Date of Award

Fall 1998

Document Type

Thesis - Restricted

Degree Name

Master of Science (MS)


Biomedical Engineering

First Advisor

Merker, Marilyn P.

Second Advisor

Linehan, John H.

Third Advisor

Olson, Lars E.


The information disclosed in the following pages is preliminary work of a project to ultimately identify by sequencing the reductase or family of reductases that reside on the luminal surface of the endothelium in the lung. The presence of these reductases was discovered first, to the best of our knowledge, in the laboratories of Dr. Merker and Dr. Dawson of the Medical College of Wisconsin. The methods and results of multiple protocols for isolation and characterization of the plasma membranes of lung endothelial cells and the reductases they contain are described herein. Chapter one introduces the concept of transplasma membrane electron transport and the role these reductases may participate in physiologically. The importance of this research and previous related work is discussed along with an introduction of three areas that were investigated. Chapter two explains the methods and results of the first of three parts of the thesis. Two plasma membrane isolation techniques were attempted along with multiple enzyme markers for testing the adequacy of the isolation methods. Methods and results of DCIP reduction/NAD(P)H oxidation to further characterize the plasma membrane fractions are discussed in Chapter 3. Inhibitors of prosthetic groups of known redox enzymes were reacted with lung fractions to help identify the method of DCIP reduction/NAD(P)H oxidation. The methods and results of the third and final area of investigation are discussed in Chapter 4. Two protein separation and isolation techniques, native electrophoresis and biotin tagging of luminally exposed proteins, were combined in an attempt to identify the protein. Chapter 5 draws conclusions from the combined results and is a final discussion and explanation of the data. Future work is also discussed in the final chapter.



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