Presence of mast cell precursors in the yolk sac of mice

Concentration of mast-cell precursors in hematopoietic tissues of mouse embryos was evaluated by a limiting dilution method. Cells from yolk sacs, livers, and bodies of (WB x C57BL/6)F1 (hereafter called WBB6F1)- +/+ embryos were injected directly into the skin of adult WBB6F1-W/Wv mice which were genetically depleted of tissue mast cells. Concentration of mast-cell precursors was calculated from the proportion of injection sites at which mast cells did not appear. Since the concentration of mast-cell precursors in the yolk sac was about 30 times as great as that of embryonic body at Day 9.5 of the pregnancy, the mast-cell precursors seemed to be generated within the yolk sac. The concentration in the yolk sac reached the maximum level at Day 11, and then dropped markedly at Day 13. In contrast, mast-cell precursors increased from Day 11 to Day 15 in the fetal liver. As a result, the concentration of 11-day yolk sacs was comparable to that of 15-day fetal liver. Although intravenous injection of 15-day fetal liver cells (2 x 10(6)) rescued the general mast-cell depletion of WBB6F1-W/Wv mice, the intravenous injection of the same number of 11-day yolk sac cells did not rescue it. In contrast with fetal livers, yolk sacs scarcely contained hematopoietic stem cells which were measured by spleen colony formation. Therefore, the mast-cell precursors of the yolk sac may not originate from such stem cells.     

Strain differences in the teratogenicity induced by sodium valproate in cultured mouse embryos

Strain differences in the teratogenicity of valproic acid (VPA) have been reported in mice. Finnell and Chernoff (Proc. Grnwd. Genet. Ctr. 5:162-163, 1985) showed that 300 mg/kg of VPA twice a day on days 6-8 of gestation induced exencephaly in 82% of SWV embryos but in 0% of C57BL/6J embryos. In the present experiment, we have collected similar results and investigated this strain difference using whole embryo culture in an attempt to determine whether maternal or embryonic factors are responsible for the difference. Mouse embryos were explanted on day 8.5 (plug day 0), and embryos at the 6-8-somite stage were cultured for 48 hours in rat serum containing various doses of sodium valproate (NaVP). All the embryos died within 24 hours with 4.5-mM and higher doses of NaVP in C57BL/6NCr1BR (C57) and with 3.0-mM and higher doses in SWV. Unfused brain folds were recognized in embryos treated with 3.0-mM and higher doses in C57, and with 1.0-mM and higher doses in SWV. Irregular somite formation was observed in many embryos treated with 1.6-mM and higher doses in C57 and with 1.0-mM and higher doses in SWV. These results indicate that SWV embryos have 1.5-3 times the sensitivity of C57 embryos to the embryolethal and teratogenic effects of NaVP. Furthermore, the results suggest that the basis of the strain difference resides within the embryo rather than the mother.     

Strain dependency of TGFbeta1 function during embryogenesis

There is incomplete penetrance to Tgfb1 knockout phenotypes. About 50% of Tgfb1 homozygous mutant (Tgfb1-/-) and 25% of Tgfb1 heterozygous (Tgfb1+/-) embryos die during embryogenesis. In a mixed NIH/Ola x C57BL/6J/Ola x 129 background partial embryonic lethality of the Tgfb1-/-embryos occurs due to defective yolk sac vasculopoiesis and/or hematopoiesis. We show here that on a predominantly CF-1 genetic background, lack of TGFbeta1 causes a pre-morula lethality in about 50% of the null embryos. This partial lethality is not reversed by transfer of Tgfb1-/- embryos to Tgfb1-/+ hosts. The extent of embryonic lethality in Tgfb1-/- embryos ranges in a background dependent manner from 20% to 100%. Based on these and other studies it is clear that TGFbeta1 acts at two distinct phases of embryogenesis: pre-implantation development and yolk sac vasculogenesis/hematopoiesis. The susceptibility for the pre-implantation lethality depends on a small number of genetic modifiers since a small number of backcrosses onto the high susceptibility strain C57BL/6 leads to complete penetrance of the lethality.     

