Biochimica et Biophysica Acta, i 109 (1992) 59-64 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2736/92/${15.01~ 59 BBAMEM 75721} Subcellular distribution of carbonic anhydrase in the enterocyte of the rabbit ileum and the Caco-2 cell. Evidence for the presence of two isozymes bound to brush-border membranes Hassan Aguenaou and Didier A Colin Laboratou'e tit" Toximdogie Bactt;riemw t't des Agents Anti-infi'ctiett~ h~stitut de Bact~:riologie. l:acultt: &' Ah:~h'cim', Unicersitd Louis Pastern; Strasbourg, (France) (Received 16 March 1992) Key words: Carbtmic anhydrase; l~lrush-bordcr membrane; Intestine; Caco-2 The activity of CA has bccn dctcrmincd in the membranes of cntcrocytes from rabbit ileum and of Caco-2 cells. No ('A activity was detected in the BLM, but the activity in lhe BBM (43 and 7 WAU/mg protein for rabbit and Caco-2, respectively) was doubled by the addition of Triton. These two types of activity could bc distinguished in rabbit ileum by their different ICst j in the presence of acetazolamidc (111-5 and 5.10-7 M) and their different sensitivities to heat. They were not modified by inhibitors of cytoplasmic isozymes and seem to correspond to two forms of CA, one situated in the extracellular leaflet of the BBM and the other one in the intracellular leaflet. Introduction In mammals, carbonic anhydrase (EC 4.2.1.1) is an ubiquitous zinc metalloenzyme first isolated by Mel- drum and Roughton [1] from red blood cells. To date, 7 isozymes of carbonic anhydrase (CA) have been described, differing in subcelhalar localization, suscep- tibility to inhibitors, physical kinetic properties and molecular structure. Among them, CA IV has been described as a membrane bound isozyme in the en- dotheliurn of bovine t-] and human lung [3] and the epithelium of dog [4,5] and human kidney [6,7]. In the digestive tract, the cytoplasmic CA,~ have been studied for quantity evaluation and immunohisto- chemical localization [8-10] and regulation by steroids [11] and thyroxin [13]. In contrast, the membrane bound CA is poorly documented for this tissue [13]. Several authors have suggested that the metabolism of CO 2 is closely related to the cellular absorption of salt [14-17], but the role of CA is not clearly understood, although it seems implicated in the coupling of Na + and CI- Correspondence to: D.A. Colin, Institut de Bact6riologie, 3 rue KoeberlC F-67000 Strasbourg, France. Abbreviations: BBM, brush-border membrane; BLM, basolateral membrane; CA, carbonic anhydrase; EGTA, [ethylenebis(oxyethy- lenenitrilo)]tetraacetic acid; Hepes~ N-2-hydroxyethyl-piperazine-N'- 2-ethanes:,.!fonic acid; PMSF, phenylmethylsulfony fluoride; WAU, Wilbur-Anderson unit. absorption through Na+/H + and Ci-/OH-(HCO~) antiports [13]. Nevertheless, it has been shown that CA mediates ionic absorptive responses to variations in acid-base balance [18]. The present study was designed to locate the CA activity in cells of the small intestine more precisely and to evaluate the importance of the membrane-bound enzyme in the overall CA activity of the whole cell. Preliminary results have been presented at the 9th Rgunion of the CECED (Montpellier, 1990). Materials and Methods Isolation of membranes fi'om rabbit intestine. Male New Zealand White rabbits weighing 3 to 4 kg were killed by rapid intravenous injection of 6 sodium pento- barbital (9.5 mi/kg). The distal third of the intesline was carefully removed, divided into three portions and rinsed with 50 ml of cold NaC! (0.9%). After eversion, the intestine was gcnt!y blotted and the mucosa was scraped off. Brush-border membranes wer~ prepared according to a method modified from Kessler et al. [19]. Mucosal scrapings were homogenized using a tissue grinder (E. Biihler, Tubingen, Germany) at 25000 rpm for 2 rain in 125 ml of a buffer solution containing 350 mM manni- tol, 1 mM EGTA, 0.1 mM PMSF, 1 /zg/ml aprotinin and 10 mM Hepes adjusted to pH 7.4 with Tris-base. After addition of 10 mM MgSO4, the suspension was