Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Biomineralization of the spicules of sea urchin embryos

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.     

Control of flatfish sperm motility by CO2 and carbonic anhydrase

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.     

Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO₃ crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO₃ nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO₃ from a saturated solution containing CaCl₂ and NaHCO₃, indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.     

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.     

Biosilica formation in spicules of the sponge Suberites domuncula: synchronous expression of a gene cluster

The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.     

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 μM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO₃ deposition in the spicule.     

Locomotion and contraction in an asconoid calcareous sponge

Various large‐scale behaviors (e.g., locomotion, shape changes, contractions) have been documented numerous times in intact sponges of the class Demospongiae. However, little is known about such motile events in calcareous sponges (Class Calcarea). Here, we report on whole‐sponge behaviors of the calcareous asconoid sponge Leucosolenia botryoides, as revealed by time‐lapse videos. These behaviors included locomotion and contraction. Locomotion in these sponges appeared as an outward movement (25–130 μm h^?1) of the asconoid tubes away from the sponge's center; such translocations were always accompanied by extensive movements of protruding spicules, which appear to act as anchoring hooks for the sponge's translocations. This is the first report of whole‐sponge locomotion in the Calcarea. Contractile waves also were propagated in these sponges at speeds of 50–150 μm h^?1, and they involved systemic contraction, then re‐extension of the asconoid tubes. The observations suggest that, like the more complex demosponges, these simple calcareous sponges are capable of adaptive whole‐animal behaviors (changes in flow, shape, and location), which occur in response to environmental stimuli such as crawling intruders.     

Carbonic anhydrase activity in a calcium-mobilizing epithelium of the crustacean Orchestia cavimana during molting

We investigated the involvement of the enzyme, carbonic anhydrase, in the calcification-decalcification processes occurring in the posterior caeca of the midgut of the terrestrial crustacean, Orchestia cavimana, before and after exuviation. This enzyme was ultrahistochemically localized throughout the membranes of the caecal epithelium as well as extracellularly, i.e., within pre-exuvial calcareous concretions and postexuvial calcified spherules. During the molt cycle, the pattern of carbonic anhydrase activity in the posterior caeca was correlated with the calcium content at this level. Acetazolamide treatment in vivo inhibited about 50% of the calcium uptake during both pre-exuvial secretion and postexuvial reabsorption. The role of carbonic anhydrase in this mineralizing-demineralizing epithelium is discussed and compared with that of other mechanisms involved in this calcium turnover.     

Nano-cluster composite structure of calcitic sponge spicules--a case study of basic characteristics of biominerals

Spicules of calcareous sponges are elaborately shaped skeletal elements that nonetheless show characteristics of calcite single-crystals. Our atomic force microscopic and transmission electron microscopic investigation of the triradiate spicules of the sponge Pericharax heteroraphis reveals a nano-cluster structure with mostly well-aligned small crystal domains and pockets with accumulated domain misalignments. Combined high-resolution and energy-filtering transmission electron microscopy revealed carbon enrichments located in between crystal domain boundaries, which strongly suggests an intercalated network-like proteinaceous organic matrix. This matrix is proposed to be involved in the nano-clustered calcite precipitation via a transient phase that may enable a 'brick-by-brick' formation of composite and yet single-crystalline spicules with elaborate morphologies. This composite cluster structure reduces the brittleness of the material by dissipating strain energy and deflecting crack propagation from the calcite cleavage planes, but the lattice symmetry and anisotropic growth properties of calcite still play a major role in the morphogenesis of these unusual calcite single-crystals. Our structural, crystallographic, textural, and chemical analysis of sponge spicules corroborates the view that nano-clustered crystal growth, induced by organic matrices, is a basic characteristic of biomineralisation that enables the production of composite materials with elaborate morphologies.     

The choanoderm of Sycettusa hastifera (Calcarea,Porifera) is able to generate new individuals

One of the main characteristics of sponges is their capacity for cell dedifferentiation. This capability can allow an impressive amount of asexual reproduction in these animals, because they are able to develop new individuals from just a few somatic cells. Studies of dedifferentiation, however, have focused mainly on sponges of the class Demospongiae. Therefore, we investigated here whether individuals of three different species of Calcarea are able to reconstitute new individuals following artificial fragmentation. We observed that fragmentation releases clumps of choanoderm able to initiate somatic embryogenesis. In Borojevia brasiliensis (asconoid aquiferous system, subclass Calcinea) and Paraleucilla magna (leuconoid aquiferous system, subclass Calcaronea), these clumps started to develop, but they did not pass through the first developmental phases. In Sycettusa hastifera (syconoid aquiferous system, subclass Calcaronea), the choanoderm was reorganized into primmorphs that fused to each other and formed an exopinacoderm. The first primmorphs’ spicules were triactines. Despite a large mortality rate, the primmorphs developed into olynthus stages. The somatic embryogenesis and the metamorphosis of the olynthus were similar to those observed during the sexual development of this and other calcareous sponge species. Our results show that in S. hastifera, and perhaps in other syconoid calcareous sponges, somatic embryogenesis occurs mainly from choanocytes, at least in vitro. However, primmorph development does not follow the same pattern observed in post‐metamorphic sexual development, as in that case diactines are always the first spicules to be synthesized in calcaronean species.     

