THE ULTRASTRUCTURE OF THE NEUROMUSCULAR JUNCTIONS OF MAMMALIAN RED, WHITE, AND INTERMEDIATE SKELETAL MUSCLE FIBERS

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.     

On the relationship of ultrastructural and cytochemical features to color in mammalian skeletal muscle

Summary The semitendinosus muscle of the albino rat is divided grossly into two clearly distinguishable parallel longitudinal bands, one red (anterior) and the other white (posterior). By using mitochondrial content as a criterion for distinguishing fiber types, it is demonstrated that the red portion of the muscle is composed predominantly of red (52%) and intermediate (40%) fibers, while the white portion consists primarily of white fibers (82%). Red fibers have the smallest and white fibers have the largest average diameter. Ultrastructural characteristics of the three fiber types resemble closely those previously described for the rat diaphragm. Red fibers are rich in large mitochondria with abundant cristae, and possess the widest Z lines. In red fibers, the H-band region of the sarcoplasmic reticulum consists of an elaborate network of narrow tubules. In white fibers, mitochondria are smaller, less numerous, and have fewer cristae; Z lines are about half as wide as in red fibers. In the H-band region of the sarcoplasmic reticulum there is a more compact arrangement of broad more or less parallel tubules. Intermediate fibers are similar to red fibers except that their diameters are larger; mitochondria are somewhat smaller and cristae are less abundant; the width of the Z lines is close to that of white fibers. The consistent difference in Z line width establishes this dimension as an important criterion for distinguishing fiber types and facilitates ultrastructural identification, especially of the intermediate fiber.The clear relationship between color of the semitendinosus and cytological features of its component fibers supports the use of the terms red, white, and intermediate as simple and valid designations for fiber types in mammalian skeletal muscle. Measurement of the cross-sectional area contributed by each fiber type to the total area indicates that both red and intermediate fibers may contribute to redness in mammalian skeletal muscle.An early portion of this work was carried out with MissSharon Whelan (Mrs.Bernard Weiss). The author acknowledges the important contribution of Mr.Richard Stearns through his skillful work on the photographic illustrations and the technical assistance of MissAnn Campbell and Mrs.Joan Normington. — This study was supported by Grant No. HD 01026-04 from the United States Public Health Service.     

CYTOCHEMICAL STUDIES OF ADENOSINE TRIPHOSPHATASES IN SKELETAL MUSCLE FIBERS

Mitochondrial ATPase and myosin ATPase have been localized in the muscle fibers of the rat diaphragm. The principal fiber type possesses a structure favorable for making this cytochemical separation with the light microscope. This small red fiber has numerous large, nearly spherical, mitochondria (ca. 1.5 µ) which are aggregated beneath the sarcolemma. In the interior of the fiber, smaller paired filamentous mitochondria (ca. 0.2 µ diameter) are aligned with the I band. Distribution of mitochondria was determined by sudanophilia, succinic dehydrogenase activity, and by direct examination with the electron microscope. ATPase activity at pH 7.2 is located in the large peripheral mitochondria and in the smaller mitochondria associated with the I band. The alignment of the small mitochondria results in a discrete cross-striated appearance in fibers stained for this enzymic activity. This mitochondrial ATPase does not cleave adenosine diphosphate or adenosine monophosphate; it is not sulfhydryl dependent and, in fact, is enhanced by the mercurial, p-hydroxymercuribenzoate. It requires magnesium ion and is stimulated by dinitrophenol. It is inhibited after formol-calcium fixation, but the residual activity is demonstrable by lengthening the incubation time. At pH 9.4 the ATPase is myofibrillar in origin and is located in the A bands. This myosin ATPase activity is sulfhydryl-dependent. Mercurial at this high pH has an interesting dual effect: it suppresses myosin ATPase but evokes mitochondrial ATPase activity. A third type of ATPase activity can be demonstrated, especially in the large white fibers. This activity occurs at pH 7.2 in the presence of cysteine. Its position is manifested cytochemically as a fine reticular pattern which surrounds individual myofibrils. The distribution suggests that it may originate in the sarcoplasmic reticulum.     

