Carbonic anhydrase activity in skeletal muscle fiber types, axons, spindles, and capillaries of rat soleus and extensor digitorum longus muscles

Carbonic anhydrase (CA) activities were studied in soluble extracts and cryostat sections of skeletal muscles from prepubertal and postpubertal rats. Acetazolamide inhibition was utilized to distinguish between activities of the acetazolamide-sensitive (CA I and II) and acetazolamide-resistant (CA III) forms of the enzyme. The inhibition studies indicated that fast-twitch oxidative-glycolytic muscle fibers contained both the sensitive and resistant forms of CA. Acetazolamide-sensitive activity was localized within muscle fibers, axons, myelin, and capillaries. Axoplasmic staining was restricted to subpopulations of myelinated axons in both the dorsal and ventral roots. Soleus muscles exhibited significantly greater activity of CA III than extensor digitorum longus muscles at all ages examined. CA III was richest in slow-twitch oxidative and intrafusal fibers. During puberty, soleus muscle fibers matured and converted from fast-twitch oxidative-glycolytic to slow-twitch oxidative fibers. There was a shift from the sensitive to the resistant form of CA; CA III activity increased about sevenfold. This activity peaked earlier in the muscles of female rats than male rats. These results demonstrated a complex distribution of CA isozymes in the neuromuscular system and pointed out that isozyme content depends on both the type of muscle and the age and sex of the animal.     

Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

Summary 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.     

Quantification of carbonic anhydrase III and myoglobin in different fiber types of human psoas muscle

Summary Carbonic anhydrase (CA III) and myoglobin contents from isolated human muscle fibers were quantified using a sensitive time-resolved fluoroimmunoassay. Human psoas muscle specimens were freeze-dried, and single fibers were dissected out and classified into type I, IIA and IIB by myosin ATPase staining. Fiber typing was further confirmed by SDS-PAGE. CA III and myoglobin were found in all fiber types. Type I fibers contained higher concentrations of CA III and myoglobin than type IIA and IIB fibers. The relative concentrations of CA III in type IIA and IIB fibers were respectively 24% and 10% of that in type I fibers. The relative concentrations of myoglobin in type IIA and IIB fibers were 60% and 28% of that in type I fibers. Anti-CA III immunoblotting results from fiber-specific pooled samples agreed well with quantitative measurements. The results indicate that CA III is a more specific marker than myoglobin for type I fibers.     

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span

Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.     

Thyroidectomy significantly alters carbonic anhydrase III concentration and fiber distribution in rat muscle

Thyroidectomy has a dramatic effect on rat muscle, greatly increasing the number of Type I fibers and the concentrations of carbonic anhydrase III (CAIII) in the muscle. Carbonic anhydrase III is not confined to the Type I fibers, as was previously believed, but also occurs in fibers that exhibit a level of ATPase staining less than that of 2A fibers but greater than 2B. These fibers are rare in normal muscle but become numerous after thyroidectomy, when they stain heavily for CAIII.     

Carbonic anhydrase isozyme distribution and characterization in metabolic fiber types of the dorsal levator muscle of the blue crab, Callinectes sapidus

Carbonic anhydrase (CA) distribution and characterization were examined in white and light pink fibers of the dorsal levator muscle of the blue crab. White fibers were structurally and metabolically characterized as fast twitch glycolytic, while the light pink fibers were fast oxidative. All subcellular fractions of both fiber types had significant levels of CA activity; cytoplasmic and microsomal activity was significantly higher in light pink vs white fibers. Cytoplasmic CA from both fiber types was highly sensitive to the inhibitors acetazolamide and chlorzolamide, with Ki values of approximately 2 and 0.4 nM, respectively. Further analysis confirmed that cytoplasmic CA from both fiber types was kinetically similar to the high turnover Type II isoform. It appears that the evolution of the CA Type III isoform, found in vertebrate red muscle, did not occur with the differentiation of metabolic fiber types in crustaceans. Membrane-associated CA, which was also kinetically similar to the Type II isoform, was 20-fold higher in light pink fibers, suggesting a physiological role in facilitated CO2 efflux from the muscle fiber during periods of prolonged maximal activity.     

