Studies on the Metabolic Activity of Muscle Proteins

Transcription

1 [Agr. Biol. Chem., Vol. 31, No. 11, p `1378, 1967] Studies on the Metabolic Activity of Muscle Proteins Part III. The Time-Course of Changes in the Protein Components of Muscle in Rat during Protein Depletion By Michio YAMAGUCHI* and Makoto KANDATSU Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo Received July 2, 1967 The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others. INTRODUCTION In the previous papers1,2) we reported the over-all aspects of the metabolic activity of muscle proteins with respect to protein nutri tion, and indicated that the enzyme activities, or presumably the enzyme proteins, of both Ca++ ion- and Mg++ ion-activated ATPases and glutamic oxaloacetic transaminase of muscle were reduced via the similar patterns to reserve proteins on the 4 to 8th days of protein depletion. Before the further precise investigations on such a metabolic response of enzyme systems, it is necessary to assess the corresponding changes in the main protein fractions that are characteristic of muscular * This report is a part of the doctorate thesis of the author. 1) M. Yamaguchi and M. Kandatsu, This Journal, 31, 776 (1967). 2) M. Yamaguchi and M. Kandatsu, J. Agr. Chem. Soc. Japan, 41, 521 (1967). tissue. The present study was undertaken using rats at the earlier stages of protein depletion in order to examine the time-course of changes in the amount of protein fractions such as sarcoplasm, myofibril and stroma, and also of changes in the proportion of the muscles distributed in several regions of body. Fur thermore, for reference those changes in rats at the time of death owing to protein deple tion and in rats fed a 60% casein diet were also examined. In two separate experiments, the second one was undertaken as a part of the experiment which was aimed at investigating the over-all catobolic pattern of body proteins under protein depletion. EXPERIMENTAL PROCEDURES Animals and diets. Male adult rats of Wister strain weighing about 350 to 450g were used in both experiments; 12 animals for 4 groups in the first experiment (Experiment 1), and the same number of