Capillarization,mitochondrial densities,oxygen diffusion distances and innervation of red and white muscle of the lizard Dipsosaurus dorsalis

Summary The white and red regions of the iliofibularis muscle of the lizard Dipsosaurus dorsalis were analyzed using histologic and morphometric analysis. These regions are composed of fast glycolytic (FG) and both fast oxidative, glycolytic (FOG) and tonic fibers, respectively. Endplate morphology and number of endplates per fiber were estimated from fibers from both areas. Capillary volume densities of the red and white regions were quantified from transverse sections. Mitochondrial volume of fibers from the red and white regions were estimated from electron micrographs.All fibers from the white region of the iliofibularis possessed a single, well defined endplate, as did most red region fibers. The remaining red fibers (28±5%) possessed an average of 14.7±3 endplates each, distributed along the entire length of the fiber at intervals of approximately 1124 m.Red fibers possessed twice the mitochondrial volume of white fibers (7.6±0.4%, red; 3.8±0.3%, white). Mitochondria were distributed uniformly through the fibers from both regions. Capillary anisotropy was low ( = 1.018) in both regions. Capillary densities of the red region (629±35 mm^-2) were much greater than those of the corresponding White region (73±8 mm^-2).The data indicate that capillary densities, mitochondrial volumes and theoretical diffusion distances correlate well with the oxidative capacity of lizard muscle fibers. Tonic fibrs of this species appear oxidative and therefore metabolically capable of functioning during locomotion. The similar mitochondrial volumes and capillary densities of reptilian and mammalian muscles suggest that the greater oxidative capacity of mammalian muscle is due in part to possession of more oxidatively active mitochondria rather than to possession of more mitochondria per se.     

CYTOLOGICAL STUDIES OF FIBER TYPES IN SKELETAL MUSCLE : A Comparative Study of the Mammalian Diaphragm

A comparative investigation of the mammalian diaphragm has revealed a correlation between certain cytological aspects of red and white muscle fibers and functional activity. This skeletal muscle presents the advantage of a similar and constant function among the mammals, but its functional activity varies in a quantitative manner. Both the rate of breathing (and hence the rate of contraction of the diaphragm) and metabolic activity are known to be inversely related to body size; and this study has demonstrated a relationship between cytological characteristics of the diaphragm and body size of the animal. Small fibers rich in mitochondria (red fibers) are characteristic of small mammals, which have high metabolic activity and fast breathing rates; and large fibers with relatively low mitochondrial content predominate in large mammals, which have lower metabolic activity and slower breathing rates. In mammals with body size intermediate between these two groups (including the laboratory rat), the diaphragm consists of varying mixtures of fiber types. In general, the mitochondrial content of diaphragm fibers is inversely related to body size. It appears, then, that the red fiber reflects a high degree of metabolic activity or a relatively high rate of contraction within the range exhibited by this muscle.     

Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure and arrangement of mitochondria in the red, white and intermediate striated muscle fibers of the rat were examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by means of the Osmium-DMSO-Osmium procedure.Beneath the sarcolemma, spherical or ovoid subsarcolemmal mitochondria show accumulations. The mitochondria are numerous and large in size in the red fibers, intermediate in the intermediate fibers, and few and small in the white fibers. Paired, slender I-band-limited mitochondria were located on both sides of the Z-line and partly embraced the myofibrils at the I-band level; they occurred in all three types of fibers. In the intermyofibrillar spaces, numerous mitochondria formed mitochondrial columns. These columns were classified into two types: 1) thick mitochondrial columns, formed by multiple mitochondria each with an intermyofibrillar space corresponding to one sarcomere in length, and 2) thin mitochondrial columns, established by single mitochondria corresponding to one sarcomere in length. In the red fibers mitochondrial columns were abundant and the ratio of the thick and thin columns was almost the same, while in the intermediate fibers most of the columns belonged to the thin type. The white fibers displayed rare, very thin columns.     

