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Rigor mortis pattern of skeletal muscle and sarcomere length of the myofibril
309
SHORT COMMUNICATIONS
sc 43 048
Rigor mortis pattern of skeletal muscle and sarcomere length of the myofibril The extent of muscle contraction (isotonic) m a y be estimated from the sarcomere length of the myofibril 1-~. The onset of rigor morris, marked by a decrease in muscle extensibility, is frequently accompanied b y shortening or contraction 5, which has been shown to be ATP related ~s. From the recent work of INFANTE et al?, it appears that ATP breakdown occurs when the developed tension causes the relative inward movements of the actin and myosin filaments (resulting in sarcomere shortening). Little information exists, however, on the variations in sarcomere length which m a y be attributed to post-mortem changes in the muscle. The present study was conducted to determine the relationship between post-rigor sarcomere length and the time course of rigor mortis. Twenty-five experimental animals (Sus domesticus) averaging IOO kg and similar in genetic and nutritional backgrounds were used in this investigation. A sample of the longissimus dorsi muscle was excised from the lumbar region of the right side of the carcass immediately after exsanguination. Parallel strips of fibers were taken from this excised sample to estimate the time course of rigor mortis at 37 °, and IOO % relative humidity in a nitrogen atmosphere as described by BRISKEY et al. ~°. From the record of the rigor mortis pattern, the durations of the delay and onset phases were ascertained (Figs. I a and Ib). The delay phase was the period during which there was essentially no loss in extensibility. The onset of rigor mortis was considered to be the point at which extensibility started to diminish and the duration of the onset phase was considered to be the entire period of diminishing extensibility. A sample from the longissimus dorsi muscle of the left side of the carcass held at 4 ° was taken 24 h post-mortem to determine the sarcomere length of undisturbed
Fig. I. lZigor morris patterns with super-imposed phase-contrast photomicrographs showing the relationship between sareomere length of the myofibril and the delay-phase duration of rigor mortis. The rigor patterns show the relative loss of extensibility with respect to the loaded (upper dotted line) and unloaded (lower line) condition. (a) Delay phase duration is 3° rain, sarcomere length is 1. 7/~. (b) Delay phase duration is 135 rain, sarcomere length is 2.2/*.
Biochim. Biophys. Acta, lo2 (1965) 3o9-311
31o
SHORT COMMUNICATIONS
muscle according to the procedure of LOCKER11. The sarcomere length of 25 different myofibrils in each sample was determined with a phase-contrast microscope and reported as a mean value. The correlation coefficients were calculated by the method of SNEDECOR12. The data are presented (delay phase only) in a scatter diagram (Fig. 2), which also shows the linear regression, least-squares line of best fit ~3. 24[
•
~ 2 01--
• •
u~ 1.61 20
I 40
I I I I I I 60 80 100 120 140 160 180 Delay phase duration of rigor rnootis (rain) Fig. 2. Scatter d i a g r a m of sarcomere length in the myofibril of the longissimus dorsi muscle verse, s delay-phase d u r a t i o n of rigor mortis for 25 e x p e r i m e n t a l a n i m a l s (S~ts domeslic**s). Sarcomere length was determined on left-side muscles 24 h p o s t - m o r t e m while delay-phase d u r a t i o n was determined on right-side muscles i m m e d i a t e l y after e x s a n g u i n a t i o n . The regression line of best fit is shown (Y = 0.90 (P < o . o i ) ; b = 0.004).
