scholarly journals Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

1992 ◽  
Vol 282 (1) ◽  
pp. 165-171 ◽  
Author(s):  
C Geers ◽  
D Krüger ◽  
W Siffert ◽  
A Schmid ◽  
W Bruns ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Cardiac Muscle ◽  
High Activity ◽  
Skeletal Muscles ◽  
Carbonic Anhydrases ◽  
Striated Muscles ◽  
Membrane Bound ◽  
Triton X ◽  
Dextran Solution ◽  
Low Activity

We have studied the distribution of carbonic anhydrases (CA) in several skeletal muscles of the hindlimb of rabbits and rats and in cardiac muscle of the rabbit. To remove erythrocyte CA, hindlimbs and hearts were thoroughly perfused with dextran solution, and the effectiveness of the perfusion was in most cases assessed by determining the contamination of the muscles with radioisotopes that had been used to label the erythrocytes before the perfusion was started. We observed three forms of CA: (1) cytosolic (sulphonamide-resistant) CA III; (2) a cytosolic sulphonamide-sensitive CA, probably isoenzyme II; (3) a membrane-bound form that was extracted from the particulate fraction using Triton X-100. These CA isoforms were distributed as follows. (1) CA III is located in the cytoplasm of slow, oxidative skeletal muscles and is absent from or low in fast skeletal and cardiac muscle; this holds for rabbits and rats and is identical with the pattern previously described for several other species. (2) The cytosolic sulphonamide-sensitive CA is present in fast rabbit muscles and absent from slow muscles of this species. In contrast, all skeletal muscles of the rat studied here lack, or possess only very low, activity of this isoenzyme. (3) The membrane-bound form of CA is present in all rabbit muscles studied; its activity appears somewhat higher in fast than in slow skeletal muscles. (4) Cardiac muscle constitutes an exception among all striated muscles of the rabbit as it possesses no form of cytosolic CA but a high activity of the membrane-bound form.

scholarly journals The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

1970 ◽  
Vol 120 (4) ◽  
pp. 797-808 ◽  
Author(s):  
M. J. Carter ◽  
D. S. Parsons
Keyword(s):  
Gastrointestinal Tract ◽  
Carbonic Anhydrase ◽  
Guinea Pig ◽  
High Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Carbonic Anhydrases ◽  
Low Activity

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (`high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (`low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). `High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly `low activity' carbonic anhydrases from those tissues were indistinguishable. `High activity' carbonic anhydrase was markedly different from the `low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.

scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

Renal carbonic anhydrase

1982 ◽  
Vol 243 (4) ◽  
pp. F311-F324 ◽  
Author(s):  
D. C. Dobyan ◽  
R. E. Bulger
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Mammalian Species ◽  
Carbonic Anhydrase Activity ◽  
The Body ◽  
Carbonic Anhydrases ◽  
Duct System ◽  
Renal Carbonic Anhydrase ◽  
Membrane Bound ◽  
Editorial Review

Carbonic anhydrase is a zinc metalloenzyme widely distributed throughout the tissues of the body. This enzyme exists in a number of isozymic forms in most mammalian species. Significant advances over the past decade have been made in characterizing the nature of renal carbonic anhydrase. In the kidney, this enzyme is thought to play a pivotal role in urinary acidification and bicarbonate reabsorption. Two distinct isozymes of carbonic anhydrase have now been identified in the mammalian kidney. A soluble cytoplasmic form, similar if not identical to human erythrocyte carbonic anhydrase C, accounts for the bulk of the renal carbonic anhydrase activity. In addition, a membrane-bound form constituting only about 2--5% of the renal activity has been found in the brush border and basolateral fractions of kidney homogenates. The histochemical and immunocytochemical localization of these isozymes along the nephron and collecting duct system of various mammalian species suggests that marked heterogeneity exists. The Editorial Review examines the biochemical and morphological approaches that have been used to elucidate the nature of renal carbonic anhydrase and to assess its distribution along the urinary tubule. Possible physiological roles for the renal carbonic anhydrases are considered for the different segments of the nephron and collecting duct system.

scholarly journals Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis

2019 ◽  
Vol 317 (5) ◽  
pp. C922-C931 ◽  
Author(s):  
Han-Zhong Feng ◽  
J.-P. Jin
Keyword(s):  
Carbonic Anhydrase ◽  
Transgenic Mice ◽  
Intracellular Ph ◽  
Skeletal Muscles ◽  
Ex Vivo ◽  
Physiological Role ◽  
Wild Type Mouse ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Carbonic Anhydrase Iii

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and [Formula: see text] in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

Membrane-bound carbonic anhydrase in the heart

1992 ◽  
Vol 262 (2) ◽  
pp. H577-H584 ◽  
Author(s):  
W. Bruns ◽  
G. Gros
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Rabbit Heart ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
The One ◽  
Triton X