PGE measurement in mouse embryos and uterine/embryo tissue

Embryonic tissue of rodents and other species has been reported to produce prostaglandins (PG) of the E series during gestation. We attempted to establish the presence of PGE in C57BL/6J mouse embryos and peri-embryonic tissue as an initial step in examining the role of maternal ethanol treatment on PG production. Gestation day 10 embryos were found not to produce or degrade PGE. However, a tissue complex which included embryonic tissue, peri-embryonic membranes, placenta and uterus was capable of producing PGE from both endogenous and exogenous arachidonic acid. Furthermore, and aspirin was able to suppress PGE production from this tissue. It is concluded that gestation day 10 C57BL/6J mouse embryonic tissue, unlike that of rat, is not capable of measurable PGE production. However, uterine and peri-embryonic tissues, needed to support pregnancy, are capable of significant PGE production.     

Strain differences between C57BL/6 and SWV mice in time of palate closure and induction of palatal slit and cleft palate

Palatal slit, which occurs spontaneously in C57BL/6 (C57BL) mice, is increased in frequency among C57BL fetuses from dams treated with triamcinolone acetonide, but is not induced in SWV fetuses. On the other hand, C57BL is more resistant than SWV to cleft palate induction by triamcinolone. Using these C57BL and SWV mice, the relation of palate stage and chronological age was examined from 1 P.M. on day 14 to 9 A.M. on day 16 in untreated embryos, and the condition of the palate after triamcinolone treatment on day 12 was examined at 9 A.M. on day 16. In untreated embryos, horizontalization and fusion of the palatal shelves occurred earlier in C57BL than in SWV embryos, but fusion of the primary palate with the secondary palate occurred later. After triamcinolone treatment, the development of the palate was delayed in both C57BL and SWV embryos. These results suggest that the times of normal palate closure are related to the differences between C57BL and SWV mice in their susceptibilities to palatal slit and cleft palate induction and that triamcinolone produces palatal slit and cleft palate by delaying palate closure.     

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.     

PGE measurement in mouse embryos and uterine/embryo tissue

Embryonic tissue of rodents and other species has been reported to produce prostaglandins (PG) of the E series during gestation. We attempted to establish the presence of PGE in C57BL/6J mouse embryos and peri-embryonic tissue as an initial step in examining the role of maternal ethanol treatment on PG production. Gestation day 10 embryos were found not to produce or degrade PGE. However, a tissue complex which included embryonic tissue, peri-embryonic membranes, placenta and uterus was capable of producing PGE from both endogenous and exogenous arachidonic acid. Furthermore, in vivo and in vitro aspirin was able to suppress PGE production from this tissue. It is concluded that gestation day 10 C57BL/6J mouse embryonic tissue, unlike that of rat, is not capable of measurable PGE production. However, uterine and peri-embryonic tissues, needed to support pregnancy, are capable of significant PGE production.     

A two-locus model for experience-conditioned direction of paw usage in the mouse is suggested by dominant and recessive constitutive paw usage behaviours.

Left-right direction of paw usage in the mouse depends on the genotype and the directional nature of the test. There are two phenotypic classes; in some strains, direction of paw usage is learned or conditioned by the direction of the initial test chamber and the experience of reaching and, in other strains, paw usage is a constitutive behaviour not affected by previous experience. We report the evidence for locus heterogeneity in the cause of constitutive versus experience-conditioned paw usage from a phenotypic analysis of F1 hybrid generations from the experience-conditioned C57BL/6J, C3H/HeHa, and SWV strains and the constitutive CDS/Lay and DBA/2J strains. The F1 hybrids between strains of different phenotypic classes provide evidence of locus heterogeneity. Constitutive paw usage in CDS/Lay is phenotypically dominant to experience-conditioned behaviour in both C57BL/6J and SWV. However, constitutive paw usage in DBA/2J is phenotypically recessive to experience-conditioned behaviour in C57BL/6J and dominant to experience-conditioned behaviour in SWV. Among the experience-conditioned strains, C57BL/6J is highly lateralized but SWV is only weakly lateralized. Our data suggest a model in which C57BL/6J may have a "strong" allele that identifies a functional difference between the constitutive paw usage of CDS/Lay and DBA/2J. DBA/2J may have a loss-of-function mutation at the same locus that is recessive to the strong C57BL/6J allele. SWV may have a "weak" allele and the (SWV x D2)F1 compound heterozygote may be below a threshold for detectability of experience-conditioned behaviour, making the constitutive behaviour of DBA/2J appear to be dominant to the experience-conditioned behaviour of SWV. CDS/Lay may have a dominant allele at a second locus that suppresses experience-conditioned behaviour in all F1 hybrids.     

