Hypothesis Anti-Oxidative Response of Carbonic Anhydrase III in Skeletal Muscle Un-Jin P. Zimmerman, Ping Wang, Xiangming Zhang, Sasha Bogdanovich and Robert E. Forster Department of Physiology, University of Pennsylvania School of Medicine A201 Richard Building, 37th and Hamilton Walk, Philadelphia, PA 19104, USA Summary We propose that carbonic anhydrase III (CAIII) functions as an anti-oxidant agent in skeletal muscle. To explore this hypothesis, we analyzed the gene expression profile of skeletal muscle in mice deficient in CAIII gene utilizing the murine genome U74Av2 set microarray. Pairwise comparison between CAIII knockout mice and their wild-type littermates revealed that more than 500 of 12,000 genes in the array showed an altered level of transcription. Of particular note were transcriptional alterations among genes associated with the glutathione redox cycle, suggesting a possible involvement of CAIII in the glutathione-mediated anti-oxidant activity. We therefore investigated S-glutathiolation and irreversible oxidation of the 2 reactive sulfhyryls of CAIII in skeletal muscle under oxidative stresses of ischemia, or exhaustive exercise. Analysis by isoelectric focusing followed by Western blot revealed that the two sulfhydryls were differentially and progressively oxidized. Brief ischemia of 10 – 20 min provoked partial (one of the suflhydryls) modification of CAIII via reversible S-glutathiolation. Protracted 60 min ischemia yielded equal amounts of both partially and completely (both sulfhydryls) modified CAIII due to irreversible oxidization. Twenty minutes of repetitive electrical stimulation, simulating exhaustive exercise, produced a mixed yield: partial modification by reversible S- glutathiolation and complete modification by irreversible oxidation. Thus CAIII responds to oxidative stress with a distinctive sulfhydryl oxidation patterns reflecting duration and severity that may prove sensitive indices of extent and type of damage in muscle injury. IUBMB Life, 56: 343–347, 2004 Keywords Transcription profile; CAIII knockout mice; S-glutathio- lation; irreversible oxidation; ischemia; exhaustive exercise. INTRODUCTION Carbonic anhydrase III (CAIII: 4.2.1.1) is something of a functional enigma. It is one of 13 isozymes belonging to a class of zinc metalloenzymes that reversibly catalyzes hydration of carbon dioxide to bicarbonate and a proton. The importance of carbonic anhydrases I and II for efficient transport and elimination of CO 2 from tissues and lungs is well documented (1). However, the physiological role of CAIII is not well understood. CAIII is distinguished by its very low (*1%) specific activity for CO 2 hydration-dehydration reaction when compared with CAI and CAII isozymes (2, 3). The relative lack of activity is largely due to a sterically constricted catalytic cavity and to the presence of Lys 64 in place of His 64, an important residue for rapid proton transport out of the active site of CAII (4). CAIII is located predominantly in skeletal muscle where it constitutes *8% of soluble protein. It is present in high concentrations (2% of wet weight) in the cytoplasm of slow twich fibers such as soleus muscle, but is largely absent from white muscles such as the anterior tibialis and the pectoral muscle (5). Why such large amounts of this inefficient enzyme have come to play a physiological role in skeletal muscle remains a mystery. The fact that CAIII is an ‘inefficient’ enzyme abundantly present in muscles of high oxidation potential, led some to suggest that CAIII may operate in a oxidizing environment that provokes its own modification to function as a cellular antioxidant (6). Indeed, it has been shown that cells over- expressing CAIII in NIH-3T3 cells display a lower basal oxidized state than non-transfected cells (7). Furthermore, the rapid increase in reactive oxygen species (ROS) when exposed to H 2 O 2 observed in non-transfected cells is absent in CAIII transfected cells. Additionally, cells with overexpressed CAII did not block H 2 O 2 -induced ROS increase as does CAIII. S-glutathiolation, the reversible formation of a mixed disulfide between protein sufhydryls and glutathione, is regarded as one of the earliest and most important compo- nents of cellular defence mechanisms that prevent irreversible oxidation of proteins (8 – 11). CAIII has 2 reactive surface cysteines, Cys 183 and Cys 188, that can be S-glutathiolated in vitro and in vivo (4, 12, 13). These cysteines are not present in CAI and CAII. Previous in vitro studied have shown that Received 18 March 2004; accepted 6 July 2004 Address correspondence to: Un-Jin P. Zimmerman, PhD, C203, Richard Building, Dept. of Physiology, University of Pennsylvania School of Medicine, 37th and Hamilton Walk, Philadelphia, PA 19104, USA. E-mail: unjin@mail.med.upenn.edu IUBMB Life, 56(6): 343–347, June 2004 ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB DOI: 10.1080/1521-6540400000850