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-c alled cores) built in higher plant peroxisomes by high resolution scanning electron microscopy (HRSEM). Purification of peroxisomal inclusions from su nflower (Helianthus annuus L.) cotyledons led to highly enriched preparatio ns suitable for HRSEM. Isolated peroxisomal cores occurred mostly as quadra ngular blocks, but sometimes also cube-like particles were found. In all ca ses, two edges of the blocks had similar lengths resulting in two square si des. The third edge was shorter than the two edges of the square sides. Edg e 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 t hree edge lengths of individual cores allowed for the first time to determi ne the volume of peroxisomal cores from plants. Core volumes ranged from ab out 0.01 mu m(3) to almost 0.1 mu m(3) indicating that cores represent a si gnificant part of the total peroxisomal volume. As revealed by HRSEM, the s urface layers of cores were built of regularly arranged, repeating square u nits with an edge length of about 20 nm. Between these units, interstices o f about 4 nm appeared. Light optical diffraction analysis of HRSEM microgra phs of cores revealed diffraction patterns up to the second order indicatin g that peroxisomal cores grown in vivo have a high degree of regularity. HR SEM on immunogold-labelled samples verified that isolated cores contained t he 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 partic les. Another form of catalase, differing in amino acid sequence from core c atalase, obviously does not participate in the crystallization process, alt hough it is present in the peroxisomal matrix during the formation of cores in vivo.