Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.     

Purification and characterization of liver catalase in acatalasemic beagle dog: comparison with normal dog liver catalase

Catalase from acatalasemic dog liver was purified to homogeneity and its properties were compared with those of normal dog liver catalase. The purified acatalasemic and normal dog liver catalases were found to have the same molecular weight (230,000 Da) and isoelectric point (pI: 6.0-6.2) and both enzymes contained four hematins per molecule. The catalytic activity of catalase from acatalasemic dog was normal. Furthermore, there was no difference between the acatalasemic and normal dog catalases in the binding affinity to NADPH (apparent Kd: 0.11-0.12 microM) and in the sensitivity to oxidative stress by hydrogen peroxide, the normal substrate of catalase. The acatalasemic dog enzyme was stable only in a narrow pH range (pH 6-9) although the normal enzyme was stable in a wide pH range (pH 4-10). Acatalasemic dog liver catalase also showed a slight low thermal stability at 37 degrees C and the heat-lability was remarkable at 45 degrees C, compared to the normal dog enzyme. These results indicated that the acatalasemic dog catalase is catalytically normal although it is associated with an unstable molecular structure.     

Tissue and organ expression of catalase in acatalasemic beagle dogs.

Acatalasemic Beagle dogs which were maintained in our laboratories showed no sign of catalase activity at all in the erythrocytes, and glutathione peroxidase and superoxide dismutase were at normal levels. Immunoblotting analysis demonstrated that no catalase protein is detectable in their erythrocytes. On the other hand, catalase activity was detected in other tissues and organs, albeit at varying, lower levels than in normal dogs. Quantitative immunoblotting analysis consistently demonstrated that the catalase protein is expressed in the liver and kidneys of acatalasemic dogs in proportion to the activity in these organs. The catalase mRNA expressions in the blood, liver and kidneys in acatalasemic dogs were almost the same as those in normal dogs. These results suggested that catalytically normal catalase protein is translated from mRNA in the tissues and organs including erythrocytes, but in erythrocytes this enzyme protein is disposed of by an unknown mechanism.     

The Philadelphia variant of galactokinase.

We have previously reported the existence of a polymorphism that causes black populations to have lower mean RBC galactokinase activity than comparable white populations. We have designated this allele the Philadelphia variant, GALKP, and have suggested that it is common in blacks and rare in whites. GALKP individuals have normal WBC GALK activity, in contrast to the half normal WBC GALK activities of heterozygotes for the allele (GALKG) that causes the galactokinase-deficient form of galactosemia. In one family, we have presented evidence for the existence of two sisters heterozygous for both GALKG and GALKP alleles. These individuals have 50% normal WBC GALK activity and less than 50% normal red cell activity. The latter finding indicates that the two variant GALK alleles additively affect RBC activity. The WBC results suggest that the low activity of GALK in RBC of individuals with the GALKP allele is due to its relative instability. We could obtain no evidence for such instability from studies of high reticulocyte bloods or RBC fractionation. Furthermore, we could not demonstrate that the GALK in WBC from GALKP individuals has altered electrophoretic migration.     

Establishment and cellular characteristics of a hepatocyte cell line (oums-31) derived from an acatalasemic mouse

Summary Liver cell lines with very low catalase activity were established from an acatalasemic mouse. Hepatocytes isolated by a collagenase-liver-perfusion technique were cultured in Williams’ E medium supplemented with 10% fetal bovine serum. The acatalasemic liver cell line showed approximately 20% of the catalase activity of a normal mouse liver cell line, whereas its glutathione peroxidase activity was approximately equal to that of the normal liver cell line. DNA sequence analysis of this cell line showed the same mutation in the catalase gene as is seen in the acatalasemic mouse. Our observation of intracellular content of hydrogen peroxide (H₂O₂) radical and increased susceptibility of the cells to H₂O₂ were compatible with the existence of low catalase activity in the acatalasemic mouse. This hepatocyte cell line should be useful for studying effects of oxidative radical stress at the cellular level.     

