Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

Low bundle sheath carbonic anhydrase is apparently essential for effective c(4) pathway operation

Bundle sheath cells from leaves of a variety of C₄ species contained little or no carbonic anhydrase activity. The proportion of total leaf carbonic anhydrase in extracts of bundle sheath cells closely reflected the apparent mesophyll cell contamination of bundle sheath cell extracts as measured by the proportion of the mesophyll cell marker enzymes phosphoenolpyruvate carboxylase and pyruvate,Pi dikinase. Values of about 1% or less of the total leaf activity were obtained for all three enzymes. The recorded bundle sheath carbonic anhydrase activity was compared with a calculated upper limit of carbonic anhydrase activity that would still permit efficient functioning of the C₄ pathway; that is, a carbonic anhydrase level allowing a sufficiently high steady state [CO₂] to suppress photorespiration. Even before correcting for mesophyll cell contamination the activity in bundle sheath cell extracts was substantially less than the calculated upper limit of carbonic anhydrase activity consistent with effective C₄ function. The results accord with the notion that a deficiency of carbonic anhydrase in bundle sheath cells is vital for the efficient operation of the C₄ pathway.     

Complete amino acid sequence of ovine salivary carbonic anhydrase

The primary structure of the secreted carbonic anhydrase from ovine salivary glands has been determined by automated Edman sequence analysis of peptides generated by cyanogen bromide and tryptic cleavage of the protein and Staphylococcus aureus V8 protease, trypsin, and alpha-chymotrypsin subdigests of the large cyanogen bromide peptides. The enzyme is a single polypeptide chain comprising 307 amino acids and contains two apparent sites of carbohydrate attachment at Asn-50 and Asn-239. The protein contains two half-cystine residues at 25 and 207 which appear to form an intramolecular disulfide bond. Salivary carbonic anhydrase shows 33% sequence identity with the ovine cytoplasmic carbonic anhydrase II enzyme, with residues involved in the active site highly conserved. Compared to the cytoplasmic carbonic anhydrases, the secreted enzyme has a carboxyl-terminal extension of 45 amino acids. This is the first report of the complete amino acid sequence of a secreted carbonic anhydrase (CA VI).     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Hanssons' enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mesencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Summary Hansson's enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mecencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.     

A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes

A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.     

Mitochondrial carbonic anhydrase in osteoclasts and two different epithelial acid-secreting cells

Summary Acid secreting cells are rich in mitochondria and contain high levels of cytoplasmic carbonic anhydrase II. We have studied the ultrastructural distribution of a mitochondrial isoenzyme, carbonic anhydrase V, in two different acid-secreting epithelial cells, gastric parietal cells and kidney intercalated cells as well as in osteoclasts, which are the main bone resorbing cells. The mitochondria differ in carbonic anhydrase V content in these three acid-producing cells: gastric parietal cell mitochondria show strong immunolabelling for this isoenzyme, osteoclast mitochondria faint labelling and kidney intercalated cell mitochondria no labelling. The immunolabelling was located in the mitochondrial matrix, often in close contact with the inner mitochondrial membrane. These results show that mitochondrial carbonic anhydrase levels are not related to acid-transporting activity.     

碳酸酐酶在红细胞分化发育过程中的功能研究

目前红系分化调控相关的研究主要集中在细胞因子、转录因子、lncRNA及表观遗传方面,为了对红系分化调控机制进行更加深入的解析,研究了碳酸酐酶在红系分化中的功能。碳酸酐酶可以高效催化二氧化碳的水合,但它在红细胞发育过程中的功能尚不清楚。利用脐带血来源的CD34⁺细胞在体外进行红细胞诱导分化,在分化过程中通过慢病毒介导的基因敲降的方法能够降低碳酸酐酶1和碳酸酐酶2的表达,并使用流式细胞仪检测红细胞的生成和分化效率。研究结果表明,与对照组相比,碳酸酐酶1的表达缺陷使红细胞的晚期分化明显受阻,而碳酸酐酶2的表达缺陷则将红细胞的分化阻滞在早期阶段。研究结果表明,虽然作用窗口不同,但碳酸酐酶1和碳酸酐酶2在红系分化的过程中均发挥着重要的调控作用,这一发现对将来在体外红细胞生成具有指导意义。     

Carbonic anhydrase supports electrolyte transport in Drosophila Malpighian tubules. Evidence by X-ray microanalysis of cryosections

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO(3)-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO(3)(-) ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.     

Hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.     

An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted ratfish (Hydrolagus colliei)

Separated plasma and whole blood non-bicarbonate buffering capacities, together with plasma and gill carbonic anhydrase activities and endogenous plasma carbonic anhydrase inhibitor activity were investigated in three species of fish: the brown bullhead (Ameirus nebulosus), a teleost; the longnose skate (Raja rhina), an elasmobranch; and the spotted ratfish (Hydrolagus colliei), a chimaeran. The objective was to test the hypothesis that species possessing gill membrane-bound carbonic anhydrase and/or plasma carbonic anhydrase activity would also exhibit high plasma nonbicarbonate buffering capacity relative to whole blood non-bicarbonate buffering capacity and would lack an endogenous plasma carbonic anhydrase inhibitor. Separated plasma non-bicarbonate buffering capacity constituted > or = 40% of whole-blood buffering in all three species. In addition, all species lacked an endogenous plasma carbonic anhydrase inhibitor. Separated plasma from skate and ratfish contained carbonic anhydrase activity, whereas bullhead plasma did not. Examination of the subcellular distribution and characteristics of branchial carbonic anhydrase activity revealed that the majority of branchial carbonic anhydrase activity originated from the cytoplasmic fraction in all species, with only 3-5% being associated with a microsomal fraction. The microsomal carbonic anhydrase activity of bullhead and ratfish was significantly reduced by washing, indicating the presence of carbonic anhydrase activity that was not integrally associated with the membrane pellet, microsomal carbonic anhydrase activity in skate was unaffected by washing. In addition, microsomal carbonic anhydrase activity from skate and ratfish but not bullhead gills was released to a significant extent from its membrane association by treatment with phosphatidylinositol-specific phospholipase C. The results obtained for skate are consistent with published data for dogfish, suggesting that the possession of branchial membrane-bound carbonic anhydrase activity may be a generalised elasmobranch characteristic. Ratfish, which also belong to the class Chondrichthyes, exhibited a similar pattern. Unlike skate and ratfish, bullhead exhibited high plasma non-bicarbonate buffering capacity and lacked an endogenous carbonic anhydrase inhibitor in the absence of plasma and gill membrane-bound carbonic anhydrase activities.     

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.     

Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.     

The use of prontosil for the visualization of carbonic anhydrase bands in polyacrylamide gels. Application to the carbonic anhydrase isozymes of erythrocytes and white skeletal muscle of the rabbit

Prontosil, a carbonic anhydrase inhibitor of orange-red colour, is used to visualize carbonic anhydrase bands during isoelectric focusing in polyacrylamide gels. 5–60 ng of the sulfonamide Prontosil are added to the 100–200 μl samples before application to the gels. Bound Prontosil moves into the gel together with carbonic anhydrase and stains the enzyme bands formed there, while unbound Prontosil remains on top of the gels. The method is specific, no proteins other than carbonic anhydrase were observed to be stained, and it requires no special equippment. It was applied to chloroform/ethanol extracts of erythrolysates and while muscle homogenates from rabbits. Densitometric evaluation of the Prontosil-stained bands obtained with these extracts showed that rabbit red cells contain roughly equla amounts of carbonic anhydrase isoenzymes B and C while in rabbit white skeletal muscle isoenzyme C is predominant and little B enzyme occurs. These results confirm previous findings obtained by affinity chromatography of erythrolysates and muscle homogenates.