When sarcomere length at 24 h post-mortem was correlated with the durations of both the delay and onset phases of rigor mortis, correlation coefficients (r) of 0.90 and 0.35, respectively, were obtained. Although the relationship of sarcomere length with duration of the onset phase was not significant, the association between sarcomere length and duration of the delay phase was highly significant (P < o.oi). The coefficient of determination (#) showed that 81% of the variation in sarcomere length of the myofibri] in the longissimus dorsi m a y be explained as due to, or associated with, the duration of the delay phase of rigor mortis. The small scatter of points about the regression line (Fig. 2) brings into prominence the small deviation from the calculated correlation which emphasizes the high degree to which these two muscle properties are related. The studies described in this paper, under the stated conditions, quantitatively demonstrate the amount of contraction which m a y be related to the post-mortem changes in muscle. The data presented show that shortening, which has been reported to occur with the onset of rigor mortisS, 14 is quite severe when the delay phase of rigor mortis is of short duration (Fig. Ia). However, when the delay phase of rigor morris is of long duration, the sarcomere shortening that occurs is much less (Fig. Ib). Consequently, it can be stated that the amount of sarcomere shortening or contraction, coincident with rigor mortis onset, is highly dependent upon the time course of rigor morris. This study was supported in part by a Public Health Service Research Grant E F - 8 I (C6), from the Division of Environmental Engineering and Food Protection, United States Department of Health, Education and Welfare, National Institutes of Health. This paper, Department Manuscript No. 402, is published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Biochim. Biophys. ~4cta, lO2 (1965) 3o9-31I
SHORT COMMUNICATIONS
311
Dr. J. D. SINK is a National Science Foundation Postdoctoral Fellow on leave from The Pennsylvania State University, University Park, Pennsylvania (U.S.A.).
Departme~#s o] Meat and Animal Science and o/Biochemistry, The University o/Wisconsin, Madison, Wise. (U.S.A.)
J. D. SINK R . 6 . CASSENS W . G. HOEKSTRA
E. J. BRISKEY 1 F. BUCHtTHAL AND G. G. KNAPPEIS, Acta Physiol. Scand., 5 (1943) 256. F. BLICHTHAL AND G. G. I~NApPEIS, Acta Physiol. Scand., 6 (1943) 123. 3 B. HORVATI-I, Bioehim. Biophys. Acta, 8 (1952) 257. 4 A. J. HODGE, J. Biophys. Biochem. Cytol., I (1955) 361. 5 E. C. BATE-SMITH AND J. I~. BENDALL, J. Physiol., i i o (1949) 47. 6 J. R. BENDALL, J. Physiol., 114 (1951) 71. 7 E. C. BATE-SMITH, Advan. Food Res., I (1948) I. 8 B, B. MARSH, Biochim. Biophys. Acta, 9 (1952) 127. 9 A, A. INFANTE, D. KLUAPIKS AND R. E. DAVIES, Nature, 2ol (1964) 620. Io E. J. BRISKEY, R, N. SAYEE AND R. G. CASSENS, J. Food Sci., 27 (1962) 560. i i R. H. LOCKER, ]. Biophys. Biochem. Cytol., 6 (1959) 419 . 12 C*, W. SNEDECOR, Statistical Methods, I o w a State College Press, Ames, 5th ed., 1956, p. 6o-174. 13 M. EZEKIEL AND 1~. A. Fox, Methods of Correlation and Regression Analysis, J o h n Wiley a n d Sons, New York, 1949, p. 55-15o. 14 B. B. MARSH, Biochim. Biophys. Acta, 12 (1953) 478. 2
Received November 23rd, 1964 Biochim. Biophys. Acta, lO2 (1965) 3o9-311
sc 43o45
Binding of acridine orange to yeast ribosomes When BRADLEY AND WOLF 1 showed how the spectral changes associated with the binding of the cationic dye acridine orange to various polyanions afforded an estimate of the number of sites on the polymers available for dye binding, it occurred to us to use this method to answer the question : " H o w much of the RNA of ribosomes is available for binding acridine orange ?". In order that their method be applied to a complex of polynucleotide and protein, such as a ribosome, it is necessary to take into account how much dye the ribosomal protein might bind. We have not answered this question experimentally but offer the following reasoning: As far as is known, acridine orange will bind preferentially to anionic sites. Typical ribosomal protein contains about 17 % of acidic amino acids, of which some 7 % are in the form of amides ~, leaving