Red Skeletal Muscle Research Articles

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles.

We employed solid-phase immunochemical methods to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscle. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

The cytosolic redox is coupled to VO2. A working hypothesis.

In aerobic tissues the match between energy demand (ATPase activity) and energy supply (ATP production) requires close integration of the glycolytic and mitochondrial metabolic controls. Since most ATPase activity occurs in the cytosol this integration must be mediated by cytosolic signals. While there is uncertainty about the mechanism, there is general agreement that recruitment of mitochondrial ATP production is strongly coupled to changes in cytosolic phosphorylation state. Although the role of mitochondrial redox has been identified, cytosolic redox has been left out of models of mitochondrial control. Results from three different kinds of studies suggest that in the cell there is a strong interaction between the mitochondrial and cytosolic redox states and mitochondrial metabolism. These results include studies on: 1) intact tissues such as liver (Berry et al, 1980), heart (Kauppinen et al, 1983) and red skeletal muscle (Connett et al, 1986); 2) reconstituted glycolytic and mitochondrial systems (Jong & Davis, 1983) and 3) metabolic models of heart metabolism that include kinetic submodels of most of the individual enzymes (Kohn, 1983). The objective of this paper is to describe a distributed equilibrium hypothesis that accounts for a cooperative interaction between the glycolytic and oxidative metabolic systems under aerobic conditions.

Protein synthesis in tissues of fed and starved carp, acclimated to different temperatures

The temperature dependence of the rates of protein synthesis in the red and white skeletal muscle of the carp (Cyprinus carpio) was measured using a method which involved a single injection of tritiated phenylalanine. Plasma and muscle-free phenylalanine quickly reached a plateau level at all temperatures. During the plateau phase the incorporation of label into protein was liner. Muscle from fish previously acclimated to either a low temperature (8°C) or a high temperature (28°C), showed marked differences in the rates of protein synthesis. The results show that cold acclimation is associated with significantly higher rates of protein synthesis (p<0.001) in both red and white muscle. Arrhenius activation energies, derived from the rates of protein synthesis at the different experimental temperatures, were similar for both red and white muscle in fish acclimated to warm or cold temperatures. Measurements for both acclimated groups over the temperature range 8-34°C showed that the activation energy for the process of protein synthesis was 86.7 kJ/mol and 78.7 kJ/mol for the red and white muscle respectively.

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.

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.

Induction of an increase in mitochondrial matrix enzymes in muscle of iron-deficient rats.

Young rats maintained on an iron-deficient diet developed severe anemia and had large decreases in the levels of the iron-containing flavoproteins and cytochromes of the mitochondrial respiratory chain in skeletal muscle. In contrast, the levels of a number of mitochondrial matrix marker enzymes, including citrate synthase, isocitrate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and aspartate aminotransferase, increased in red skeletal muscle but not in white muscle. Phosphocreatine concentration was decreased and inorganic phosphate concentration was increased in soleus muscle frozen in situ. We hypothesize that the increase in mitochondrial matrix enzymes reflects a stimulus to mitochondrial biogenesis in posture-maintaining and weight-bearing red muscle fibers in severely iron-deficient rats. It is our working hypothesis that this stimulus to mitochondrial biogenesis arises from mild activity of the red fibers and is due to the same perturbation in cellular homeostasis that is normally caused by vigorous exercise or hypoxia. In iron deficiency, the stimulus to mitochondrial biogenesis can induce an increase in only those enzymes not prevented from increasing by iron deficiency, resulting in formation of mitochondria of grossly abnormal composition.

