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Purification and properties of pig muscle carbonic anhydrase III
Abstract:
Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.
Insights
Pig muscle carbonic anhydrase III, a 30 kDa protein, exhibits CO2 hydratase, acetate esterase, and phosphatase activities. Its amino acid composition shows high homology with other mammalian muscle carbonic anhydrase III isoenzymes.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Carbonic anhydrase III (CA III) is a key enzyme found in mammalian muscle tissue.
- Muscle isoenzymes of CA III exhibit distinct enzymatic activities compared to erythrocyte forms.
- Understanding the properties of CA III from different species aids in comparative enzymology.
Purpose of the Study:
- To isolate and purify pig muscle carbonic anhydrase III.
- To characterize the enzymatic activities and physical properties of pig muscle CA III.
- To compare the pig muscle CA III with homologous isoenzymes from other species.
Main Methods:
- Protein isolation and purification using chromatographic techniques.
- Enzymatic activity assays for CO2 hydratase, acetate esterase, and p-nitrophenyl phosphatase.
- Determination of kinetic parameters (Km, Vmax), extinction coefficient, and amino acid composition.
Main Results:
- Pig muscle carbonic anhydrase III was purified to homogeneity as a 30 kDa protein.
- The purified enzyme displayed CO2 hydratase, acetate esterase, and phosphatase activities, similar to rabbit muscle CA III.
- Kinetic parameters for CO2 hydratase activity were determined (Km = 83 mM, Vmax = 6000 s-1).
- Analysis revealed five free half-cystine residues, with four accessible to titration.
- High homology was observed between the amino acid composition of pig muscle CA III and homologous isoenzymes from rabbit and bovine.
Conclusions:
- Pig muscle carbonic anhydrase III shares functional and structural similarities with rabbit muscle CA III.
- The characterized enzymatic activities and kinetic properties provide insights into the physiological role of muscle CA III.
- Comparative analysis supports conserved structural and functional features of mammalian muscle CA III isoenzymes.