ISOLATION AND BIOCHEMICAL PROPERTIES OF 2 TYPES OF MICROBODY FROM NEUROSPORA-CRASSA CELLS

  • 1 January 1978
    • journal article
    • research article
    • Vol. 18  (1) , 132-144
Abstract
Cells of the N. crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS) and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole C source (acetate medium) induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble ICL activity increased rapidly after a lag phase of about 45 min. Addition of 0.1 mM cycloheximide to the acetate medium 3 h after transfer of the cells halted the rise of ICL activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 h. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of slime cells by sucrose density gradient centrifugation yielded 2 major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activities of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of 2 types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activites were recovered at 1.24 kg/l. A similarly different sedimentation behavior of the 2 enzyme systems was observed when crude extracts of another slime mutant of N. crassa (arg-, FGSC No. 326) and of a wild type strain (ATCC 10816) were resolved by sucrose density gradient centrifugation. Transfer of sucrose grown cells of the slime mutant to acetate medium induced a 3-5 fold increase in specific activities of ICL and MS in the light microbody fraction but had no effect on enzyme activities in the heavy microbody fraction. The data are taken for evidence that, in contrast to higher plant cells, in N. crassa the glyoxysomal and the peroxisomal enzyme system are localized in different cell compartments represented by 2 types of microbodies which are regulated independently of each other.