The peroxidatic and catalatic activity of catalase in normal and acatalasemic mouse liver

Monomeric, dimeric and tetrameric forms of mouse liver catalase have been shown to express peroxidatic activity while the tetrameric form expresses the catalic activity. Autosomally inherited acatalasemia, produced by X-ray irradiation of mice results in almost complete loss of catalic activity of catalase but has no effect on the peroxidatic activity. Liver catalase from normal and acatalasemic mice was purified by following the catalic and peroxidatic activity, respectively. Antiserum produced in rabbit against catalase from normal mouse completely precipitated the catalatic and peroxidatic activity from normal liver, and peroxidatic activity from the acatalasemic liver homogenate. Similar results were obtained when antiserum against peroxidase from acatalasemic mice was used. These studies indicate that acatalasemia in mice is due to a structural gene mutation which leads to synthesis of structurally altered catalase subunits. The altered subunits express peroxidatic activity but do not combine to form a tetramer which expresses catalatic activity.     

Catalase activity measured with a micro oxygen electrode in a pressurized reaction vessel

The assembly and the use of a simple airtight pressurized reaction vessel are described for the measurement of catalase activity with a micro oxygen electrode in an optically heterogenous medium. The oxygen concentration is expressed as the ratio of observed current to the current in an air-saturated solution. Thus, an individual standard can be obtained for each measurement and the calibration is less affected by changes in the amplification factor. Different procedures for calibration of the oxygen electrode were compared. Specific activities of crystalline catalase, of red blood cells from humans and from normal or acatalasemic mice, and of liver homogenates from normal or amino-triazole-pretreated rats were determined. The specific catalase activity of human erythrocytes, as found by this method, agrees with that obtained photometrically.     

Characterization of acatalasemia detected in two Hungarian sisters.

Acatalasemia was detected in 2 sisters of a Hungarian family. The pedigree of the family showed hypocatalasemia in the children of the patients and in 1 of their brothers, while the other members of the family had normal blood catalase activity. The biochemical characterization (catalase activity, electrophoretic migration, isoelectric point and enzyme stability) of the blood as well as tissue catalase of the acatalasemic patients yielded a catalase form which did not differ from normal.     

Detection of hunter heterozygotes by enzymatic analysis of hair roots.

We have developed a procedure for testing iduronate sulfatase, the enzyme deficient in Hunter syndrome, in single hair roots. Beta-Hexosaminidase was used as the reference enzyme. The ratio of iduronate sulfatase to beta--hexosaminidase, expressed in arbitrary units of activity, is near zero for Hunter patients and greater than 0.6 in almost all roots of normal individuals. Hair roots of Hunter heterozygotes show a characteristic continuum of activity ratios, ranging from totally deficient up to and including the normal range. The results are consistent with the origin of hair roots from a small number of progenitor cells which obey the Lyon hypothesis. The proportion of roots with low activity can be used to discriminate between normal and heterozygous individuals.     

Alpha-L-iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Using phenyl-α-l-iduronide as substrate, we have examined the level of α-l-iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α-l-iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α-l-iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α-l-iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α-l-iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.     

Methanol metabolism in acatalasemic mice

The methanol metabolism in acatalasemic mice was studied by administering [14C]methanol and [14C]formic acid to acatalasemic and normal mice and determining the radioactivity of exhaled carbon dioxide. Methanol metabolism was also studied in acatalasemic and normal mice treated with 3-amino-1,2,4-triazole (AT), which is known to be an inhibitor of catalase (EC 1.11.1.6). The metabolism of methanol and formic acid was inhibited in acatalasemic mice as seen by reduced [14C]CO2 production. Similar results were obtained when AT was given prior to the methanol injection into the normal and acatalasemic mice. The results indicate the peroxidative activity of catalase plays the major role in the methanol metabolism in mice. On the other hand similar studies with [1-14C] ethanol showed that the metabolism of ethanol was not inhibited in acatalasemic mice.     

Effects of post low-dose X-ray irradiation on carbon tetrachloride-induced acatalasemic mice liver damage

The catalase activities in the blood and organs of the acatalasemic (C3H/AnLCsb-Csb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCSa-Csa) mouse. We examined the effects of post low-dose (0.5 Gy) X-ray irradiation which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in acatalasemic or normal mice. As a result, the 0.5 Gy irradiation after carbon tetrachloride administration decreased the glutamic oxaloacetic and glutamic pyruvic transaminase activity in the acatalasemic mouse blood to a level similar to that of the acatalasemic mouse blood not treated with carbon tetrachloride; this is in contrast to a high-dose (15 Gy) irradiation. In the same manner, pathological disorder was improved by 0.5 Gy irradiation. The fat degeneration in normal mice was quickly reduced, in contrast to acatalasemic mice. These findings suggest that low-dose irradiation after carbon tetrachloride administration accelerates the rate of recovery and that catalase plays an important role in the recovery from hepatopathy induced by carbon tetrachloride, in contrast to high-dose irradiation.     