2 Studies on the Metabolic Activity of Muscle Proteins. Part III 1373 TABLE I. COMPOSITION OF DIETS IN EXPERIMENT 2 distrophy. Treatments of tissue for assay. At the end of a experimental period, the animals were killed by giv ing a blow to skull and rapidly exsanguinated by decapitation. But those in the group of death owing to protein depletion were only offered with the ex sanguination immediately after their last breath; one of them, however, being treated at ƒ time within 6 * Powder of filter paper (40 to 80 mesh) man ufactured by Toyo Roshi Company, Ltd., Tokyo. ** Prepared as follows to conform to Salts IV of Hegsted et al.3) with a modification of addition of cobalt chloride: (mg per each 100g of diet) CaCO3, 1200; K2HPO4, 1290; CaHPO4 E2H2O, 300; MgSO4 E7H2O, 408; NaCl, 669; FeC6H5O7 E6H2O, 110.0; KI, 3.18; MnSO4 E4H2O, 19.98; ZnCl2, 1.02; CuSO4 E5H2O, 1.20; CoCl2 E6H2O, 0.18 *** (per each 100g of diet) vitamin A, 500IU; vitamin D, 40IU; ƒ -tocopherol, 30mg; vitamin K, 40ƒÁ; thiamine, 200ƒÁ; riboflavine, 300ƒÁ; pyridoxine hydrochloride, 200ƒÁ; cobalamin, 2.2ƒÁ; nicotinamide, 2.0mg; folic acid, 100ƒÁ; calcium pantothenate, 2.0mg; biotin, 10ƒÁ; p-aminobenzoic acid, 100ƒÁ; inositol, 10 mg; choline chloride, 100mg; ascorbic acid, 7.5mg animals for 5 groups in the second experiment (Ex periment 2). All the animals were preliminarily fed a stock diet containing 20% of casein for 8 days. The animals of control group were killed for assey at the end of this period, and others were devided into the following groups: in Experiment 1, those fed on a protein-free diet for 8 and 24 days and on a high-protein diet containing 60% of casein for 12 days respectively, and in Experiment 2, those fed on a protein-free diet for 8, 16 and 32 days and also up to time of death. The composition of diets in Experiment 1 was the same as shown in a previous report,2) except that some of vitamins were varied in content* and that the protein content of the 60% casein diet was made up at the expense of the equal amount of wheat starch of the stock diet. The com positions of diets in Experiment 2 are shown in Table I, in which 5% of cellulose and a larger amount of ƒ -tocopherol were newly added, the latter being expected for the protective effect on muscular * (per each 100g of diet) ƒ -tocopherol acetate.2 mg; pyridoxine hydrochloride, 0.2mg; cobalamin, 2.01ƒÁ; calcium pantothenate, 1.5mg; biotin, 10ƒÁ; p- aminobenzoic acid, 0.1mg; and inositol, 10mg. 3) D. M. Hegsted, R. C. Mills, C. A. Elvehjem and E. E. Hart, J. Biol. Chem., 138, 459 (1941)., 3 hours after death. The muscles and the liver were removed and weigh ed, the latter being analyzed only for total nitrogen. The samples of muscle for protein fractionation were dissected out from the two regions of body: one the muscles of abdomen consisting of obliquus abdominis, transversus abdominis and quadratus lumborum, the other those of hind-limb. They were cut into as small pieces as possible with scissors, the resulting pieces being about 1 to 2 mm cube. These pieces were well mixed and analyzed for protein components. In Experiment 2, moreover, the total amount of muscles was determined in the four regions of body; the dorsum including shoulder, the thoraco-abdomen, the fore-limb and the hind-limb. These muscles were, beforehand, quantitatively removed from the skeleton by immersing in the hot solution of 2% oxalic acid. Protein fractionation. The minced muscles above, each three of 1 g in Experiment 1 and each two of 3 g in Experiment 2, were fractionated by the method of Helander4) with the appropriate modifications as illustrated in Fig. 1. The sample was extracted three times with 10 volumes of each buffer solution. A single course of the extraction was done as follows: first the sample was placed in a porcelain mortar together with about one-fifth of the buffer solution required, and ground with a pestle, secondly the ground muscle was transferred thoroughly to the glass vessel (50ml) of a blender with the rest of the buffer solution and gently homogenized for three minutes cooling with ice, and thirdly the homogenized sample was centrifuged at about 2,500 ~g for five minutes. The soluble fraction was saved for joining to the same fractions followed, and the residue was reextracted in another course. The total nitrogen of each fraction was determined by semi-micro Kjeldahl procedure. The nitrogen of each protein and nonprotein fraction was evaluated as illustrated under the diagram of Fig. 1. 4) E. Helander, "On Quantitative Muscle Protein Determination," Acta Phys. Scandinavia, 41, sup. 141 (1957).

3 1374 Michio YAMAGUCHI and Makoto KANDATSU FIG. 1. Diagram Showing the Analytical Methods for Determination of Nitrogen of Each Protein and Nonprotein Fraction in Muscle. Solid lines denote the processes in Experiment 1, and doted ones those in Experiment 2. RESULTS All the animals seemed normal in their ap pearance throughout the experiment, except those in the group of death owing to protein depletion; in which all the animals became inactive accompanied with manifestations such as alopecia and perioral sore towards the final stage of the depletion and thus survived up to the 106, 114, 122 and 128th days respective ly, the average survival period being 118 days. Body weight of the protein-deficient groups decreased almost linearly in the earlier stages, while that of the 60% casein group increased about 10g for 12 days but with a slight decre ase at first because of a reduction in food intake. The total amount of muscle nitrogen decreased nearly in an exponential manner, its decreasing rate being a little more than that of body weight but far less than that of liver nitrogen (Table II).

