Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry.
Current research into the function of carbonic anhydrases (CAs) in cell physiology emphasizes the role of membrane-bound CAs such as CA IX, which has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. Here we present a mass spectrometric method to determine the extent to which total CA activity is due to extracellular CA in whole cell preparations. The method is based on the biphasic rate of depletion of (18)O from CO(2) measured by membrane inlet mass spectrometry. The slopes of the biphasic depletion are a sensitive measure of the presence of CA inside and outside of the cells. This property is demonstrated here using suspensions of human red cells in which external CA was added to the suspending solution. It is also applied to breast and prostate cancer cells, both of which express exofacial CA IX. Inhibition of external CA is achieved by the use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethylene glycol polymer.
Delacruz
Joannalyn
J
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA.
Mikulski
Rose
R
Tu
Chingkuang
C
Li
Ying
Y
Wang
Hai
H
Shiverick
Kathleen T
KT
Frost
Susan C
SC
Horenstein
Nicole A
NA
Silverman
David N
DN
D001943:Breast Neoplasms / Q000201:enzymology; D002245:Carbon Dioxide / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D045744:Cell Line, Tumor; D004912:Erythrocytes / Q000201:enzymology; D005110:Extracellular Space / Q000201:enzymology*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000201:enzymology; D012680:Sensitivity and Specificity
D002257:Carbonic Anhydrase Inhibitors; D002245:Carbon Dioxide; D011092:Polyethylene Glycols; D002256:Carbonic Anhydrases