Extremely low-frequency electromagnetic fields affect lipid-linked Carbonic anhydrase

In the last years, the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on the activity of different enzymes were investigated. Only the membrane-anchored enzymes did decrease their activity, up to 50%. In this work, the effect of ELF-EMF on bovine lung membrane carbonic anhydrase (CA) were studied. Carbonic anhydrases are a family of 14 zinc-containing isozymes catalyzing the reversible reaction: CO₂+H₂O = HCO₃^? +H⁺. CA differ in catalytic activity and subcellular localization. CA IV, IX, XII, XIV, and XV are membrane bound. In particular, CA IV, which is expressed in the lung, is glycosyl phosphatidyl inositol-linked to the membrane, therefore it was a candidate to inhibition by ELF-EMF. Exposure to the membranes to a field of 75 Hz frequency and different amplitudes caused CA activity to a reproducible decrease in enzymatic activity by 17% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. When the source of enzyme was solubilized with Triton, the field lost its effect on CA enzymatic activity, suggesting a crucial role of the membrane, as well as of the particular linkage of the enzyme to it, in determining the conditions for CA inactivation. Results are discussed in terms of the possible physiologic effects of CA inhibition in target organs.     

Effects of extremely low frequency electromagnetic fields on membrane-associated enzymes

The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54-61% with field amplitudes above a threshold of 73-151 microT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.     

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.     

Effects of extremely low frequency electromagnetic fields on the adenylate kinase activity of rod outer segment of bovine retina

Extremely low frequency electromagnetic fields (ELF-EMFs) of 75 Hz with amplitudes above a threshold of about 125 microT have a dramatic effect on the adenylate kinase (AK) activity of the rod outer segment (ROS) membranes. In fact, the ATP production by ROS membranes or by purified disk membranes placed in the field decreased by approximately 54%. The decrease in enzymatic activity was independent of the time of exposure to the field and was completely reversible. When disk membranes were solubilized with Triton or a soluble isoform of AK was used, negligible effects of the field were obtained on the enzymatic activity, suggesting that the membrane has an important role in determining the conditions for the enzyme inactivation.     

Sinusoidal ELF magnetic fields affect acetylcholinesterase activity in cerebellum synaptosomal membranes

The effects of extremely low frequency magnetic fields (ELF‐MF) on acetylcholinesterase (AChE) activity of synaptosomal membranes were investigated. Sinusoidal fields with 50 Hz frequency and different amplitudes caused AChE activity to decrease about 27% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. Identical results were obtained with exposure to static MF of the same amplitudes. Moreover, the inhibitory effects on enzymatic activity are spread over frequency windows with different maximal values at 60, 200, 350, and 475 Hz. When synaptosomal membranes were solubilized with Triton, ELF‐MF did not affect AChE activity, suggesting the crucial role of the membrane, as well as the lipid linkage of the enzyme, in determining the conditions for inactivation. The results are discussed in order to give an interpretation at molecular level of the macroscopic effects produced by ELF‐MF on biological systems, in particular the alterations of embryo development in many organisms due to acetylcholine accumulation. Bioelectromagnetics 31:270–276, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of ELF-EMF on antioxidant status and micronuclei in K562 cells and normal lymphocytes

The effect of ELF-EMF on DNA through changes in antioxidative enzyme activities has not been sufficiently explored yet. The aim of this study was to determine ELF-EMF effect on antioxidative enzymes in cancer cell line and genotoxic potential on normal human lymphocytes. K562 cells were exposed to 50 Hz ELF-EMF (40 μT, 100 μT; 3 h, 24 h) and spectrophotometric determination of lipid peroxidation and antioxidative enzyme activities was conducted. Genotoxicity of ELF-EMF (50 Hz, 100 μT) was investigated by cytokinesis-block micronucleus assay in a normal human lymphocytes (exposure 24 h and 48 h). Results demonstrated that ELF-EMF did not alter the process of lipid peroxidation and superoxide dismutase activity. Catalase activity was increased only after application of 100 μT EMF for 24 h. Glutathione-S-transferase and -reductase activities were increased. Treatment with 100 μT ELF-EMF (24 h, 48 h) significantly reduced micronuclei incidence, while cell proliferation was significantly increased. Results indicate that 50 Hz ELF-EMF (40 μT, 100 μT) are week stressors which alone cannot generate enough ROS to induce process of lipid peroxidation in cancer cell line but strong enough to induce response of antioxidative system. Furthermore, 100 μT ELF-EMF in human lymphocytes did not exhibit genotoxic potential during 24 h and 48 h treatment, but stimulated cell proliferation.     

