s. 561., ,. .II... — ~v11. IV.- 1. 1‘. .......Is3=.fi. as by? . .5... .w .4 w? yaw—1...: 13:. “r a... II......‘) :UJIIJI. IIIIYI 1h . vi .lIihrIH. .1..le- l - I‘M .~,, , . 4 .911:th i-Iiuh- This is to certify that the thesis entitled Rat Liver Microsomal Structure and the Mixed-Function Oxidases presented by Ann F. Welton has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry Mqior professor 0-7639 U S' 3‘. am mom mc. LIBRARY BINDERS H gmsrnnmcmm Alh“ -‘. .\‘ ABSTRACT RAT LIVER MICROSOMAL STRUCTURE AND THE MIXED-FUNCTION OXIDASES BY -,.c-~ ‘ Ann Ff Welton The objectives of this research were: (1) to investigate the general arrangement of proteins within the rat liver endoplasmic reticulum (microsomes), and (2) to investigate the mechanism by which multiple mixed-function oxidase activities are achieved in this membrane. This latter objective was studied by investigating the possibility that multiple forms of NADPH-cytochrome c reductase and cytochrome P450 might be present in this membrane and that different forms of these proteins might be inducible by treating rats with phenobarbital and 3-methy1cholanthrene, compounds which are known to induce different mixed-function oxidase activities. The general arrangement of proteins within the microsomal membrane was examined by the techniques of sodium dodecyl sulfate- polyacrylamide gel electrophoresis and 1actoperoxidase-catalyzed protein iodination. The membranes used in this study were washed free of ribosomes and adsorbed proteins. Polyacrylamide gel electrophoresis protein profiles indicated that the major protein constituents of the microsomal membrane have molecular weights ranging from 40,000 to Ann F. Welton 60,000 daltons while the minor protein components have molecular weights ranging between 10,000 to over 200,000 daltons. Enzymatic protein iodination was conducted in the presence of an antioxidant, butylated hydroxytoluene, to prevent the peroxidation of membrane lipids. This procedure preserved the general structure of the membrane during iodination and doubled the incorporation of 125I into membrane proteins. It also prevented the destruction of cytochrome P Polyacrylamide 450' gel electrophoresis, following enzymatic iodination, demonstrated that the minor polypeptide components of this membrane, having both low and high molecular weights, and major polypeptide components having mole- 1251 and hence are cular weights of approximately 50,000 incorporate located on the membrane's exterior (cytoplasmic face). To determine if multiple forms of NADPH-cytochrome c reductase are present in rat liver microsomes, the molecular weights of the detergent-solubilized reductases from control and phenobarbital- or 3-methylcholanthrene-treated rats were compared by sodium dodecyl 1251_ sulfate-polyacrylamide gel electrophoresis. These enzymes were labeled in microsomes and then isolated by immunoprecipitation from sodium deoxycholate-solubilized microsomal proteins. The immunopre— cipitation was carried out using antibody prepared against a purified, protease-solubilized fragment of NADPH-cytochrome c reductase. The immunoprecipitate was electrophoresed on polyacrylamide gels and the molecular weight of the enzyme determined from the 125I-distribution in the gel. Using this technique the molecular weight of the NADPH- cytochrome c reductases from the liver microsomes of control and phenobarbital or 3-methylcholanthrene-treated rats were each shown to be 79,000 daltons. By this criteria, it was concluded that the Ann P. Welton NADPH-cytochrome c reductase enzymes present in all three types of microsomes are identical. A comparison of the molecular weight of detergent-solubilized NADPH-cytochrome c reductase (79,000) with that of the proteolytically solubilized fragment used for antibody production (71,000) suggests that this enzyme is an amphipathic membrane protein. Thus this protein appears to consist of a single polypeptide chain with a large hydrophilic segment which contains the active site and is exposed to the exterior of the membrane and a smaller hydrophobic segment of approximately 70 amino acids which is buried within the phospholipids of the membrane. The existence of multiple forms of cytochrome P450 was investi- gated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare the 40,000 to 60,000 dalton polypeptides present in the rat liver microsomes from control and phenobarbital- or 3-methylcholanthrene treated rats. Since cytochrome P 50 is thought to be a major microsomal 4 protein having a molecular weight of approximately 50,000 the induction of different forms of this cytochrome by these compounds was observable by this technique. 3—Methylcholanthrene induced a 53,000 dalton pro- tein while phenobarbital induced protein(s) having molecular weights slightly lower than 50,000 daltons. The induced proteins co-purified with cytochrome P450 fractions prepared from the three types of micro- somes. A method was developed by which benzidine and H20 could be 2 used to stain for the peroxidase activity of cytochrome P 0 on poly- 42 acrylamide gels. Three hemoproteins were observed in rat liver micro- somes using this technique and these had molecular weights of 53,000, 50,000, and 45,000 daltons. 3—Methylcholanthrene induced the 53,000 dalton hemoprotein while phenobarbital induced the 45,000 dalton Ann P. Welton hemoprotein. These hemoproteins were also present in partially purified fractions of this cytochrome from the three types of microsomes. These results suggest that multiple cytochrome P450 hemoproteins are present in rat liver microsomes. The spatial position in the microsomal membrane of the hemo- proteins induced by phenobarbital and 3-methylcholanthrene was investi- gated by combining the techniques of sodium dodecyl sulfate-polyacry- lamide gel electrophoresis and lactoperoxidase-catalyzed protein iodination. The liver microsomes isolated from rats pretreated with these compounds incorporated more 1251 into proteins of approximately 50,000 than did control of microsomes. This suggests that the hemo- proteins may be inserted onto the exterior of the microsomal membrane during the induction process. The 45,000 dalton hemoprotein present in rat liver microsomes was found to be resistant to proteolysis by trypsin and this character- istic was used in its purification. In the procedure used, a sodium cholate-solubilized preparation of cytochrome P 0 from the liver micro- 4S somes of phenobarbital-treated rats was digested with trypsin and the hemoprotein was the purified from proteolytic degradation products by Sephadex G-100 column chromatography. The isolated hemoprotein appeared, on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophore- sis, to be homogenous and by spectral assay to be a cytochrome P420 hemoprotein. Antibody was prepared against this hemoprotein and immunoprecipitation studies were conducted using detergent-solubilized partially purified cytochrome P450 preparations from control and phenobarbital- or 3-methylcholanthrene-treated rats. The antibody specifically immunoprecipitated the 45,000 dalton hemoprotein from Ann F. Welton these cytochrome P450 preparations. This antibody therefore will be useful in studying the microsomal hydroxylation reactions catalyzed by the 45,000 dalton hemoprotein and in studying the orientation of this protein in the microsomal membrane. RAT LIVER MICROSOMAL STRUCTURE AND THE MIXED-FUNCTION OXIDASES BY Ann F: Welton A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1974 To My Parents ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Steven D. Aust, my graduate research advisor, for his continual advice, encourage- ment, and friendship throughout this phase of my scientific career. He has helped me through many tempests both at Michigan State University and on the banks of the Au Sable River. My thanks also go to the members of my guidance committee, Drs. William Wells, Robert Ronzio, John Boezi, and Harold Hafs for their help throughout my stay at Michigan State, and to Dr. Loren Bieber for his assistance in the final phases of the completion of my Ph.D. degree. I have enjoyed collabo- rative work with many members of Dr. Aust's laboratory both past and present, and these include Dr. David L. Roerig, Dr. Thomas Pederson, Ms. Linda Chaney, Mr. Robert Moore, Mr. John Buege, and Mr. Fred O'Neal. In addition I want to thank the members of the Department of Bio— chemistry in general for their help and friendship throughout the past five years. The financial assistance of the National Science Foundation and the Department of Biochemistry is gratefully acknowledged. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. ABBREVIATIONS INTRODUCTION. ORGANIZATION OF THE THESIS . LITERATURE REVIEW . . . . . . . . . . The Rat Liver Endoplasmic Reticulum . . . . . The Microsomal Mixed- Function Oxidase System . . . . . The Protein Constituents of the Mixed- Function Oxidase System . . . . . . . . . . . . . . . Multiplicity of the Microsomal Mixed- Function Oxidase System . . . . . . . . . . . . . . . CHAPTER ONE: THE SPATIAL ARRANGEMENT OF PROTEINS IN THE RAT LIVER ENDOPLASMIC RETICULUM . . . . . . . . . . . Abstract . . Introduction . . . . . . . . . Materials and Methods . . . . . . . . Materials . . . . . . . . . . . . . . . PB- Pretreatment of Rats. . . . . . Preparation of Microsomal Membranes. . Enzyme Assays and Analytical Methods . . . . . Preparation of Samples for Electron Microscopy . . . Iodination of Microsomes . . . . . . Assay for Malondialdehyde Levels in Iodinated Microsomes. Chloroform: Methanol (2:1) Extraction of 1251- Labeled Microsomes . . . . . . . . . . . Iodination of Lipid- -Extracted Microsomal Protein . . . Trypsin Treatment of Microsomes . . SDS- -Polyacrylamide Gel Electrophoresis. . iv Page ix xi xvi Page Results . . . . . . . . . . . . . . . . . . . 33 Enzymatic and Electron Microscopic Characterization of the Microsomal Fraction Isolated by Differential Centrifugation . . . . . . . 33 Comparison of Rough and Smooth Microsomal Membranes After Removal of Ribosomes and Adsorbed Proteins . . . . . 35 Lipid Peroxidation During Enzymatic Iodination of Rat Liver Microsomes. . . . . . . . . . . . . . . 