High resolution scanning electron microscopy of protein inclusions (cores) purified from peroxisomes of sunflower (Helianthus annuus L.) cotyledons

This paper describes the first characterization of protein inclusions (so-called cores) built in higher plant peroxisomes by high resolution scanning electron microscopy (HRSEM). Purification of peroxisomal inclusions from sunflower (Helianthus annuus L.) cotyledons led to highly enriched preparations suitable for HRSEM. Isolated peroxisomal cores occurred mostly as quadrangular blocks, but sometimes also cube-like particles were found. In all cases, two edges of the blocks had similar lengths resulting in two square sides. The third edge was shorter than the two edges of the square sides. Edge lengths of the square side were typically in the range from 200 to 600 nm, however, also values of up to 1 mu m were observed. Measurements of the three edge lengths of individual cores allowed for the first time to determine the volume of peroxisomal cores from plants. Core volumes ranged from about 0.01 mu m(3) to almost 0.1 mu m(3) indicating that cores represent a significant part of the total peroxisomal volume. As revealed by HRSEM, the surface layers of cores were built of regularly arranged, repeating square units with an edge length of about 20 nm. Between these units, interstices of about 4 nm appeared. Light optical diffraction analysis of HRSEM micrographs of cores revealed diffraction patterns up to the second order indicating that peroxisomal cores grown in vivo have a high degree of regularity. HRSEM on immunogold-labelled samples verified that isolated cores contained the peroxisomal enzyme catalase. Ln summary, the results suggest cores to be formed in sunflower peroxisomes by crystallization of a specific molecular form of catalase, which is the predominant protein component of the particles. Another form of catalase, differing in amino acid sequence from core catalase, obviously does not participate in the crystallization process, although it is present in the peroxisomal matrix during the formation of cores in vivo.