4 Studies on the Metabolic Activity of Muscle Proteins. Part III 1375 TABLE II. BODY WEIGHT, NITROGEN OF MUSCLE AND LIVER *1) Number of rats; *2) Mean }standard error; *3) Determined on the m uscles of abdomen and hind limb; *4) Mean of the two rats; *5) This means "initial," but in case of the control group "final"; *6) C alculated from the values of mg/100 g Body weight; *7) Determined on the muscles of whole body. The nitrogen component of each protein and non-protein fraction of muscle showed different responses in the different stages of protein depletion. The sarcoplasmic and the myofibrillar fractions, which are generally con sidered to constitute the so-called intracellular fraction of muscle, decreased much more than the stromal fraction in the earlier stages follow ing the same pattern as seen in reserve pro teins. Furthermore, there seems to exist a slight difference in the rate of reduction be tween these two intracellular components, in which the sarcoplasmic fraction is suggested to have somewhat greater rate of reduction than the myofibrillar fraction as early as 8 days of the depletion. However, it should be noted that the relative proportion between these two fractions did not differ so much as compared with that of the control group on and after the 16th day of protein depletion and even at the time of death about three months later. The changes in the stromal fraction showed a

5 1376 Michio YAMAGUCHI and Makoto KANDATSU TABLE III. CHANGES IN NITROGEN COMPOSITION OF PROTEIN AND NONPROTEIN FRACTIONS OF MUSCLE * Mean }standard error determined on the muscles of abdomen and hind-limb. FIG. 2. Time-course of Percentage Change in Total Amount of Each Protein Fraction of Muscle. \ Groups of protein-free diet, ---- Group of 60% casein diet; Sarcoplasmic protein N, Myofibrillar protein N, ~ Stromal N: Calculat ed from the percentage change in nitrogen com ponent of each fraction (Table III, Values of Conrtol group were taken as 100) respectively multiplied by the percentage change in total a- mount of muscle nitrogen (Table II). much different pattern, in which its relative amount increased at all the stages of depletion, while its absolute amount remained almost unchanged as long as a month of the deple tion but with the subsequent decrease at about the same proportional rate as other fractions (Table III and Fig. 2). In the 60% casein group, on the other hand, the change in total amount of protein of each fraction showed a contrary pattern against that observed in the deficient groups. The sarco plasmic fraction showed a preferential increase in contrast to such a decrease observed on the 8th day of protein depletion, and vice versa in case of the stromal fraction. But the my ofibrillar fraction did not increase so much as expected from the changes of the sarcoplasmic fraction (Fig. 2). As to the proportion of the muscles distribut ed in the four main regions of body under protein depletion, no appriciable differences were detected in the earlier stages of the depletion. But, at the time of death the muscles of dorsum rather occupied the larger part as compared with the ones of the control group, while the muscles of thoraco-abdomen

