Histochemical identification of fibre types in the hamstring muscles of phosphorylase kinase-deficient ICR/IAn and normal C3H mice.

The low-activity sulphonamide resistant isozyme of carbonic anhydrase 111 has been shown to be skeletal-muscle-specific in man (Carter et al., 1979), and preliminary experiments have demonstrated high activities of the homologous molecule in rabbit, bovine, sheep, baboon, pig, chicken, rat and mouse muscle (Jeffery & Carter, 1980). Several years ago a sulphonamide(Diamox)-resistant isoenzyme of carbonic anhydrase was identified in male rat liver, but only in trace amounts in the female (Garg, 1974). The genetic and molecular relationship of the liver enzyme t o the well characterized erythrocyte forms of carbonic anhydrase (CAI and CAII) was not defined at that time. Here we present evidence that this Diamox-resistant enzyme present in male rat liver is a carbonic anhydrase I11 isoepzyme similar to that found in other mammals, and we describe some of its characteristics and tissue distribution. Homogenates (2 : 1) of rat tissues were prepared in water and centrifuged at 100oOg. Electrophoresis was carried out at pH9.1 in Cellogel (Whatman) (Carter et al., 1979). Location of carbonic anhydrase aRer electrophoresis was carried out by spraying the gel, presoaked in Bromothymol Blue, with carbon dioxide, when the enzyme zones appeared as bright yellow bands. Tissues initially examined were blood lysate, liver, kidney, testis, prostate, heart, brownand white-adipose tissue, skeletal muscle (slow and fast fibre types). All of these tissues showed the high-activity blood isoenzymes of carbonic anhydrase (CAI and CAII). In addition, most tissues, (with the exception of kidney and fast-fibre-type muscle), showed a faster-moving (anodal) group of carbonic anhydrase bands (see Fig. 1). Liver from the male rat and red muscle (soleus) from both male and female showed the highest specific activity of this fast-moving carbonic anhydrase. Addition of mercaptoethanol (10mM) to homogenates decreased the fast-moving groups of isoenzymes to a single zone (Fig. 1). All of these fast-moving bands retained activity aRer immersion of electrophoresed gels in the inhibiting sulphonamide (Diamox) at 1OmM. From these criteria and amino-acid analysis of the pure enzymes (J. Batten & N. D. Carter unpublished work), in comparison to other mammalian carbonic anhydrases, this anodal group of isoenzymes is thought to represent rat carbonic anhydrase I11 (CAIII) locus gene products. Male livers were found to contain about five times more CAIII than females, and this was confirmed by titrating carbonic anhydrase activity against Diamox over the concentration range 100nM-10 mM, which differentiated CAI, CAII 0 I Origin