Proc. Nati Acad. Sci.
USA
Vol. 80, pp. 2752-2756, May 1983
Medical Sciences
Carbonic anhydrase II deficiency -identified as the primary defect
in the autosomal recessive syndrome of osteopetrosis with renal
tubular acidosis and cerebral calcification
(erythrocytes/bone resorption/parathyroid hormone/isozymes/heterozygote detection)
WILLIAM S. SLY*, DAVID HEWETT-EMMETTt, MICHAEL P. WHYTEt, YA-SHIOU L. Yut,
RICHARD E. TASHIANt
*Departments of Pediatrics, Medicine, and Genetics, Washington University School of Medicine, Division of Medical Genetics, St. Louis Children's Hospital, St.
Louis, Missouri 63110; tDivision of Bone and Metabolism, Departments of Medicine and Pediatrics, Washington University School of Medicine and the Jewish
Hospital of St. Louis, St. Louis, Missouri 63110; and tDepartment of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109
Communicated by Donald C. Shreffler, January 24, 1983
ABSTRACT The clinical, radiological, and pathological find-
ings in three siblings affected with the autosomal recessive syn-
drome of osteopetrosis with renal tubular acidosis and cerebral
calcification have been reported. In an effort to explain the pleio-
tropic effects of the mutation producing this disorder, we pos-
tulated a defect in carbonic anhydrase H (CA II), the only one of
the three soluble isozymes of carbonic anhydrase that is.known to
be synthesized in kidney and brain. We report here biochemical
and immunological evidence for the virtual absence of CA II in
erythrocytes of patients affected with this condition, whereas CA
I level is not reduced. Levels of CA II in erythrocyte hemolysates
from asymptomatic obligate heterozygotes are about half of nor-
mal. These findings: (i) elucidate the basic defect in one form of
inherited osteopetrosis; (ii) provide genetic evidence implicating
CA II in osteoclast function and bone resorption; (iii) explain pre-
vious observations that carbonic anhydrase inhibitors block the
normal parathyroid hormone-induced release of calcium from bone;
(iv) clarify the role of renal CA H in urinary acidification and bi-
carbonate reabsorption; and (v) suggest a method to identify het-
erozygous carriers for the gene for this recessively inherited syn-
drome.
Osteopetrosis is an inherited metabolic bone disease in which
a generalized accumulation of bone mass prevents normal de-
velopment of marrow cavities and the enlargement of osseous
foramena (1-3). It has been called "marble bone disease" be-
cause the bones are very dense radiographically, although the
bones typically have an increased susceptibility to fracture (2,
3). Multiple genetic defects produce osteopetrosis but the
mechanism common to all the known forms of osteopetrosis is
a failure of bone resorption (4). In man, two principal types of
osteopetrosis have been described. One is a dominantly in-
herited, relatively benign condition which is often detected ra-
diologically in asymptomatic adults (2, 3). A second type is the
recessive, lethal, malignant form of osteopetrosis. In this form,
osteopetrosis is usually present at birth, becomes symptomatic
early in infancy, and leads to death in infancy or early childhood
from infection or bleeding (3). Forms of osteopetrosis with clin-
ical courses of intermediate severity have also been recognized
(3). Various animal models of osteopetrosis have been identified
including four different mutations that produce osteopetrosis in
mice (4).
In 1972, three separate reports described a distinct form of
osteopetrosis that occurred in association with renal tubular aci-
dosis (5-7). The pattern of inheritance was autosomal recessive.
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2752
AND
The clinical course was not entirely benign, but the disease was
compatible with long survival and the hematologic abnormal-
ities that dominate the clinical picture in the recessive lethal
form of osteopetrosis were absent. One of these families was
not described in a complete report until 1980 (8), by which time
two of the three affected siblings had been found to have cal-
cification of the basal ganglia. In the same year, Ohlsson et al.
(9) independently reported three Saudi Arabian families in-
volving first-cousin marriages that produced offspring with os-
teopetrosis, renal tubular acidosis, and cerebral calcification, a
syndrome for which they proposed the name "marble brain dis-
