Robert N. Feinstein

Acatalasemic and hypocatalasemic mouse mutants: II. Mutational variations in blood and solid tissue catalases

A strain of mice genetically deficient in blood catalase should be of considerable value in studying the possible role of hydrogen peroxide in carcinogenesis and cancerolysis, as suggested by WARBURG, GAWEHN, and GEISSLER (1957) and by HOLMAN (1957), and in assessing the possible role of H,O, in radiation lethality. That such a strain could be viable appeared probable from the work of TAKAHARA (1952), AEBI et al. (1961 ) , and SZEINBERG et al. (1963), with humans, and RADEV (1958) with guinea pigs. Therefore a large scale search for low-catalase mutants among the progeny of irradiated mice was initiated. A preliminary note announced (FEINSTEIN et al. 1964b) the successful production of a mouse litter containing individuals with blood catalase levels approximately 1 % of normal. The present communication details the search for mutants and describes five mutant lines which have been obtained. MATERIALS AND METHODS A. Initial source of mutants: Mice used as the initial source of possible mutants were discards from two experiments at Oak Ridge National Laboratory. In these experiments, designed to obtain radiation-induced visible mutations at specific loci (RUSSELL 1951), mice examined were offspring of irradiated fathers, who had received a total dose of 600R in fractionated exposures. B. Analytical techniques: Blood was drawn from the orbital sinus (RILEY 1960) and laked in an appropriate volume of cold water. In the initial screening, a recently published (FEINSTEIN et al. 1964.a) rapid, semi-quantitative blood catalase assay was used. After this stage, the quantitative perborate assay (FEINSTEIN 1949) was employed. Mice were weaned and sexed at four weeks of age. and blood catalase was assayed at six weeks.Abstract In a series of one acatalasemic and four hypocatalasemic mouse strains, the catalase of blood, liver, and kidney has been compared with regard to sensitivity to heat, pH extremes, ionizing radiation, and a variety of chemical reagents. It is concluded that the catalase of the normal, or wild type, strain differs qualitatively from all the mutants, and that the catalase of the five mutants all differ amongst themselves. The catalase of one pair of hypocatalasemic mutants shows so little difference in behavior that the two molecular species are possibly identical.

Activity and stability of catalase in blood and tissues of normal and acatalasemic mice

Catalase activity in blood, liver, and kidney of a mutant strain Csb has been found to be decreased as compared to the level in normal mice. However, the extent of the reduction largely depends on the conditions used for activity determination, in particular, temperature and duration of the incubation period. In liver, this effect is most pronounced, the observed activity in mutants varying between 21 and 85% of the normal level. This dependence on the assay conditions is mainly due to the unusual heat lability of the variant enzyme, which undergoes rapid inactivation when incubated at 37 C.

Purification and immunochemical characterization of aldehyde dehydrogenase from 2-acetylaminofluoreneinduced rat hepatomas

1. A series of aldehyde dehydrogenase isozymes (aldehyde:NAD (P)+ oxidoreductase, EC 1.2.1.5), has been purified from hepatomas induced in Sprague-Dawley rats by 2-acetylaminofluorene. 2. The functional hepatoma-specific aldehyde dehydrogenase isozymes exist as 105 000-dalton dimers composed to two subunits of 53 000 daltons. Isoelectric points of the purified isozymes are 6.9-7.2. 3. Antiserum to these purified hepatoma-specific aldehyde dehydrogenases has been produced and the immunological relationships of these isozymes to their normal liver counterpart have been studied. Results of Ouchterlony double diffusions, agar-gel immunoelectrophoresis and polyacrylamide gel and agar immunoelectrophoresis indicate that anti-hepatoma aldehyde dehydrogenase antiserum cross-reacts with normal liver aldehyde dehydrogenase.

Acatalasemia in the mouse and other species.

