Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Engberg, Paul; Millqvist, Erik; Pohl, Gunnar; Lindskog, Sven

doi:10.1016/0003-9861(85)90589-2

Cited by 81 publications

(56 citation statements)

References 35 publications

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“…Thus, for BCAIII, these differences in Phe content might, in addition to the extra Trp, interfere with the peptide CD bands and thereby further obscure the calculations of secondary structure content. Even though there are five Cys residues in BCAIII (Engberg et al, 1985), no disulfide bridges are found in the enzyme, hence no interference from the Cys residues is to be expected.…”

Section: Secondary Structure Estimationsmentioning

confidence: 99%

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

Borén¹,

Freskgård²,

Carlsson³

1996

Protein Science

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The CD spectra of human carbonic anhydrase I and I1 and bovine carbonic anhydrase 111 were recorded and analyzed. The 3D structures of these isoenzymes are known, showing very similar secondary structure and polypeptide-chain fold. The tryptophan content, however, differs between the isoenzymes, i.e., isoenzymes I, 11, and 111 possess 6, 7, and 8 tryptophans, respectively. All of the tryptophans except the additional tryptophans in isoenzymes I1 and 111, i.e., W245 and W47, are conserved. Despite the fact that X-ray structure determinations showed that the isoenzymes had highly similar secondary structure, the contents of a-helix and p-sheet structure differed considerably when using different CD algorithms for estimation of the fractions of various secondary structural elements. This shows that aromatic amino acids also interfere in the wavelength region (far-UV) used to calculate the amount of secondary structure. Such interference is especially problematic when analyzing proteins like carbonic anhydrase, which consist mainly of p-structure that gives rise to weak ellipticity bands, compared to the bands arising from a-helical structure.Keywords: aromatic amino acids; bovine carbonic anhydrase 111; p-structure; human carbonic anhydrase I and I1Proteins exhibit characteristic CD spectra in the far-UV region and the appearance of these spectra are related to the presence of secondary structure (Greenfield & Fasman, 1969 Compton & Johnson, 1986;Manavalan &Johnson, 1987;Perczel et al., 1991). Knowledge of the secondary structure is of great importance in the understanding of the function, stability, and folding of a protein, and CD estimations of the amount of a-helices have been particularly accurate for many proteins. On the other hand, it has been more difficult to predict the contents of p-structure (Sreerama & Woody, 1994). One obvious reason for this is that the a-helix spectrum dominates the protein far-UV CD spectrum (Johnson, 1990;Sreerama & Woody, 1993), and the CD signal arising from p-structure is of lower intensity. This means that calculations of the amount of this type of secondary structure are more sensitive to perturbations from other contributing structural elements. Based on theoretical calculations and experimental evidence from peptide models, it has been suggested that aromatic amino acids make contributions to the far-UV CD spectrum (Strickland, 1974; ManReprint ning & Woody, 1989;Manning et al., 1992). Because this wavelength region is used. for estimation of the amount of secondary structure and the models used do not consider aromatic contributions explicitly, aromatic amino acids could seriously interfere with such calculations. In a recent study, we used site-directed mutagenesis to replace one Trp at a time in human carbonic anhydrase I1 (HCA 11) in order to assign the contribution of each of the Trp residues to the CD spectrum (Freskgkd et al., 1994). We found that the individual Trp residues contributed to the CD spectrum not only in the near-UV wavelength region, but also...

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Section: Secondary Structure Estimationsmentioning

confidence: 99%

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

Borén¹,

Freskgård²,

Carlsson³

1996

Protein Science

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“…No increase in CA II activity can be observed at 48 h (not shown). CA III migrates to another position in this system, but because of its substantially lower carbonic anhydrase activity [28], it was not included in this experiment.…”

Section: Ca H Appears To Be the Only Ca In Hl-60 Cellsmentioning

confidence: 99%

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation

Shapiro

Venta

et al. 1989

FEBS Letters

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Carbonic anhydrase II (CA II) generates the H ÷ required for osteoelast-mediated bone resorption in humans. We have developed the human promyelocytic cell line HL-60 as a model system with which to study the osteoclast-specific expression of the CA II gene. Treatment of the cell line with 1,25-dihydroxyvitamin D 3 resulted in a dramatic de novo induction of CA II at both the protein and mRNA levels. CA II mRNA was also induced to a lesser extent by 12-O-tetradecanoyl phorbol 13-acetate. Treatment with dimethyl sulfoxide did not increase CA II mRNA. These findings indicate that the HL-60 cell line will be a useful model system to study the osteoclast-speeific expression of the CA II gene.Carbonic anhydrase II; Osteoclast; Dihydroxyvitamin D3, 1,25-; (HL-60 cell)