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

Mitochondrial carbonic anhydrase is involved in rat renal glucose synthesis

1989 ◽  
Vol 257 (6) ◽  
pp. E791-E796 ◽  
Author(s):  
S. J. Dodgson ◽  
K. Cherian
Keyword(s):  
Carbonic Anhydrase ◽  
Glucose Production ◽  
Carbonic Anhydrase Activity ◽  
Proximal Tubules ◽  
High Rate ◽  
Male Rats ◽  
Co2 Production ◽  
Carbonic Anhydrases ◽  
Glucose Synthesis ◽  
Membrane Bound

At 37 degrees C, pH 7.4, carbonic anhydrase activity (kenz) of disrupted rat renal proximal tubules and cortical mitochondria was 2.5 +/- 0.8 (n = 3) and 0.15 +/- 0.40 (n = 3) ml.mg-1.s-1, respectively. Turnover number for renal mitochondrial carbonic anhydrase (CA V) was 24,000 s-1. CA V activity of intact mitochondria was completely inhibited by 0.15 microM ethoxzolamide (EZ). Intact proximal tubules, prepared from 48-h starved male rats, were incubated at 37 degrees C in 10 mM pyruvate in Krebs-Henseleit bicarbonate saline buffer, 5% CO2-95% O2. The rate of glucose synthesis over 60 min was reduced 50% by including 0.6 microM EZ in the incubation solution. The concentration of NaHCO3 was doubled to 50 mM (with a corresponding decrease in NaCl) and the solution gassed with 10% CO2-90% O2; 2.4 microM EZ no longer decreased glucose synthesis. It was concluded that inhibition of glucose synthesis by EZ was directly a result of inhibiting the carbonic anhydrases. The rate of glucose production was subsequently determined with tubules incubating in a HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) buffer; this rate was decreased 50% by 0.6 microM EZ. These data support the hypotheses that CA V provides HCO3- for pyruvate carboxylase and that CO2 can be provided by tubular metabolism. Intact tubules were incubated in from 5 to 20 mM pyruvate in either 25 or 50 mM HCO3-; in either buffer, the rate of glucose synthesis was similar, increasing with increasing pyruvate concentration. At no pyruvate concentration was there a change in the rate of glucose production when tubules were incubated in 50 mM HCO3- buffer with 1.6 microM EZ. These data also support the hypothesis that CA V provides the HCO3- substrate for pyruvate carboxylation when there is a high rate of intracellular CO2 production and external CO2 is low. It is further concluded that the cytosolic carbonic anhydrase (CA II) and the membrane-bound carbonic anhydrase (CA IV) are not involved in glucose synthesis from pyruvate.

scholarly journals Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes

2000 ◽  
Vol 278 (4) ◽  
pp. H1335-H1344 ◽  
Author(s):  
Anja Sylvia Knüppel-Ruppert ◽  
Gerolf Gros ◽  
Wolfgang Harringer ◽  
Hans-Peter Kubis
Keyword(s):  
Carbonic Anhydrase ◽  
Microsomal Fraction ◽  
Cell Types ◽  
Human Cardiomyocytes ◽  
Ca Ix ◽  
Specific Distribution ◽  
Carbonic Anhydrase Isozyme ◽  
Membrane Bound ◽  
Membrane Anchoring ◽  
Triton X

To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti-human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme.

Distribution of c-protein isoforms in sarcomeres of developing chick skeletal muscle

1988 ◽  
Vol 46 ◽  
pp. 226-227
Author(s):  
D. A. Fischman ◽  
J. E. Dennis ◽  
T. Obinata ◽  
H. Takano-Ohmuro
Keyword(s):  
Skeletal Muscles ◽  
Thick Filament ◽  
Protein Isoforms ◽  
Actin Binding ◽  
Striated Muscles ◽  
C Protein ◽  
Sarcomeric Protein ◽  
Thick Filaments ◽  
Cross Bridges ◽  
Bridge Bearing

C-protein is a 150 kDa protein found within the A bands of all vertebrate cross-striated muscles. By immunoelectron microscopy, it has been demonstrated that C-protein is distributed along a series of 7-9 transverse stripes in the medial, cross-bridge bearing zone of each A band. This zone is now termed the C-zone of the sarcomere. Interest in this protein has been sparked by its striking distribution in the sarcomere: the transverse repeat between C-protein stripes is 43 nm, almost exactly 3 times the 14.3 nm axial repeat of myosin cross-bridges along the thick filaments. The precise packing of C-protein in the thick filament is still unknown. It is the only sarcomeric protein which binds to both myosin and actin, and the actin-binding is Ca-sensitive. In cardiac and slow, but not fast, skeletal muscles C-protein is phosphorylated. Amino acid composition suggests a protein of little or no αhelical content. Variant forms (isoforms) of C-protein have been identified in cardiac, slow and embryonic muscles.

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