ease.
In an effort to explain the pleiotropic effects of the mutation
underlying this disorder by a single enzyme defect, we pos-
tulated a defect in, one of the three isozymes of carbonic an-
hydrase (CA I, CA II, CA III) which are known to be under sep-
arate genetic control in humans (10-14). This hypothesis seemed
attractive for two reasons: (i) metabolic acidosis can be pro-
duced by sulfonamide inhibitors of CA (12), and (ii) several re-
ports have shown that CA inhibitors can block the parathyroid
hormone-induced release of calcium from bone, suggesting a
role for CA in bone resorption (15-17).
The relationship of CA deficiency to cerebral calcification was
less apparent, although it is known that CA II is present in brain
(18) and that CA inhibitors inhibit cerebral spinal fluid pro-
duction (19) and affect electrical activity of the brain (20). A de-
fect in the CA II isozyme seemed most likely because this is the
most widely distributed of the three known soluble isozymes
of CA in human tissues (10, 11) and CA II is the only soluble
isozyme so far identified in renal and brain tissue (18, 21, 22).
In addition, a genetically determined, virtually complete ab-
sence of CA I in mature erythrocytes has been found to have
no clinical consequences (23). Because both CA I and CA II are
expressed in human erythrocytes, it was possible to test this
hypothesis by examining these isozymes in hemolysates of pe-
ripheral blood from the family we reported previously (8).
In this report, we describe studies showing what appears to
be an almost complete absence of CA II in erythrocytes of pa-
tients affected with the syndrome of osteopetrosis, renal tu-
bular acidosis, and cerebral calcification. Furthermore, we re-
port that in asymptomatic normal parents of affected patients
the levels of CA II are half of normal, which supports the inter-
pretation that the CA II deficiency is the basic defect under-
lying this clinical disorder and suggests a means to identify het-
erozygote carriers.
Abbreviations: CA, carbonic anhydrase; PTH, parathyroid hormone.
 Medical Sciences: Sly et al.
MATERIALS AND METHODS
Preparation of Erythrocyte Hemolysates. Whole blood (about
10 ml) was collected in heparinized tubes (lithium heparin) and
shipped by overnight carrier in ice. Immediately on arrival, the
erythrocytes were washed three times in 2 vol of 0.85% NaCl
at room temperature. Hemolysates were prepared by lysing the
cells with 1 vol of distilled water and extracting with 0.4 vol of
toluene. This mixture was shaken on a Vortex mixer for 1 min
and then centrifuged at 2,250 X g for 20 min. After aspiration
of the toluene layer and membrane interface, the aqueous layer
was centrifuged in a Microfuge at 12,000 X g for 5 min to re-
move remaining cellular debris.
Electrophoresis, Staining, and Immunodiffusion. Vertical
starch gel electrophoresis of the hemolysates was carried out (8
V/cm for 18 hr) with a borate buffer system at pH 8.6 (24). The
esterase activities of the electrophoretically separated CA I and
CA II isozymes were detected with a mixture of 4-methylum-
belliferyl acetate for CA I and fluorescein diacetate for CA II
(25); the electrophoretograms were stained with 0.4% nigrosin
to detect protein (24).
The antisera to the CA isozymes were prepared as described
(26) by injecting rabbits with human CA I and CA II purified
from hemolysates by affinity chromatography on sulfonamide-
bound CM-Sephadex columns (27). Double immunodiffusion
on agar plates (1.5% agar in 0.2 M sodium citrate-buffered sa-
line, pH 6.7) was carried out by standard methods.
Quantitation of CA I and CA II. The ratio of CA I and CA
II in individual hemolysates was determined by measuring the
amounts of CA I and CA II after their separation by reverse-
phase HPLC as described (28). Two hundred microliters of fresh
hemolysate was extracted with 200 ,ul of 40% ethanol (kept at
-20°C) and 100 .ul chloroform (kept at -20°C) by vortexing in
a polypropylene Microfuge tube for 1 min at 40C. A longer ex-
traction time results in some loss of CA II (and a smaller loss
of CA I); shorter extraction can result in a pink aqueous layer
due to retention of some hemoglobin components. Samples were
centrifuged at 12,000 x g for 2 min and the aqueous layer was
collected. Fifty-microliter samples were injected into an Altex-
Beckman HPLC apparatus containing a Waters ,Bondapak C18