Regulation of carbonic anhydrase III by thyroid hormone: opposite modulation in slow- and fast-twitch skeletal muscle

This laboratory previously reported that a major 30 kilodalton (kDa) protein of the soluble cytoplasmic fraction of the rat slow-twitch soleus muscle is modulated by thyroid hormone. This protein has been purified and a portion of the primary structure has been determined. The sequence analysis suggested that the 30-kDa protein is carbonic anhydrase III (CA III; EC 4.2.1.1). The reaction of the protein with a CA III specific antibody and the similar modulation of CA III by thyroid hormone also support this conclusion. Immunochemical quantification of CA III and measurement of CA activity were performed in skeletal muscles of defined fiber-type composition from rats that were rendered hyperthyroid by treatment with 3,3',5-triiodo-L-thyronine. These experiments revealed that CA activity and CA III content are deinduced in the soleus muscle (primarily type I fibers) and induced in the superficial vastus lateralis muscle (primarily type IIb), whereas no changes were detected in the tibialis anterior muscle (primary type IIa). These results show that the modulation of CA III by thyroid hormone in rat skeletal muscle is not limited to the slow-twitch soleus muscle and that the amplitude and direction of this modulation are directly related to the initial fiber-type composition of the skeletal muscle.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.     

Carbonic anhydrase inhibitors: inhibition of human and bovine isoenzymes by benzenesulphonamides,cyclitols and phenolic compounds

Carbonic anhydrase inhibitors (CAIs) are a class of pharmaceuticals used as anti-glaucoma agents, diuretics and anti-epileptics. We report here the inhibitory capacities of benzenesulphonamides, cyclitols and phenolic compounds 1–11 against three human CA isozymes (hCA I, hCA II and hCA VI) and bovine skeletal muscle carbonic anhydrase III (bCA III). The four isozymes showed quite diverse inhibition profiles with K_i values ranging from low micromolar to millimolar concentrations against all isoenzymes. Compound 5 and 6 had more powerful inhibitory action against hCA I and very similar action against hCA II and hCA VI as compared with acetazolamide (AZA) and sulphapyridine (SPD), specific CAIs. Probably the inhibition mechanism of the tested compounds is distinct of the sulphonamides with RSO₂NH₂ groups and similar to that of the coumarins/lacosamide, i.e. binding to a distinct part of the active site than that where sulphonamides bind. These data may lead to drug design campaigns of effective CAIs possessing a diverse inhibition mechanism compared to other sulphonamide/sulphamate inhibitors.     

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.     

Carbonic anhydrase III: the phosphatase activity is extrinsic

The carbonic anhydrases reversibly hydrate carbon dioxide to yield bicarbonate and hydrogen ion. They have a variety of physiological functions, although the specific roles of each of the 10 known isozymes are unclear. Carbonic anhydrase isozyme III is particularly rich in skeletal muscle and adipocytes, and it is unique among the isozymes in also exhibiting phosphatase activity. Previously published studies provided evidence that the phosphatase activity was intrinsic to carbonic anhydrase III, that it had specificity for tyrosine phosphate, and that activity was regulated by reversible glutathionylation of cysteine186. To study the mechanism of this phosphatase, we cloned and expressed the rat liver carbonic anhydrase III. The purified recombinant had the same specific activity as the carbonic anhydrase purified from rat liver, but it had virtually no phosphatase activity. We attempted to identify an activator of the phosphatase in rat liver and found a protein of approximately 14 kDa, the amount of which correlated with the phosphatase activity of the carbonic anhydrase III fractions. It was identified as liver fatty acid binding protein, which was then purified to test for activity as an activator of the phosphatase and for protein-protein interaction, but neither binding nor activation could be demonstrated. Immunoprecipitation experiments established that carbonic anhydrase III could be separated from the phosphatase activity. Finally, adding additional purification steps completely separated the phosphatase activity from the carbonic anhydrase activity. We conclude that the phosphatase activity previously considered to be intrinsic to carbonic anhydrase III is actually extrinsic. Thus, this isozyme exhibits only the carbon dioxide hydratase and esterase activities characteristic of the other mammalian isozymes, and the phosphatase previously shown to be activated by glutathionylation is not carbonic anhydrase III.     

Carbonic anhydrase III--a marker of the myoepithelial cells of human salivary glands

Carbonic anhydrase III (CAIII) is the isoenzyme purified from the human skeletal slow muscle immunohistochemically revealed in smooth muscle cells of uterus, myoepithelial cells of salivary, lactiferous and prostatic glands of man. Immunohistochemical determination of CAIII is a useful approach to correct the identification of myoepithelial cells in human salivary glands.     