8-Oxoguanosine and uracil repair of nuclear and mitochondrial DNA in red and white skeletal muscle of exercise-trained old rats.

Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.     

Alteration in skeletal muscle mitochondria of cold-acclimated rats: Association with enhanced metabolic response to noradrenaline

This paper reports a search for structural changes in skeletal muscle mitochondria of cold-acclimated rats. Histochemical studies (succinic dehydrogenase) show that there appears to be a higher proportion of red fibers in the semitendinosus muscle of the cold-acclimated rat and that the white region of this muscle contains fibers which resemble intermediate fibers. Electron micrographs show an apparently larger number of small mitochondria in both red and white fibers. Counts of mitochondria isolated from skeletal muscle show that there are more mitochondria per gram of both red and white muscle in the cold-acclimated rat than in the non-acclimated control rat. Each mitochondrion contains less protein and less cytochrome oxidase. Thus the mitochondrial mass per gram of red and white muscle is not altered, as indicated by the unchanged content of mitochondrial protein and of cytochrome oxidase per gram of muscle. Thus there appears to be a repackaging of mitochondrial material into smaller units in the skeletal muscle of the cold-acclimated rat. The alteration is shown to be associated with the adaptive state of the rat. No change occurs in muscle mitochondria of cold-acclimated rats in which the development of the enhanced metabolic response to noradrenaline, a measure of the extent of adaptation, is inhibited by treatment with oxytetracycline. The change in skeletal muscle mitochondria disappears when the enhanced metabolic response to noradrenaline in rats which are already cold-climated is reversed by treating the rats with oxytetracycline while they continue to live in the cold. The change in muscle mitochondria also disappears when the cold-acclimated rat undergoes deacclimation after return to room temperature. The alteration in muscle mitochondria is thus not associated either with shivering or with a high metabolic rate. Skeletal muscle of the cold-acclimated rat is known to be an important site of heat production in the course of nonshivering thermogenesis; that is, it can undergo a considerable increase in metabolic rate in the absence of shivering on exposure of the cold-acclimated rat to cold. The metabolic basis of nonshivering thermogenesis is in an enhanced capacity of the tissues of the cold-acclimated rat, principally skeletal muscle, to respond by an increase in metabolic rate to the large amounts of noradrenaline secreted by the nerve endings of the sympathetic nervous system as a consequence of cold-exposure. The mechanism by which the metabolic response to noradrenaline in the cold-acclimated rat can be enhanced is unknown. The structural alteration observed in the skeletal muscle mitochondria of the cold-acclimated rat may indicate a functional alteration responsible for the enhanced capacity of the muscle to respond to noradrenaline by an increase in metabolic rate.     

Ultrastructural identification of muscle fiber types by immunocytochemistry

In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.     

PLURIVESICULAR SECRETORY PROCESSES AND NERVE ENDINGS IN THE PINEAL GLAND OF THE RAT

The pineal body of white normal rats, 1.5 to 3 months old, was studied under the electron microscope. A single type of parenchymal cell—the pinealocyte—is recognized as the main component of the tissue, and some of the structural characteristics of the nucleus and cytoplasm are described. The main morphological characteristic of the pinealocytes is represented by club-shaped perivascular expansions connected to the cell by thin pedicles. They are found lying in a large, clear space surrounding the blood capillaries. The name plurivesicular secretory processes is proposed, to emphasize the main structural feature and the probable function of these cellular expansions. A tubulofibrillar component is mainly found in the pedicle, and within the expansion there are numerous small mitochondria and densily packed vesicles of about 425 A. Two types of vesicles, one with a homogeneous content and another with a very dense osmium deposit, are described. Between the two types there are intermediary forms. In these processes, mitochondria show profound changes which may lead to complete vacuolization. The significance of this plurivesicular secretory component is discussed in the light of recent work on the biogenic amines of the pineal body and preliminary experiments showing the release of the vesicles containing dense granules after treatment with reserpine. These vesicles are interpreted as the site of storage of some of the biogenic amines. Bundles of unmyelinated nerve fibers and endings on large blood vessels which also contain a plurivesicular content are described and tentatively interpreted as adrenergic nerve terminals.     

