A modified colorimetric estimation of carbonic anhydrase

Philpot, Flora J.; Philpot, J. St. L.

doi:10.1042/bj0302191

Cited by 150 publications

(26 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The compound, Cl (14) and expressed in units according to Maren et al (15). For the inhibition studies the acetazolamide was added immediately before the buffer so that equilibrium was not attained.…”

Section: Methodsmentioning

confidence: 99%

Carbonic Anhydrase Function and the Epithelial Organization of H+ Secretion in Turtle Urinary Bladder

Schwartz¹,

Rosen²,

Steinmetz³

1972

J. Clin. Invest.

View full text Add to dashboard Cite

A B S T R A C T The function of carbonic anhvdrase in H+ secretion by the turtle bladder was studied in vitro. Dose response curves were obtained for the carbonic anhydrase inhibitors, acetazolamide and ethoxzolamide, with and without addition of C02 to the system. In addition, carbonic anhydrase was assayed in hoilogenates of mucosal cells. The activity in the homogenates was 155±16 U/g dry wt, of which only 11 U represented contamination from erythrocytes; after addition of 5 X 10-M acetazolamide, no enzyme activity was detectable.In the intact preparation free of exogenous C02, the dose response curve for acetazolamide showed two plateaus of inhibition, one at 50% and one at more than 80% inhibition. At 50% inhibition (from 5 X 10-to 5 X 10-M acetazolamide), H+ secretion was restored or enhanced by C02 addition to the same extent as bladders not exposed to acetazolamide. At concentrations of more than 1 X 10-4 M, H+ secretion was no longer restorable by C02. Unlike acetazolamide, ethoxzolamide caused progressive inhibition of H+ secretion in the C02-free system. The maximal extent of inhibition with ethoxzolamide and the behavior of inhibition in the presence of 2.5% C02 were the same as for acetazolamide. Evidence is presented that all surface epithelial cells secrete H+ and generate OH-within the cell interior. The capacity of cells to dispose of OH-by C02 hydroxylation varies with the availability of cytoplasmic carbonic anhvdrase. A small population of cells contains abundant carbonic anhydrase and secretes at high rates even when C02 is in short supply.On the basis of these results and histochemical data on the distribution of carbonic anhydrase within the mucosa, an analysis is presented of the epithelial organization of acidification by the turtle bladder.

show abstract

Section: Methodsmentioning

confidence: 99%

Carbonic Anhydrase Function and the Epithelial Organization of H+ Secretion in Turtle Urinary Bladder

Schwartz¹,

Rosen²,

Steinmetz³

1972

J. Clin. Invest.

View full text Add to dashboard Cite

show abstract

“…385,386 This method consists of adding CA to a solution that is saturated with CO 2 and monitoring the change in the color of a pH indicator. 387 The change in pH results from the enzymatic activity of CA.…”

Section: Absorption Spectrophotometrymentioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

View full text Add to dashboard Cite

I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

show abstract

“…The enzyme activities in the tissue homogenates and in the chromatographic fractions were assayed by a pH-changing method of Philpot and Philpot [8]. The enzyme activity of the separated bands after electrophoresis on the cellulose acetate strips was assayed as the rapid change in colour when incubating the strips in bromthymol blue under CO, atmosphere 191.…”

Section: Enzyme a S S A~ ~mentioning

confidence: 99%

Human Renal Carbonic Anhydrase. Purification and Properties

1975

View full text Add to dashboard Cite

Carbonic anhydrase was isolated from fresh human donor kidneys which had been thoroughly perfused free from blood. The isolation procedure involved biospecific affinity chromatography on a sulfanilamide -agarose column and yielded one soluble form of the enzyme, which was homogenous with respect to sedimentation in the ultracentrifuge, electrophoresis, isoelectric focusing and immunodiffusion. The renal enzyme had an amino acid composition and behaved chromatographically, electrophoretically, and immunochemically like the erythrocyte form human carbonic anhydrase C, isolated by the same technique. The kinetic behaviour of the renal enzyme was similar to that of human carbonic anhydrase C when compared by the stopped-flow pH-indicator technique.The results therefore suggest that the cytoplasmic carbonic anhydrase of the human kidney is very similar, if not identical, to the high-activity erythrocyte form of human carbonic anhydrase C.Carbonic anhydrase (carbonate hydro-lyase) is needed for the acidification of the urine, and inhibitors of the enzyme are used therapeutically as diuretic agents [l]. It would, therefore, appear important to know the properties of the enzyme. However, it has not yet been purified to an extent which would allow a more detailed characterization. This would seem even more necessary in view of recent reports which indicate that various tissues contain different forms of the enzyme with different properties and perhaps functional significance [2]. Even a single cell like the human erythrocyte contains two forms of the enzyme, which are very different with respect to amino-acid composition and kinetic activities [3]. It is not yet known whether the kidney and erythrocytes use the same or different forms of the enzyme.The present paper describes the isolation and properties of the major human renal enzyme in comparison with the erythrocyte enzymes. Advantage has been taken of the access to virtually blood-free donor kidneys and a new affinity chromatographic technique for the purification of carbonic anhydrase.Abbreviations. HCA C and HCA B are isoenzymes of carbonic Enzyme. (CBN Recommendations 1972). Carbonic anhydrase anhydrase from human erythrocytes.

show abstract

A modified colorimetric estimation of carbonic anhydrase

Cited by 150 publications

References 2 publications

Carbonic Anhydrase Function and the Epithelial Organization of H+ Secretion in Turtle Urinary Bladder

Carbonic Anhydrase Function and the Epithelial Organization of H+ Secretion in Turtle Urinary Bladder

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Human Renal Carbonic Anhydrase. Purification and Properties

Contact Info

Product

Resources

About