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Carbonic anhydrase in guinea pig skeletal muscle mitochondria
B T Storey1, L C Lin, B Tompkins
1Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Abstract:
The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.
Insights
Guinea pig skeletal muscle mitochondria contain carbonic anhydrase (CA) activity, primarily located in the matrix. This enzyme exhibits properties consistent with the CA II isozyme class.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Carbonic anhydrase (CA) enzymes are crucial for various physiological processes, including CO2 transport and pH regulation.
- Mitochondria are known to contain specific CA isozymes, but their presence and function in skeletal muscle mitochondria require further elucidation.
Purpose of the Study:
- To investigate the presence and characteristics of carbonic anhydrase activity in guinea pig skeletal muscle mitochondria.
- To determine the subcellular localization and kinetic properties of this mitochondrial carbonic anhydrase.
Main Methods:
- Purification of guinea pig skeletal muscle mitochondria using Percoll gradient centrifugation.
- Assay of carbonic anhydrase activity in purified mitochondria and sarcoplasmic reticulum vesicles.
- Enzyme kinetics studies, including inhibition by ethoxzolamide and determination of activation energy.
Main Results:
- Carbonic anhydrase activity was detected in purified guinea pig skeletal muscle mitochondria with minimal contamination from sarcoplasmic reticulum.
- Enzyme activity was significantly enhanced by Triton X-100, indicating a matrix localization.
- The enzyme demonstrated high sensitivity to ethoxzolamide (Ki = 8.7 nM) and an activation energy of 12.8 kcal/mol for CO2 hydration.
Conclusions:
- Guinea pig skeletal muscle mitochondria possess carbonic anhydrase activity, predominantly located in the mitochondrial matrix.
- The characterized properties align with those of the carbonic anhydrase II (CA II) isozyme, distinguishing it from other known CA forms.