Judith B. Howard

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.

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.

Molecular location of the genetic lesion in the acatalasemic mouse

Antibody to normal mouse catalase will stabilize blood and liver catalase of the acatalasemic mouse against a variety of agents which damage protein tertiary structure (urea, guanidine, trypsin) but not against agents which affect the heme group (azide, hydroxylamine). The antibody will also stabilize catalase against inhibition by 3-amino 1,2,4-triazole (AT), the specific site of action of which is known. The antibody is able also to protect normal mouse catalase from urea denaturation, but it is without effect on AT inhibition of normal catalase. A hypothesis is proposed which explains these results and which helps localize the site of the mutation on the catalase molecule.

Acatalasemia and Hypocatalasemia in the Dog and the Duck

Summary The dog is shown to have very low blood catalase activity levels, ranging from an immeasurably low blood catalase to a level which in other species would be considered hypocatalasemic. Although some tissue levels of catalase activity are lower than, say, those of the normal mouse, the dog and the duck are not acatalatic.

Sensitivity of acatalasemic mice to acute and chronic irradiation and related conditions.

Acatalasemic mice are no more sensitive, as measured by lethality, than normocatalasemic mice to acute whole-body x-irradiation, chronic whole-body gamma irradiation, or exposure to one or several atmospheres of 100% oxygen. If the animals receive a preliminary injection of aminotriazole, which reduces the catalase activity of all solid tissues but does not affect erythrocyte catalase, the acatalasemic mice are rendered essentially acatalatic, but are still no more sensitive to the agents and procedures listed. In fact, the data suggest that low body catalase, far from being deleterious, may actually be protective to an animal receiving a single dose of whole-body x-irradiation. On the other hand, the acatalasemic mouse is considerably more sensitive to injected hydrogen peroxide than is the normal mouse, and prior injection of aminotriazole further increases this sensitivity.

A RAPID BLOOD CATALASE SCREENING TECHNIQUE ADJUSTABLE TO ANY LEVEL OF ACTIVITY.

Abstract A procedure is described for rapidly screening whole blood samples for any desired level of catalase activity. With this test, one individual can nondestructively screen perhaps 125 mice per day.

Catalase levels and radiation resistance in three species of wild duck

SummaryThree species of wild duck said to have very different LD50s of x-radiation do not differ significantly in catalase activity per gramme body-weight. It is suggested that the wide variation in LD50 may be a reflection of such factors as age, adaptability to capture and penning, etc. A secondary finding of interest is that the breast muscle of these wild ducks has a significantly higher level of catalase activity than does the leg muscle. This relationship is reversed in domestic chickens, and it is suggested that muscle catalase activity may be a reflection of functional usage.

Stoichiometry of Antibody Stabilization of a Labile Enzyme

Summary Guanadrel was evaluated for its cardiovascular profile and was found to possess properties which are desirable in the treatment of hypertension. The compound causes blockade of the sympathetic nerve ending and depletion of peripheral and “hypothalamic” catecholamines. Guanadrel does not deplete intestinal norepinephrine, which may be the reason for the fact that it does not produce diarrhea when administred experimentally or clinically.