The Journal of Heredity 65: 28-32. 1974.
Subcellular Localization of Catalase Variants Coded by Two Genetic Loci During Maize Development

CATALASE (H2O2:H2O2 oxidoreductase, EC of maize is a tetramer9 that is controlled by at least two genetic loci, Ct1 and Ct2, which are expressed at different stages in development12. In a given inbred strain homozygous for the Ct1 gene (which is expressed during kernel development) the second ygene, Ct2, is also expressed but only after the seed matures and during early sporophytic development. The general developmental pattern is one in which the Ct1 catalase gradually disappears as the Ct2 catalase gradually becomes the predominant form; this is dramatically apparent in the scutellum during the first 10 days of sporophytic development. Thus, there is a point in sporophytic development when the two genes overlap in their expression and when their products (subunit polypeptides) interact to generate intergenic hybrid catalase molecules with characteristic physicochemical properties13. Quail and Scandalios8 have shown that the Ct1 and Ct2 catalases differ in their rates of synthesis and degradation and that this post-translational control partly accounts for their differential expression.

In this investigation, I have attempted to determine the subcellular location of catalase in maize and whether there is differential compartmentation of the different catalase isozymes. Catalase is known to be the common factor in microbodies from a variety of organisms spanning the evolutionary scale10. However, whether the catalase in such organelles is unique with respect to the total catalase complement within a given organism has, to my knowledge, not been determined.

Materials and Methods

The highly inbred maize (Zea mays L.) strain 229 was used. This strain is homozygous for the allelic gene Ct1v and undergoes the kind of developmental shift discussed above to allow for the expression of the Ct2 gene in early sporophytic development.

Seeds were soaked in running tap water at 25°C for 24 hours, surface sterilized with 1 percent sodium hypochlorite for 10 minutes, and washed twice with sterilized deionized water. The seeds were then placed on a layer of germination paper (Kimpak) in sterilized plastic trays and germinated in darkness at a constant temperature of 25°C and 60 percent humidity.

For the preparation of the glyoxysomes, 15 g of maize scutella were minced with a razor blade and gently homogenized with a mortar and pestle with 3 volumes of the grinding medium described by Breidenbach and Beevers1, Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was used as a buffering agent, however, instead of tris. The homogenate was filtered through four layera of cheesecloth. The particulate fraction, sedimenting between 480 and 10,500 g after 10 minutes, was carefully homogenized with a Potter-Elvehjem homogenizer in 2 ml of 35 percent (w/v) sucrose. An aliquot of this suspension containing 15 mg of protein was layered on a continuous 35-65 percent (w/v) sucrose gradient and spun in the SW-25.1 Spinco rotor at 25,000 rpm and 0°C for 4 hours. Under these experimental conditions, the glyoxysomes form a yellow-green pellet (density = 1.25 g/cc), while the mitochondrial band comes to equilibrium midway down the tube (density 1.19 g/cc); nearly 100 percent of the cytochrome oxidase activity is in this band.

The mitochondrial band was repelleted by collecting and resuspending it in grinding medium and spinning it at 23.500 g for 20 minutes. The mitochondrial and glyoxysomal pellets were then resuspended in 0.5 ml of 0.5 M sucrose dissolved in 10 mM Hepes buffer, pH 7.5. At this stage of fractionation, malate synthetase was associated exclusively with the glyoxysomes. Catalase and malate synthetase were totally absent from the mitochondria, suggesting that cross-contamination between glyoxysomes and mitochondria was not a problem.

Protein was determined according to the method of Lowry et al.7, malate synthetase according to Hock and Beevers4, and catalase according to Chance and Maehly4 (unless stated otherwise). Qualitative assays of catalase were done according to methods described by Scandalios11.

Electron microscopy was very generously performed by Drs. Giovanna and Claudio Longo. University of Milan. Italy, according to their procedures6.