IO % free anionic residues. One yeast 8o-S ribosome, of particle weight 4-IO 6, will contain 5300 nucleotide residues (of average weight 340) and 200o0 total amino acid residues (of average weight 11o) or 2000 anionic amino acid residues. While all the nucleotide residues are in the form of a polyanion, it is highly probable that only a small proportion of the anionic amino acids in the ribosomal protein are neighbored b y other anionic amino acids. Therefore, we have assumed that the amount of dye bound to ribosomal protein will be only a few hundredths of that bound to the ribosomal RNA, and have equated "polymer sites" with "nucleotide residues". Biochim. Biophys. Acta, lO2 (I965) 311-313
SHORT COMMUNICATIONS
sc 43 048
Rigor mortis pattern of skeletal muscle and sarcomere length of the myofibril The extent of muscle contraction (isotonic) m a y be estimated from the sarcomere length of the myofibril 1-~. The onset of rigor morris, marked by a decrease in muscle extensibility, is frequently accompanied b y shortening or contraction 5, which has been shown to be ATP related ~s. From the recent work of INFANTE et al?, it appears that ATP breakdown occurs when the developed tension causes the relative inward movements of the actin and myosin filaments (resulting in sarcomere shortening). Little information exists, however, on the variations in sarcomere length which m a y be attributed to post-mortem changes in the muscle. The present study was conducted to determine the relationship between post-rigor sarcomere length and the time course of rigor mortis. Twenty-five experimental animals (Sus domesticus) averaging IOO kg and similar in genetic and nutritional backgrounds were used in this investigation. A sample of the longissimus dorsi muscle was excised from the lumbar region of the right side of the carcass immediately after exsanguination. Parallel strips of fibers were taken from this excised sample to estimate the time course of rigor mortis at 37 °, and IOO % relative humidity in a nitrogen atmosphere as described by BRISKEY et al. ~°. From the record of the rigor mortis pattern, the durations of the delay and onset phases were ascertained (Figs. I a and Ib). The delay phase was the period during which there was essentially no loss in extensibility. The onset of rigor mortis was considered to be the point at which extensibility started to diminish and the duration of the onset phase was considered to be the entire period of diminishing extensibility. A sample from the longissimus dorsi muscle of the left side of the carcass held at 4 ° was taken 24 h post-mortem to determine the sarcomere length of undisturbed
Fig. I. lZigor morris patterns with super-imposed phase-contrast photomicrographs showing the relationship between sareomere length of the myofibril and the delay-phase duration of rigor mortis. The rigor patterns show the relative loss of extensibility with respect to the loaded (upper dotted line) and unloaded (lower line) condition. (a) Delay phase duration is 3° rain, sarcomere length is 1. 7/~. (b) Delay phase duration is 135 rain, sarcomere length is 2.2/*.
Biochim. Biophys. Acta, lo2 (1965) 3o9-311
31o
SHORT COMMUNICATIONS
muscle according to the procedure of LOCKER11. The sarcomere length of 25 different myofibrils in each sample was determined with a phase-contrast microscope and reported as a mean value. The correlation coefficients were calculated by the method of SNEDECOR12. The data are presented (delay phase only) in a scatter diagram (Fig. 2), which also shows the linear regression, least-squares line of best fit ~3. 24[
•
~ 2 01--
• •
u~ 1.61 20
I 40
I I I I I I 60 80 100 120 140 160 180 Delay phase duration of rigor rnootis (rain) Fig. 2. Scatter d i a g r a m of sarcomere length in the myofibril of the longissimus dorsi muscle verse, s delay-phase d u r a t i o n of rigor mortis for 25 e x p e r i m e n t a l a n i m a l s (S~ts domeslic**s). Sarcomere length was determined on left-side muscles 24 h p o s t - m o r t e m while delay-phase d u r a t i o n was determined on right-side muscles i m m e d i a t e l y after e x s a n g u i n a t i o n . The regression line of best fit is shown (Y = 0.90 (P < o . o i ) ; b = 0.004).