column. Buffer A [0.1% trifluoroacetic acid (Pierce) in HPLC-
grade water (Burdick and Jackson, Muskegon, MI)] and buffer
B [0.05% trifluoroacetic-acid in UV-grade acetonitrile (Burdick
and Jackson)] were used to develop, over 40 min, a 38-46%
linear gradient of buffer B. The peaks were monitored at 215
nm, and those corresponding to CA I and CA II were integrated
by using a Hewlett-Packard 3390A integrator. The CA I/CA II
ratios were calculated without any correction for the small (<10%)
differences in extinction coefficients at 215 nm.
RESULTS
Fig. 1 presents an abbreviated pedigree of the family with the
recessive syndrome of osteopetrosis, renal tubular acidosis, and
cerebral calcification that we reported (8). It includes the three
affected sisters, their unaffected sister, and the parents who are
obligate heterozygotes. Hemolysates from these individuals were
used for the electrophoretic, immunologic, and HPLC studies
of the CA I and CA II isozymes that are presented below.
The soluble isozymes of CA in erythrocyte hemolysates have
high affinities for certain esters and can be assayed on the basis
of their esterase activity (24, 25). Fig. 2 presents the patterns
of electrophoretically separated CA I and CA II isozymes on
starch gels stained for esterase activity and for protein. No CA
II isozyme was detected by either stain in the hemolysates of
the three affected sisters. The two obligate heterozygotes (II-
14, II-15) and the unaffected sister (III-3) showed lower levels
Proc. Natl. Acad. Sci. USA 80 (1983) 2753
FIG. 1. Abbreviated pedigree of a family with osteopetrosis, renal
tubular acidosis, and cerebral calcification. Solid symbols indicate af-
fected sisters.
of protein and esterase staining than did the controls. These
results indicate virtual absence of both CA II esterase activity
and stainable CA II protein in the hemolysates of affected pa-
tients and decreased levels in the heterozygotes. CA I levels
from the same hemolysates were not reduced. In fact, CA I lev-
els appeared to be slightly greater in the patterns of the three
affected sisters, suggesting the possibility of a small compen-
satory increase in CA I in CA II-deficient patients (note that
levels of HbA2 remained essentially the same).
Fig. 3 presents results of double immunodiffusion studies on
this family with specific antisera to CA I and CA II. With anti-
serum to CA I, immunodiffusion patterns were normal for all
individuals. By contrast, with antiserum to CA II there was no
crossreacting material in hemolysates from the three affected
individuals (III-1, III-5, and III-6). Immunoprecipitin bands in
hemolysates from the two obligate heterozygotes (11-14 and II-
15) and their clinically unaffected daughter were not different
from those of controls (the spouses of 111-3 and III-6). Thus, the
results of the immunodiffusion studies were consistent with the
data in Fig. 2 showing the virtual absence of CA II in the af-
fected sisters but did not detect quantitative differences be-
tween heterozygotes and controls.
We next attempted quantitation of the CA I and CA II iso-
zyme levels in individual hemolysates by measuring the amounts
of each isozyme after their separation by reverse-phase HPLC.
Fig. 4 presents the elution patterns for hemolysates from one
of the three affected sisters (III-6), her husband, and the ob-
ligate heterozygote mother (II-14) and father (II-15). The CA I/
CA II ratios for these individuals were >104, 9.0, 16.8, and 16.3,
respectively. The virtual absence of CA II in the affected pa-
tient was confirmed by this method. Because the higher-than-
control ratios in the obligate heterozygote parents suggested
that CA I/CA II ratios determined by this method might be
'
HbA---
--
HbA2 --
CAI
-
Origin
-- CA If---
8 7 6 5 4 3 2 1
FIG. 2. Patterns of erythrocyte CA I and CA II of individuals shown
in Fig. 1 and of controls, stained for esterase (A) and protein (B) after
starch gel electrophoresis for 18 hr in 20 mM sodium borate pH 8.6 buff-
er at 40C. Lanes: 1, unaffected control (spouse of m-3); 2, unaffected
father (11-15); 3, unaffected mother (11-14); 4, affected proband (EI-1);
5, unaffected sister (1-3); 6, affected sister (I11-5); 7, affected sister (III-
6); 8, unaffected control (spouse of m-6). See Figs. 1 and 5 for individual
pedigree designations.
2754 Medical Sciences: Sly et al. Proc. Natl. Acad. Sci. USA 80 (1983)

A R

FIG. 3. Ouchterlony double-diffusion tests. Center wells: A, anti-

serum against human erythrocyte CA I; B, antiserum against human 030 0 15 30
erythrocyte CA II. Outer wells in both A andB contained hemolysates
from the following sources: 1, affected sister (E1-6); 2, unaffected control
(spouse of III-3); 3, unaffected mother (11-14); 4, unaffected father (11- O'4 i c.IAI 16.3
15); 5, unaffected control (spouse of III-6); 6, unaffected sister (II-3); 7, 012 44 012 44
affected proband (III-1); 8, affected sister (III-5). 43
000 ~~43 0910 -

used to determine heterozygosity for the gene for this condi- 006 42 0.06 42
tion, blood was obtained from additional members of this family I06 4.0
-a 41 41
and CA I/CA II ratios were determined by HPLC. The ratios
fell into three groups: >104 for affected patients; 6.2-9.8 for ol04 7 40 0.04 CAnl 40

one group which included four controls and five unaffected 6); 39ther i
3II15)9NtCA
I 9
members at risk for heterozygosity; and 13.4-17.2 for a group
which included two obligate heterozygotes and five unaffected [0 0 155 30
38 0- 1~05 30
people at risk for heterozygosity (Fig. 5). TIME(minutes)
The mean value for the CA I/CA II ratio for controls ob-
tained by using HPLC in this study is somewhat higher than FIG. 4. Separation of CA II and CA I from hemoglobin-extracted
that obtained by radioimmunoassay in a prior study [mean ± hemolysates by reverse-phase HPLC. (Upper Left) Control (spouse of
SEM: 6.32 ± 0.94 for 58 white control subjects and 6.64 ± 1.37 mI-6); (UpperRight) mother (11-14); (LowerLeft) affected daughter (Ill-
for 79 black control subjects (24)]. This higher value may reflect 6); (LowerRight) father (11- 15). Note the absence of CA II in the affected
daughter. The CA I/CA 11 ratios were determined by integration of the
the preferential denaturation of CA II by the chloroform/ethanol HPLC peaks (see text). The value >104 for the affected daughter rep-
extraction used to prepare hemolysates for the HPLC analysis. resents a minimum obtained by using the smallest genuine peak in-
However, the absence of overlap in CA I and CA II ratios de- tegrated (no peak was detected in the CA 11 region).
termined by HPLC between the small number of controls and
the presumed heterozygotes in this study suggests that the CA 14). Reaction HI is an ionic dissociation that is virtually instan-
I/CA II ratio can be used to determine heterozygosity for the taneous and is not subject to enzymatic acceleration.
gene producing the inherited syndrome of osteopetrosis with
renal tubular acidosis and cerebral calcification. I II
CO2 + H20=±H2CO3 HCO3 HC. +
DISCUSSION The direction of these reactions depends on the relative con-
All three soluble isozymes of CA in humans, CA I, CA II, and centrations of CO2 and HCO3 and on the pH. There is also a
CA III, are monomeric, =29,000-dalton, zinc metalloenzymes distinctive membrane-bound CA in lung, which .may represent
which catalyze the reversible hydration of CO2 (reaction I) (10- a fourth enzyme and tentatively is designated CA IV (29). The