42 Determination of Optimum H202 and Lactoperoxidase Concentrations for Maximum 125I-Incorporation into Microsomes. . . . . . . . . . . . . . . . . 43 The Effect of Iodination on Microsomal Cytochromes and Enzymatic Activities Associated with the Mixed-Function Oxidases . . . . . . . . . . . . . . . . 49 1251- -Labeling Pattern for the Proteins from the Microsomes of a PB- Pretreated Rat. . . . . . . . . . . 52 Recover of 1251 from SDS- —Polyacrylamide Gels . . . . SS Iodination of Microsomal Protein Extracted with Chloroform: Methanol (2:1) . . . . . . . . . . . 62 Trypsin Treatment of 1251- Labeled Microsomes . . . . 65 A Comparison of the Iodination Profiles of Rough and Smooth Microsomes. . . . . . . . . . . . . . . . . 65 Discussion . . . . . . . . . . . . . . . . . . 70 CHAPTER TWO: MULTIPLICITY OF THE NADPH-CYTOCHROME C REDUCTASE ENZYMES IN RAT LIVER MICROSOMES . . . . . . . . . . . 77 Abstract. . . . . . . . . . . . . . . . . . . 77 Introduction . . . . . . . . . . . . . . . . . 78 Materials and Methods . . . . . . . . . . . . . . 81 Materials. . . . . . . . . . . . . . . . . . 81 Drug Pretreatment of Animals . . . . . . . . . 81 Preparation of Bromelain-Solubilized NADPH- Cytochrome c Reductase from the Liver Microsomes of PB- Pretreated Rats . 82 Preparation of Antibody to Bromelain—Solubilized NADPH- Cytochrome c Reductase. . . . . . . . . . . . 82 Ouchterlony Double Diffusion Analysis . . . . . . . . 82 Enzymatic Assays . . . . . . . . . . . 83 Lactoperoxidase- Catalyzed Iodination of Microsomal Membranes and l°o SDS-Polyacrylamide Gel Electrophoresis. . 83 Immunoprecipitation of "Native” NADPU-Cytochrome c Reductase from Detergent-Solubilized Rat Liver Microsomal Proteins. . . . . . . . . . . . . . 83 Results . . . . . . . . . . . . . . . . . . Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between the Antibody to Bromelain Reductase, the Bromelain Reductase, and Detergent-Solubilized Liver Microsomal Proteins from a PB-Pretreated Rat . . . . Enzymatic Characterization of the Immunoprecipitate Formed Between the Antibody and Detergent-Solubilized Microsomes. . . . . . . . . . SDS- -Polyacrylamide Gel Electrophoresis of the Protein Components of the Immunoprecipitate Formed Between the Antibody to Bromelain Reductase and Detergent-Solubilized PB- Microsomes. . . . . . . . . . . A Comparison of the Molecular Weights of "Native" Reductase and Bromelain Reductase . . . . . . . Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between the Antibody to Bromelain Reductase and Detergent-Solubilized Liver Microsomal Proteins from Control, PB-, and 3—MC-Pretreated Rats . . . . . . . Enzymatic Characterization of the Immunoprecipitates Fromed Between the Antibody and Detergent-Solubilized Microsomal Proteins from the Livers of Control, PB-, and 3-MC- Pretreated Rats . . . . . . . . . . . . . . Comparison of the Molecular Weights of the "Native" Reductases Present in the Liver Microsomes from Control, PB-, and 3-MC-Pretreated Rats . . . . . . . . . . Discussion . . . .. . . . . . . . . . . . . . CHAPTER THREE: MULTIPLICITY OF CYTOCHROME P450 HEMOPROTEINS IN RAT LIVER MICROSOMES. . . . . . . . . Abstract. . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Materials. . . . . . . . . Drug Pretreatment of Animals . . . . . . . . Isolation of Microsomal Membranes. . . . . . . . . Partial Purification of Cytochrome P450 Fractions from Liver Microsomes of Control and PB- or 3-MC-Pretreated Rats. . . . . . . . l°o SDS- Polyacrylamide Gel Electrophoresis . . . . . 0.1% SDS- Polyacrylamide Gel Electrophoresis . . . Use of Benzidine and H202 to Stain for Cytochrome P450 Hemoproteins . . . . . . . . . . . . Lactoperoxidase- -Catalyzed Protein Iodination . Enzyme Assays . . . . . . vi Page 84 84 85 89 92 99 100 100 106 109 109 110 115 115 115 115 116 117 118 118 119 119 Results . . . . . . . . . . . . . . . Comparison of the 1% SDS-Polyacrylamide Gel Electrophoresis Protein Patterns of the Rat Liver Microsomes from Control and PB- or 3- MC- Pretreated Rats. . 1% SDS- -Polyacrylamide Gel Electrophoresis Protein Profiles of the Liver Microsomes from Control and PB- or 3- MC- Pretreated Rats and the Cytochrome P450 Fractions Purified from the Microsomes. . . . . Use of Benzidine and H202 to Stain for Cytochrome P450 Hemoprotein(s) on SDS- Polyacrylamide Gels . . . Hemoprotein Profiles of Partially Purified Cytochrome P450 Preparations Isolated from the Liver Microsomes of Control and PB- or 3- MC— Pretreated Rats . . . . . . Comparison of the SDS- Polyacrylamide Gel Electrophoresis Protein and 125I- Incorporation Profiles of the Liver Microsomes from Control and PB- or 3- MC- Pretreated Rats. Discussion . . . . . . . . . . . . . . . . CHAPTER FOUR: PREPARATION OF ANTIBODY TO THE CYTOCHROME P450 HEMOPROTEIN INDUCED IN LIVER MICROSOMES BY PRETREATMENT OF RATS WITH PHENOBARBITAL. . . . . . . . . . . . . Abstract. Introduction Materials and Methods Materials. . . . . . . . . . . . Drug Pretreatment of Animals . . . . . . . Isolation of Hemoprotein 3 from the Liver Microsomes of a PB- Pretreated Rat. . . . . . . . . . . . Immunological Techniques. . . . . . . . Iodination of Partially Purified Cytochrome P450 Fractions from Control, PB-, and 3- MC— Pretreated Rats Immunoprecipitation of Hemoprotein 3 from Detergent- Solubilized Partially Purified Cytochrome P450 Fractions from Control, PB-, and 3-MC-Pretreated Rats. . . . . SDS-Polyacrylamide Gel Electrophoresis . Results . . . . . . . . . . . . . . . Isolation of Hemoprotein 3 . . . Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between the Antibody to Hemoprotein 3, the Hemoprotein 3 Antigen, and Partially Purified Cytochrome P450 Preparations from Control, PB-, or 3—MC- Pretreated Rats . . . . . . . vii Page 120 130 128 137 144 149 154 163 163 164 165 165 166 166 167 168 169 170 170 170 182 Page SDS-Polyacrylamide Gel Electrophoretic Analysis of the Immunoprecipitates Formed Between the Antibody and Detergent-Solubilized Partially Purified Cytochrome P450 Preparations from Control and PB- or 3-MC-Pretreated Rats . . . . . . . . . . . . . . . . . . 185 Discussion . . . . . . . . . . . . . . . . . 193 SUMMARY . . . . . . . . . . . . . . . . . . . 203 REFERENCES . . . . . . . . . . . . . . . . . . 207 APPENDIX. . . . . . . . . . . . . . . . . . . 217 viii Table II III IV VI VII VIII IX LIST OF TABLES Specific Activity of Organelle Marker Enzymes in the Microsomal Fraction Relative to That of the Total Liver Homogenate . . . . . . . . . . . . The Levels of Various Constituents in the Total Microsomal Fraction Isolated from Control Rats Before and After Washing the Membranes with 0.3 M Sucrose Containing 0.1 M Sodium Pyrophosphate, pH 7.5 . . . . . The Effect of Inhibitors of Lipid Peroxidation on the Formation of Malondialdehyde and Incorporation of 1251 Into Microsomes. . . . . . . . . . The Correlation Between Loss of Cytochrome P450 and the Peroxidation of Microsomal Lipid. . . . . Effect of Iodination on Lipid Peroxidation, Cytochromes, and Enzymatic Activities in the Liver Microsomes from PB-Pretreated Rats . . . . . . . . . . . Distribution of 1251 in Fractions Resulting from a Chloroforszethanol (2:1) Extraction of 25I-Labeled Liver Microsomes from a PB-Pretreated Rat. . . . . NADPH-Cytochrome c and NADPH-Ferricyanide Reductase Activities in the Immunoprecipitate Formed Between Antibody to Bromelain Reductase and Detergent- solubilized Liver Microsomal Protein from a PB- Pretreated Rat. . . . . . . . . . . . NADPH-Ferricyanide Reductase Activities in the Immunoprecipitates Formed Between Antibody to Bromelain Reductase and Detergent—solubilized Liver Microsomal Proteins from Control, PB-, and 3-MC- Pretreated Rats . . . . . . . . . . . . . The Molecular Weights of the Major Protein Constituents of Rat Liver Microsomes. ix Page 34 38 44 50 51 61 88 103 127 Table Page X Partial Purification of Cytochrome P450 (448 from the Liver Microsomes of Control and PB- or 3-M - Pretreated Rats . . . . . . . . . . . . . 129 XI The Percent of 125I Incorporated into Microsomal Proteins Which Appears in Proteins of 40,000 to 60,000 Daltons on SDS—Polyacrylamide Gels . . . . 153 Figure l. 2. LIST OF FIGURES Electron Micrograph of Microsomal Membranes . SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of Rough, Smooth and Total Liver Microsomal Fractions Before and After Washing the Membranes with Sucrose Containing Sodium Pyrophosphate . . . . . Incorporation of 1251 into Liver Microsomes from 3 PB- Pretreated Rat Using Varying Concentrations of H Incorporation of 125 Pretreated Rat Using Varying Lactoperoxidase Concentrations . . . . . . . . . . . SDS-Polyacrylamide Gel Electrophoresis Protein and 2 02. I into Liver Microsomes from a PB- I-incorporation Profiles Obtained for the Liver Microsomal Fraction from a PB-Pretreated Rat . SDS-Polyacrylamide Gel Electrophoresis Protein and incorporation Profiles Obtained for the Liver Specific Activity of was Varied . . Microsomal Fraction fig? a PB-Pretreated Rat When the A Comparison of the SDS-Polyacrylamide Gel Electro- phoresis Protein and 125I-Inco oration Profiles 125 I in the Iodination Reaction Obtained by Electrophoresing 12 I-Labeled Microsomes Before.and After Lipid Extraction with Chloroform: Methanol (2:1) . . . . . . . . . . . SDS-Polyacrylamide Gel Electrophoresis Protein and 1 25 I- I- Incorporation Profiles for Rat Liver Microsomes Which Were Iodinated Before and After Extracting Lipids from the Membranes. . . . . . . . . . Protein Solubilized from Rat Liver Microsomes by Trypsin Treatment at 25°C for Varying Periods of Time. xi Page 37 41 46 48 54 57 59 64 67 Figure 10. 11. 12. 13. 14. 15. 16. 17. The Effect of Trypsin Treatment on the SDS-Polyacrylamide Gel Electrophoresis Protein and 125I-Incorporation Profiles of the Liver Microsomal Fraction from PB- Pretreated Rats . . . . . . . . . . . SDS-Polyacrylamide Gel Electrophoresis Protein and 125I- Incorporation Profiles of Rough and Smooth Liver Microsomal Fractions from a Control Rat . . . . Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between Antibody to Bromelain-Solubilized NADPH-Cytochrome c Reductase, the Bromelain-Reductase, and Detergent-Solubilized Liver Microsomal Proteins from a PB-Pretreated Rat . . . ,, . . . . . . SDS-Polyacrylamide Gel Electrophoresis Protein and 1251-- Distribution Profiles of Total PB-Microsomal Proteins and the Immunoprecipitate Formed From Sodium Deoxycholate-Solubilized PB-Microsomal Proteins and the Reductase Antibody. . . . . . . . . . . A Comparison of the SDS—Polyacrylamide Gel Electrophoresis Protein Profiles of IgG Isolated from Antiserum to Bromelain Reductase and the Immunoprecipitate Formed Between Detergent-Solubilized Liver Microsomes from a PB-Pretreated Rat and This IgG . . . . . . . A Comparison of the SDS-Polyacrylamide Gel Electorphoresis Protein Profiles of the Liver Microsomal Proteins from a PB-Pretreated Rat and the Immunoprecipitate Formed Between Detergent-Solubilized PB-Microsomal Proteins and Anti—Reductase IgG. . . . . . . . . . . A Comparison of the SDS-Polyacryclamide Gel Electrophoresis Protein Profiles of Purified Bromelain-Solubilized NADPH- Cytochrome C Reductase and the Immunoprecipitate Formed Between Detergent-Solubilized Microsomal Proteins from a PB-Pretreated Rat and Antibody to the Bromelain Reductase . . . . Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between Antibody to Bromelain-Solubilized NADPH-Cytochrome c Reductase, the Bromelain Reductase, and Detergent-Solubilized Liver Microsomal Proteins from Control, PB-, and 3-MC-Pretreated Rats. xii Page 69 72 87 91 94 96 98 102 Figure Page 18. A Comparison of the SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of the Immunoprecipitates Formed Between Antibody Prepared to Purified Bromelain- Solubilized NADPH-Cytochrome c Reductase and Detergent- Solubilized Liver Microsomes from Control, PB-, and 3-MC-Pretreated Rats . . . . . . . . . . . . 105 19. 1% SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of the Rat Liver Microsomes from Control and PB- or 3-MC-Pretreated Rats . . . . . . . . . . . . 122 20. 1% SDS-Polyacrylamide Gel Electrophoresis Profiles of the Protein Constituents Having Molecular Weights Larger than 40,000 Which are Present in the Rat Liver Microsomes from Control and PB- or 3-MC-Pretreated Rats . . . . 124 21. 1% SDS-Polyacrylamide Gel Electrophoresis Pattern of the Protein Constituents Having Molecular Weights Greater than 40,000 Which are Present in a Mixture of the Liver Microsomes from 3-MC and PB-Pretreated Rats . . . . 126 22. 1% SOS-Polyacrylamide Gel Electrophoresis Protein Profiles of Rat Liver Microsomes from Control and PB- or 3-MC- Pretreated Rats and the Cytochrome P450 Fractions Purified from the Microsomes . . . . . . . . . 132 23. 1% SDS-Polyacrylamide Gel Electrophoresis Profiles of the Protein Constituents Having Molecular Weights Larger then 40,000 Which are Present in the Rat Liver Microsomes from Control and PB- or 3-MC-Pretreated Rats and in the Cytochrome P450 Fractions Prepared from the Microsomes. 134 24. 1% SDS-Polyacrylamide Gel Electrophoresis Pattern of the Protein Constituents Having Molecular Weights Greater than 40,000 Which can be Resolved from a Mixture of the Partially Purified Cytochrome P450 ( 448% Fractions Isolated from the Liver Microsomes of 3- C-Pretreated Rats and PB-Pretreated Rats. . . . . . . . . . 136 25. 0.1% SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of the 50,000 Molecular Weight Region of Gels Run on Liver Microsomes from Control and PB- or 3-MC- Pretreated Rats. . . . . . . . . . . . . . 140 26. Hemoprotein Profiles Obtained by Staining 0.1% SDS- Polyacrylamide Gels of Liver Microsomes from Control and PB- or 3-MC-Pretreated Rats with Benzidine and H202. 142 xiii Figure Page 27. Hemoprotein Profiles of Cytochrome P450 448) Fractions Isolated from the Liver Microsomes of ontrol and PB- or 3-MC—Pretreated Rats . . . . . . . . . . . 146 28. A Comparison of the Coomassie Blue Protein Patterns Obtained by Electrophoresing Cytochrome P450 Fractions Isolated from the Liver Microsomes of Control and PB- or 3-MC-Pretreated Rats on 1% and 0.1% SDS- Polyacrylamide Gels . . . . . . . . . . . . 148 29. A Comparison of the 1% SDS- Polyacrylamide Gel Electro- phoresis Protein and 1251- Incorporation Profiles Obtained from the Liver Microsomes of Control and PB- or 3-MC-Pretreated Rats . . . . . . . . . . . 152 30. Sephadex G-100 Column Chromatography of a Trypsin-Treated Cytochrome P450-Enriched Fraction from the Liver Microsomes of a PB-Pretreated Rat. . . . . . . . 173 31. 1% SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of the Cytochrome P450-Enriched Fraction Before Trypsin Treatment and the Cytochrome P420 Preparation Resulting from Trypsin Digestion and Sephadex G-100 Column Chromatography . . . . . . . . . . . . . . 176 32. 0.1% SDS-Polyacrylamide Gel Electrophoresis Hemoprotein Profiles of the Cytochrome P450-Enriched Fraction Before Trypsin Treatment and the Cytochrome P420 Preparation Resulting From Trypsin Digestion and Sephadex G-100 Column Chromatography. . . . . . . 178 33. SDS- Sephadex G-200 Column Chromatography of the Cytochrome P420 Preparation . . . . . . . . . 181 34. Ouchterlony Double Diffusion Analysis of the Precipitin Reactions Between the Hemoprotein 3 Antibody, Its Antigen, and Partially Purified Cytochrome P450 Preparations from the Liver Microsomes of Control and PB- or 3-MC—Pretreated Rats. . . . . . . . . . 184 35. A Comparison of the 1% SDS-Polyacrylamide Gel Electro— phoresis Protein Profiles of Partially Purified Cytochrome P450 Preparations Isolated from the Liver Microsomes of Control, and PB- or 3-MC-Pretreated Rats and the Immunoprecipitate Formed Between Each Cytochrome P450 Preparation and the Hemoprotein 3 Antibody . . - 187 xiv Figure Page 36. A Comparison of the 1% SDS-Polyacrylamide Gel Electro- phoresis Protein Profiles of the Immunoprecipitates Formed Between the Hemoprotein 3 Antibody and Detergent—Solubilized Partially Purified Cytochrome P450 Preparations from the Liver Microsomes of Control and PB- or 3-MC-Pretreated Rats . . . . . . 190 37. Spectral and Electrophoretic Analysis of the Immuno- precipitates Formed Between the Hemoprotein 3 Antibody and Detergent-Solubilized Partially Purified Cytochrome P450 Preparation from the Liver Microsomes of a PB-Pretreated Rat. . . . . . . 192 XV ADP ATPase BHT CPM DEAE DDT EDTA 'IgG 3-MC NADH NADP NADPH PB Reductase RNA SDS TBA Tris ABBREVIATIONS adenosine-5'-diphosphate adenosine-S'-triphosphatase butylated hydroxytoluene or 2,6-ditert-butylated-cresol counts per minute diethylaminoethyl 1,1,1-trichloro-2,2-tris(p-chlorophenyl)ethane ethylenediaminetetraacetate immunoglobulin G 3-methylcholanthrene reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate reduced nicotinamide adenine dinucleotide phosphate phenobarbital NADPH-cytochrome c reductase ribonucleic acid sodium dodecyl sulfate thiobarbituric acid Tris(hydroxymethyl)aminomethane xvi INTRODUCTION The rat liver endoplasmic reticulum (microsomes) serves as the matrix within which are found the enzymes of a mixed-function oxidase system. These enzymes utilize NADPH and O2 in the hydroxylation of drugs, steroids, fatty acids, and a wide variety of xenobiotics. The work reported in this thesis was initiated upon the premise that a better understanding of the structure of the endoplasmic reticulum would in turn lead to a better understanding of the proteins involved in this electron transport system. Indeed this was thought to be true because components of the mixed-function oxidase system at times con- stitute nearly 20% of the total microsomal protein. At the time of initiation of this work, two important techniques in the field of membrane biochemistry had just been developed. These were SDS-polyacrylamide gel electrophoresis and lactoperoxidase- catalyzed iodination of membrane proteins. SDS-polyacrylamide gel electrophoresis for the first time provided a means by which membrane proteins could be completely solubilized and separated from one another. Lactoperoxidase-catalyzed protein iodination provided a mechanism by which only those proteins exposed to the exterior of the 25 membrane could be 1 I-labeled, since this enzyme is impermeable to membranes. In combination, these two provided a simple means by which to study the spatial arrangement of proteins within a membrane. Thus the experimental work presented in the first chapter of this thesis describes the use of these two techniques for studying the topographical arrangement of proteins in the rat liver endoplasmic reticulum. The remaining chapters of this thesis then describe studies in which these two techniques were used to investigate questions which have been the subject of much current research in the area of microsomal mixed-function oxidases. The mixed-function oxidase system is known to catalyze the hydroxylation of a wide variety of lipophilic compounds with greatly differing structures. Because such apparent nonspecifi- city is contrary to many of our current theories concerning enzyme catalysis, many investigators have attempted to reconcile this apparent contradictory property by providing evidencefor the existence of multiple mixed-function oxidase activities. One of the best arguments for this hypothesis is the fact that different microsomal hydroxylation activities can be induced in rat liver microsomes by pretreating animals with different types of substrates for the mixed-function oxidase system. Thus pretreatment of rats with the compounds pheno- barbital (PB) or 3-methylcholanthrene (3-MC) induces different liver microsomal hydroxylation activities. But it is not known how this induction is accomplished at the molecular level. At present the mixed-function oxidase electron transport system is thought to be made up of at least two components, a flavoprotein, NADPH-cytochrome c reductase, and the substrate-binding, terminal oxidase, cytochrome P450. It is possible that different electron transport chains exist in microsomes for each class of substrate hydroxylated and that PB and 3-MC then induce different chains. Indeed spectral and catalytic evidence already suggests the presence of multiple forms of cytochrome P450 in microsomes. But if PB and 3-MC induce different electron transport chains, one would expect that multiple forms of NADPH- cytochrome c reductase should also exist in these microsomes. Since this protein has not been purified from microsomes, however, this is a difficult problem to investigate. By combining the techniques of SDS- polyacrylamide gel