High -resolution scanning electron-microscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog

Summary The three-dimensional structure of the mitochondria and sarcoplasmic reticulum (SR) in the three types of twitch fibers, i.e., the red, white and intermediate skeletal muscle fibers, of the vastus lateralis muscle of the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) was examined by high resolution scanning electron microscopy, after removal of the cytoplasmic matrices.The small red fibers have numerous mitochondrial columns of large diameter, while the large white fibers have a small number of mitochondrial columns of small diameter. In the medium-size intermediate fibers, the number and diameter of the mitochondrial columns are intermediate between those of the red and white fibers.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of each Z-line. The thick terminal cisternae continue into much thinner flat intermediate cisternae, through a transitional part where a row of tiny indentations can be observed. Numerous slender longitudinal tubules originating from the intermediate cisternae, extend longitudinally or obliquely and form elongated oval networks of various sizes in front of the A-band, then fuse to form the H-band collar (fenestrated collar) around the myofibrils. On the surface of the H-band collar, small fenestrations as well as tiny hollows are seen. The three-dimensional structure of SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a smaller total volume of SR than the mitochondria-poor white fiber. The volume of SR of the intermediate fiber is intermediate between other the two.     

Protein composition and function of red and white skeletal muscle mitochondria

Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PAGE, two-dimensional differential in gel electrophoresis (2D DIGE), optical spectroscopy, and isobaric tag for relative and absolute quantitation (iTRAQ). Complex IV and V activities were compared using BN-PAGE in-gel activity assays, and maximal mitochondrial respiration rates were assessed using pyruvate (P) + malate (M), glutamate (G) + M, and palmitoyl-carnitine (PC) + M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon removal of contamination, very few protein differences were observed between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I-V or the activities of Complexes IV and V. iTRAQ analysis detected 358 mitochondrial proteins, 69 statistically different. Physiological significance may be lower: at a 25% difference, 48 proteins were detected; at 50%, 14 proteins were detected; and 3 proteins were detected at a 100%. Thus any changes could be argued to be physiologically modest. One area of difference was fat metabolism where four β-oxidation enzymes were ～25% higher in red mitochondria. This was correlated with a 40% higher rate of PC+M oxidation in red mitochondria compared with white mitochondria with no differences in P+M and G+M oxidation. These data suggest that metabolic demand differences between red and white muscle fibers are primarily matched by the number of mitochondria and not by significant alterations in the mitochondria themselves.     

Age-related alterations of mitochondria from muscle tissue

The ultrastructure of mitochondria of cross-striated muscles during aging was studied by electron microscopy. Mitochondrial ultrastructure was analyzed in the flight muscle of D. melanogaster (1- and 36-day-old) and in the cardiomyocytes and skeletal muscle of young and senile Wistar and OXYS rats (3- and 25-month-old). The mitochondria in the flight muscle samples of senile D. melanogaster flies were shown to have several types of peculiar age-related mitochondrial abnormalities corresponding to those described previously. Previously unknown changes were revealed in the ultrastructure of cardiomyocyte mitochondria in senile rats (both Wistar and OXYS). Substantial changes in the ultrastructure of subsarcolemmal mitochondria were found in the fibers of red skeletal muscle of senile OXYS rats. It has been shown that the subsarcolemmal mitochondria of red muscle fibers are a peculiar population of mitochondria with atypical ultrastructure. Initial changes in the ultrastructure of subsarcolemmal mitochondria were revealed even in 3-month-old OXYS rats. At the same time, the skeletal muscle mitochondria of senile Wistar rats maintain their morphological characteristics, and their ultrastructure corresponds to that of skeletal muscle mitochondria in 3-month-old Wistar rats.     