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“…Umei Universitet, S-90187 Umeb, Sweden isoenzyme I1 [2]. While the pH activity profile of isoenzyme I1 displays a pKa value near 7, the C 0 2 hydration activity of isoenzyme 111 is nearly independent of pH between pH 5.5 and 9 [2 -51.…”

Section: (3)mentioning

confidence: 99%

“…Carbonic anhydrase 111 was prepared from bovine muscle tissue by ion-exchange chromatography and gel filtration according to method 1 of Engberg et al [2]. Co(I1)-substituted carbonic anhydrase 111 was prepared by adding the chelator 2-carboxy-1 ,lo-phenanthroline (synthesized according to Corey et al [7]) to a solution of the enzyme followed by dialysis against an excess of CoClz as described by Engberg and Lindskog [6].…”

Section: Enzyme and Chemicalsmentioning

confidence: 99%

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Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase

Ren

Sandström

Lindskog

1988

European Journal of Biochemistry

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The binding of N; to Co(I1)-substituted bovine carbonic anhydrase 111 was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of C 0 2 hydration by N; was found to be essentially uncompetitive at all investigated pH values (pH 6.3 -8.9). The Ki values for the inkbition of k,,, are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N;, presumably having a metal-bound H20, accumulates in the steady state at saturating COz concentrations. Assuming that the low pH limit of Ki = 9 pM for the inhibition of k,,, represents the affinity of N; for the Co(I1)-OHz form, a pKa value near 5 can be estimated for Co(I1)-bound water from the pH dependence of N; binding in the absence of c02.Measurements of time-resolved absorption spectra during C 0 2 hydration in the presence of a low N; concentration showed the transient appearance of the characteristic spectrum of the enzyme-N; adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N;. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(I1)-OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(I1)-OH-species. Our results strongly support the hypothesis that the rate-limiting step in C 0 2 hydration catalyzed by carbonic anhydrase I11 is the protolysis of metal-bound water.Three cytosolic isoenzyme forms of carbonic anhydrase are known to occur in higher vertebrates [l]. They are genetically distinct and have different kinetic and inhibitorbinding properties but are structurally homologous. The highactivity carbonic anhydrase 11, with a maximal C 0 2 hydration turnover number of lo6 s -l at 25"C, has been studied extensively. Two functional groups withpK, values near 7 at normal ionic strengths are thought to be involved in catalysis. These groups are presumably a metal-bound water molecule and the imidazole group from His-64 [I]. Kinetic studies have shown that the interconversion between C 0 2 and HCO; is temporally separate from the transfer of H + between the active site and the reaction medium as shown in Eqn (1) and (2). E-Zn2'-OH-+ C 0 2 E-Zn2+-HCO; n20(1 1 (2) J E-Zn2+-OH2 + HCO; E-Zn2+-OHz + E-Zn2+-OH-+ H + .In carbonic anhydrase 11, reaction 2 occurs in two steps. First, a rate-limiting intramolecular proton transfer between metalbound water and His-64 followed by proton transfer between His-64 and buffer as indicated in Eqn (3).His-E-Zn2+-OHz + B + +H-His-E-Zn2+-OH-+ B + His-E-Zn2+-OH-+ BH'. (3)At pH 8, the C 0 2 hydration activity (kCa,/Km) of the muscle-specific carbonic anhydrase 111 is only 0.3% of that of Correspondence to Sven Lindskog, Avdelningen for Biokemi, Enzyme. Carbonic anhydrase (EC 4.2.1.1).Umei Universitet, S-90187 Umeb, Sweden isoenzyme I1 [2]. Whi...

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Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Cited by 81 publications

References 35 publications

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation

Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase

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Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Cited by 81 publications

References 35 publications

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D3: A model for osteoclast gene regulation

Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase

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Carbonic anhydrase II is induced in HL‐60 cells by 1,25‐dihydroxyvitamin D₃: A model for osteoclast gene regulation