A Comparison of Plasma, White Blood Cell, Red Blood Cell, and Tissue Distribution of Amiodarone and Desethylamiodarone in Anesthetized Dogs

Desethylamiodarone (DA) is a major metabolite of amiodarone (AM), a Class III antiarrhythmic drug. The plasma pharmacokinetics and tissue distribution of AM and DA (10 mg/kg i.v.) were compared in anesthetized dogs. Plasma, white blood cell (WBC), red blood cell (RBC), liver, and skeletal muscle samples were obtained at frequent intervals up to 6 h after a single i.v. bolus of the two drugs. Drug concentrations in these and other tissues, i.e., lung, kidney, heart (right and left atrium, right and left ventricle, Purkinje fibers, and AV node), and femoral nerve were measured by a highly sensitive and specific high-pressure liquid chromatographic technique developed in our laboratory. Four different patterns of AM and DA uptake and washout could be identified in these experiments. The first pattern is biexponential decline in plasma drug levels with a rapid distribution phase (t1/2 alpha = 5.1 +/- 2.1 min for AM and 5.5 +/- 1.2 min for DA, respectively) and a slower elimination phase (t1/2 beta = 3.7 +/- 1.3 h for AM and 4.96 +/- 0.8 h for DA, respectively). The volume of distribution of DA was significantly larger than that of AM. The second pattern is that both WBCs and RBCs showed an initial uptake within 5 min followed by a biexponential decrease in drug levels, with t1/2 alpha similar to that in plasma but t1/2 beta significantly longer than in plasma. In both these types of cells, the elimination half-life for DA was significantly longer than that of AM. The third pattern is that in the liver there was a rapid uptake of both drugs with peak concentrations at 15 min; the decline in hepatic levels of AM was biexponential, but that of DA appeared to be monoexponential. In addition, in dogs given AM alone, the metabolite (DA) was easily detected in the liver from the earliest time of measurement, suggesting that the parent drug is rapidly metabolized to DA. In the experiments where DA was injected, two new peaks were also identified in the liver suggesting that DA was metabolized further in the liver. The fourth pattern was in the skeletal muscle, where AM uptake was relatively slow, reaching peak concentrations between 1.5-2 h followed by a monoexponential decline; however, DA was rapidly taken up by skeletal muscle, but the rate of decline appeared to be slower as compared to that of AM.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue-Specific Controls on Carbohydrate Catabolism during Anoxia in Goldfish

Tissue-specific controls on carbohydrate metabolism regulate the response to anoxia in goldfish (Carassius auratus) and bring about the overall metabolic rate depression that is a key feature of anoxia tolerance. The effect of anoxia (24 h at 7 C under N2/ CO atmosphere) on glycogen phosphorylase activity and on the content of fructose2,6-bisphosphate was determined in eight tissues (liver, brain, kidney, gill, spleen, heart, and red and white skeletal muscle) of goldfish. Anoxia resulted in an increase in the percentage of phosphorylase in the active a form in brain but significantly reduced phosphorylase-a content in liver, kidney, spleen, and heart; total phosphorylase activity (a + b) was also reduced during anoxia in liver and gill. Levels of the phosphofructokinase activator, fructose-2,6-P2, dropped 10-fold in liver during anoxia and were also reduced in gill and spleen. Brain and heart showed the opposite response, a 3.5-fold rise in fructose-2,6-P2 during anoxia. The data support a relative decrease in glycolytic flux in tissues such as liver, spleen, gill, and kidney (in line with a general metabolic rate depression) and a relative increase in glycolysis in brain and heart. In liver, the sharp drop in fructose-2,6-P2 content is probably also important in directing glycogenolysis toward glucose export into the blood.

Carbonic anhydrase III: a new histochemical marker for myoepithelial cells.

Carbonic anhydrase III, a major protein of red skeletal muscle, was localized immunohistochemically in smooth muscle cells of human uterus and in myoepithelial cells of mammary and prostate glands. The presence of this antigen in myoepithelial cells could be used to detect these not easily recognizable cells in paraffin-embedded tissue sections. Some epithelial cells of larger mammary ducts revealed occasional strongly positive cells. These cells may represent a new epithelial cell type transitional between myoepithelium and ductal epithelium.

