AN ABSTRACT OF THE THESIS OF Marilyn L. Walsh for the degree of Doctor of Philosophy in Biochemistry and Biophysics presented on May 3..2001. Title: Protocols. Pathways. Peptides and Redacted for Privacy Wilbert Gamble The vascular system transports components essential to the survival of the individual and acts as a bamer to substances that may injure the organism. Atherosclerosis is a dynamic, lesion producing disease of the arterial system that compromises the functioning of the organ by occlusive and thrombogenic processes. This investigation was undertaken to elucidate some of the normal biochemical processes related to the development of atherosclerosis. A significant part of the investigation was directed toward developing and combining methods and protocols to obtain the data in a concerted manner. A postmitochondnal supernatant of bovine aorta, usingmevalonate-2-14C as the substrate, was employed in the investigation. Methods included paper, thin layer, and silica gel chromatography; gel filtration, high performance liquid chromatography (HPLC), and mass spectrometry. This current research demonstrated direct incorporation of mevalonate-2- 14Cinto the trans-methyiglutaconic shunt intermediates. The aorta also contains alcohol dehydrogenase activity, which converts dimethylallyl alcohol and isopentenol to dimethylacrylic acid, a constituent of the trans-methylgiutaconate Small, radioactive peptides, named Nketewa as a group, were biosynthesized using mevalonate-2-'4C as the substrate. They were shown to pass through a 1000 D membrane. Acid hydrolysis and dabsyl-HPLC analysis defined the composition of the Nketewa peptides. One such peptide, Nketewa 1, had a molecular weight of 1038 and a sequence of his-gly-val-cys-phe-ala-ser-met (HGVCFASM), with afarnesyl group linked via thioether linkage to the cysteine residue. Methods were developed for the concerted investigation of the trans- methylglutaconate shunt, the isolation of mevalonate-2-'4C labeled peptides, and characteristics of neutral and acidic metabolites of mevalonate. The question as to whether or not mevalonate was the direct precursor was answered in the affirmative. These results contribute to the understanding of the biochemistry of the vessel wall and the associated atherogenic processes. Mevalonate-derived volatile and acidic compounds may represent an alternate metabolic pathway. The prenylated Nicetewapeptide may be, as are other prenylated peptides, participants in the intracellularsignaling process, release of cytokines, expansion of extracellular matrix, and calcium release. Protocols, Pathways, Peptides and the Aorta: Relationship to Atherosclerosis by Marilyn L. Walsh A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented May 3, 2001 Commencement June 2001 Doctor of Philosophy thesis of Marilyn L. Walsh presented May 3. 2001. APPROVED: Redacted for Privacy Major Professor, representing Biochemistry and Biophysics Redacted for Privacy Chair of Department of Biochemistry and Biophysics Redacted for Privacy Dean of thGraduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy Marilyn L. Walsh, Author Acknowledgments This project would not have been possible without the support, guidance and encouragement of many individuals. I would like to acknowledge my mentor and guide though this process, Wil Gamble. He has taught me to love science, to "let the data speak to me", and to find the right questions to ask. His enthusiasm for research is an inspiration to me and to the many students who find their way to his office and laboratory for advice and support. I would like to acknowledge my graduate committee: Dr. Robert Becker, Dr. Sonia Anderson, Dr. Daniel Arp and Dr. Thomas Savage. Dr. Savage, in particular, for guiding me through several editions of this work. His patience and standards are to be commended.I wish to thank both Dr. Becker and Dr. Anderson for introducing me into the study of proteins; and to Dr. Arp for helping me appreciate the intricacies of the plant and bacterial world. Undergraduate students in the Gamble laboratory have also contributed to this work and I wish to thank them. The Department of Biochemistry and Biophysics has provided ongoing financial and moral support, and I wish to thank in particular Dr. Christopher Mathews for his role in providing me the opportunities as a graduate teaching assistant. The Oregon Heart Association and the National Institutes of Health provided financial and technical help. Kate McGaughey and Priscilla Dombek have been good friends and fellow graduate students.Karen Guzman and the Wise Women at Luther House have shared the joys and stress as this work