Developmental toxicity of methanol: Pathogenesis in CD-1 and C57BL/6J mice exposed in whole embryo culture

BACKGROUND: Methanol causes axial skeleton and craniofacial defects in both CD-1 and C57BL/6J mice during gastrulation, but C57BL/6J embryos are more severely affected. We evaluated methanol-induced pathogenesis in CD-1 and C57BL/6J embryos exposed during gastrulation in whole embryo culture. METHODS: Conceptuses with five to seven somites were exposed to 0, 1, 2, 3, 4, or 6 mg methanol/ml culture medium for 24 hr and embryonic morphology was assessed. Cell death was evaluated by histology and LysoTracker red staining, and cell-cycle distribution was evaluated by flow cytometry. RESULTS: In C57BL/6J embryos, craniofacial defects were observed at 3 mg methanol/ml and greater. The response for CD-1 embryos was different, with increased dysmorphology only at 6 mg/ml. However, protein content in CD-1 embryos was reduced at 3 mg methanol/ml and above, indicating growth retardation. Yolk sac toxicity occurred only at 6 mg methanol/ml in both strains. Methanol caused only small changes in cell-cycle distribution, while cell death was induced at 4 and 6 mg methanol/ml in both strains after 8 hr. The extent of cell death after 8 hr was greater in C57BL/6J embryos, and increased over time through 18 hr; in contrast, CD-1 embryos showed less cell death at 18 than at 8 hr, suggesting recovery. CONCLUSIONS: Cell death plays a prominent role in methanol-induced dysmorphogenesis, while cell-cycle perturbation may not. Differences in the extent of cell death between CD-1 and C57BL/6J embryos correlated with differences in the severity of dysmorphogenesis.     

Altered Circadian Period of Locomotor Activity in Carbonic Anhydrase II-Deficient Mice

This study finds lengthened circadian period in a congenic strain of mice homozygous for a null mutation in carbonic anhydrase isoenzyme-II gene on proximal Chromosome 3. Carbonic anhydrase II has the highest turnover rate of any constitutive enzyme. It catalyzes the reversible hydration of carbon dioxide to control intercellular acid/base balance. A strain of congenic mice has a carbonic anhydrase II null mutation within a DBA/2J inbred strain insert on a C57BL/6J inbred strain background. The locomotor activity levels and period of circadian rhythms were examined in the homozygous null mutants and their progenitors, mice heterozygous for the region around the carbonic anhydrase gene. The heterozygous mice siblings and the wild-type siblings served as the controls. During behavioral studies, male and female offspring and parents were housed singly in constant darkness. Locomotor activity was monitored using an infrared photobeam array. Mice homozygous for the carbonic anhydrase null mutation had a longer circadian period than either heterozygote or wild type littermates. Carbonic anhydrase null mutants also had low locomotor activity compared to either heterozygous or wild-type litter mates. This implies that either the physiological changes resulting from absence of carbonic anhydrase II isozyme or the presence of DBA/2J alleles around the carbonic anhydrase locus influence the circadian period and level of locomotor activity in laboratory mice.     

Altered Circadian Period of Locomotor Activity in Carbonic Anhydrase II-Deficient Mice

This study finds lengthened circadian period in a congenic strain of mice homozygous for a null mutation in carbonic anhydrase isoenzyme-II gene on proximal Chromosome 3. Carbonic anhydrase II has the highest turnover rate of any constitutive enzyme. It catalyzes the reversible hydration of carbon dioxide to control intercellular acid/base balance. A strain of congenic mice has a carbonic anhydrase II null mutation within a DBA/2J inbred strain insert on a C57BL/6J inbred strain background. The locomotor activity levels and period of circadian rhythms were examined in the homozygous null mutants and their progenitors, mice heterozygous for the region around the carbonic anhydrase gene. The heterozygous mice siblings and the wild-type siblings served as the controls. During behavioral studies, male and female offspring and parents were housed singly in constant darkness. Locomotor activity was monitored using an infrared photobeam array. Mice homozygous for the carbonic anhydrase null mutation had a longer circadian period than either heterozygote or wild type littermates. Carbonic anhydrase null mutants also had low locomotor activity compared to either heterozygous or wild-type litter mates. This implies that either the physiological changes resulting from absence of carbonic anhydrase II isozyme or the presence of DBA/2J alleles around the carbonic anhydrase locus influence the circadian period and level of locomotor activity in laboratory mice.     

