[Frontiers in Bioscience 3, d483-493, May 1, 1998]
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ISOLATION AND STRUCTURAL ANALYSIS OF MICROSOMAL MEMBRANE PROTEINS

F.S. Heinemann1 and Juris Ozols2

1Department of Pathology, Hoag Memorial Hospital, Newport Beach, CA, 2Department of Biochemistry, University of Connecticut School of Medicine, Farmington, CT, USA.

Received Accepted

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Isolation of microsomal proteins
3.1. Preparation of microsomes
3.2. Solubilization of microsomal membrane proteins
3.3. Fractionation of solubilized microsomes
3.3.1. Fractionation of solubilized microsomes by polyethylene glycol precipitation
3.3.2. Isolation of microsomal proteins in the 6-12% PEG fraction
<3.3.3. Purification of esterase 2, stearyl Co-A desaturase, and oligosaccharyl transferase
4. Structure analysis of microsomal proteins
4.1. N-terminal amino acid sequence analysis
4.2. Internal amino acid sequence analysis
4.2.1. Preparation of chemical and enzymatic digests
<4.2.2. Isolation and structure analysis of peptides
4.3. Structural analysis of post-translational modifications
4.3.1. Identification of N-terminal blocking groups
4.3.2. Glycosylation
4.3.3. Disulfide bonds
5. Perspective
6. Acknowledgments
7. References
8. Entire manuscript

Key words:11 beta-Hydroxysteroid Dehydrogenase, Endoplasmic Reticulum, Microsomes, Membrane Proteins, Cytochrome B5, Cytochrome B5 Reductase, Cytochrome P450, Epoxide Hydrase, Heat Shock Proteins, Esterases, Flavin Monooxygenases, Protein Degradation, Glucose-6-Phosphate-Dehydrogenase, Stearyl Coa Desaturase, Membrane Post-Translational Modifications

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