Bilirubin inhibition of enzymes involved in the mitochondrial malate-aspartate shuttle

The effect of increasing bilirubin concentrations upon the catalytic activity of a series of dehydrogenases and aminotransferases was examined. The particular enzymes were chosen to examine the effect of bilirubin upon the activity of enzymes responsible for the indirect transfer of reducing equivalents across the inner mitochondrial membrane. Malate dehydrogenase was inhibited at very low concentrations of bilirubin and showed competitive inhibition with respect to coenzyme of 2 μM, while the cytosolic form of this enzyme exhibited a 15 μM inhibition constant. Cytosolic glycerol-3-phosphate dehydrogenase was not appreciably inhibited by bilirubin. Both the mitochondrial and cytosolic forms of aspartate aminotransferase showed moderate competitive bilirubin inhibition with respect to substrates with a K_i of 30 μM with respect to 2-oxoglutarate and a K_i of 80 μM with respect to aspartate. Preincubation studies indicated that inhibition was reversible for all enzymes examined. These results are interpreted in terms of the inhibition of the malate-aspartate shuttle by relatively low concentrations of bilirubin.     

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.     

Monospecific polyclonal antibodies directed against purified cinnamate 4-hydroxylase from Helianthus tuberosus. Immunopurification, immunoquantitation, and interspecies cross-reactivity.

We recently reported the purification of cinnamic acid 4-hydroxylase (CA4H), a cytochrome P-450 catalyzing the second reaction of the general phenylpropanoid pathway, from Jerusalem artichoke (Helianthus tuberosus L.) (B. Gabriac, D. Werck-Reichhart, H. Teutsch, F. Durst [1991] Arch Biochem Biophys 288: 302-309). Rabbit polyclonal antibodies were raised against the native and denaturated nitrocellulose-bound enzyme. Only the immunoglobulins G (IgGs) elicited upon immunization with native enzyme produced strong inhibition of catalytic activity and good cross-reactivity on western blots. In microsomes from H. tuberosus tissues induced by wounding and various chemicals, a positive correlation between catalytic activity and amounts of immunoreactive protein on western blots was observed. When coupled to cyanogen bromide-activated Sepharose, purified IgGs selectively retained CA4H activity from solubilized plant microsomes. Acid elution from the immunoaffinity matrix provided a rapid procedure for high-yield purification of the CA4H protein. The same IgGs immunoprecipitated a single protein from the in vitro translation products of mRNA isolated from wounded tissues. The apparent molecular weight (57,000) of this polypeptide was identical to that of CA4H purified from tuber microsomes. Immunochemical relatedness between CA4H from different plant species was demonstrated by strong inhibition of catalytic activity and immunopurification of several orthologous enzymes, using IgGs directed against CA4H from H. tuberosus. However, only limited interspecies cross-reactivity was observed on western blots. A careful immunochemical analysis indicates that CA4H immunoreactivity significantly differs from plant to plant. Results are discussed in terms of antibody specificity, enzyme glycosylation, and CA4H regulation.     

Total CA activity in isolated perfused guinea pig lung by 18O-exchange method

The rate of exchange of 18O between alveolar CO2 and lung water was measured in isolated perfused guinea pig lungs to quantify carbonic anhydrase (CA) activity. The average lung CA activity, with a reaction velocity constant of 5.32 +/- 2.2 s-1, is sufficient to accelerate CO2 reactions in lung water by two orders of magnitude over the uncatalyzed rate at 22 degrees C and a PCO2 of 40 Torr. Three sulfonamide inhibitors of CA with different human erythrocyte membrane permeabilities were used to determine the availability of the enzyme to the perfusate. Ethoxzolamide, the most permeable at 0.1 microM (100 times its inhibition constant, of Ki) inhibited 85% of enzyme activity after exposure of the lung for 3 min and 94% of enzyme activity after 30 min, whereas 1.25 microM (320 times its Ki) acetazolamide (1/165 as permeable) only inhibited CA 28% at 3 min and 75% at 30 min. Benzolamide (less than 1/1,000 as permeable) at 4 microM (1,000 times its Ki) inhibited only approximately 17% of control CA activity by 5 min and 48% by 30 min after the start of perfusion. These data indicate the CA available to pulmonary capillary plasma is approximately 10% of the total lung CA activity, in agreement with published measurements on the homogenized lung.     

