No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Données physiques comparées sur les anhydrases carboniques érythrocytaires humaines A et B
The carbonic anhydrases are extremely efficient catalysts of the reversible hydration of carbon dioxide with maximum turnover numbers among the highest known for any enzyme. They have also been found to catalyze the hydrolysis of certain esters and related compounds and the hydration of aldehydes. They play an important role in respiration as well as in other physiological processes, where the rapid interconversion of carbon dioxide and bicarbonate ion is essential to the organism. Carbonic anhydrase is very widespread in nature and occurs in animals, plants, and certain bacteria. In addition to their respiratory role of facilitating the transport of metabolic CO2, the enzymes are involved in the transfer and accumulation of H+ or HCO3– in a wide variety of organs. The enzyme is present in gills and secretory organs of various types, such as the parotid and pancreatic glands, the rectal and alkaline glands, and the swim bladders of various species of fish. Carbonic anhydrase has been stated to occur exclusively in the cytoplasm of the plant cell. However, several recent reports show that the enzyme can also occur in the chloroplasts. This seems to be the situation in plants carrying out CO2 fixation according to the Calvin cycle, where ribulose diphosphate becomes carboxylated with CO2 as the reactive species. The carbonic anhydrases can be liberated from the red cells together with hemoglobin by simple hemolysis in hypotonic solutions. It is generally necessary to remove the vast excess of hemoglobin before attempting the separation of the carbonic anhydrase isoenzymes.
Les deux formes majeures de l'anhydrase carbonique érythrocytaire bovine ont été désignées par CI et CII en raison de leur haute activité de type C. La séparation de ces deux formes et l'isolement de CI à partir de l'extrait éthanol chloroforme de l'hémolysat ont été obtenus aussi bien par chromatographie sur DEAE-cellulose DE 23 que sur DEAE-sephadex A-50. Cependant l'obtention de préparations purifiées de la forme CII n'est possible que par la chromatographie sur DEAE-sephadex A-50 qui seule sépare CII d'un constituant mineur CIv1.
L'étude comparée des formes CI et CII et du constituant mineur CIv1, suggère fortement que CIv1 n'est qu'un variant conformationnel de CI et démontre que CI et CII sont des variants génétiques. Ces derniers diffèrent dans leur structure primaire par au moins une substitution Arg → Gln en position 56 à partir de l'extrémité N-acétylée terminale. Sur la base de la très grande variabilité de proportion des formes CI et CII constatée chez les individus hétérozygotes, les modalités de la transmission génétique de ces enzymes sont discutées.
Zusammenfassung
1.Die zwei Molekularformen A und B der Kohlensäureanhydrase in den Rindererythrozyten waren das Objekt einer hydrodynamischen Studie, die die Bestimmung der Sedimentations- und Diffusionskonstanten, der teilweisen anhydrierten und hydrierten spezifischen Volumina, der inneren Viskosität und der Molekularmasse durch Sedimentationsgleichgewicht umfasst.2.Innerhalb der experimentellen Fehlergrenzen, die bei der Berechnung verschiedener Konstanten verstärkt in Erscheinung treten, scheint es, dass die Kohlensäureanhydrasen A und B beim Rind die gleichen physikalischen Konstanten besitzen und dass ihre Moleküle eine identische Masse von 30,0.103und, im hydratisierten Zustand, eine sphärische Form mit einem Nachbarschaftsdiameter von 48besitzen.3.Die physikalischen Konstanten der Kohlensäureanhydrasen in den Rindererythrozyten A und B denen der Form B beim Menschen ähnlich.
Limiting viscosity numbers of bovine and ovine erythrocytes carbonic anhydrase variants were calculated by the objective method of comparing viscosimetric data obtained from low-activity-human erythrocyte carbonic anhydrase and its natural variant. Shifts of mobilities and isoelectric points are shown for all species variants, but variations of limiting viscosity numbers were only detected for human and bovine variants. Results of the study are consistent with the observation that variants arise by deamidation of erythrocyte carbonic anhydrases, and that deamidation is responsible for changes in structure and hydration (i. e. "conformational" modifications). Thus, all the variants so far investigated are stable conformational variants or erythrocyte carbonic anhydrases.
The model system, carbonic anhydrase to which dansyl sulphonamide is specifically adsorbed, was chosen to test the fundamentals of the fluorescence depolarisation method. Fluorescence decay was shown to be satisfactorily represented by a single exponential decay term and the derived lifetime did not seem to vary significantly with mean excitation or emission wavelength. Depolarisation measurements were quite reproducible and those made with varying temperature were consistent with those performed with varying solution viscosity, thus excluding localised dye rotation. The calculated relaxation time, which was independent of wavelength of excitation or emission, was in conformity with a rigid spherical molecule of molecular weight 29 000 with 30% hydration.
Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.
L'étude spectrale comparée des anhydrases carboniques érythrocytaires humaines A, B, C et des dérivés stables b1 et b2 obtenus par traitement alcalin contrôlé de B permet de distinguer aussi bien par DOR que par DC le groupe A = B = b1 = b2 de l'enzyme C.
L'hypothèse, suggérée par la comparaison des données actuelles sur les structures primaires et par l'identité des structures tertiaires de l'enzyme natif A et du variant conformationnel b1 de B, selon laquelle A pourrait dériver de B in vivo de la même manière que b1 dériverait de B in vitro, ne saurait être infirmée par l'identité des propriétés spectrales de A et de b1.
