Download Isoenzymes of carbonic anhydrase III in rat tissues

592nd MEETING, LONDON
73
Isoenzymes of carbonic anhydrase I11 in rat tissues
NICHOLAS CARTER,* JANE BATTEN,'
MALCOLM JACKSON,? STEPHEN JEFFERY* and
SARAH MOYLE*
*Department of Child Health, St George's Hospital Medical
School, London SWI 7 ORE, U.K.,and ?Department of Human
Metabolism, University College Hospital Medical School,
London W.C.2. U.K.
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, brown- and 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
In presence of 1pM-Diamox
lsoenzyme
CAI I I
CAI
0
I
0
I
Origin
Origin
la)
16)
Fig. 1. Electrophoretic separation of rat carbonic anhydrase on
Cellogel,pH 9.1
(a) Rat haemolysate; (b) muscle homogenate; (c) haemolysate
(no activity with inhibiting Diamox); (d) muscle homogenate:
CAI and CAII inhibited; (e) muscle homogenate and mercaptoethanol.
and CAIII activity. Castration of male rats produced, after 40
days, decreased CAIII activity at a value intermediate between
that for male and female. Treatment of a female rat with
testosterone produced markedly elevated liver CAIII activity
similar to that of the male. Activity of CAIII in muscle showed
no sex difference and was apparently unaffected by testosterone
administration or by castration.
Because of the marked difference in liver CAIII in male and
female, and the known zinc content of the carbonic anhydrase
molecule, we assayed liver zinc. The levels were: male,
1.39pmol/g dry wt.; female, 1.45 pmol/g dry wt.
In summary, many rat tissues contain a unique carbonic
anhydrase 111 isoenzyme, a product of a third carbonic
anhydrase gene locus. Liver from male rats contains very high
activity of CAIII, in contrast with low activity in female liver
and this activity in males was found to be controlled by ambient
testosterone. Since the contents of zinc in male and female livers
were found to be identical, there is likely to be some stringent
control of zinc content that compensates for the different sized
pools of zinc contained in CAIII. Since CAIII is totally absent
from human liver, the role of this molecule in the male rat liver
may give a clue to its unusual catalytic function, which in most
mammals is confined to red skeletal muscle.
Carter, N. D., Jeffery, S., Shiels, A., Edwards, Y., Tipler, T. &
Hopkinson, D. A. (1979)Biochem. Genet. 17,837-854
Garg, L. C. (1974)J. Phannacol. Exp. Ther. 180,557-562
Jeffery, S. & Carter, N. D. (1980) Comp. Eiochem. Physiol. 66B,
439441
Histochemical identification of fibre types m the hamstring muscles of phosphorylase
kinase-deficient ICR/IAn and normal C3H mice
RITA SINGH and JOHN R. GRIFFITHS
Department of Biochemistry, St. George's Hospital Medical
School, Cranmer Terrace, London S W17 ORE, U.K.
Enzyme histochemical techniques have been used to characterize the muscle-fibre types of human skeletal muscles and to
diagnose specific enzyme deficiencies (Dubowitz, 1974). Fibre
VOl. 9
typing has also been performed by various mammalian species
(Davies, 1972; Lobley et al., 1977; Pullen, 1977; Taylor &
Calvery, 1971). The aim of the present investigation was to
ascertain the proportion of glycolytic (type-I) and oxidative
(type-11) fibres in the muscles of normal (C3H) and phosphorylase kinase-deficient (ICR/IAn) mice. Glycogenolysis in ICR/
IAn muscle appears to be achieved by non-covalent activation