Carbonic anhydrase activity in a calcium-mobilizing epithelium of the crustacean Orchestia cavimana during molting

Summary We investigated the involvement of the enzyme, carbonic anhydrase, in the calcification-decalcification processes occurring in the posterior caeca of the midgut of the terrestrial crustacean, Orchestia cavimana, before and after exuviation. This enzyme was ultrahistochemically localized throughout the membranes of the caecal epithelium as well as extracellularly, i.e., within pre-exuvial calcareous concretions and postexuvial calcified spherules. During the molt cycle, the pattern of carbonic anhydrase activity in the posterior caeca was correlated with the calcium content at this level. Acetazolamide treatment in vivo inhibited about 50% of the calcium uptake during both pre-exuvial secretion and postexuvial reabsorption. The role of carbonic anhydrase in this mineralizing-demineralizing epithelium is discussed and compared with that of other mechanisms involved in this calcium turnover.     

Protein folding by domain V of Escherichia coli 23S rRNA: specificity of RNA-protein interactions

The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed.     

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.     

Biomineralisation markers during a phase of active growth in Pinctada margaritifera

To search for the biochemical parameters involved in calcium and carbonate transport during crystal formation and biomineralisation in nacreous molluscs, the carbonic anhydrase activity, the levels of calciotropic hormones in hemolymph and in tissues and the circulating concentration of calcium were measured in pearl oysters (Pinctada margaritifera) during a phase of active growth. Activity of carbonic anhydrase in gill tissue increased linearly with age of the animals, while no age variation in activity was noted for the mantle. The circulating level of total calcium increased during the growth of the animals. Calciotropic hormones were radioimmunoassayed in gill, mantle and hemolymph. Only a calcitonin-gene related peptide (CGRP) could be detected and its concentration decreased as a function of growth, both in hemolymph and mantle. No variation in CGRP concentration with age was observed in gill tissue. Our data demonstrate that carbonic anhydrase and a molecule biologically and immunologically related to CGRP are involved during growth of the animals. In addition, this study shows the presence of three main calcium compartments, gill, hemolymph and mantle, involved in the biomineralisation process.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China

Botting, J.P., Muir, L.A., Xiao, S., Li, X. & Lin, J.‐P. 2012: Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China. Lethaia, Vol. 45, pp. 463–475. The relationships of the extant sponge classes, and the nature of the last common ancestor of all sponges, are currently unclear. Early sponges preserved in the fossil record differ greatly from extant taxa, and therefore information from the fossil record is critical for testing hypotheses of sponge phylogenetic relationships that are based on modern taxa. New specimens of the enigmatic sponge Lenica sp., from the Early Cambrian Hetang Biota of South China, exhibit an unusual spicule structure. Each spicule consists of a siliceous core with an axial canal, an organic outer layer and a middle layer interpreted to have been originally calcium carbonate. This finding confirms previous work suggesting the existence of biminerallic spicules in early sponges. Combined with data from other early sponges, the new findings imply that the two fundamental spicule structures of modern sponges were derived from a compound, biminerallic precursor. Spicules are therefore homologous structures in Calcarea and Silicea, and if sponges are paraphyletic with respect to Eumetazoa, then spicules may also have been a primitive feature of Metazoa. □Calcarea, Early Cambrian, Hetang Biota, phylogeny, Silicea, taphonomy.     

SPICULES IN SILURIAN TABULATE CORALS FROM CANADA, AND IMPLICATIONS FOR THEIR AFFINITIES

Abstract:  Specimens of Favosites from upper Llandovery strata of Anticosti Island show three types of calcite structures, herein interpreted as spicules, preserved within their calices and on top of the last tabula. This is stratigraphically younger material, some 50 m higher than fossils described two decades earlier, in which calcified polyps, each with 12 retracted tentacles, were noted. These more recently found structures show striking similarities in form and position to point, collaret and capstan spicules found in the soft tissues of modern pipe corals, i.e. the Octocorallia (Alcyonacea). Where preserved in a distinct pattern on top of the calcite tabulae, the spicular sclerites in Favosites occur in a particular sequence. Twelve individual, or sometimes six pairs of, triradiate point spicules have shrunk to a circlet near the middle of the calice (resting on the last, outermost, tabula). Surrounding the point spicules are 3–6 circlets of curved, usually perforated, lenticular collaret spicules; and surrounding these are scattered, much smaller, capstan spicules. The spicules display variability, probably ontogenetic, in their form and relative sizes; and they are more similar in form to calcareous spicules of alcyonacean corals than to those known from calcareous sponges. Structures with 12-fold radial symmetry in Heliolites, originally described by one of us as 'septal florets', consist of elements that are considered comparable with the point spicules found in Favosites . They have been recognized in ten species of Heliolites from Silurian (Wenlock–Ludlow) strata in the Canadian Arctic islands.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.