electrophoresis and lactoperoxidase-catalyzed protein iodination with various immunochemical methods, I was able to investigate this problem further without purifying this enzyme. In chapter two, evidence is presented to suggest that a single form of this flavoprotein exists in microsomes. This observation then argues against the theory that the different microsomal hydroxylation activi- ties result from multiple electron transport chains. In light of existing data it would therefore appear that the site of multiplicty must lie in a single component of the electron transport chain. That is, the different hydroxylation activities must be due to the existence of multiple forms of the substrate-binding, terminal cytochrome. Thus in the final two chapters of this thesis, the mechanism of cytochrome P multiplicity in rat liver microsomes was 450 investigated. While spectrally and catalytically different forms of this cytochrome have been observed in microsomes, there are at least two mechanisms which could explain these observations. Either multiple cytochrome P hemoproteins are present in microsomes or there is but 450 a single hemoprotein whose spectral and catalytic properties are modi- fied by as of yet unidentified "regulatory" components within the membrane. My work provides evidence for the former mechanism and hopefully future work from Dr. Aust's laboratory will establish the exact relationship between the multiple cytochrome P450 hemoproteins observed in these studies and the different rat liver microsomal mixed- function oxidase activities. ORGANIZATION OF THE THESIS Four areas of experimental endeavor are presented in this thesis in the form of separate chapters. Each chapter is organized using a format similar to that used for most scientific papers. That is, each chapter has its own Abstract, Introduction, Materials and Methods, Results, and Discussion sections. The references for each chapter, however, have been combined at the end of the thesis. These chapters are preceded by a literature review which is designed to provide a broad overview of current knowledge on the structure and function of the rat liver endoplasmic reticulum and the microsomal mixed-function oxidases. The introduction to each chapter then provides a more specific background on the subject matter of that chapter. Also it should be noted that in this thesis when the term protein is used in reference to SDS-polyacrylamide gels it refers to those species which stain with Coomassie blue on the gels. While SDS- gel electrophoresis is known to separate complex proteins into their polypeptide chains, I have made no differentiation between the terms protein and polypeptide in this context because currently there is little information about the arrangement of polypeptide subunits into proteins within membranes. LITERATURE REVIEW The Rat Liver Endoplasmic Reticulum In electron micrographs of cross sections of rat liver paren- chymal cells, the endoplasmic reticulum appears as a series of parallel membranes forming tubules, cisternae, and vesicles throughout the cyto— plasm of the cell (1,2). By studying serial sections of such cells it has been concluded that the membranes of these various structures are connected with one another and that the endoplasmic reticulum is actually a continuous tubular network of membranes extending throughout the cellular cytoplasm (3,4). Often the membranes of the endoplasmic reticulum appear to be connected with the outer membranes of other sub- cellular organelles, such as nuclei (5) and peroxisomes (2), suggesting that all these membranes may have a common origin. Morphologically the endoplasmic reticulum can be divided into two classes: the rough endOplasmic reticulum, so named because ribo- somes stud its cytoplasmic surface; and the smooth endoplasmic reticu- lum, which is free of ribosomes (2,3). When liver tissue is homoge- nized, the tubules are "pinched off" into sealed vesicles which retain the normal orientation of the membrane (2,3). That is, the outer face of these vesicles is the side normally exposed to the cellular cytoplasm while the luminal contents of the endoplasmic reticulum is preserved within the vesicular cavity. A mixture of rough and smooth endoplasmic reticulum vesicles can be relatively easily isolated from a liver homogenate by differential centrifugation. These vesicles pellet as the microsomal fraction by centrifugation of a post-mitochondrial super- natant at 105,000 xg for 90 minutes (2,3). If a relatively mild and short homogenization is performed on the liver tissue, there is usually very little contamination of this fraction with other membranes such as plasma membranes, lysosomes, Golgi, and mitochondria (6,7). It is also possible to separate the total microsomal fraction into rough (ribosome-bound) and smooth subfractions. The most common procedure for this separation is to add CsCl to a post-mitochondrial supernatant of a liver homogenate to aggregate rough microsomes (8). This super- natant is then placed over a discontinuous sucrose density gradient and during centrifugation the aggregated rough membranes pellet while the smooth membranes float within the gradient (8). There have been many attempts to determine how smooth and rough microsomes differ from one another, outside of the fact that ribosomes are attached to the rough membranes. With regards to the chemical composition of the membranes, both fractions appear to have similar phospholipid to protein ratios after ribosomes and adsorbed or luminal proteins are removed from the membranes (9). Both types of membranes also appear to have similar phospholipid compositions but the smooth membranes appear to have relatively higher levels of neutral lipids (9). Some subtle differences have also been reported with regards to the enzymatic contents of these two membrane populations but in general the membranes have strikingly similar enzyme compositions (10-12). The endoplasmic reticulum of the liver has a wide variety of functions within the cell. One of the most well-known concerns the synthesis and intracellular transport of proteins, such as albumin, which are destined to be exported to the blood (12,13). In addition, the membrane of this organelle contains two electron transport chains, one involved in the oxidation of NADPH and the other in the oxidation of NADH. The metabolic function of the NADPH-linked sequence is to participate in the hydroxylation of a wide variety of lipophilic com~ pounds (14-16). Two microsomal proteins are known to be associated with this electron transport chain, NADPH-cytochrome c reductase, a flavoprotein (17), and cytochrome P450, a hemoprotein (18). Studies concerning these two proteins constitute a major portion of this thesis and the properties of these two proteins will be described in more detail later. The NADH-linked electron transport chain is thought to be involved in fatty acid desaturation (19,20). The components of this system are the flavoprotein, NADH-cytochrome bS reductase (21), the other microsomal hemoprotein, cytochrome bS (21,22), and an as of yet unidentified "cyanide sensitive enzyme" (19,20). In this system, electrons are thought to be transferred from NADH to cytochrome b5 via NADH-cytochrome b5 reductase. Cytochrome bS in turn transfers electrons to the "cyanide-sensitive" enzyme which then catalyzes the desaturation (19,20). Besides electron transport enzymes, liver microsomes also con- tain a number of phosphatases, such as glucose-G-phosphatase, which catalyzes the final step in hepatic glycogenolysis (23). In addition, a Mg+2-activated ATPase has been found in rat liver microsomes (24). Enzymatic activities associated with phospholipid biosynthesis (25) and cholesterol biosynthesis (26) have also been localized in the micro— somal subfraction. Very little is known about the specific arrangement of phospho- lipid and protein constituents within the microsomal membrane. In accordance with current theories of membrane structure (27,28), the phospholipid components of this membrane are most likely arranged in a bilayer which is penetrated to various extents by hydrophobic portions of membrane proteins. Since proteases such as trypsin (29), chymo- trypsin (29), and cathepsins (30), have been observed to release catalytically active fragments of specific microsomal proteins, such as NADH-cytochrome bS reductase (30), cytochrome b5 (29), and NADPH- cytochrome c reductase (29), it is assumed that these proteins are located in the outside of the microsomal vesicle. This is true because it has been shown that the proteases cannot diffuse through the micro~ somal membrane (29,31,32). Recently the "native" forms of two of these proteins, NADH-cytochrome b reductase (33) and cytochrome b5 (34), 5 have been isolated by Strittmatter and co-workers. Each protein is a single polypeptide chain consisting of a large globular hydrophilic segment, containing the catalytic site, and a smaller hydrophobic polypeptide segment at one end of the protein. It is hypothesized that the hydrophilic portion of each protein is located on the exterior surface of the membrane while the hydrophobic "tail" portion is buried within the phospholipid bilayer of the membrane, functioning to anchor the rest of the protein to the membrane (33,35). But while a great deal is known about the arrangement of NADH-cytochrome b reductase and 5 cytochrome b in the microsomal membrane, these proteins constitute 5 only a small portion of all the microsomal proteins (33,35). Recently techniques have become available for more general investigations of membrane structure and in the first chapter, two of these techniques, 10 SDS-polyacrylamide gel electrophoresis and lactoperoxidase-catalyzed protein iodination, have been used to more thoroughly examine the arrangement of all the proteins within the rat liver endoplasmic reticulum. The Microsomal Mixed-Function Oxidase System As previously discussed, the rat liver endOplasmic reticulum _ contains an electron transport system which utilizes both NADPH and O2 in the hydroxylation of various lipOphilic compounds (14-16). Because this system utilizes both NADPH and 02 it is often referred to as the microsomal mixed-function oxidase system (36). Substrates for