1 2
I + +

E 1-13 1 51

16.8 16.3 7.8

2 3 4 5 6

Air>o10 1.7 6.4 >10' >10' 9

HOMOZYGOUSCA II DEFICIENCY. OSTEOPETROSIS

12 Cbb1
(
14.5 13.4
[ O PRESUMED HETEROZYGOTE UNAFFECTED
D O PRESUMED NORMALS AND CONTROLS

FIG. 5. Extended pedigree of family with syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Values beneath in-
dividuals in the pedigree indicate CA I/CA II ratios determined by HPLC. (See legend to Fig. 4 and text for details of the HPLC analysis.) Family
members II-14 and II-15 are obligate heterozygotes. The other heterozygotes indicated are presumed to be heterozygotes on the basis of similarities
of their CA I/CA 11 ratios to those of obligate heterozygotes.
 Medical Sciences: Sly et al.
kidney also contains a membrane-bound CA that is an intrinsic
component of the brush border of the proximal tubule (30-32).
Genetic and structural evidence suggests that at least the sol-
uble isozymes comprise a multilocus enzyme family derived from
a common ancestral gene by gene duplications (11). However,
the kinetic parameters of these isozymes and their sensitivity
to inhibitors can differ markedly (33, 34). These findings have
suggested that the physiological roles for the different isozymes
are diverse, which is also suggested by their different tissue
distributions.
The human CA II isozyme, whose turnover number for the
CO2 hydration reaction under physiological conditions (1.3-1.9
X 10' sec') is the highest known for any enzyme (35, 36), has
been identified (immunologically or by purification) in a wide
variety of cells, tissues, and organs including erythrocytes, brain,
eye, kidney, cartilage, liver, lung, skeletal muscle, pancreas,
gastric mucosa, and anterior pituitary body (10, 11). The other
isozymes, whose activities toward CO2 and HCO3 are lower
than those of CA II in the order CA II > CA IV > CA I > CA
III (29, 33, 34), appear to have a more limited distribution. CA
I is found primarily in erythrocytes, CA III mainly in red skel-
etal muscle, and CA IV in lung.
The finding of a quantitative defect in CA II in these patients
provides us with an unusual opportunity to assess the impor-
tance and function of this isozyme. In view of the high CO2 hy-
drase activity of CA II and its wide tissue distribution, one might
expect widespread effects of this deficiency in organs in which
CAII plays an important role. However, it should be noted that
the finding of a virtual absence of CA II in erythrocytes does
not necessarily imply a comparable deficiency in other tissues
and organs in which CA II has been reported. CA II levels in
cells with considerably more rapid turnover than erythrocytes
could be appreciably higher. Also, there may be additional, still
unidentified, CA genes contributing to enzyme levels in tissues
spared by this mutation. However, what is clear from these pa-
tients is that the quantitative deficiency of CA II that we have
demonstrated in erythrocytes has important clinical conse-
quences for bone, for kidney, and for brain that merit discus-
sion.
Bone Metabolism. All known forms of osteopetrosis are as-
sociated with failure to resorb bone (4). Studies of several pa-
tients with osteopetrosis have demonstrated impaired hyper-
calcemic responses to infused parathyroid hormone (PTH) (37,
38). The cause for this impairment might differ in the different
forms of osteopetrosis. Studies showing inhibition of PTH-in-
duced release of calcium from bone by CA inhibitors have sug-
gested a role for CA in bone resorption (15-17). Also, CA has
been demonstrated histochemically in chick and hen osteoclasts
(39). On the basis of these and other observations (40, 41), it
has been suggested that PTH activates CA in certain bone cells
where it might aid the resorptive process by mediating secre-
tion of H' (16, 39). The genetic evidence presented here pro-
vides strong support for a role of CA in bone resorption, which
was suspected from the pharmacological and histochemical evi-
dence cited above, and specifically implicates the CA II iso-
zyme in bone resorption.
Renal Tubular Acidosis. There is general agreement that renal
reabsorption of bicarbonate is a major factor in the maintenance
of acid-base homeostasis (42). Most of the bicarbonate recla-
mation takes place in the proximal tubule and depends on CA
(43). Only recently has it become clear how both a soluble (cy-
tosolic) and a membrane-bound (luminal) CA play separate roles
in the proximal tubule (21, 42-46). Bicarbonate reclamation de-
pends on H+ secretion, the major mechanism for proximal tu-
bular acidification (42). The H' secreted into the lumen of the
proximal tubule is titrated by the HCO3 in the glomerular fil-
Proc. Natl. Acad. Sci. USA 80 (1983) 2755
trate to produce H2CO3 which is in contact with the membrane-
bound CA. The luminal CA then catalyzes the dehydration of
H2CO3 to CO2 and H20 (42, 43). The CO2 diffuses freely into
the proximal tubular cell, where it can be hydrated by the cy-
tosolic CA to H2CO3. Dissociation of this product into H' and
HCO3 allows HCO3 to be transported by unknown mecha-
nisms into interstitial fluid or the peritubular capillary, com-
pleting the reclamation of filtered bicarbonate (42, 43). The re-
generated H' can be secreted in exchange for Na+ to initiate
another cycle (42). From the above, it is clear how two different
CAs operate at different sites to participate in bicarbonate rec-
lamation.
The enzymatic dehydration of H2CO3 in the lumen appears
to be mediated entirely by the membrane-bound CA present
in the brush border of proximal tubular cells (42, 45, 46). Al-
though we have no direct information on the status of this
enzyme in the patients described here, we have no reason to
suspect that this enzyme, which is biochemically and im-
munologically distinct from CA II and the other soluble iso-
zymes (21, 22, 31, 32), is defective in these patients. On the
other hand, the enzymatic hydration of intracellular CO2 is pre-
sumably mediated entirely by the soluble enzyme CA II, for
which these patients are deficient. The renal tubular acidosis
present in patients with this syndrome must be explained in
this context.
Although the renal tubular acidosis in the different pedi-
grees has been variable in severity, and somewhat heteroge-
neous in type, most of the patients reported have a significant
distal defect (47). In those cases in which HCO3 reabsorption
has been adequately studied, a proximal component, evidenced
by HCO3 wasting at normal plasma HCO3 concentrations,
has also been found (47). Thus, the patients appear to have a
mixed or hybrid type of renal tubular acidosis which includes
both a proximal component and a distal component. The hy-
dration of CO2 in cells of the proximal tubule presumably is
mediated by CA II which appears to be the major (and perhaps
only) soluble isozyme present in the kidney (21, 22, 48). Be-
cause this reaction generates some of the H' secreted by the
proximal tubule, and bicarbonate reclamation in the proximal
tubule depends almost entirely on H' secretion (42-44), we
can understand why patients with CA II deficiency might have
a renal tubular acidosis that includes a proximal component.
The prominent distal component of the renal tubular acidosis
in CA II-deficient patients, evidenced by inappropriately high
urine pH values when patients were quite acidotic (47), initially
was more difficult to understand. A possible explanation was
provided recently by immunohistochemical evidence showing
much more intense reaction for CA II in the distal tubules (and
even in collecting ducts) than in proximal tubules of human kid-
neys (48). These results suggest that CA II plays a more im-
portant role in the distal tubule than was previously suspected
(22), either in generating H+ or in titrating OH- produced by
the proton-translocating ATPase.
The report (23) that a genetically determined, virtually com-
plete, absence of erythrocyte CA I in man is not associated with
renal tubular acidosis is consistent with biochemical and im-
munological evidence that CA II is the only soluble isozyme
present in kidney (21, 22, 48). In view of this evidence, and of
the findings presented here, it is difficult to understand the sig-
nificance of the abnormalities in erythrocyte CA I that have been
reported in a few patients with distal type renal tubular acidosis
(49, 50).
Brain Metabolism. The function of CA II in brain and the
reasons for brain calcification in patients with defects in CA II
are less well understood. In the central nervous system, CA II
is primarily a glial enzyme and occurs predominantly in oh-
2756 Medical Sciences: Sly et al.
godendrocytes (18). CA II has been identified in brain homog-
enates, with up to 50% of the activity in a membrane-bound
form (51). Although the functions of CA in brain are still spec- 12.
ulative, it is worth noting that many of the patients with the 13.
syndrome of osteopetrosis with renal tubular acidosis and ce-
rebral calcification have significant mental retardation (9). The 14.
patients in the family reported here are exceptional in this re- 15.
gard because their IQ scores are in the low normal range (8).
Like the mechanism of the cerebral calcification in this syn- 16.
drome, the mechanism of the mental retardation is not yet clear. 17.
18.
Erythrocyte Function. One might expect some secondary
consequences of CA II deficiency in tissues in which the CO2 19.
produced must be delivered to circulating erythrocytes and dis- 20.
charged from the lungs. This transport depends on the ability
of the CA activity in erythrocytes to convert metabolic CO2 to 21.
HCO3 rapidly in the tissues and to catalyze the reverse re- 22.
action in lung capillaries. Although CA II normally accounts for
23.
only 14-17% of the CA in erythrocytes (CA I accounts for the 24.
rest), it has been estimated that CA II accounts for about 90%
of the CA activity of erythrocytes in vivo (36, 52). This estimate
is based on the much greater specific activity of CA II compared 25.
to CA I and on the much greater sensitivity of CA I to inhibition
26.
by the normal chloride concentration of erythrocytes (52). This
estimate is in general agreement with the findings by W. R. 27.
Chegwidden (personal communication), in a preliminary ex-
periment on the family described here, that the relative CA ac- 28.
tivities for the HCO3 dehydration reaction in hemolysates from
an affected homozygote (III-1), an obligate heterozygote (II-14), 29.
and an unrelated control were 0.17, 0.43, and 1.0, respectively,
30.
after correction for the nonenzymatic control reaction. Thus, all
of the available evidence indicates that CA II is more important 31.
than CA I for the CO2 hydrase reaction in the erythrocyte.
However, Wistrand (36) has estimated that only 2% of normal 32.
levels of CA activity in erythrocytes would be required for un- 33.
loading CO2 in lung capillaries at rest, and 4% would be re- 34.
quired at work. If all of these estimates are correct, CA I ac-
tivity alone may be sufficient for this erythrocyte function. The 35.
fact that the patients described here have no disability that we 36.
can ascribe to the virtual absence of CA II in their erythrocytes 37.
supports this conclusion.
38.
We thank Patrick J. Venta and Dr. Alan Robson for helpful sugges-
tions, Mrs. Sabra Lovejoy for typing the manuscript, and Mrs. Dorothy 39.
Chesnut, RN, for obtaining blood samples. This research was supported 40.
by National Institutes of Health Grants GM 21096, GM 31988 (to W. S. S.),
and GM 24681 (to R.E.T.) and by the National Institutes of Health 41.
Clinical Research Center sponsored by Grant RR00036.
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2. Johnston, C. C., Jr., Lavy, N., Lord, T., Vellios, F., Merritt, A.
D. & Deiss, W. P., Jr. (1968) Medicine 47, 149-167. 43.
3. Beighton, P., Hamersma, H. & Cremin, B. J. (1979) S. Afr. Med.
J. 55, 659-665. 44.
4. Marks, S. C. (1982) Am. J. Anat. 163, 157-167.
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Fr. Pediatr. 29, 269-286.
6. Sly, W. S., Lang, R., Avioli, L., Haddad, J., Lubowitz, H. & 46.
McAlister, W. (1972) Am. J. Hum. Genet. 24, 34 (abstr.).
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