When sarcomere length at 24 h post-mortem was correlated with the durations of both the delay and onset phases of rigor mortis, correlation coefficients (r) of 0.90 and 0.35, respectively, were obtained. Although the relationship of sarcomere length with duration of the onset phase was not significant, the association between sarcomere length and duration of the delay phase was highly significant (P < o.oi). The coefficient of determination (#) showed that 81% of the variation in sarcomere length of the myofibri] in the longissimus dorsi m a y be explained as due to, or associated with, the duration of the delay phase of rigor mortis. The small scatter of points about the regression line (Fig. 2) brings into prominence the small deviation from the calculated correlation which emphasizes the high degree to which these two muscle properties are related. The studies described in this paper, under the stated conditions, quantitatively demonstrate the amount of contraction which m a y be related to the post-mortem changes in muscle. The data presented show that shortening, which has been reported to occur with the onset of rigor mortisS, 14 is quite severe when the delay phase of rigor mortis is of short duration (Fig. Ia). However, when the delay phase of rigor morris is of long duration, the sarcomere shortening that occurs is much less (Fig. Ib). Consequently, it can be stated that the amount of sarcomere shortening or contraction, coincident with rigor mortis onset, is highly dependent upon the time course of rigor morris. This study was supported in part by a Public Health Service Research Grant E F - 8 I (C6), from the Division of Environmental Engineering and Food Protection, United States Department of Health, Education and Welfare, National Institutes of Health. This paper, Department Manuscript No. 402, is published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Biochim. Biophys. ~4cta, lO2 (1965) 3o9-31I
SHORT COMMUNICATIONS
311
Dr. J. D. SINK is a National Science Foundation Postdoctoral Fellow on leave from The Pennsylvania State University, University Park, Pennsylvania (U.S.A.).
Departme~#s o] Meat and Animal Science and o/Biochemistry, The University o/Wisconsin, Madison, Wise. (U.S.A.)
J. D. SINK R . 6 . CASSENS W . G. HOEKSTRA
E. J. BRISKEY 1 F. BUCHtTHAL AND G. G. KNAPPEIS, Acta Physiol. Scand., 5 (1943) 256. F. BLICHTHAL AND G. G. I~NApPEIS, Acta Physiol. Scand., 6 (1943) 123. 3 B. HORVATI-I, Bioehim. Biophys. Acta, 8 (1952) 257. 4 A. J. HODGE, J. Biophys. Biochem. Cytol., I (1955) 361. 5 E. C. BATE-SMITH AND J. I~. BENDALL, J. Physiol., i i o (1949) 47. 6 J. R. BENDALL, J. Physiol., 114 (1951) 71. 7 E. C. BATE-SMITH, Advan. Food Res., I (1948) I. 8 B, B. MARSH, Biochim. Biophys. Acta, 9 (1952) 127. 9 A, A. INFANTE, D. KLUAPIKS AND R. E. DAVIES, Nature, 2ol (1964) 620. Io E. J. BRISKEY, R, N. SAYEE AND R. G. CASSENS, J. Food Sci., 27 (1962) 560. i i R. H. LOCKER, ]. Biophys. Biochem. Cytol., 6 (1959) 419 . 12 C*, W. SNEDECOR, Statistical Methods, I o w a State College Press, Ames, 5th ed., 1956, p. 6o-174. 13 M. EZEKIEL AND 1~. A. Fox, Methods of Correlation and Regression Analysis, J o h n Wiley a n d Sons, New York, 1949, p. 55-15o. 14 B. B. MARSH, Biochim. Biophys. Acta, 12 (1953) 478. 2
Received November 23rd, 1964 Biochim. Biophys. Acta, lO2 (1965) 3o9-311
sc 43o45
Binding of acridine orange to yeast ribosomes When BRADLEY AND WOLF 1 showed how the spectral changes associated with the binding of the cationic dye acridine orange to various polyanions afforded an estimate of the number of sites on the polymers available for dye binding, it occurred to us to use this method to answer the question : " H o w much of the RNA of ribosomes is available for binding acridine orange ?". In order that their method be applied to a complex of polynucleotide and protein, such as a ribosome, it is necessary to take into account how much dye the ribosomal protein might bind. We have not answered this question experimentally but offer the following reasoning: As far as is known, acridine orange will bind preferentially to anionic sites. Typical ribosomal protein contains about 17 % of acidic amino acids, of which some 7 % are in the form of amides ~, leaving IO % free anionic residues. One yeast 8o-S ribosome, of particle weight 4-IO 6, will contain 5300 nucleotide residues (of average weight 340) and 200o0 total amino acid residues (of average weight 11o) or 2000 anionic amino acid residues. While all the nucleotide residues are in the form of a polyanion, it is highly probable that only a small proportion of the anionic amino acids in the ribosomal protein are neighbored b y other anionic amino acids. Therefore, we have assumed that the amount of dye bound to ribosomal protein will be only a few hundredths of that bound to the ribosomal RNA, and have equated "polymer sites" with "nucleotide residues". Biochim. Biophys. Acta, lO2 (I965) 311-313