Immunogold studies on peroxisomes: Review of the localization of specific proteins in vertebrate peroxisomes

Abstract

Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A-gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research.

The Utility Value of High Voltage Electron Microscopy for X-Ray Microanalysis

“…Then, we used the same procedure for quantifying cerium contents in enzyme histochemical staining in specimens of spleen and kidney of mice demonstrating acid phosphatase activity [67][68][69][70][71]. Finally we used a similar method to quantify the gold content of gold particles in specimens stained with immunogold staining [82][83][84][85][86][87][88][89][90]. The respective procedures will be described in detail in the following.…”

“…However, the trial was not carried out at that time. We later developed immunostaining of peroxisomal enzymes by protein A-colloidal gold complex technique using cryo-fixation and freeze-substitution embedding for electron microscopy in correlation with the intracellular localization of peroxisome proliferators [82][83][84][85][86][87][88][89][90] [82][83][84][85][86][87][88][89][90]. The results demonstrated that the peroxisome of rat hepatocyte is a spherical organelle with a single limiting membrane, containing a homogeneous matrix and a high electron dense core.…”

“…The electron-lucent subcompartment of the matrix contains only D-amino acid oxidase while the electrondense subcompartment contains all the other enzymes. The immunogold techniques were also applied to the livers of different species [89,90]. It was demonstrated that species specific differences exist in the size and shape of the peroxisomes and the ultrastructure of the core in man, monkey, cow, cat, dog, rat, mouse, frog and so on [82][83][84][85][86][87][88][89][90].…”

“…The immunogold techniques were also applied to the livers of different species [89,90]. It was demonstrated that species specific differences exist in the size and shape of the peroxisomes and the ultrastructure of the core in man, monkey, cow, cat, dog, rat, mouse, frog and so on [82][83][84][85][86][87][88][89][90]. In order to quantify the gold content in immunogold staining for catalase by X-ray microanalysis, we first used hepatocytes of a normal adult male Wistar rat, fixed in paraformaldehyde and glutaraldehyde mixture, embedded in Lowicryl K4M, sectioned and stained with anti-catalase antibody by the protein A-gold technique as the models.…”

Multiple Targeting Modules on Peroxisomal Proteins Are Not Redundant: Discrete Functions of Targeting Signals within Pmp47 and Pex8p

“…The peroxisome has two principal compartments, the matrix, which itself can comprise several subcompartments, and a unit membrane surrounding it (Masters and Crane, 1995;Usuda et al, 1995). The targeting of matrix proteins from cytoplasm to peroxisomes is becoming well understood Holroyd and Erdmann, 2001;Purdue and Lazarow, 2001;Gould and Collins, 2002): most matrix proteins contain a short targeting signal at their carboxy terminus (termed PTS1) that recruits a shuttling cytosolic receptor, and the resulting complex then binds to a docking element on the peroxisomal surface.…”