| This dissertation presents data on peroxisomal biogenesis with the aim of developing an in vitro peroxisomal import assay. The first chapter represents an examination of the different forms of malate synthase (MS) found in maturing and germinated cotton (Gossypium hirsutum L.) seeds. A cytosolic 5 Svedberg unit (S) form and a glyoxysomal 20S form of MS were detected, with the latter becoming more prevalent with increasing seed age. No detectable differences in subunit ;The second chapter represents an investigation of the development and regulation of MS, isocitrate lyase (ICL), and catalase (Cat) in maturing and germinated cotton seeds. Enzyme activity, immunoselected protein, and mRNA for all three enzymes occurred together as early as 17 days postanthesis (DPA) in a coordinate manner; however, subsequent nonconcerted increases in protein, activity, and mRNA for each enzyme indicated that developmental expression in cottonseed embryos was regulated in a noncoordinate fashion by unidentified control mechanism(s).;The third chapter describes a method for isolating highly-purified peroxisomes from cotton cotyledons, castor bean endosperm, and spinach leaves using iso-osmotic metrizamide gradients. Intactness of these peroxisomes ranged from a high of 89% (castor bean) to a low of 69% (cotton). Light and electron microscopy confirmed both the purity and intactness of these organelles. Collectively, the data showed that these isolated peroxisomes met three important criteria (highly purified, intact, and in an iso-osmotic condition) for organelles in in vitro import studies.;The fourth chapter describes two cDNA clones encoding full-length amino acid sequences of cottonseed MS and ICL. MS and ICL have carboxy terminal tripeptides consisting of S-K-L and A-R-M, respectively, which are similar to a putative peroxisomal targeting sequence in animals (S/A/C-K/H/R-L). In vitro transcription/translation of the MS and ICL clones produced authentic MS and ICL protein. Preliminary results from an in vitro import assay with in vitro transcribed/translated MS and isolated glyoxysomes (castor bean) showed a temperature sensitive accumulation of MS into glyoxysomes. |