Measurement of rat heart fatty acid binding protein by ELISA. Tissue distribution, developmental changes and subcellular distribution

A class of soluble, low molecular weight proteins collectively called fatty acid binding proteins (FABP) are thought to function in the intracellular movement of fatty acids. To understand more clearly the role of FABP in cardiac metabolism, we used ELISA and immunoblotting techniques to study the distribution of heart FABP in several rat tissues, compare male and female rat heart content, quantitate developmental changes, and determine its subcellular distribution. Immunoreactive protein was found in appreciable amounts in rat heart, red skeletal muscle and kidney. Adult rat heart contained about 1.5 mg FABP/g tissue wet weight with the atrial content being approximately 50% of the ventricular concentration. No significant difference was detected between the sexes. The amount of FABP increased progressively during development from fetal to adult animals, and measureable amounts were found in 17-day-old fetal tissue. Comparisons between myoglobin and FABP showed that FABP appeared earlier than myoglobin in development, but myoglobin was more abundant than FABP at birth. Using immunoblots it was determined that rat heart FABP was localized in the cytosol with no detectable intramitochondrial material.

The Neonatal Piglet as a Model for Human Neonatal Carnitine Metabolism

Investigations concerning carnitine metabolism and possible requirements for exogenous carnitine in human preterm neonates are limited by ethical considerations. The neonatal piglet is a potential animal model for these investigations. Tissue carnitine concentrations were determined in fetuses from cross-bred domestic gilts at stages of gestation corresponding to those of neonates found in neonatal intensive care units. Fetal piglet plasma and red blood cell carnitine levels decreased from approximately 90 d to term. Skeletal muscle carnitine increased from 60 d to term. Temporal changes in fetal carnitine concentrations in plasma, red blood cells and skeletal muscle throughout gestation are similar to the pattern reported by our laboratory for the human neonate. Cardiac muscle carnitine increased earlier than skeletal muscle but also continued to increase to term. Carnitine concentrations in fetal liver, kidney and intestine were maximal at 90 d and decreased until term. Similarities in physiology, metabolism and profiles of tissue carnitine concentration between the newborn piglet and the human neonate indicate that the neonatal piglet is an appropriate animal model for investigations concerning neonatal carnitine metabolism.

Demonstration of creatine kinase in myocardial and skeletal muscle using the semipermeable membrane technique

The histochemical method as described by Sjövall (1967) for the detection of creatine kinase activity in myocardium and skeletal muscle has been modified by the introduction of a semipermeable membrane method. With this technique a semipermeable membrane is interposed between incubating solution and tissue sections preventing diffusion of the enzyme into the medium during incubation. A very high enzyme activity was demonstrated along myofibrils and probably also in mitochondria. Addition of ZnCl2 or 2,4-dinitrofluorobenzene to the incubation medium prevented the production of formazan, confirming the specificity of the staining reaction. Creatine kinase activity was observed in both white (type II) and red (type I) skeletal muscle fibres without notable difference in level of activity.

Does myoglobin contribute significantly to diffusion of oxygen in red skeletal muscle?

We have examined the role of myoglobin to facilitate O2 diffusion to active mitochondria in skeletal muscle by constructing computer-simulation experiments. Steady-state mitochondrial O2 consumption under different conditions of supply partial pressure of O2 (PO2) in a system with and without myoglobin were examined for a one-dimensional slab of tissue. O2 consumption by mitochondria was saturable with the mitochondria located in bands at uniform intervals throughout the tissue. Under these conditions, myoglobin provides a measurable increase in O2 transport for supply PO2 below 10 Torr and diffusion lengths expected for skeletal muscle fibers. We conclude that under circumstances where hypoxia lowers PO2 below 10 Torr that myoglobin begins to provide a measurable increase in O2 delivery to mitochondria.