progressed. Barbara Hansen helped immensely in manuscript prepartion, I wish to thank Priscilla Dombek and Kate McGaughey for their friendship and advice through this process as well as Karen Guzman and others in the community. TABLE OF CONTENTS INTRODUCTION Atherosclerosis Structure of the vessel wall 2 Atherosclerosis development 3 Mevalonic Acid 6 The mevalonate pathway 6 Isoprenoid biosynthesis 8 Cellular functions of mevalonate-derived products 14 Protein prenylation 17 Mevalonate and vascular biochemistry 19 Research Goals 22 Use of the bovine model 22 Research focus 22 Research goals 23 MATERIALS AND METhODS 24 Introduction 24 Preparation of the crude homogenate of bovine aorta 25 Preparation of the postmitochondrial fraction of the crude homogenate 25 Preparation of mevalonic acid-2-'4C 26 Methods of detecting radioactivity in metabolites 26 The concerted protocol 27 Method of incubation of the postmitochondrial supernatant with 27 mevalonic acid-2-14C Extraction of neutral lipids from the incubation mixture 32 Extraction of the acid lipid fraction from the incubation mixture 32 TABLE OF CONTENTS (Continued) Precipitation of protein from the incubation mixture 33 Preparation of the aqueous extract and the soluble proteins 34 Determination of protein concentration 35 Gel filtration chromatography 36 Bio-Gel P100 chromatography 37 Bio-Gel P6 chromatography 37 Bio-Gel P2 chromatography 38 Ion exchange chromatography 38 Anion exchange chromatography 38 Cation exchange chromatography 39 Membrane filtration 39 Thin layer chromatography 40 Thin layer chromatography of carboxylic acids 41 Thin layer chromatography of amino acids and peptides 46 Preparative thin layer chromatography 46 Paper chromatography 47 General procedures 47 Paper chromatography of organic acids 48 Paper chromatography of amino acids and peptides 49 Preparative isolation of peptides 56 Performic acid oxidation of peptides 56 Acid hydrolysis of peptides 57 High performance liquid chromatography 58 Preparation of Dabsyl derivatives of amino acids 59 Mass spectroscopy of soluble peptides 60 TABLE OF CONTENTS (Continued) EXPERIMENTAL RESULTS 63 Incubation of the postmitochondrial supematant 63 Characterization of the neutral lipid fraction 65 The acid petroleum ether extract 67 Thin layer chromatography of the acid petroleum ether extract 67 Paper chromatography of the acid petroleum ether extract 73 The trans-methylglutaconate shunt 75 Silica gel column chromatography 76 Paper chromatography of silica gel chromatography fractions 79 Thin layer chromatography of the trans-methyiglutaconate shunt 80 intermediates Determination of alcohol dehydrogenase activity 80 in the postmitochondrial supernatant Isolation and characterization of proteins and peptides 84 Identification of the peptide fraction 85 Paper and thin layer chromatography to identify the peptide fraction 86 Isolation and characterization of the aqueous extract peptides 86 Characterization of peptides from thin layer chromatography 86 Membrane filtration of the aqueous extract 88 Paper chromatography of Band I from preparative thin layer 96 chromatography Amino acid analysis by dabsylation 100 Characterization of the Soluble Protein Fraction 103 Gel filtration of the soluble peptide fraction 104 Ion exchange chromatography of the soluble fraction 104 HPLC of the soluble peptides 109 Mass spectrometry of the soluble peptides 112 TABLE OF CONTENTS (Continued) DISCUSSION AND CONCLUSIONS 120 The neutral and acid petroleum ether extracts 121 The trans-methylgiutaconatic acid shunt intermediates 124 Mevalonate derived radioactivity in peptides 127 Summary statement 130 BIBLIOGRAPHY 133 LIST OF FIGURES Figure 1. A. Isoprene 7 B. The pathway for the biosynthesis of mevalonic acid 7 2. Biosynthesis of farnesyl pyrophosphate, squalene, and lanosterol 10 from mevalonic acid 3. Biosynthesis of cholesterol from lanosterol 11 4. The trans-methylgiutaconate shunt 17 5. Isolation of various fractions from the postmitochondrial 28 supernatant 6. Protocols for the isolation and characterization of lipid and acid 29 components in the incubation mixture 7. Protocols for the characterization of peptides in both the 30 soluble and aqueous extract fractions 8. Thin layer chromatography of the trans-methyiglutaconate shunt 42 intermediates in an acid solvent 9. Thin layer chromatography of the trans-methyiglutaconate shunt 44 intermediates in a basic solvent 10. Paper chromatography of the trans-methylglutaconate shunt 50 intermediates in an acid solvent 11. Paper chromatography of the trans-methylgiutaconate shunt 52 intermediates in a basic solvent 12. Two dimensional chromatography of amino