6 Studies on the Metabolic Activity of Muscle Proteins. Part III 1377 TABLE IV. CHANGES IN PROPORTION OF MUSCLES OF SEVERAL REGIONS OF BODY UNDER PROTEIN DEPLETION * Mean }standard error. and fore-limb the less part and those of hindlimb the equal part (Table IV). DISCUSSION The previous experiments dealing with the changes in protein composition of muscles have, in most cases, been conducted on the muscles under the so-called dystrophic or the atrophic conditions. Although the results of these experiments were not in good accord with one another, Helander's systematic in vestigation4) on the atrophic muscles clearly indicated that all forms of atrophy that were induced by neurotomy, tenotomy and im mobilization gave rise to a diminution of my ofibrillar protein and also to an increase of stromal protein, the latter being most marked after denervation atrophy. On the other hand, those fractional changes in muscle under malnutritional conditions are somewhat different from the above. Mendes and Waterlow5) demonstrated in rats fed a 6.4% protein diet a considerable increase in the amount of collagen, both relative and absolute, regardless of a greater decrease in the total amount of muscle, and they suggest ed that the ratio of non-collagen nitrogen to DNA in muscle might be a useful index of the degree of protein depletion. The similar results were obtained in the avian muscle by Dickerson et al.,6) in which the intracellular 5) C. B. Mendes and J. C. Waterlow, Brit. J. Nutrition, 12, 74 (1958). 6) J. W. T. Dickerson and R. A. McCance, Brit. J. Nutrition, 14, 331 (1960). proteins, especially those of sarcoplasmic frac tion, were clearly more affected than others by underfeeding. Since these two observa tions were made on the young animals under a prolonged malnutrition, the relationship be tween those changes and reserve proteins is not clear. Hagan et al.7) demonstrated the similar changes in the weaning rats completely fasted for 3 to 7 days. Our present study was undertaken to in vestigate the time-course of changes in protein components of muscle under protein depletion during which time at first the so-called re serve proteins might be lost. Therefore, the experiments were conducted in adult rats on and after the 8th day of protein depletion. The results showed that the different protein fractions had the different responses, in which the intracellular fraction, apparently the sar coplasm slightly more than the myofibril, diminished representing the behavior of reserve proteins in muscle. It should be noted that in the 60% casein group the sarcoplasmic fraction increased much more than other frac tions in marked contrast to the case of protein depletion, apparently demonstrating the nature of reserve protein. The sarcoplasmic fraction contains many kinds of enzymes chiefly concerning glycolysis and participates in the intermediate meta bolism for muscular function; while the myofibrillar fraction consisting of myosin and 7) S. H. Hagan and R. 0. Snow, Am. J. Physiol., 188, 91 (1957).

7 1378 Michio YAMAGUCHI and Makoto KANDATSU actin etc. is responsible for the contractility. For this reason, those metabolic disturbance might have any metabolic significance in the relationship between the muscular func tion and the dietary protein level, or pos sibly reserve proteins. A slight difference in metabolic activity between the sarcoplasmic and the myofibrillar fractions is considered partly due to a difference in their metabolic turnover rate. Recently Funabiki and Kanda tsus8) demonstrated some differences in the pattern of metabolic turnover between several fractions of muscle proteins, in which the sarcoplasmic fraction was classified as a type of rapid turnover. The stromal fraction, on the other hand, showed the special pattern perhaps representing the chronological type of metabolic turnover. These results afford a certain evidence for our previous experimental results1,2) that the activities of the enzymes in muscle, such as Granular and Myosin ATPases and Glutamic oxaloacetic transaminase, were reduced in the earlier stages of protein depletion. The fact that the intracellular proteins of muscle in cluding some of enzymes diminish in the exponential manner would support the Bor sook's metabolic concept9,10) in which the 8) R. Funabiki and M. Kandatsu, J. Agr. Chem. Soc. Japan, 39, 109, 157, 257, 307, 501 (1965). 9) H. Borsook and G. L. Keighley, Proc. Roy. Soc. (B), 118, 488 (1935). 10) H. Borsook and J. W. Dubnoff. Ann. Rev. Biochem., 12, 183 (1943). cytoplasmic proteins constituting reserve pro teins undergo breakdown at a rate depending on its mass. But, the real significance of these facts with respect to dietary protein would be clarified by further precise investigations on such a response at the various levels of protein intake. Although many observations on such a frac tional change in liver protein have been made, no distinctive responses have been found except those concerning with enzyme proteins. Therefore, those fractional changes of tissue proteins might be rather specific for muscle, probably because the muscullar tissue is constructed from the different proteins which are structurally and chemically much different from one another. Since muscles distribute in almost the entire part of body, it is necessary to acertain whether or not any part of them is more affected under these experimental conditions. From our results no appriciable differences in pro portion were detected among the muscles distributed in the four main regions of body during the earlier period of protein depletion. Dickerson,6) however, demonstrated that in the undernourished cockerels the pectoral muscles were much more seriously affected than the sartorims. This discrepacy may have arised from the difference in protein components or physiological functions of each muscle between the two species of animals.