specific radioactivity of the extracted protein. Extracting with solutions of progressively increasing ionic strength and eventually with dilute alkali progressively solubilizes the more viscous contractile proteins. These proteins reported to have ha!f-lives ranging from 4o to I20 days would not be expected to show- either high baseline incorporation or an insulin effect in a I-h incubation i ~ vitro. The connective tissue proteins, such as elas{in and collagen whose half-lives m a y indeed approach the life of the animal36, a7 would least likely be expected to show an insulin effect. Nevertheless, a substantial incorporation of [l~C]leucine into peptide linkage and an insulin stimulation of this process in all the reported protein fractions have been observed. FuI th.ermore, the percentage stimulation is about the same in all fractions. I t is possible t h a t insulin is influencing messenger RNA production, translocation, or activation in mediating its effect. However, an effect on messenger RNA production de ~ovo seems improbable with the observation that incorporation of labelled amino acid into protein proceeds undiminished in the presence of actinomycin concentrations sufficient to b!ock messenger RNA synthesis as. An insulin effect at the level of translocation or activation seems unlikely in that it would not be expected to influence equally messengers corresponding to all of the fractions. I t m a y be argued that insulin is simultaneously promoting the synthesis of some proteins while retarding the synthesis of others within each fraction. Our crude fractionaiion would then cancel these subtle changes wrought b y insulin. The data in Fig. 2 do no*_ support such a hypothesis. An insulin effect to alter the ratio of a specific fraction was not observed. The simultaneous promoting-retarding ef~ee~ resulting in cancellation appears remote, and more credible is a non-specific insulin effec~ on all prozeins. The data can be reconciled with an insulin effect mainly, if not entirety, on transport. In this way, insulin could accelerate the presentation of rate-limiting precursor amino acids to the synthetic machinery and enhance protein synthesis perhaps via mass action or perhaps b y stabilizing the polysome. The difference in synthetic potential between diabetic and normal muscle polysomes reported by W o o l s may merely be secondary to the increased influx of amino acid brought about b y adequate insulin levels. The recent report a9 of enhanced protein synthesis in ribosomes prepared after hearts from diabetic donors were perfused with insulin in substrate-free m e d m m could also be explained on the basis of augmented retention of effluent amino acids consequent to the stimulating effect of insulin transport. It is implicitly assumed in the discussion above and that of others that the [l~C]leucine in the medium equilibrates with the [~2C]leucine in the intrace!iular pool equally in the presence or absence of insulin. In other words, it is assumed that during the incubation, nascent proteins are drawing upon a ]~4C]leueine pool of identical specific radioactivity in both contro]s and insulin-treated samples. [t is probable that insulin in stimulating transport of labelled amino acids including [l~C]teucine is merely providing the peptide-forming machinery, here operating az constanz velocity. with a precursor pool of higher specific radioactivity than controls. This pe~, se would result in the formation of proteins of higher specific radioactivity and the as:ensible insulin effect. This phenomenon is still consistent with a ~ranspor* locus of insulin Biochim. Biophys. Acts, x4I (I967) 3~o-318
INSULIN AND PROTEIN SYNTHESIS
317
action and could well account for the insulin effect evident in the present observations and those of others. Experiments concerned with controlling and measuring specific radioactivity of intracellular 14C-labelled amino acids in this system are now in progress. ACKNOWLEDGEMENTS
We acknowledge the generosity of Dr. MARY A. ROOT of the Eli Lilly Company for her assistance in providing us with insulin, Lot P J 4609 and the assistance of Dr. DAVID P. LAULER and Mr. J. H. M0WBRAY who provided the chloride analysis and Mr. EDWARD BOYLE and Miss MARYANNE BRIENZ0 for their technical assistance. This research was supported by a grant from National Institutes of Health AM 09262. Dr. S. GOLDSTEINwas a postdoctoral fellow of the Diabetes Training Grant National Institutes of Health AM 05077.