Carbonic anhydrases IV and IX: subcellular localization and functional role in mouse skeletal muscle

The subcellular localization of carbonic anhydrase (CA) IV and CA IX in mouse skeletal muscle fibers has been studied immunohistochemically by confocal laser scanning microscopy. CA IV has been found to be located on the plasma membrane as well as on the sarcoplasmic reticulum (SR) membrane. CA IX is not localized in the plasma membrane but in the region of the t-tubular (TT)/terminal SR membrane. CA IV contributes 20% and CA IX 60% to the total CA activity of SR membrane vesicles isolated from mouse skeletal muscles. Our aim was to examine whether SR CA IV and TT/SR CA IX affect muscle contraction. Isolated fiber bundles of fast-twitch extensor digitorum longus and slow-twitch soleus muscle from mouse were investigated for isometric twitch and tetanic contractions and by a fatigue test. The muscle functions of CA IV knockout (KO) fibers and of CA IX KO fibers do not differ from the function of wild-type (WT) fibers. Muscle function of CA IV/XIV double KO mice unexpectedly shows a decrease in rise and relaxation time and in force of single twitches. In contrast, the CA inhibitor dorzolamide, whether applied to WT or to double KO muscle fibers, leads to a significant increase in rise time and force of twitches. It is concluded that the function of mouse skeletal muscle fibers expressing three membrane-associated CAs, IV, IX, and XIV, is not affected by the lack of one isoform but is possibly affected by the lack of all three CAs, as indicated by the inhibition studies.     

Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.     

Angiotensin I-converting enzyme from guinea pig lung and serum. A comparison of some kinetic and inhibition properties.

The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar.     

Inhibition and derivatization of the renal Na,K-ATPase by dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Reversible and irreversible effects of nitric oxide on the soluble hydrogenase from Alcaligenes eutrophus H16.

The effects of NO on the H2-oxidizing and diaphorase activities of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. With fully activated enzyme, NO (8-150 nM in solution) inhibited H2 oxidation in a time- and NO-concentration-dependent process. Neither H2 nor NAD+ appeared to protect the enzyme against the inhibition. Loss of activity in the absence of an electron acceptor was about 10 times slower than under turnover conditions. The inhibition was partially reversible; approx. 50% of full activity was recoverable after removal of the NO. Recovery was slower in the absence of an electron acceptor than in the presence of H2 plus an electron acceptor. The diaphorase activity of the unactivated hydrogenase was not affected by NO concentrations of up to 200 microM in solution. Exposure of the unactivated hydrogenase to NO irreversibly inhibited the ability of the enzyme to be fully activated for H2-oxidizing activity. The enzyme also lost its ability to respond to H2 during activation in the presence of NADH. The results are interpreted in terms of a complex inhibition that displays elements of (1) a reversible slow-binding inhibition of H2-oxidizing activity, (2) an irreversible effect on H2-oxidizing activity and (30 an irreversible inhibition of a regulatory component of the enzyme. Possible sites of action for NO are discussed.     

The role of alkyl chain length in the inhibitory effect n-alkyl xanthates on mushroom tyrosinase activities

Sodium salts of four n-alkyl xanthate compounds, C2H5OCS2Na (I), C3H7OCS2Na (II), C4H9OCS2Na (III), and C6H13OCS2Na (IV) were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) in 10 mM sodium phosphate buffer, pH 6.8, at 293 K using UV spectrophotometry. 4-[(4-Methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed, competitive or uncompetitive inhibition for the four xanthates. For the cresolase activity, I and II showed uncompetitive inhibition but III and IV showed competitive inhibition pattern. For the catecholase activity, I and II showed mixed inhibition but III and IV showed competitive inhibition. The synthesized compounds can be classified as potent inhibitors of MT due to their Ki values of 13.8, 11, 8 and 5 microM for the cresolase activity, and 1.4, 5, 13 and 25 microM for the catecholase activity for I, II, III and IV, respectively. For the catecholase activity both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail of these compounds. The length of the hydrophobic tail of the xanthates has a stronger effect on the Ki values for catecholase inhibition than for cresolase inhibition. Increasing the length of the hydrophobic tail leads to a decrease of the Ki values for cresolase inhibition and an increase of the Ki values for catecholase inhibition.     

Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino acids

The effect of aminooxyacetic acid (AOAA), an inhibitor of pyridoxal phosphate-dependent enzymes (including the aminotransferases), on the K⁺-evoked release of amino acids was studied during microdialysis of neostriatum in anesthetized rats. K⁺-evoked (100 mM) release of asparatate, glutamate, and GABA was inhibited by 74%, 70%, and 63%, respectively, by 20 mM Mg²⁺ and are therefore reflecting release from the transmitter pools of these amino acids. Treatment with AOAA decreased the K⁺-evoked release of aspartate, glutamate, and GABA instantly, with a delayed decrease in the efflux of glutamine and alanine, arguing that the synthesis of transmitter amino acids in particular is sensitive to the activity of pyridoxal phosphate-dependent enzymes. Interestingly, GABA release increased severalfold following the initial decrease, probably reflecting inhibition by AOAA on GABA aminotransferase, the enzyme most sensitive to inhibition by AOAA, and responsible for enzymatic inactivation of transmitter GABA.Special issue dedicated to Dr. Claude Baxter.     

Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.