this system include drugs, carcinogens, pesticides, and many other foreign compounds (xenobiotics) which by various mechanisms end up within the body. The mixed-function oxidase system functions in the removal of these compounds from the body by oxidizing various functional groups of these lipophilic compounds making them more water soluble and hence more easily excreted into the urine, bile or air. Thus this system is usually associated with detoxification, but, ironically, its oxidative reactions have also been implicated in the activation of various toxins, such as carcinogens (37). Endogenous body constituents, such as fatty acids and steroids are also substrates for this system (14-16). A biochemically unique and somewhat baffling property of the mixed-function oxidase system is its apparent non-specificity with regard to the reactions it catalyzes. For example, it catalyzes such widely diverse reactions as the oxidation of saturated and aromatic compounds, the dealkylation of secondary and tertiary amines, the oxidative cleavage of ethers, the sulfoxidation of thio-ethers, the 11 epoxidation of aromatic hydrocarbons, halogenated aromatic hydrocarbons, and alkenes, and the conversion of phosphothionates to their phosphate derivatives (38). Brodie gt_§l, (39) observed one unifying property of these reactions, however, in that most of them can be visualized as hydroxylation reactions, which in some instances, form unstable inter- mediates. Nevertheless, the apparent nonspecificity of this system has been a consideration of much interest to workers in the field of xenobiotic metabolism because such a property is contrary to the common biochemical concept of substrate specificity as found with other enzyme systems. Another unusual property of the mixed-function oxidase system is the inducibility of its hydroxylation activity by in_vivg.pretreat- ment of animals with various lipophilic xenobiotics. This property was first described by Brown ethal. (40) in 1954 when it was found that treatment of animals with polycyclic hydrocarbons enhanced the hydro- xylation of these compounds by the mixed-function oxidases. Subsequent investigations indicated that this enhanced activity was most probably the result of an increased synthesis of the enzyme constituents of this system (14,41-43). This induction phenomenon is important to pharmacologists since it explains the often observed ability of animals to develop tolerance to certain drugs. Tolerance is developed because the drugs are capable of inducing the enzymes which catalyze their detoxification. While over two hundred drugs, insecticides and carcinogens have been reported to stimulate the mixed-function oxidase system, the inducers can be divided into two classes: general inducers which increase hydroxylation activity towards most substrates and specific inducers which selectively stimulate activity towards 12 particular substrates only (16). In the laboratory, the induction of the mixed-function oxidase enzymes by phenobarbital (PB), a general inducer, and 3-methylcholanthrene (3-MC), a specific inducer, has been the most widely studied. The Protein Constituents of the Mixed-Function Oxidase System The association of the microsomal proteins, NADPH-cytochrome c reductase and cytochrome P450, with the mixed-function oxidase electron transport chain is now well established (14-16). Cytochrome P450, a hemoprotein, is so named because its reduced, CO-difference spectrum exhibits an absorbance maximum at 450 nm. NADPH-cytochrome c reductase, a flavoprotein, derives its name from its ability to transfer electrons from NADPH to exogenous cytochrome c. Pretreatment of animals with the general inducer, PB, induces the levels of both of these proteins in microsomes while pretreatment with the specific inducer, 3-MC, only induces the cytochrome (14-16). Cytochrome P was discovered in liver microsomes in 1958 by 450 Klingenberg (44) and Garfinkel (45). It was subsequently characterized as a hemoprotein by Omura and Sato (46,47). Two lines of evidence suggested the involvement of this cytochrome in microsomal hydroxyla- tions. First of all, the levels of this hemoprotein were increased by compounds, such as PB and 3-MC, which were known to induce microsomal hydroxylation activities (48,49). Secondly, Estabrook and co-workers showed that CO inhibited mixed-function oxidase hydroxylations and that the photochemical action spectrum for the reversal of this inhibition was identical to the reduced, CO-difference spectrum of cytochrome P450 (50). Because of the requirement for both 02 and NADPH in 13 microsomal hydroxylations, it was hypothesized that this microsomal cytochrome reacted with oxygen to form an "active oxygen" intermediate which in turn was reduced by NADPH during the hydroxylation sequence (39). Evidence that NADPH-cytochrome c reductase was the enzyme catalyzing the transfer of electrons from NADPH to cytochrome P450 included: the observed increase in the level of this enzyme in micro- somes after pretreatment of animals with PB (14), the observation that addition of cytochrome c to microsomes inhibited microsomal hydroxy- lations (51), the finding that solubilization of this enzyme from microsomes paralleled the loss of hydroxylation activity (52), and the observed ability of antibody prepared to a proteolytically solubilized form of this enzyme to inhibit both cytochrome P450 reduction by NADPH and microsomal hydroxylations (53-55). The following is a schematic summary of the current hypothesized mechanism for the involvement of cytochrome P450 and NADPH-cytochrome c reductase in the mixed-function oxidase electron transport systems, taken from Gillette g£_al, (56). +3 P -Fe R 450 ér//// e' e- L(//<:;”‘{ <’\‘\\\\ +3 P450“Fe NADPH—-—9——?»flavoprotein ;-? .1; P4SO-Fe - l 1. R exogenous cytochrome c OH 02‘\$ +2 Fe+3 P450-Fe —02 P450—ROH R //2fl ? -2;--1y P F +396 -e .- 2H+ H20 4f0 R 14 In this system, substrate (R) is hypothesized to combine with the oxidized form of cytochrome P450. This substrate-cytochrome P450 complex then is reduced by an electron from NADPH—cytochrome c reductase to form a reduced substrate-cytochrome P450 complex. This complex in turn reacts with oxygen to form a substrate-cytochrome P4SO-oxygen complex. The sequence of events after this stage is still uncertain but it is believed that a second electron reduces the substrate-cytochrome P4SO-oxygen complex resulting in the formation of an "active oxygen" intermediate which then decomposes to form hydro- xylated product and oxidized cytochrome P450. The source of the second electron required in this hydroxylation scheme is at present unclear. It is most likely the NADPH-cytochrome c reductase transfers the electron in this step also. This point will be discussed in more detail below, however. Attempts at isolating the native forms of either NADPH- cytochrome c reductase or cytochrome P450 have been unsuccessful. The reductase appears to be located on the external surface of the micro- somal membrane, since treatment of microsomes with proteases which cannot permeate this membrane (29,31,32) readily solubilizes a fragment of the reductase (29). This fragment is enzymatically active with respect to its ability to reduce cytochrome c and has been isolated in several laboratories (57-59). Cytochrome P450 appears to be much more intimately associated with the phospholipids of the membrane since solubilization of this cytochrome from the microsomal membrane requires either the use of detergent or treatment of the membrane with phospho- lipase A (47,60,61). Cytochrome P450 is also a very labile hemoprotein and upon perturbation of the microsomal membrane with salts (62), 15 proteases (29,63), or phospholipases (47,61,64), or upon solubilization of this cytochrome from the membrane with detergents (47,60), it is often converted to a spectrally distinct and catalytically inactive form called cytochrome P420. This cytochrome is so named because the absorbance maxima of its reduced CO-difference spectra is shifted from 450 nm to 420 nm. Neither form of this cytochrome has been purified to homogeneity. Based upon the fold purifications obtained in isolations of a trypsin-solubilized form of NADPH-cytochrome c reductase (57-59,65) it can be suggested that this enzyme constitutes about 0.6% of the micro- somal protein. Partial purification of cytochrome P 0 has suggested 45 that this hemoprotein appears to have a molecular weight of 50,000 (61,66,67) and, on the basis of its specific activity in microsomes, probably constitutes 4-5% of the microsomal protein in untreated animals and up to 20% of the protein in PB-treated animals (67,68). Thus it appears that during microsomal hydroxylations, one molecule of _ reductase must reduce a number of cytochrome P450 molecules. One of the major problems in using whole microsomes to study the mixed-function oxidase hydroxylation reactions is that microsomes contain many other enzymatic activities which often interfere. Ulti- mately it is hoped that both NADPH-cytochrome c reductase and cyto- chrome P 450 hydroxylations can be studied by recombining these proteins to recon- can be isolated in their native forms and that microsomal stitute hydroxylation activity. Some success at such reconstitution studies has been reported in the laboratories of Coon (69-72) and Lu (73-75). These investigators have succeeded in resolving solubilized microsomes into three fractions, a NADPH-cytochrome c reductase 16 fraction, a cytochrome P450 fraction, and a phospholipid fraction, which when recombined catalyze the hydroxylation of many of the sub- strates of the mixed-fUnction oxidase system. The phospholipid fraction can be replaced by pure phosphatidylcholine, which appears to be required for the reduction of cytochrome P NADPH-cytochrome c 450 by reductase (76). The reductase and cytochrome P 0 fractions used in 45 these experiments are very impure, however, and although this system represents in improvement over microsomes for studying hydroxylation mechanisms, definitive studies will have to await a more refined system. Because neither NADPH-cytochrome c reductase nor cytochrome P450 has been purified to homogeneity and used to reconstitute hydro- xylation activity, it is possible that other undiscovered components may also be involved in the mixed-function oxidase electron transport system. In particular, much attention has been given to the possible involvement of other components in the transfer of electrons from NADPH-cytochrome c reductase to cytochrome P450 (hence the inclusion of the ? at this step in the reaction scheme presented above). Such a component has been found in at least two other mixed-function oxidase systems—-the system found in adrenal cortex mitochondria which is responsible for steroid metabolism (77), and the soluble mixed-function oxidase of Pseudomonas putida which catalyzes the hydroxylation of D-camphor and certain camphor analogues (78). In both cases electron transfer from the reductase to a cytochrome P450 occurs via a non-heme iron protein, adrenodoxin in the adrenal system (77) and putidaredoxin in the bacterial system (78). Such a non-heme iron component has not been found in the liver microsomal system. 