Student Theses and Dissertations
Date of Award
1972
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Christian de Duve
Keywords
catalase biosynthesis, peroxisomes, immunoprecipitation, 3H-leucine labeling, biosynthetic intermediates, liver cell fractionation
Abstract
Rat liver peroxisomal catalase was purified to homogeneity. The starting material was purified peroxisomes, The purity of the final preparation was judged by Sephadex G-200 chromatography, SDS gel electrophoresis, spectral analysis, and immunochemical means. Two sera containing anticatalase antibodies were prepared. The first, a rabbit serum, contained a minor impurity. The anticatalase was purified forty times by affinity chromatography and the contaminating antibodies were thereby removed. The second serum, prepared in a goat, was proved to be monospecific by immunodiffusion and immunoelectrophoresis. The standard methods of immunoprecipitation were modified to allow the specific precipitation of a minor component (catalase) out of a crude cell homogenate and fractions thereof. The use of siliconized, glass, conical tubes and the omission of a 37° incubation were found to be essential. Immunochemical and biochemical controls were employed. The incorporation of 3H-leucine into catalase was determined in rats receiving the isotope by intraportal injection, in order to effect a nearly pulse-chase situation. Under these conditions about 10% of the injected isotope is incorporated into TCA-insoluble material in the liver within minutes (the apparent half-life of the liver's free leucine pool is 2 minutes). The kinetics of incorporation of 3H-leucine into material precipitated from the liver by anticatalase was identical to the kinetics of labeling of total protein just described. However, when newly synthesized, this material does not accompany catalase through chemical purification. Its migration on SDS polyacrylamide gels is identical to that of catalase. It is quantitatively converted into purifiable, authentic catalase in two hours or more. We interpret these results as the synthesis of a biosynthetic intermediate of catalase. Analogous experiments were carried out using 3H-δ-aminolevulinic acid (ALA) to label the heme moiety of catalase. Pulse-labeling was not achieved using this precursor. Nevertheless it was possible to identify a catalase biosynthetic intermediate with this label as well. Detailed analysis of the incorporation kinetics revealed that the intermediates identified with leucine and with ALA were not the same and in fact there are two leucine-containing intermediates: one without heme, representing about 2% of the total liver catalase, and one containing heme, representing about 0.5% of the liver catalase. A method was designed for fractionating postnuclear supernatants on sucrose density gradients, taking advantage of both differences in density and differences in size of the various cell organelles. By means of this procedure a reasonable separation of most of the cell organelles can be effected within two hours. This technique may be applied to localizing enzymes within liver cells and can be adapted to swinging-bucket rotors. The locations of labeled catalase and its biosynthetic intermediates within the liver cells were investigated by applying the above fractionation method to the livers of rats double-labeled with 3H-leucine and 14C-ALA. The overall labeling kinetics in these double-labeled rats confirmed the existence of two biosynthetic intermediates: one without, and one containing heme. The leucine-containing intermediate, still lacking its heme, was found to be transported rapidly to the peroxisomes, with a transport half-time of approximately 12 minutes. Inside the peroxisomes it was equipped with heme and converted to authentic, purifiable catalase. In addition, some heme appeared to be added to apocatalase outside of the peroxisomes, and some of this extraperoxisomal catalase even appeared to become purifiable. The significance of these latter findings was difficult to assess, and the possibility of an artifact could not be excluded. The labeled catalase intermediates which had not yet reached the peroxisomes were found partly in the microsome-rich fractions and largely at the top of the gradients where the layer was applied. Many experiments were performed to clarify the early events in catalase biosynthesis and transport, but they did not succeed in completely solving the problem. A participation of the rough endoplasmic reticulum is strongly implicated. The very considerable label found at the top of the gradients is considered to arise, either from the synthetic activity of free polyribosomes, or from a homogenization artifact whereby a fragile particle is broken open.
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Recommended Citation
Lazarow, Paul Bernard, "The Biogenesis of Peroxisomal Catalase in Rat Liver" (1972). Student Theses and Dissertations. 545.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/545
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy