Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates.
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.
Brion
L P
LP
Albert Einstein College of Medicine, Bronx, New York 10461.
Schwartz
J H
JH
Zavilowitz
B J
BJ
Schwartz
G J
GJ
D000818:Animals; D001639:Bicarbonates; D002256:Carbonic Anhydrases / Q000032:analysis*; D002460:Cell Line; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007093:Imidazoles; D007668:Kidney / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D008832:Microchemistry / Q000379:methods*; D011817:Rabbits; D051381:Rats; D014325:Tromethamine
D001639:Bicarbonates; D007093:Imidazoles; D014325:Tromethamine; C029899:imidazole; D002256:Carbonic Anhydrases