'It has recently been reported, however, that an electron acceptor distinct from the 17 heme-iron atom of liver microsomal cytochrome P450 has been found in the partially purified preparation of this cytochrome which is used in the reconstituted system of Coon and co-workers (79). This suggests the possible involvement of a component other than non-heme iron in the liver mixed-function oxidase system. In addition, Pederson et a1, (57) have presented evidence for the existence of a microsomal component other than cytochrome P which appears to be reduced by NADPH- 450 cytochrome c reductase during the NADPH-dependent peroxidation of microsomal lipid. It is possible that this component is also involved in microsomal mixed-function oxidase hydroxylations. In early studies of the mixed-function oxidase system it was observed that NADH in the presence of saturating concentrations of NADPH increased the activity of the mixed-function oxidase system (80). This observation has led to speculation in the past that components of the NADH-linked fatty acid desaturase electron transport system may also be involved in microsomal hydroxylations, perhaps by providing the second electron to the oxygenated-cytochrome P4SO-substrate complex (81). Specifically it has been suggested that the transfer of the second electron to the oxygenated-cytochrome P4so-substrate complex may be the rate limiting step in the hydroxylation sequence and that cytochrome b5 may function to transfer this electron from either NADPH-cytochrome c reductase or NADH-cytochrome bS reductase to cytochrome P450 (81,82). Since the transfer of electrons from NADH to cytochrome bS via NADH-cytochrome bS reductase is a much more rapid reaction than the transfer of electrons from NADPH to cytochrome bS via NADPH-cytochrome c reductase, this hypothesis would explain the observed ability of NADH to increase microsomal hydroxylation activity. 18 Several lines of evidence have been recently presented which argue against this mechanism, however. First of all, antibodies directed against either NADH-cytochrome bS reductase or cytochrome b5 do not inhibit microsomal hydroxylations (83). Secondly, preparations of NADPH-cytochrome c reductase and cytochrome P450 have been prepared free of cytochrome b5 and will reconstitute microsomal hydroxylation activity, indicating that cytochrome bS is not an obligation component of the hydroxylation system (75,84). In fact, addition of cytochrome bs to this reconstituted system actually has been observed to inhibit the hydroxylation activity. This inhibition is reversible if NADH- cytochrome b reductase and NADH are then added to the system. Thus, 5 it appears that the best explanation for the synergistic effect of NADH on microsomal hydroxylations is that NADH, via NADH-cytochrome b5 reductase, functions to keep cytochrome bS in a reduced form and hence prevent the transfer of electrons from NADPH to cytochrome b5. Actually, cytochrome b5 in the absence of NADH may be acting as an electron sink tunneling electrons away from rather than to cytochrome P450. Multiplicity of the Microsomal Mixed-Function Oxidase System As previously stated, one of the most intriguing aspects in the study of the rat liver microsomal mixed-function oxidase electron transport system is the apparent lack of specificity this system displays with regard to the substrates it hydroxylates. Many studies, utilizing microsomal suspensions, have suggested that this property may be explained by the existence of multiple mixed-function oxidase activities. The existence and differential inducibility of these activities would explain the ability of some compounds, such as PB, to 19 stimulate the hydroxylation of many compounds while others, such as 3-MC, only stimulate the metabolism of a few (16). Investigations in which the kinetics of the metabolism of specific compounds by microsomes have been studied have provided one type of evidence for this idea. For example, Alvares g£_al: (85) have reported that while pretreatment of rats with both PB and 3-MC increased the Vmax for microsomal benzpyrene hydroxylation, 3-MC-pretreatment also lowered the Km for this reaction while PB—pretreatment did not. Such data suggest that 3-MC was inducing a different metabolizing activity than was PB. Wada gt_§l, (86) have shown that if a Lineweaver-Burk plot were made for the hydroxylation of aniline by rat or mouse liver microsomes, a biphasic line resulted which was indicative of a reaction catalyzed by two enzymes having different Km values. Pretreatment of animals with P8 not only increased the vmax for this reaction but also appeared to increase the relative amount of the low Km component. Pederson and Aust (87) observed similar non-linear Lineweaver-Burk plots when studying the metabolism of aminopyrine by the rat liver microsomal mixed-function oxidase system. Their data too suggested that amino- pyrine was hydroxylated by two different enzyme systems with two differ- ent Km values. Pretreatment of animals with PB both increased the vmax for microsomal aminopyrine demethylation and increased the relative amount of the low Km component. Pretreatment of rats with 3-MC did not change the Vmax for aminopyrine demethylation but did cause a relative increase in the amount of the high Km component. Studies using various inhibitors of mixed-function oxidases have provided another type of evidence for the existence of multiple microsomal hydroxylating activities. For example, Sladek and 20 Mannering (88) have reported that the inhibitor SKF-SZSA inhibited the demethylation of 3-methy1-4-monomethy1-aminoazobenzene in liver micro- somes from PB-induced or untreated rats, but not in the microsomes from 3-MC-induced rats. This too suggests that PB and 3-MC induce different mixed-function oxidases. A similar inhibitory pattern with SKF-SZSA was also observed by Pederson and Aust (87) while studying aminopyrine demethylation in the microsomes from control and PB- or 3-MC-pretreated rats. Aust and Stevens (89) used another inhibitor, DDT, to further investigate the existence of multiple microsomal aminopyrine demethylase activities. DDT is believed to inhibit aminopyrine metabolism because it is an alternate substrate for the microsomal oxidases. A plot of demethylase activity versus DDT concentration was seen to be made up of three linear segments which in the microsomes from control, PB- or 3-MC-induced rats divided total activity into three components, one not inhibited by DDT, one moderately inhibited, and a third which is extremely sensitive to DDT inhibition. These three components were suggested to represent three separate hydroxylation activities. PB- pretreatment appeared to induce the extremely sensitive component while 3-MC induced the component insensitive to DDT inhibition. It should be noted that this was one of the first studies to suggest the existence of at least three microsomal hydroxylating systems instead of two. Thus, kinetic studies employing the use of various substrates and inhibitors have led to the conclusion that rat liver microsomes appear to contain multiple mixed-function oxidase activities. The question then becomes: How is this multiplicity manifested in micro- somes? Are there different electron transport chains for each class of compound to be metabolized or does multiplicity exist somewhere 21 within the components of the electron transport chain? It would seem unnecessarily redundant to require both a different NADPH-cytochrome c reductase enzyme and a different cytochrome P450 hemoprotein for each class of compound, but this question has not yet been adequately answered. The studies presented in the last three chapters of this thesis were designed to further investigate this question. CHAPTER ONE THE SPATIAL ARRANGEMENT OF PROTEINS IN THE RAT LIVER ENDOPLASMIC RETICULUM Abstract SDS-polyacrylamide gel electrophoresis and lactoperoxidase- catalyzed protein iodination were used to study the topographical arrangement of proteins within the rat liver endOplasmic reticulum (microsomes). The membranes were isolated by differential centrifu- gation and washed with 0.3 M sucrose containing 0.1 M sodium pyro- phosphate, pH 7.5 to remove ribosomes and adsorbed proteins. On the basis of SDS-polyacrylamide gel electrophoretic analyses, rough and smooth microsomal subfractions had identical protein compositions after this treatment, indicating that it was not necessary to subfractionate the total microsomal fraction if the membranes were first washed with sucrose containing sodium pyrophosphate. Iodination of microsomal membranes was carried out in the presence of butylated hydroxytoluene (BHT), an antioxidant, to avoid the peroxidation of membrane lipids. This procedure not only protected the membranes from lipid peroxidation, but also doubled the incorpo- ration of 125I into membrane proteins. The use of BHT was especially important in studies conducted to determine the effect of iodination on microsomal enzymatic activities, since destruction of cytochrome 22 23 P450 was shown to be associated with the peroxidation of microsomal lipids. In the presence of BHT, iodination only slightly decreased the level of cytochrome P450 and aminopyrine demethylase activity. Cytochrome b5 and NADPH-cytochrome