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 i6 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32
J. J. CASTLES AND I. G. WOOL, Bioehem. dr., 91 (1964) I I C . D. M. KIPNIS AND M. W. NOALL, Biochim. Biophys. Acta, 28 (1958) 226. D. M. 1KIPNIS AND J. E. PARRISH, Federation Proc., 24 (1965) lO51. K. L. MANCHESTER AND M. ]~. I~RAHL, J. Biol. Chem., 234 (1959) 2938. I. G. WOOL AND M. E. •RAHL, Nature, 183 (1959) 1399. R. SCHARFF AND I. G. WOOL, Biochem. J., 97 (1965) 257. R. SCHARFF AND I. G. WOOL, Biochem. J., 97 (1965) 272. I. G. WOOL, Federation Proe., 24 (1965) lO6O. I. G. WOOL, in P. KARLSON, Mechanisms of Hormone Action, Academic Press, N e w York, 1965, p. 98. P. P. COHEN, in W. W. UMBREIT, R. H. BIIRRIS AND J. F. STAUFFER, Manometric Techniques, Burgess, Minneapolis, Minn., 1957, p. 148. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. R. R. PORTER, in L. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 4, Academic Press, N e w York, 1957, p. 221. E. N. SASSENRATH AND D. M. GREENBERG, Cancer Res., 14 (1954) 563. D. S. ROBINSOI;, Biochem. dr., 52 (1952) 621. A. SZENT-GY6RGI, in G. H. BOURN~, The Structure and Function of Muscle, Academic Press, N e w York, 196o, p. i. M. E. KRAHL, Science, 116 (1952) 524 . M. E. KRAHL, dr. Biol. Chem., 200 (1953) 99F. M. SINEX, J. MACMULLEN AND A. B. HASTINGS, dr. Biol. Chem., 198 (1952) 615. E. BAR AND M. C. BLANCHAER, Am. dr. Physiol., 209 (1965) 905. C. H. BEATTY, R. D. PETERSON AND R. M. BOCEK, Am. J. Physiol., 204 (1963) 939M. C. BLANCHAER, A~n. dr. Physiol., 206 (1964) lO15. K. HAMA, dr. Biophys. Biochem. Cytol., 7 (196o) 575. D. L. ODOR, Am. J. Anat., 95 (1954) 433D. L. ODOR, dr. Biophys. Biochem. Cytol., 2 (1956) lO5, Suppl. R. D. PETERSON, C. H. BEATTY AND R. M. BOCEK, Am. J. Physiol., 200 (1961) 182. D. M. tKIPNIS AND C. F. CORI, jr. Biol. Chem., 224 (1957) 681. D. M. KIPNIS, Ann. N. Y. Acad. Sei., 82 (1959) 354. P. J. RANDLE AND G. H. SMITH, Biochem. dr., 7 ° (1958) 5Ol. P. J. RANDLE AND G. H. SMITH, in F. G. YOUNG, W. A. BROOM AND F. W. WOLFF, The Mechanism of Action of Insulin, Blackwell, Oxford, 196o, p. 65. E. C. GREENE, Anatomy of the Rat, Hafner, Philadelphia, Pa., 1935; Trans. Am. Phil. Soe., 27 (1935). W. BLOOM AND D. W. FAWCET~, A Textbook of Histology, 8th ed., Saunders, London, 1962, p. 192. W. H. DANFORTI-IAND J. B. LYON, .J. Biol. Chem., 239 (1961) 4047 .
Biochim. Biophys. 2tcta, 14i (1967) 31o-318
3!8 33 34 35 36 37 38 39
s. GOLDSTEIN,
W. ~, R E D D Y
R. M. BOCEK, R. D. PETERSON ANn C. H. BEATTY, Am. f . Physiol, 2 i o (I966) ~r~ol /E. C. BATS-SMITH,J. SOt;. Chem. fn& (London) Trans., 53 (I934) T 3 5 I . D. PETTE AND T. B0CHER, Z. Physiol. Chem., 331 (z963) I8o. A. NEUBERGZR, J. C. P~RRONE AND H. G. B. SLACK, Biochem. J., 49 (I95 I) I99. W. VAN B. ROBERTSON, J. Biol. Chem., I97 (I952) 495. I). EBOUA-BONIS, A. M. CHAMBAUT, P. VOLFIN AND H. CLAUSER, Nature, I99 (I963) zi83. W . S. STIREWALT AND I. G. W'OOL, Physiologist, 9 (I966) 297.
Biochim. Biophys. Aeta, z4x (I967) 310-318