Distribution of CAIII in fetal and adult human tissue

Carbonic anhydrase III (CAIII), an enzyme recently shown by conventional electrophoresis to be muscle specific, has been quantitated by “rocket” immunoelectrophoresis. This more sensitive technique has shown that the enzyme is virtually specific to skeletal muscle, where it occurs at a level of 5mg/g, with trace levels in smooth muscle, cardiac muscle, and lung. In man there does not appear to by any correlation between CAIII levels and the proportion of red and white muscle fibers. The fetal development of CAIII has also been examined using immunoelectrophoresis, and the enzyme can be detected at 11 weeks' gestation. The CAIII level rises gradually up to 25 weeks, and there is then a more dramatic increase to reach approximately half adult level at birth.     

The 30 kDa abnormal protein in avian dystrophic muscle is indistinguishable from carbonic anhydrase III by physicochemical, immunological, and enzymological criteria

In the accompanying paper, we described the existence, molecular characterization, and ontogeny of a 30 kDa abnormal protein in chicken dystrophic muscles. In this study, we have purified chicken carbonic anhydrase III and the 30 kDa protein and directly compared them. In terms of its enzymological features, the 30 kDa protein is a typical carbonic anhydrase III. Like carbonic anhydrases, it contains one mole zinc per mole of protein. The protein selectively cross-reacted with a chicken carbonic anhydrase III antibody. Antibody to the 30 kDa protein cross-reacted with chicken skeletal muscle carbonic anhydrase III. Moreover, the distribution of the abnormal protein is exactly identical to that of carbonic anhydrase III; however, there is a possibility that the 30 kDa protein is a variant of carbonic anhydrase III. Slight differences were found in antigenicities and in the apparent molecular weights of the two proteins. We have compared the two proteins by 125I-labeled two-dimensional peptide mapping. Tryptic maps have shown that the two proteins are highly homologous. Combined, these results strongly indicate that the 30 kDa protein and carbonic anhydrase III are similar, if not identical.     

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Evaluation of acetylcholinesterase and carbonic anhydrase inhibition profiles of 1,2,3,4,6‐pentasubstituted‐4‐hydroxy‐cyclohexanes

Carbonic anhydrase (CA; EC 4.2.1.1) is used for remedial purposes for several years, as there is significant focus on expanding more new activators (CAAs) and high affinity inhibitors. Alzheimer′s disease and other similar ailments such as dementia and vascular dementia with Lewy bodies reduce cholinergic activity in the important areas involved in cognition and memory. Prevalent drugs for the symptomatic therapy of dementia are significant in increasing the associated cholinergic deficiency by inhibiting acetylcholinesterase (AChE). These six‐membered carbocycles showed nice inhibitory action against AChE and human carbonic anhydrase (hCA) II and I isoforms. The hCA I, II, and AChE were efficiently inhibited by these molecules, with K_i values in the range of 6.70–35.85 nM for hCA I, 18.77–60.84 nM for hCA II, and 0.74–4.60 for AChE, respectively.     

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

Carbonic anhydrase III in skeletal muscle fibers: an immunocytochemical and biochemical study

The objectives of the present study were to determine if carbonic anhydrase III (CA III) demonstrated a specific association for any particular organelle or structure of the skeletal muscle cell and to quantify the activity and content of this enzyme in different types of skeletal muscle fibers. Ultrastructural localization of CA III in the soleus (SOL), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL), composed of predominantly type I, IIa, and IIb fibers, respectively, was performed using a high-resolution immunocytochemical technique and antibody specific for CA III on ultra-thin sections of skeletal muscle embedded in the water-soluble medium polyvinyl alcohol (PVA). The results indicated a uniform distribution of CA III within the sarcomere. Mitochondria, nuclei, triads, Z-, and M-bands were not specifically labeled. Immunoblotting of washed myofibril preparations did not show any detectable CA III associated with this structure. In addition to quantification of the immunogold labeling, CA III activity and content were assayed in the post-mitochondrial supernatant of the three muscles. In the SOL, these values were found to be 3.6-7.6 times higher than in the DVL. The SVL showed a labeling intensity slightly higher than background level, while the enzyme activity and content were indistinguishable from background levels. We therefore conclude that CA III is randomly distributed in the cytoplasm of the three muscle fiber types and that the relative CA III content and activity in the three muscles studied is SOL greater than DVL greater than SVL approximately equal to 0.