Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

Abstract

Current research into the function of carbonic anhydrases in cell physiology emphasizes the role of membrane-bound carbonic anhydrases, such as carbonic anhydrase IX that has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. We present here a mass spectrometric method to determine the extent to which total carbonic anhydrase activity is due to extracellular carbonic anhydrase 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 carbonic anhydrase outside and inside of the cells. This property is demonstrated here using suspensions of human red cells in which external carbonic anhydrase was added to the suspending solution. It is also applied to breast and prostate cancer cells which both express exofacial carbonic anhydrase IX. Inhibition of external carbonic anhydrase is achieved by use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethyleneglycol polymer.Keywords carbonic anhydrase; mass spectrometry; carbon dioxide; stable isotope; enzyme activityThe zinc metalloenzyme carbonic anhydrase (CA) catalyzes the hydration of carbon dioxide to form bicarbonate and a proton [1,2]. There are 16 isozymes of CA in the α-class of mammalian carbonic anhydrases that play a role in respiration, formation of secretory fluids, acid-base balance, and other physiological functions [3]. The isozyme CA IX is of particular interest since it is found in few normal tissues but its expression is induced in certain tumor cells by hypoxia [4,5]. CA IX is a transmembrane protein the catalytic site of which is external to the cell and is shown to play a role in extracellular acidification associated with cell Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. proliferation and metastasis [6][7][8][9]. Its inhibition may be of benefit in anticancer therapies for several types of cancer, including breast and prostate cancer. NIH Public AccessThe inhibition of carbonic anhydrases by sulfonamides is well studied in terms of its biochemical, physiological, and medical aspects [3,10,11]. Oxygen-18 and carbon-13 labeled bicarbonate was prepared by dissolving KH 13 CO 3 (99% 13 C) in enriched water (up to 90% 18 O enrichment). This solution was allowed to come to isotopic equilibrium overnight, after which water was removed by vacuum distillation. The resulting product was stored at room temp...

Quantification of Extracellular Carbonic Anhydrase Activity in Two Marine Diatoms and Investigation of Its Role

“…We used the long-term rate of 18 O removal or "phase II slope" (calculated as the slope of a linear fit through natural log-transformed 18 O atom fraction data) as an empirical measure of eCA activity to establish that eCA effectively inhibited the CA inhibitors ( Fig. 3; Delacruz et al, 2010). Application of increasing concentrations of AZ or DBAZ to cells expressing eCA reduces the phase II slope to near background, uncatalyzed exchange rates, verifying that eCA activity was effectively eliminated.…”

“…Our approach to quantify eCA activity is an adaptation of a common method to measure CA activity based on the enzyme's acceleration of 18 O removal from labeled C i . This technique has been used to measure the kinetics of isolated CA enzymes (Silverman, 1982), iCA activity (Tu et al, 1978), and eCA activity (Palmqvist et al, 1994;Delacruz et al, 2010). The advance presented here is to extract a quantitative, intrinsic measure of surface eCA activity by applying a simple box model of 18 O-exchange kinetics to the data that accounts for the localization of the enzyme.…”

“…The advance presented here is to extract a quantitative, intrinsic measure of surface eCA activity by applying a simple box model of 18 O-exchange kinetics to the data that accounts for the localization of the enzyme. In contrast, previous methods to measure eCA activity have effectively treated the activity as dispersed throughout the solution and, in some cases, are semiquantitative (Palmqvist et al, 1994;Elzenga et al, 2000;Delacruz et al, 2010). Previous approaches based on 18 O exchange use the long-term rate of 18 O removal as a quantitative but empirical measure of eCA activity (Palmqvist et al, 1994;Delacruz et al, 2010).…”

“…In contrast, previous methods to measure eCA activity have effectively treated the activity as dispersed throughout the solution and, in some cases, are semiquantitative (Palmqvist et al, 1994;Elzenga et al, 2000;Delacruz et al, 2010). Previous approaches based on 18 O exchange use the long-term rate of 18 O removal as a quantitative but empirical measure of eCA activity (Palmqvist et al, 1994;Delacruz et al, 2010). Another technique uses the rate of equilibration of 14 C between CO 2 and HCO 3 2 , quantifying eCA activity as an increase in the rate of CO 2 hydration in the bulk solution (Elzenga et al, 2000;Martin and Tortell, 2006).…”

Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

“…We have recently adapted the 18 O exchange between CO 2 and water measured by membrane inlet mass spectrometry (MIMS) to detect exofacial CA activity in cancer cells (22) and herein apply this technique to characterize CAIX in its native state. We have selected the MDA-MB-231 breast cancer cells for our studies for several reasons.…”