c reductase were not affected at all, but NADH-ferricyanide reductase was inhibited nearly 100%. To determine the distribution of 125I into microsomal proteins (and hence which proteins are located in the exterior of the membrane), 125I-labeled microsomes were electrophoresed on SDS-gels. Many minor constituents of this membrane, having both low and high molecular weights, and major polypeptide components having molecular weights of approximately 50,000 were 125I-labeled. Chloroform:methanol (2:1) 125 extraction of lipids from I-labeled microsomes, prior to electro- phoresis, did not alter this pattern. When lipids were extracted from the membranes prior to iodination, more membrane protein were 1251- labeled, suggesting that in the intact membrane, only proteins located on the exterior of the membrane are substrates for lactoperoxidase. This interpretation was confirmed by studies with trypsin, a protease which cannot permeate the microsomal membrane. Treatment of 1251- labeled microsomes with this protease removed 125I from microsomal proteins again indicating that lactoperoxidase was only iodinating exterior proteins. During experimentation with trypsin it was also observed that if membranes which were iodinated in the absence of BHT were subsequently digested with trypsin, more membrane proteins were susceptible to proteolytic digestion. This further suggested the importance of using BHT during membrane protein iodination since lipid peroxidation, occurring during iodination in the absence of BHT, would otherwise cause extensive breakdown in the structure of the membrane. 24 The results of these studies suggest that within the cell, many minor protein components of the endoplasmic reticulum membrane are in direct contact with the cellular cytoplasm while many of the major protein components are either buried within the membrane or oriented on its luminal face. Introduction Until the late 19605 the study of membrane proteins was retarded by the lack of a convenient method by which they could be separated from one another. Because these proteins are very hydro- phobic, even when solubilized from the membrane by most detergents, they tend to aggregate together. The advent of polyacrylamide gel electrophoresis in the presence of the ionic detergent, sodium dodecyl sulfate (SDS), was therefore an important breakthrough in membrane biochemistry. This provided a simple means by which membrane proteins could be solubilized and separated on the basis of their molecular weights and led to the development of other techniques which could be used in combination with SDS-gel electrophoresis to study membrane structure. One such technique was lactoperoxidase-catalyzed iodination of membrane proteins. This technique was first introduced by Phillips and Morrison (90,91), who used it to study the arrangement of proteins in the human erythrocyte membrane. Lactoperoxidase catalyzes the following reaction at pH 7.4: H202 + 1251- + Protein Lactoperox1da§e¢protein _ 125I 25 Iodide is incorporated predominantly into tyrosine residues of proteins, however some incorporation into histidine residues can also occur. Iodination of tyrosine occurs in the aromatic ring ortho to the hydroxyl group (92). It was originally reasoned by Phillips and Morrison that since lactoperoxidase iodinates proteins via an enzyme- substrate complex and since this enzyme is impermeable to membranes because of its high molecular weight (78,000), only those proteins exposed to the exterior of a membrane would be 125I-labeled by this enzyme. After iodination, membrane proteins can then be separated by SDS-gel electrophoresis and the position of the exterior proteins determined by fractionating the gel and analyzing the fractions for 125I by gamma counting. This technique has been widely accepted and has been used to study the structure of many membranes including the surface membranes of erythrocytes (90,91,93), lymphocytes (94), blood platelets (95), and mouse fibroblasts (96), and the inner mitochondria membrane (97). In this chapter results will be reported of experiments designed to use SDS-polyacrylamide gel electrophoresis in combination with lact0peroxidase-catalyzed protein iodination to study the spatial position of proteins in the microsomal membrane. Materials and Methods Materials Male Sprague-Dawley rats, weighing between 200 and 250 g were obtained from Spartan Research Animals, Inc., Haslett, Michigan. The reagents used for electron microscopy (i.e., glutaraldehyde, uranyl acetate, osmium tetroxide, and lead citrate) were obtained from 26 the Electron Microscope Lab located in the Pesticide Research Center, Michigan State University, East Lansing, Michigan. Lactoperoxidase, trypsin (type III), B-galactosidase (grade IV), carbonic anhydrase, alcohol dehydrogenase, ribonuclease-A (type III-A), soybean trypsin inhibitor (type I-S), cytochrome c (type VI), and NADP+-isocitrate dehydrogenase (type IV) were obtained from the Sigma Chemical Company, St. Louis, Missouri. Bovine serum albumin (Pentex) was obtained from Miles Lab., Kankakee, Illinois. Sodium dodecyl sulfate (SDS), butylated hydroxytoluene (BHT), dithiothreitol, ADP (Fermentation grade), EDTA, NADH, NADPH, thio- barbituric acid, nicotinamide, DL-sodium isocitrate, Tris base, and Brillant Blue R (Coomassie blue) were obtained from the Sigma Chemical Company, St. Louis, Missouri. Aminopyrine was purchased from K and K Laboratories, Plainview, New York. 2,4-Pentanedione and dichlorodi- methylsilane were obtained from the Aldrich Chemical Co., Milwaukee, Wisconson. Phenobarbital (PB) was obtained from Merck 5 Co., Inc., Rahway, New Jersey. Trichloroacetic acid, sodium pyrophosphate, and H202 were obtained from Mallinckrodt, St. Louis, Missouri. Sucrose was obtained from the Swartz/Mann Division of Becton-Dickinson and Company, Orangeburg, New York. All electrophoresis reagents were obtained from Canalco, Inc., Rockville, Maryland. Nalzsl (carrier- free) was obtained from New England Nuclear, Boston, Massachusetts. 1411 other reagents were analytical grade. All aqueous solutions were prepared with water which had been distilled and passed through a mixed bed resin ion exchange column. 27 PB-Pretreatment of Rats Rats were pretreated by including 0.1% PB in their drinking water for 10 days prior to sacrifice. Preparation of Microsomal Membranes Rats were fasted 18 hours before killing by decapitation. The total microsomal fraction was isolated by differential centrifugation as previously described (98). Rough and smooth microsomal subfractions were isolated according to the method of Bergstrand and Dallner (8). In some cases the isolated membranes were stored by suspension in 0.05 M Tris-HCI, pH 7.5 containing 50% glycerol to a protein concentra- tion of approximately 50 mg/ml and freezing at -15°C under N2 in the presence of 0.01% BHT. Before use the membranes were washed in 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5 by suSpension with homogenization to a protein concentration of 1-2 mg/ml and centrifu- gation at 105,000 xg for 90 minutes. All isolation and washing procedures were carried out at 0-4°C. Enzyme Assays and Analytical Methods Cytochrome oxidase (99), catalase (100), 5'nuc1eotidase (101), NADPH-cytochrome c reductase (98), NADH-ferricyanide reductase (30), cytochrome b5 (46), and cytochrome P450 (46) were assayed by previously described techniques. All assays were performed at 25°C. Microsomal NADPH-dependent hydroxylation activity catalyzed by cytochrome P450 was assayed as aminopyrine demethylation by measuring the formation of formaldehyde using the method of Nash (102). The complete details of the aminopyrine demethylase assay have been described by Pederson (98). 28 Protein was determined by the method of Lowry g£_al, (103) and 1% standardized with bovine serum albumin using Ecm at 280 nm equal to 6.6 (104). RNA was determined by the method of Munroe and Fleck (105). Lactoperoxidase concentrations were determined spectrophotometrically using a millimolar extinction coefficient at 412 nm equal to 114 (91). Hydrogen peroxide concentrations were determined similarly using molar extinction coefficient of 72.4 at 230 nm (91). ’Preparation of Samples for Electron Microscopy_ At 0-4°C: Membrane suspensions were fixed in 0.1 M sodium phosphate buffer, pH 7.5 containing 2% glutaraldehyde. After 1 hour the membranes were pelleted at 105,000 xg for 90 minutes and washed 3 times in 0.1 M sodium phosphate buffer, pH 7.5. The pellets were then post—fixed in 1% 050 also in 0.1 M sodium phosphate buffer, 4 pH 7.5. After post-fixing the pellets were washed 3 times with 0.1 M sodium phosphate buffer, pH 7.5. At room temperature: After post-fixing, the pellets were dehydrated in a graded series of ethanol solutions and finally in propylene oxide. Samples were embedded in Epon 812 (70:30 mixture) for sectioning. Sections were then stained with either uranyl acetate or lead citrate. Electron microscopy was performed using the Phillips EM-lOOB electron microscope located in the Pesticide Research Center, Michigan State University, East Lansing, Michigan. 29 Iodination of Microsomes Sucrose containing sodium pyrophosphate-washed microsomal membrane preparations (usually 2.5 mg of microsomal protein) were suspended to a protein concentration of 0.5 mg/ml in 0.1 M Tris-HCl, 12517ml), 5 x 10-7 M pH 7.5 (at 25°C) containing 10'6 M KI (2-10 uC lactoperoxidase, and 0.0001% BHT. The iodination was usually carried out in a five ml volume in a cellulose nitrate centrifuge tube (1" x 3 1/2"). The reaction temperature was kept at 25°C by incubation in a Dubnoff shaker. After a two minute equilibration period, 5 nmoles H202/ml (10 ul of 0.5 mM H202/ml) were added at 1 minute intervals over a three minute reaction period. The mixture was then diluted approximately 7-fold with cold 0.1 M Tris-HCl, pH 7.5 (at 25°C) and centrifuged at 105,000 xg for 90 minutes. This centrifugation was performed at 0-4°C. The pelleted microsomes were either suspended in buffer for subsequent use (i.e., enzymatic assay or trypsin digest) or directly prepared for polyacrylamide gel electrophoresis. (For electrophoresis, 2.5 mg of 125I-labeled microsomes were usually suspended in 1 m1 of 1% SDS-buffer as described below.) Assay for Malondialdehyde Levels in Iodinated Microsomes After iodination, microsomal pellets were resuspended in 0.05 M Tris-HCl, pH 7.5 (at 25°C) to a protein concentration of (0.5 mg/ml). They were then incubated at room temperature with 2 mM ADP and 0.12 mM Fe (N03)2 for 2 minutes to decompose all peroxides present in the membranes to malondialdehyde. Malondialdehyde was then measured by assaying the chromogen formation with thiobarbituric acid (TBA) by a method similar to that described by Pederson (98). One ml aliquots 30 of the membrane suspensions were mixed with 2.0 ml of TBA reagent (0.375% TBA and 15% trichloroacetic acid in 0.25 N HCl). The mixture was heated for 15 minutes in a boiling water bath. After cooling, the assay mixtures were centrifuged at 1000 xg and the absorbance of the supernatant was measured at 535 nm using a Coleman Jr. Spectrophoto- meter. The content of malondialdehyde was calculated using a standard curve prepared as described by Pederson (98). Chloroforszethan 1 (2:1) Extraction of 12SI-Labeled Microsomes All procedures were performed at room temperature. 