Date of Award

5-1988

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Environmental Sciences

First Advisor

Robert L. Carter

Second Advisor

Bettina Harrison

Third Advisor

Anne E. McElroy

Abstract

The filamentous fungus Cladosporium resinae grows on n-alkanes as well as on substrates such as glucose. Growth on n-alkanes is associated with production of intracellular organelles known as microbodies. This work was undertaken in order to isolate and characterize microbodies from C. resinae.

Cells were first converted to spheroplasts. Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support. Techniques used to disrupt spheroplasts also ruptured a significant fraction of the microbodies. Of eight techniques used to disrupt microbodies, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min.

Microbodies were partially purified by a combination of differential and density gradient centrifugation. Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched with microbodies and one enriched in mitochondria. The microbody-enriched fraction was enriched in catalase, a marker enzyme for microbodies, but another marker enzyme, urate oxidase, was not found in this fraction or in crude cell extracts. The mitochondria-enriched fraction was enriched in cytochrome c oxidase, a marker for mitochondria. Fractions enriched in microbodies or mitochondria were relatively free of contamination from the endoplasmic reticulum or the cytosol as indicated by analysis of other marker enzymes.

Electron microscopy showed that the microbody fraction contained intact microbodies, with mitochondria as a minor contaminant. Catalase was localized in microbodies in intact cells or in the microbody fraction. Mitochondria were present in the cytochrome c oxidaseenriched fraction and took up the vital stain Janus green B.

For comparison, glucose-grown cells of C. resinae were fractionated. Catalase was largely found at the top of Percell gradients and microbodies were not observed in thin sections prepared from particulate fractions. This is consistent with the observation that cells grown on glucose form few microbodies. In contrast to mitochondria isolated from hydrocarbon-grown cells, mitochondria from glucose-grown cells were more dense and were well-preserved with distinct cristae.

Use of the methods developed in this work should provide a basis for determining the physiological role of microbodies in C. resinae.

Comments

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