Alan Shiels

Characterization of human carbonic anhydrase III from skeletal muscle.

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to β-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.

Immunoassay of carbonic anhydrase III in rat tissues

Relatively high levels (i.e., up to 1% wet muscle wt) of a low-activity sulfonamide-resistant carbonic anhydrase isozyme has been reported from skeletal muscle of chicken, cat and sheep [ 11, ox [2], rabbit [3], mouse [4], human [8,9] and gorilla [7]. It has been accepted that these isozymes are all homologous forms of an enzyme designated CA111 and essentially confined to skeletal muscle. Nevertheless, trace levels of CA111 have also been detected immunologically in sheep lung 141, rabbit liver [4], and human liver, smooth muscle, lung and cardiac muscle [ 81. Assay of carbonic anhydrase III was carried out as described for human CA111 [8]. Antiserum was prepared in New Zealand white rabbits using 3 sequential weekly injections of 1 mg CA111 in Freund’s complete adjuvant, followed by an i.v. injection of 1 mg rat CA111 in 0.005 M phosphate buffer (pH 7.0). Tissue extracts (20%, w/v) were prepared in distilled water and spun at 20 000 X g for 15 mm. Extracts from soleus, anterior tibialis (AT), extensor digitorum longus (EDL), prostate, white fat and liver were made as above.

SEXUAL DIFFERENTIATION OF RAT LIVER CARBONIC ANHYDRASE III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.

Hormonal Control of Carbonic Anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III (CA III) in the livers of adult male rats was found to be approximately 30 times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver CA III concentrations that could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomized female rats induced about a 5-fold increase in liver CA III concentration. Immunoprecipitational analysis of the products of liver mRNA translation in vitro with antiserum specific for CA III showed that hormonal control of the levels of CA III in rat liver is mediated by changes in the amount of translatable CA III mRNA. Marked changes in liver CA III concentrations were also observed in developing and aging male rats. Different control mechanisms appear to operate in mouse and man.

Red cells genetically deficient in carbonic anhydrase II have elevated levels of a carbonic anhydrase indistinguishable from muscle CA III.

Adult human red cells contain appreciable levels of two carbonic anhydrase (CA) isozymes, CA I ( I 1.6 ? 2.3 mg/g Hb) and CA I1 (1.8 ? 0.3 mg/g Hb), as determined by radioimmunoassay (RIA). Recently, biochemical and immunological studies have demonstrated the presence of an additional red cell carbonic anhydrase with properties indistinguishable from skeletal muscle CA III.2*3 It is present at levels of 147 ? 17 pg/g Hb assuming that the RIA, set up for the skeletal muscle CA 111,4 is in fact detecting a CA isozyme in red cells immunologically indistinguishable from muscle CA 111. Purification of this red cell “CA 111” by the methods used for isolating skeletal muscle CA I11 (affinity chromatography and gel f i l t ra t i~n)~ has, however, resulted in yields of less than 10% of that e ~ p e c t e d . ~ The purified red cell “CA 111” and skeletal muscle CA I11 have identical elution properties from reverse-phase pBondapak CIS high performance liquid chromatography (HPLC) column^^-^ and preliminary peptide-map data indicate that the allelic polymorphism found in skeletal muscle CA I11 (31 Ile -+ Val)5 is also present in the “CA 111” purified from outdated red cells pooled from several individuals. These data and the finding that the characteristic electrophoretic mobilities of skeletal muscle CA I11 from different mammals are mimicked by their red cell “CA 111” counterparts strongly support the view that we are examining products of the same gene. Future work at the gene level should confirm this. However, we must take into account that the phenomenon of gene conversion, whereby adjacent homologous genes can evolve “in concert” (e.g. , a globin and y globin gene^)^; this provides a basis for the possibility that there are two tissue-specific CA I11 genes encoding identical or almost identical proteins. The CA I and CA I1 genes are closely linked on chromosome 3 of the mouse.8 There is good evidence for such linkage in the Old World monkey, Macaca nerne~trina,~ and other mammals.1° It seems likely that CA I and CA I1 will also be linked in humans although only CA II has been mapped (to chromosome 8) so

Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate

Carbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle.

Androgen-linked control of rat liver carbonic anhydrase III

The concentration of carbonic anhydrase IlI (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.

Synthesis of rat muscle carbonic anhydrase III in a cell-free translation system

Carbonic anhydrase III (CA III) was identified in the products of rat soleus muscle RNA translation in vitro by both a radioimmunoassay and a specific immunoprecipitation technique followed by SDS—polyacrylamide gel electrophoresis analysis of the precipitated antigen. The primary translation product has the same M r‐value as the native isoenzyme. CA III mRNA was found to represent ∼0.55% of the total mRNA present in rat soleus muscle.

Novel purification of carbonic anhydrase III from human, rat and baboon muscle

A new method of inhibitor elution from DEAE cellulose is described for carbonic anhydrase III. Highly purified fractions free of other isozymes were obtained after one column elution.