Phase-related changes in tissue energy reserves during hemorrhagic shock

In view of the well-known fact that the liver is more sensitive to ischemia than skeletal muscle, it was the purpose of the present study to determine the relationship between the hemorrhage-induced changes in plasma glucose and lactate concentrations and the status of the energy reserves of these two tissues. Sprague-Dawley rats were bled to a constant mean arterial blood pressure of 40 mm Hg and held there by removal or reinfusion of blood. The stages of shock defined on the basis of the net blood loss were early compensatory, maximal compensatory, early decompensatory, and late decompensatory phases. The results showed a depletion of hepatic ATP levels which occurred between the early compensatory and maximal compensatory phases of shock, coincident with the most dramatic increases in plasma glucose and lactate seen during the shock protocol. Hepatic ATP levels fell no further through the decompensatory phases of shock while plasma glucose declined to hypoglycemic levels and plasma lactate was maintained at the same high level attained at the maximal compensatory phase. Since hepatic sources of glucose were exhausted by the maximal compensatory phase and hepatic energy stores were depleted to a point which precludes significant gluconeogenesis, the large increase in plasma lactate was probably largely due to loss of the hepatic sink for lactate during this phase of shock. In contrast to the liver, soleus muscle showed no change in the levels of glycogen, ATP, CrP, free creatine, or total creatine compared to time-matched controls in any phase of hemorrhagic shock suggesting the absence of significant muscle ischemia. The possibility that red skeletal muscle may act as a sink for lactate is considered.

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis.

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase.

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.

A model study of intracellular oxygen gradients in a myoglobin-containing skeletal muscle fiber

A theoretical two-dimensional model is used to investigate oxygen gradients in a red skeletal muscle fiber. The model describes the steady state, free and myoglobin-facilitated diffusion of oxygen into a respiring cylindrical muscle fiber cross section. The oxygen tension at the sarcolemma is assumed to vary along the sarcolemma as an approximation to the discrete capillary oxygen supply around the fiber. Maximal oxygen gradients are studied by considering parameters relevant to a maximally-respiring red muscle fiber. The model predicts that angular variations in the oxygen tension imposed at the sarcolemma due to the discrete capillary sources do not penetrate deeply into the fiber over a range of physiological values for myoglobin concentration, diffusion coefficients, number of surrounding capillaries, and oxygen tension level at the sarcolemma. Also, the oxygen tension in the core of the fiber is determined by the average oxygen tension at the sarcolemma. The drop in oxygen tension from fiber periphery to core, however, does depend significantly on the myoglobin concentration, the oxygen tension level at the sarcolemma, and the oxygen and myoglobin diffusivities. This dependence is summarized by calculating the minimum average sarcolemmal oxygen tension for maximal respiration without the development of an intracellular anoxic region. For a myoglobin-rich muscle fiber (0.5 mM myoglobin), the model predicts that maximal oxygen consumption can proceed with a relatively flat (less than 5 mm Hg) oxygen tension drop from fiber periphery to core over a large range for diffusion coefficients.

Intracellular Buffering and Oxygen Transport in the Pacific Blue Marlin (Makaira nigricans): Adaptations to High-Speed Swimming

Blood obtained from Pacific blue marlin captured by hook and line displayed a pronounced acidosis by teleost standards (pH 7.22 ± 0.07 at 25 C), a high PACO₂ (17.3 ± 3.0 torr) and a low PAO₂ (9.7 ± 3.0 torr). Notwithstanding, these properties reflect a major anaerobic component contributing to the acid-base status of blood originating from muscle glycogenolysis during the capture period. This view is consistent with the finding that blood lactate concentration was linearly related to fight time. Marlin is exceptional in its ability to tolerate protons. The buffering capacities (β) of white and red skeletal muscles are 103.8 ± 5.2 slykes and 50.8 ± 3.9 slykes, respectively. The difference between the buffering capacities of the two muscle types likely is related to different rates of adenosine triphosphate (ATP) production and to the different fate of metabolic protons generated in each tissue. The in vitro nonbicarbonate buffering value of whole blood (ΔHC0₃⁻/ApH) also is high when compared with that of o...

Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle.

Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0.1-0.4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.) Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-1). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 X 10(-8) M and similar but not identical to that of cytosolic carbonic anhydrase II. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates (Siffert & Gros, 1982) originates from the s.r. Histochemical studies using the dansylsuphonamide method described previously (Dermietzel, Leibstein, Siffert, Zamboglou & Gros, 1985) showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre cross-sections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres. It is estimated that s.r. carbonic anhydrase accelerates CO2 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3- react fast enough to provide a rapid source and sink for protons leaving and entering the s.r. in exchange for Ca2+.

Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.