Par ailleurs, l'analogie des propriétés de DOR et de DC de A et de b1 avec celles de B ne permet pas de déceler l'incidence de cette transformation sur la structure secondaire de B.
1.1.|Physical and chemical properties of carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) purified from Neisseria sicca, strain 6021, have been investigated. Sedimentation equilibrium studies gave a molecular weight of 28 600. A molecular weight near 30 000 is also supported from the close similarity of gel filtration properties with human carbonic anhydrase B.2.2.|The enzyme contains 0.21% of Zn corresponding to 0.92 Zn2+ per molecular weight of 28 600. Determination of the amino acid composition shows that the contents of tyrosine, tryptophan, and proline are considerably lower than for the mammalian enzymes. The nitrogen content and the partial specific volume, estimated from the amino acid composition of the protein, are 17,7% and 0.731 ml/g, respectively.3.3.|Studies of the enzymatic properties show that the enzyme is a very efficient catalyst in the CO2 hydration reaction. In activity it resembles the highly active forms (the C type) of the mammalian carbonic anhydrases. It has a weak esterase function towards p-nitrophenyl acetate. Both activities are inhibitable with carbonic anhydrase inhibitors of the sulfonamide type. The inhibitors tested seem to associate with the bacterial enzyme with binding strengths comparable in magnitude to those found for human erythrocyte carbonic anhydrases.
Three different methods are described for separating hemoglobins from carbonic anhydrases in hemolysates from human erythrocytes.
The preferred method involves adsorption on diethylaminoethyl Sephadex at pH 8.7 followed by selective elution of the carbonic
anhydrases. Carbonic anhydrases A, B, and C are subsequently separated from each other on DEAE-Sephadex by elution with 0.05
m Trischloride buffer at pH 8.7. Complete amino acid analyses are reported for carbonic anhydrases B and C, with results in
good accord with those of preparations obtained in other laboratories by different procedures. The partial specific volume
of carbonic anhydrase B is found to be 0.731 ml per g and its intrinsic viscosity is 2.76 ml per g. Molecular weights from
sedimentation, diffusion, and sedimentation equilibrium are close to 28,000 for both enzymes; estimates from the amino acid
analyses are 28,730 for Enzyme B, and 30,000 for Enzyme C. The s020, w values are close to 2.75 S for both enzymes. The values for both s and D are lower than others previously reported, by a factor close to 1.2, but the resulting molecular weights are in good agreement
with others.
Studies of optical rotatory dispersion between 330 and 600 mµ are reported for the native and the acid-denatured carbonic
anhydrases B and C, and also for solutions containing the substrate bicarbonate ion and, in other experiments, the inhibitor
acetazolamide. The data in all these solvents give a good fit to the simple Drude equation, with λc values that lie below 210 mµ for native Enzyme B and average close to 210 mµ for Enzyme C. Analysis of the data in terms
of the Moffitt-Yang and Shechter-Blout equations is also reported, but a computer analysis indicates that the basis for applying
the former equation is dubious. There appears to be a small amount of helix (10 to 20%) in the acid-denatured proteins; the
complexity of the optical rotatory dispersion spectra of the native proteins makes inferences concerning helix content uncertain
(see following paper).
Studies on the esterase activity of Enzyme B, with p-nitrophenyl acetate as substrate, are reported. Esterase activity rises with increasing pH between 7 and 9.5. Acetazolamide
is a powerful inhibitor of the esterase activity, with Ki = 0.3 µm at pH 7, 0.9 µm at pH 8, and approximately 2.8 µm at pH 9. The inhibition appears to be noncompetitive.
The frictional coefficient, f0, of a polymer molecule in dilute solution is assumed to vary directly as an average linear dimension of the chain. From this assumption equations are developed, as follows, which are analogous to those used successfully in the interpretation of intrinsic viscosity measurements; f0/η0 =KfM½α and Kf=P(〈r02〉/M)½ where α represents the factor by which the actual root-mean-square end-to-end distance exceeds the unperturbed distance (〈r02〉)½, M is the molecular weight, η0 the viscosity of the medium, and P should be a universal constant.
Data in the literature on the dependence on molecular weight of sedimentation constants and diffusion coefficients, extrapolated to infinite dilution confirm the conclusions drawn from these equations.
A treatment of the hydrodynamic properties of proteins is presented wherein the molecule is assumed to possess some degree of flexibility and solvation. The configuration of the protein molecule is represented in terms of an effective hydrodynamic ellipsoid whose axial ratio and size may be determined from accurate measurements of sedimentation constant, intrinsic viscosity, molecular weight and flow birefringence, all made in the same solvent.
1.1. Amino acid analyses and determinations of elementary composition of various forms of bovine and human erythrocyte carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) have been carried out. A technique for the calculation of molecular weights from amino acid composition is described.2.2. A feature common to all of the forms of the enzyme studied is their low content of sulfur-containing amino acids which excludes the occurence of disulfide bridges in the molecules. The bovine enzyme contains no cysteine and it is thus possible to exclude a sulfhydryl group as a ligand in the binding of the zinc ion to the protein.3.3. Great differences in amino acid composition are found between forms from the different species as well as between different forms of the human enzyme. However, some of the electrophoretically separable forms of the enzyme show identical amino acid composition within the experimental errors. The possible biosynthetic relations between the forms of the human enzyme are discussed.