Molecular Characterization and Rescue of Acatalasemic Mutants of Drosophila Melanogaster

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen intermediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Cat(n4), likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Cat(n1) mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span.     

Levels of metallic mercury and mercuric ion in the venous and arterial bloods of normal and acatalasemic mice following exposure to mercury vapor

Levels of metallic mercury and mercuric ion in the arterial and venous bloods of normal and acatalasemic mice exposed to metallic mercury vapor in vitro and in vivo were investigated. Mercury uptake in venous blood from air saturated with mercury vapor with or without hydrogen peroxide in vitro was determined. Level of mercuric ion in venous blood of normal mice was significantly higher than that of acatalasemic mice. By contrast, metallic mercury in venous blood of acatalasemic mice was elevated relative to level in normal mice. Metallic mercury level in red blood cells and plasma was also significantly higher in acatalasemic mice. The ratio of metallic mercury to total mercury (Hg degrees + Hg2+) in the arterial and venous bloods of acatalasemic mice exposed to metallic mercury vapor was increased relative to normal mice. This ratio in red blood cells and plasma in the venous bloods of acatalasemic mice in vivo was also significantly higher than those of normal mice. The significance of metallic mercury in plasma for distribution of mercury in organs is discussed.     

Detection of heterozygotes for galactokinase deficiency in a human population

A simple method for assaying galactokinase in red blood cells has been described. The assay is proportional with respect to time and enzyme concentration within the limits studied. The pH optimum of the enzyme is between pH 7.8 and 8.2. In whole blood kept at 4 C, the enzyme is stable for at least 8 days, but activity is rapidly lost when the cells are washed and frozen. Employing this method, blood samples from 642 individuals were assayed for galactokinase activity. Six individuals had activity about one-half the normal value and were considered to be heterozygotes for galactokinase deficiency. Family studies on four of the individuals further substantiated this conclusion. The calculated gene frequency indicates that approximately 1 in 46,000 individuals would be expected to have galactokinase deficiency. This work was supported by a grant from the Children's Bureau.     

Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.     

A polymorphic variant of human erythrocyte carbonic anhydrase I with a widespread distribution in Australian aborigines,CAIAustralia-9 (8 Asp → Gly): Purification,properties, amino acid substitution,and possible physiological significance of the variant enzyme

Carbonic anhydrase I (EC 4.2.1.1) purified from the pooled packed red blood cells of 100 individuals typed as heterozygous for the common Australian Aboriginal carbonic anhydrase I variant CAI Australia-9 had a slightly higher specific CO2 hydratase or esterase (toward p-nitrophenyl acetate) activity than the normal component and a higher Km and Vmax using the esterase substrate. The variant enzyme was slightly more resistant to heat inactivation. The extent of inhibition of both enzymes by the specific inhibitor acetazolamide was identical, as was their immunological behavior and the lability of the active-site zinc ion. The variant enzyme was more resistant to chloride inhibition. The physiological importance of this observation is discussed in the context of a proposed adaptive advantage of the variant gene in the arid western and central regions of Australia. The amino acid substitution in the Aboriginal variant of a glycine for an aspartic acid residue has been located at residue 8 from the N terminus (i.e., 8 Asp leads to Gly), by proteolytic and partial acid hydrolyses. The possible effects of this substitution on the structure and function of the molecule are discussed.     

Acatalasemia

Summary The abnormalities in acatalasemia at the gene level as well as properties of the residual catalase in Japanese acatalasemia are historically reviewed. The replacement of the fifth nucleic acid, guanine, in the fourth intron by adenine in the acatalasemic gene causes a splicing mutation and hence a deficiency of mRNA. The guanine-to-adenine substitution was detected in two Japanese acatalasemic cases from different families. The properties of the residual catalase are similar to those of normal catalase; the exons are identical. The properties of the residual catalase and the molecular defect in the catalase gene are compared among Japanese, Swiss, and mouse acatalasemias. The physiological role of catalase, as judged from human acatalasemic blood and acatalasemic mice, is also described.