SEEDLING GROWTH AND SURVIVAL OF RED AND WHITE FIR IN A SIERRA NEVADA ECOTONE

We hypothesized that differential seedling establishment was responsible for the transition from white fir- to red fir-dominated forest. Nearly 2,000 seeds of each species were sown within six fenced gardens that spanned the ecotone (1,902–2,073 m). Germination and seedling mortality were monitored through the first growing season. Red fir germination percentage exceeded that of white fir in all gardens in sites dominated by red fir saplings and overstory trees, and a similar pattern was true for white fir. Survival at the end of the first growing season was low and statistically similar for both species, as was the average length of life for seedlings and the causes of mortality. Relative demographic performances (an integrated measure of germination and survivorship over time) suggest that: 1) open/xeric microhabitats within the ecotone favor white fir over red fir; 2) both open/mesic and shaded/xeric microhabitats favor red fir over white fir; and 3) shaded/mesic microhabitats are equally favorable for both species. Seedlings of red and white fir were grown for 6 mo in a growth chamber that imitated early summer temperature and soil moisture conditions. Red fir seedlings were consistently heavier than white fir seedlings, in part a result of the 2.5 × greater caloric content of red fir seed. Relative growth rates were similar for both. Allocation of photosynthate to roots was greater for red fir than white fir, a result contrary to the expected pattern, given that white fir occurs at more xeric, lower elevations than red fir. No consistent differences between the species occurred in root growth or morphology.     

THE ULTRASTRUCTURE OF Z DISKS FROM WHITE, INTERMEDIATE, AND RED FIBERS OF MAMMALIAN STRIATED MUSCLES

The Z disk ultrastructure of white, intermediate, and red fibers from mammalian muscle is examined. Three models are proposed that explain the differences between the three types of Z disk. The three models are all based on the same concept, i.e., looping filaments from both sides of the Z disk. The differences between the models are in terms of the spatial relationships of adjacent loops within the Z disk. In the white fiber Z disk model all the loops from one side of the Z disk are on the same plane. In intermediate fibers there are two planes of loops from both sides of the Z disk, whereas in red fibers there are three planes of loops from both sides. The implications of these three structures are discussed in relation to known physiological differences between the fiber types.     

Skeletal Muscle Contractions Induce Acute Changes in Cytosolic Superoxide,but Slower Responses in Mitochondrial Superoxide and Cellular Hydrogen Peroxide

Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.     

Changes in the ultrastructure of muscle fibers of the diaphragm in response to physical loading (morphometric analysis)

Red and white fibers have been investigated in the diaphragm of white rats, normal and under effect of physical loadings, various in intensity and duration. Relative volume and amount of lipid inclusions and mitochondria and their profiles and amount of glycogen granules have been estimated per one unit of the fiber section area. In unadapted animals acute physical loadings result in exhaustion of the reserves in the areas with a high volumetric density of mitochondria. These changes are mostly pronounced in the red fibers. Under a prolonged effect of the physical loadings of the training regimen, muscle fibers of both types have similar ultrastructural changes, demonstrating certain increase in their oxidative potential.     

Polymorphism of myosin among skeletal muscle fiber types

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.`     

STUDIES ON THE CONTENT AND ORGANIZATION OF THE RESPIRATORY ENZYMES OF MITOCHONDRIA

(1) The mathematical calculations relating spectrophotometric data with the data of Allard et al. (4, 5) on mitochondrial counts, is presented. Such a calculation indicates that an "average mitochondrion" from rat liver would contain about 17,000 molecules of each cytochrome pigment. (2) Hematocrit determinations relating respiratory pigment content for mitochondria isolated from a variety of tissues have been presented, showing a fivefold variability depending upon the source of the mitochondria. (3) Speculations on the organization of the respiratory enzymes associated with the membrane structure of the mitochondria are discussed.