Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates

Luc P. Brion; John H. Schwartz; Beth J. Zavilowitz; George J. Schwartz

doi:10.1016/0003-2697(88)90391-0

Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates

Luc P. Brion, John H. Schwartz, Beth J. Zavilowitz, George J. Schwartz

Research output: Contribution to journal › Article › peer-review

62 Scopus citations

Abstract

The kidney is responsible for the excretion of acid. Carbonic anhydrase (CA) activity facilitates H⁺ secretion by catalyzing the buffering by CO₂ of cellular-generated base. We describe a simple and inexpensive micro-method for the determination of CA activity in monolayers of cultured renal cells using imidazole-Tris buffers. Our method is twice as sensitive as that originally described by Maren and the endpoint is much less affected by other cellular proteins. It can easily determine the CA activity of a monolayer of cells grown to confluence in a 75-cm² flask. In some cases homogenates giving no detectable activity by Maren's technique had assayable CA activity by the imidazole-Tris method. A smaller reaction system providing a 10-fold reduction in volume (or increase in sensitivity) permits the determination of CA activity in 25-cm² monolayers and even in microdissected proximal tubular segments totaling less than 5 mm in length. We believe that the regulation of CA activity at the cellular level may be better understood using this more sensitive assay.

Original language	English (US)
Pages (from-to)	289-297
Number of pages	9
Journal	Analytical biochemistry
Volume	175
Issue number	1
DOIs	https://doi.org/10.1016/0003-2697(88)90391-0
State	Published - Nov 15 1988

Keywords

Tris
carbonic anhydrase
imidazole
inner medullary collecting duct
kidney
papilla acidification
proximal tubule

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/0003-2697(88)90391-0

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title = "Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates",

abstract = "The kidney is responsible for the excretion of acid. Carbonic anhydrase (CA) activity facilitates H+ secretion by catalyzing the buffering by CO2 of cellular-generated base. We describe a simple and inexpensive micro-method for the determination of CA activity in monolayers of cultured renal cells using imidazole-Tris buffers. Our method is twice as sensitive as that originally described by Maren and the endpoint is much less affected by other cellular proteins. It can easily determine the CA activity of a monolayer of cells grown to confluence in a 75-cm2 flask. In some cases homogenates giving no detectable activity by Maren's technique had assayable CA activity by the imidazole-Tris method. A smaller reaction system providing a 10-fold reduction in volume (or increase in sensitivity) permits the determination of CA activity in 25-cm2 monolayers and even in microdissected proximal tubular segments totaling less than 5 mm in length. We believe that the regulation of CA activity at the cellular level may be better understood using this more sensitive assay.",

keywords = "Tris, carbonic anhydrase, imidazole, inner medullary collecting duct, kidney, papilla acidification, proximal tubule",

author = "Brion, {Luc P.} and Schwartz, {John H.} and Zavilowitz, {Beth J.} and Schwartz, {George J.}",

note = "Funding Information: Preliminary results were presented at the 20th Annual Meeting of the American Society of Nephrology, Washington, DC, December 1987. G.J.S. was supported by USPHS Grant HD-13232 and is an Established Investigator of the American Heart Association; funds were contributed in part by the New York Chapter. J.H.S. was supported by USPHS Grants DK-37105 and 5P-50-HL 183 18. L.P.B. was supported in part by a grant from the Department of Pediatrics of the Albert Einstein College of Medicine.",

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doi = "10.1016/0003-2697(88)90391-0",

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AU - Brion, Luc P.

AU - Schwartz, John H.

AU - Zavilowitz, Beth J.

AU - Schwartz, George J.

N1 - Funding Information: Preliminary results were presented at the 20th Annual Meeting of the American Society of Nephrology, Washington, DC, December 1987. G.J.S. was supported by USPHS Grant HD-13232 and is an Established Investigator of the American Heart Association; funds were contributed in part by the New York Chapter. J.H.S. was supported by USPHS Grants DK-37105 and 5P-50-HL 183 18. L.P.B. was supported in part by a grant from the Department of Pediatrics of the Albert Einstein College of Medicine.

PY - 1988/11/15

Y1 - 1988/11/15

N2 - The kidney is responsible for the excretion of acid. Carbonic anhydrase (CA) activity facilitates H+ secretion by catalyzing the buffering by CO2 of cellular-generated base. We describe a simple and inexpensive micro-method for the determination of CA activity in monolayers of cultured renal cells using imidazole-Tris buffers. Our method is twice as sensitive as that originally described by Maren and the endpoint is much less affected by other cellular proteins. It can easily determine the CA activity of a monolayer of cells grown to confluence in a 75-cm2 flask. In some cases homogenates giving no detectable activity by Maren's technique had assayable CA activity by the imidazole-Tris method. A smaller reaction system providing a 10-fold reduction in volume (or increase in sensitivity) permits the determination of CA activity in 25-cm2 monolayers and even in microdissected proximal tubular segments totaling less than 5 mm in length. We believe that the regulation of CA activity at the cellular level may be better understood using this more sensitive assay.

AB - The kidney is responsible for the excretion of acid. Carbonic anhydrase (CA) activity facilitates H+ secretion by catalyzing the buffering by CO2 of cellular-generated base. We describe a simple and inexpensive micro-method for the determination of CA activity in monolayers of cultured renal cells using imidazole-Tris buffers. Our method is twice as sensitive as that originally described by Maren and the endpoint is much less affected by other cellular proteins. It can easily determine the CA activity of a monolayer of cells grown to confluence in a 75-cm2 flask. In some cases homogenates giving no detectable activity by Maren's technique had assayable CA activity by the imidazole-Tris method. A smaller reaction system providing a 10-fold reduction in volume (or increase in sensitivity) permits the determination of CA activity in 25-cm2 monolayers and even in microdissected proximal tubular segments totaling less than 5 mm in length. We believe that the regulation of CA activity at the cellular level may be better understood using this more sensitive assay.

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KW - carbonic anhydrase

KW - imidazole

KW - inner medullary collecting duct

KW - kidney

KW - papilla acidification

KW - proximal tubule

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Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates

Abstract

Keywords

ASJC Scopus subject areas

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