Genetic control of the level of blood catalase activity was first demonstrated in 1927. At present, such control has been demonstrated or suggested for nine different species, including man, the most studied. The development of an acatalasemic strain of mice has permitted a wide variety of experimental approaches, including most of those used in humans. Among those approaches which cannot readily be applied to man but have been used in acatalasemic mice are investigations of sensitivity to radiation lethality, mechanism of awareness to radiation, possible use as a model for replacement therapy for inborn errors of metabolism, and catalase in tissues other than erythrocytes. These are described, together with genetic, immunological, and other studies comparable to similar work on acatalasemic humans.

Aldehyde dehydrogenase activity in a rat hepatoma

Abstract A rat hepatoma, induced by feeding acetylaminofluorene (AAF), followed by phenobarbital, shows an aldehyde dehydrogenase activity that greatly exceeds that of normal liver. When tissue extracts are subjected to electrophoresis on polyacrylamide gel, a single band of activity is obtained from normal liver, but several bands are obtained from hepatoma.

Blood catalase polymorphism. Some immunological aspects.

The immunological properties of the erythrocyte catalase of mice—normal (wild type) strain, one lacking catalase (acatalasemic), and four with only slight catalase activity (hypocatalasemic strains)—have been investigated. Agardiffusion tests and antigen titration of red-cell lysates against rabbit antiserum to catalase from normal mouse blood showed that immunologically identical catalase protein was present in large amounts in the acatalasemic as well as in the hypocatalasemic mutant strains. Despite lack of catalatic activity, the erythrocytes lacking catalase as well as those with only a little catalase contain catalase protein that has been modified at the site of enzyme activity, although the antigenic determinants are identical with those of normal catalase protein. This mutation is purely structural, being characterized by modification of the enzyme active site but not of the antigenic site.

Detection of oxidases on polyacrylamide gels

Abstract Direct transfer of electrons, from the reduced flavin moiety of several oxidases to phenazine methosulfate and then to nitroblue tetrazolium, serves to detect activity of the oxidases of l -amino acids, urate, diamines, hydroxy acids (both long- and short-chain), aldehydes, and sarcosine. It is demonstrated that the method specifically detects oxidases and is more sensitive than other methods available. It is noted that, in the detection of certain dehydrogenases on polyacrylamide gels by the usual NAD-phenazine methosulfate-nitroblue tetazolium technique, one or more of the isozymes may actually represent the corresponding oxidase rather than the dehydrogenase in question.

Mechanism of inhibition of Catalase by 3-amino-1,2,4-triazole.

Abstract Injection of 3-amino-1,2,4-triazole (AT) reduces the catalase activity of six solid tissues in the same order as does the implantation of a tumor. In vitro , dilute AT, in combination with a widely distributed heat-stable factor (HSF), reduces the activity of crystalline catalase. Alcoholtreated HSF prevents catalase inhibition by an otherwise active HSF-AT system.

Comparative heat stability of blood catalase

Abstract The thermal stability of the catalase in whole blood lysates of a variety of mammalian species has been measured. A total of 26 varieties of mammal has been examined, and the T 50 (temperature at which 50% of the activity is lost under the conditions employed) varies from as low as 48.1 ° to as high as 67.1 °. The T 50 is in no way correlated with the absolute level of catalase activity. Thermal stability curves, and hence T 50 values, are sharply reproducible for individuals of a given species regardless of sex or age. Various species within a given genus tend to have similar T 50 values. Various genera, even within a single family, however, might vary widely in T 50 . In the laboratory mouse, a mutation in a structural gene, which has been shown elsewhere to produce a new molecular modification of the catalase, shows a T 50 which is significantly different from that of the catalase of the original, nonmutant strain.

Nature of the heterozygote blood catalase in a hypocatalasemic mouse mutant.

The blood catalase of a hypocatalasemic mouse mutant has been compared with that of the wild-type (normal) animal and with that of the heterozygote. Comparison is on the basis of stability to heat and to urea. Electrophoretic evidence is of no value, because all forms tested show the same mobility. Because the heterozygote heat and urea inactivation curves differ from those of the two parental forms, and because the curves are smoothly S-shaped, with no shoulders or other irregularities, it is suggested that this heterozygote produces only a single molecular form of the enzyme.