Differential teratogenesis of all-trans-retinoic acid administered on gestational day 9.5 to SWV and C57BL/6N mice: emphasis on limb dysmorphology

BACKGROUND: Mouse strain differences in teratologic response are well documented. However, because retinoids cause similar malformation syndromes across many species, the strain differences may be predicted to be minimal. The goals of this study were to characterize and explain the differences between the C57BL/6N and SWV mouse strains in terms of all-trans-retinoic acid (RA)-induced teratologic effects at the time of gestation that cause postaxial forelimb ectrodactyly. METHODS: Visceral and skeletal malformations were determined by Wilson's sectioning and double-staining techniques, respectively; developmental staging was performed according to the somite count; and retinoid concentrations were assessed by HPLC. RESULTS: C57BL/6N mice were more susceptible than SWV mice to induction of embryolethality, cardiovascular defects, and forelimb ectrodactyly, whereas the opposite was true for the induction of ear, thymus, and tail agenesis, and cleft palate, gastroschisis, and anal atresia. As determined by somite counts, 1 strain intercross was developmentally advanced compared to the parental strains and the reciprocal cross. Retinoid susceptibility was equivalent between the reciprocal crosses for some malformations and determined by the maternal genotype for others. Toxicokinetic experiments showed that whole-embryo peak retinoid concentrations did not differ between the strains, but the area under the curve (AUC) for all-trans-RA was 1.3 times higher in C57BL/6N than in SWV embryos. CONCLUSIONS: The malformation spectrum induced by RA was strain-specific, and the strain sensitivity for forelimb ectrodactyly was consistent with all previously tested teratogenic agents (i.e., C57BL/6N was more sensitive than SWV). The strain differences in teratologic effects were not explained by developmental timing differences or toxicokinetic differences at the whole-embryo level.     

The effect of antisense oligonucleoties specific to the harakiri mRNA on spontaneous and induced defects of mouse preimplantation embryo development

The effect of antisense oligonucleotides specific to mRNA of the proapoptotic gene harakiri (Hrk) on the development of mouse SAMP1 (senescence-accelerated mouse prone) and (C57BL/6J x DBA/2J)F1 preimplantation embryos cultured in vitro was investigated. The SAMP1 mice are characterized by genetically determined decrease of fertility along with the highly frequent perturbations of embryonic development. Reproduction indices of the (C57BL/6J x DBA/2J) hybrids lie within the normal range. Because of this, preimplantation abnormalities in this line were induced by the action of proapoptotic agent bleomycine. It was demonstrated that antisense inhibition of the Hrk expression had no effect on the frequency of genetically determined abnormalities of early embryonic development in SAMP1 mice. In case of induced abnormalities, addition of oligonucleotides specific to mRNA of proapoptotic Hrk gene influenced the number of abnormalities, and at the same time, improved the quality of survived embryos via increasing the blastocyst hatching.     

Identification of a murine locus conveying susceptibility to cadmium-induced forelimb malformations

The heavy metal cadmium (Cd), an environmentally ubiquitous contaminant, is a potent teratogen in mice. When administered parenterally, it induces an array of malformations that vary in scope and severity with the route, dose, time of administration, and the strain of the animal. When administered intraperitoneally on day 9.0 of gestation, 4 mg/kg cadmium chloride produces forelimb defects (predominantly ectrodactyly) in over 80% of fetuses of the C57BL/6 mouse strain, while no limb defects are observed in the identically treated SWV strain. Like other examples of strain-specific teratogenic activity, the underlying nature of the differential susceptibility remains unknown. The present study investigates the segregation of sensitivity to Cd-induced forelimb defects in crosses between C57BL/6 and SWV mice and provides evidence for the involvement of both maternal and fetal factors in the determination of defect expression. In addition, quantitative trait loci (QTL) analysis of the fetal genetic component was performed among 198 backcross progeny, utilizing a genomic linkage map of 149 informative microsatellite markers. One QTL demonstrating significant linkage to expression of the defect, designated Cadfar (cadmium-induced forelimb autopod reduction), was mapped to the distal end of chromosome 6 with a lod score of 3.1.     

Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-visceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 micrograms/ml whilst AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 microliters/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of 125I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to our previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on 125I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behaviour is observed under similar experimental conditions.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Intrauterine growth retardation in endothelial nitric oxide synthase-deficient mice is established from early stages of pregnancy

The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3(tm1Unc)/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3(-/-)), and 12 pregnant, wild-type C57BL/6J mice (group Nos3(+/+)). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3(+/+) than in Nos3(-/-) mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3(+/+) mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3(-/-) and Nos3(+/+) mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3(+/+) mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3(-/-) mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3(-/-) mice are established from early pregnancy stages.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.