Adaptation to environmental fluctuations, through sensing and appropriate physiological responses, is crucial to homeostasis. Neuroepithelial cells (NECs) are putative chemoreceptors resembling mammalian Type I (glomus) cells. They have been shown to respond in vitro to changes in O2, CO2, NH3 and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. A mutant line of zebrafish (Danio rerio) lacking functional Ca17a was generated using CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive longer than ~12-14 days post fertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signalling. Wild type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly, but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly reduced resting fH¬. This effect of knockout became more pronounced as the fish aged. Anesthesia did not contribute to the decreased fH in the ca17a-/- fish, nor did changes in cardiac adrenergic or cholinergic tone, which were probed using propranolol (-adrenergic receptor blocker) or atropine (muscarinic receptor blocker). The decrease in resting fH was prevented (“rescued”) when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this thesis support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Copper is an essential element for all living organisms. However, due to its low redox potential it can be involved in the production of reactive oxygen species; where excess amounts of copper can be exceptionally toxic.1 In humans, malfunctions in copper metabolism are linked to diseases such as Menkes syndrome, Wilson’s disease, prion disease, and Alzheimer’s disease.2 Maintenance of copper homeostasis requires a number of proteins, such as copper transporters and chaperones to deliver copper to the correct protein while limiting free copper in the cell.3 Therefore, understanding the thermodynamics of copper(II) coordination in proteins is critical to our understanding of copper homeostasis. Herein we report human carbonic anhydrase II contains a novel copper binding site with picomolar affinity.4 A full characterization of the structure and thermodynamics associated with the coordination of both Cu atoms into their respective sites is discussed. Techniques including paramagnetic nuclear magnetic resonance spectroscopy (NMR), and x-ray absorption spectroscopy (XAS) techniques provide insight into the high affinity CuA coordination environment. A detailed characterization of this high affinity binding site and related peptide-bound model complexes are included, with the results providing insights into the chemistry and physiological impact of copper binding in human carbonic anhydrase II.

Carbonic anhydrase (CA) is a well-studied, zinc dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. In the past, metal binding studies related to CA have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka= approx. 1.2 x 1012) for Zn2+. However, new methodology has allowed us to collect data using isothermal titration calorimetry (ITC), which calls that number and the association constants for many other first row transition metal ions into question. Thermodynamic parameters associated with Zn2+, Cu2+, Ni2+, and Co2+ binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with metal ion binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the as-isolated and the reconstituted ZnCA and other metalsubstituted CAs were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the metal centers in the reconstituted carbonic anhydrases offer new metal binding coordination sites that can be used as models to understand nonheme metal sites in vivo.

No description available.

Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila strain TM-1 was purified> 10,OOO-fold (22% recovery) to apparent homogeneity and a specific activity of 4,900 units mg⁻¹.The gene encoding this CA was isolated fronl a partial genomic library on a 12-kb fragment and sequenced. Comparison of the deduced anlino acid sequence with the N-terminaI sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The deduced amino acid sequence has no significant identity to any known CAs, but has, among others, 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO₂-concentrating-mechanism protein from <i>Synechococcus</i> sp. strain PCC7942. / Ph. D.

Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased chlorophyll content. Our data revealed that steady-state levels of CA and rbcS transcripts are regulated at the transcriptional level in response to external CO2 conditions, while CA and Rubisco activities are modulated at the post-transcriptional level by light. Thus CA expression in cotyledons during post-germinative growth may be to “prime” cotyledons for the transition at the subcellular level for the transition from plastids to chloroplasts, where it provides CO2 for Rubisco during photosynthesis. Furthermore, CA expression increased during embryo maturation similar to oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [14C]-acetate incorporation into total lipids in cotton embryos and tobacco leaves and cell suspensions in vivo and in vitro. Similar results were obtained in chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wildtype activity). Collectively, these results support the notion that CA plays several physiological roles in nonphotosynthetic tissues.

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pHi for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes.

The enzyme carbonic anhydrase (CA), catalysing the interconversion between CO2 and HCO3', has long been known to be present in plants as well as in animals. Several of the animal isozymes, but none of the plant CAs, have been extensively studied. When the first plant CA cDNA sequences were published in 1990, it was obvious that the animal and plant CAs represent evolutionarily distinct families with no significant sequence homology between the families. Pea CA is synthesised as a precursor and subsequently processed at the import into the chloroplast. When we purified CA from pea leaves two oligomeric forms with molecular masses around 230 kDa were obtained. One form was homogenous while the other form contained subunits of two different sizes. The larger subunit has an acidic and highly charged N-terminal extension, consisting of 37 residues. We propose that the sequence that precedes the cleavage site resulting in the large subunit represents the functional transit peptide, directing CA to the chloroplast. Neither the transit peptide nor the acidic 37-residue peptide were found to affect the folding, activity or oligomerisation of pea CA. Kinetic investigations showed that pea CA requires a reduced environment and high concentrations of buffer for maximal catalytic activity. High buffer concentrations result in a faster turnover of the enzyme (kcat) while the efficiency (kcatlKm) is not affected. This is consistent with a ping-pong mechanism with the buffer as the second substrate. Both kcat and kcatlKm increase with pH but the dependences cannot be described by simple titration curves. SCN' is an uncompetitive inhibitor at high pH and a noncompetitive inhibitor at neutral and low pH. This is in accordance with the mechanistic model, previously proposed for human CAM, involving a zincbound water molecule as a catalytic group. In this model, the carbon dioxide - bicarbonate interconversion, reflected by kcatlKm, is temporally separated from a rate limiting proton-transfer step. At high pH, solvent hydrogen isotope effects obtained for pea CA agree with this scheme, while they do not fit at neutral and low pH. Site-specific mutations of cysteine residues at positions 165, 269 and 272 were difficult to study, either because strong deviations from Michaelis-Menten kinetics were observed, or because the mutants were found in inclusion bodies. However, the mutant H208A was found to be a very efficient enzyme with the highest kcatlKm value obtained for any CA so far, 2.9-108 M'1s '1. With the H208A mutant an increased dependence on high buffer concentrations at low pH was obtained. At high pH, the mutant is more efficient than the unmutated enzyme. The H208A mutant is also more prone to oxidation than the wild-type enzyme. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser</p> / digitalisering@umu

<p><p>The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO₂-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.</p></p>

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.