1251- labeled, pelleted microsomes (2.5 mg; 740,000 cpm 125I/mg) were homogenized in 3 m1 of chloroform:methanol (2:1). This mixture was centrifuged at 1000 xg to pellet the protein residue. The residue was re-extracted with another 3 m1 of chloroform:methanol (2:1) and repelleted. The residue was dried under N2 and resuspended in 1 ml of 1% SDS-buffer for polyacrylamide gel electrophoresis. The chloroform: methanol supernatants were combined and extracted with 0.2 volumes of H O. The resulting two phases were separated. The protein residue and 2 upper and lower phases were analyzed for 125I by removing samples for gamma counting. Iodination 9f Lipid-Extracted Microsomal Protein A microsomal pellet (10 mg of protein) was homogenized in 10 ml of chloroform:methanol (2:1). After centrifugation the supernatant was removed and the protein residue re-extracted with an additional 10 ml of chloroform:methanol (2:1). Both extractions were done at room ‘temperature. The protein residue was then dried under N2 and suspended 31 in 0.1 M Tris-HCl, pH 7.5 (at 25°C). 2.5 mg of this lipid-extracted microsomal protein was iodinated as described above; however, the sample was continually agitated throughout the reaction to keep it well suspended. To terminate the reaction, the sample was diluted 6-fold with cold 0.1 M Tris-HCl, pH 7.5 (at 25°C) and centrifuged at 10,000 xg in a Sorvall centrifuge (O-4°C). The pellet was then resuspended with homogenization into 1 ml of 1% SDS-buffer of polyacrylamide gel electro- phoresis. Trypsin Treatment of Microsomes Microsomes were suspended to 6 mg/ml in 0.05 M Tris-HCl, pH 7.5 (at 25°C) containing 1 mM EDTA and 0.005% BHT. Trypsin was added in a ratio of 10 ug trypsin per mg of microsomal protein and this mixture was incubated under N at 25°C for varying lengths of time. No lipid 2 peroxidation occurred during the proteolysis if BHT was present. To terminate the reaction, soybean trypsin inhibitor was added at a ratio of 5 ug of trypsin inhibitor per ug of trypsin. The reaction mixture was then diluted 3-fold with cold 0.05 M Tris-HCl, pH 7.5 (at 25°C) containing 1 mM EDTA and centrifuged at 105,000 xg for 90 minutes at 0-4°C. The supernatants were assayed for protein released from the 125I-labeled microsomes. When this proteolysis was performed with microsomes, the pellets obtained after proteolysis were immediately resuspended in 1% SDS-buffer for polyacrylamide gel electrophoresis. SDS-Polyacrylamide Gel Electrophoresis The SDS-polyacrylamide gel electrOphoresis technique of Fair- banks, 22.9.1.3 (106) was only slightly modified. In this procedure 1% SDS was included in the electrophoresis buffer and polyacrylamide gels. 32 Samples (1-5 mg protein/ml) were also prepared in 1% SDS-buffer which in addition contained 7% sucrose, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 40 mM dithiothreitol. They were then heated at 100°C for 15 minutes. After cooling, 10 ug pyronin B tracking dye/m1 was added and the samples (usually between 10-50 ul) were applied to 5.6% poly- acrylamide gels (5 mm x 100 mm) which had been prepared in tubes coated with dichlorodimethylsilane. The gels were pre-electrOphoresed approximately 30 minutes prior to sample application. Electrophoresis was perfbrmed at room temperature with a constant voltage gradient of 5 V/cm. The current ranged between 3-4 mA/tube. The running time under these conditions was about 3 1/2 hours. After electrophoresis the gels were placed in 10% trichloroacetic acid overnight. The gels were then stained for protein overnight in a mixture of 0.4% Coomassie blue--10% trichloroacetic acid--33% methanol (107). Destaining was performed for 6 hours against a mixture of 10% trichloroacetic acid and 33% methanol in a diffusion destaining apparatus similar to that marketed by Bio-Rad Laboratories, Richmond, California. The gels were then placed in 10% trichloroacetic acid overnight to completely remove stain from the background of the gels. The protein banding pattern was visualized by scanning at 550 nm in a Gilford spectrophotometer equipped with a gel scanning attachment. After scanning for protein, gels of radioactive samples were fractionated using a Savant Autogel Divider to determine 1251 distribution. Usually 6 drop fractions were collected (1.0-1.2 mm <3f gel) into small disposable glass test tubes and counted on a quclear-Chicago gamma spectrometer. Molecular weight markers 8- galactosidase (130,000), bovine serum albumin (68,000), carbonic authydrase (29,000), alcohol dehydrogenase (37,000), trypsin (23,300) 33 and ribonuclease-A (13,700) were run in parallel with membrane protein samples. The molecular weights reported in these studies were then calculated as described by Weber and Osborn (108) from a standard curve plotted using these standards. Each value is the average of at least 3 determinations. Results Enzymatic and Electron Microsocopic Eharacterization of the Microsomal Fraction Isolated by Differential Centrifugation The subcellcular fraction used in these studies was isolated from a rat liver homogenate by differential centrifugation of a post- mitochondrial supernatant at 105,000 xg for 90minutes. This fraction has previously been characterized by many workers as being derived from the endoplasmic reticulum (2,3). To determine the purity of the membranes used in these studies, the specific activity of various sub- cellcular organelle marker enzymes in the isolated fraction was com- pared to their specific activity in the total liver homogenate. The marker enzymes assayed were cytochrome oxidase for mitochondria (99), catalase for peroxisomes (100), NADPH-cytochrome c reductase for microsomes (109), and S'nucleotidase for plasma membranes (101). It can be seen from Table I that the membrane fraction used in these studies was enriched 8-fold with respect to the microsomal enzyme, NADPH-cytochrome c reductase, while the specific activity of all other marker enzymes was much lower than that of the liver homogenate. This enzymatic comparison confirmed that the membrane fraction used in these studies was indeed enriched in endoplasmic reticulum. In addition, 34 TABLE I SPECIFIC ACTIVITY OF ORGANELLE MARKER ENZYMES IN THE MICROSOMAL FRACTION RELATIVE TO THAT OF THE TOTAL LIVER HOMOGENATE Perfused rat livers were homogenized in 1.15% KC1--0.2% nicotinamide. The homogenate was centifuged at 15,000 xg for 20 minutes to pellet mitochondria, lysosomes, nuclei, peroxisomes, plasma mem- branes, and cellcular debris. The post-mitochondrial supernatant was then centrifuged at 105,000 xg for 90 minutes to pellet the microsomal fraction. Assays were performed as described in the Materials and Methods section. Relative specific activity refers to the ratio of the specific activity of each enzyme in the microsomal fraction to its specific activity in the total homogenate. Enzyme Relative Specific Activity Catalase 0.53 Cytochrome oxidase 0.04 NADPH-cytochrome c reductase 8.10 5'-nucleotidase 0.57 35 electron microscopic examination of this subfraction confirmed this finding in that the fraction appeared to be composed of the small vesicular membrane sacs characteristics of microsomes (2,3) and appeared to be free of other subcellular organelles such as mito- chondria, nuclei, lysozomes and peroxisomes (Figure 1). Comparison of Rough and Smooth Microsomal Membranes After Removal of Ribosomes and Adsorbed Proteins The total microsomal fraction is known to be a mixture of rough (ribosome-bound) and smooth microsomes and to be contaminated by adsorbed cytoplasmic proteins. Since these studies were only to be concerned with the membrane proteins, it was desirable to establish a procedure by which ribosomes and contaminating cytoplasmic proteins could be removed from the membranes. Previous investigations have indicated that chelating agents are effective in disrupting and removing ribosomes (110-112). In this work a mixture of 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5 was used to remove both ribosomes and adsorbed proteins. As can be seen from Table II, this procedure removed approximately 30% of the protein and 80% of the RNA from the membranes. The washing procedure did not appear to be removing membrane proteins since the specific activity of such membrane-associated proteins as NADPH-cytochrome c reductase, NADH- ferricyanide reductase, cytochrome b5, and cytochrome P450 increased. Also the recovery of these proteins were nearly 100%. In addition, the ability of this washing procedure to remove 90% of the catalase from the membrane accentuates its effectiveness at removing adsorbed proteins. This was especially important for studies using Figure l. 36 ELECTRON MICROGRAPH OF MICROSOMAL MEMBRANES Rat liver microsomes were fixed in 0.1 M sodium phosphate buffer, pH 7.5 containing 2% glutaraldehyde and post-fixed in 0.1 M sodium phosphate, pH 7.4 containing 1% 0504. They were then embedded in Epon 812 for sectioning. The sections were stained with uranyl acetate and lead citrate. This micrograph was taken using a Phillips EM-lOOB electron microscope. Magnification: 35,000x. 38 TABLE II THE LEVELS OF VARIOUS CONSTITUENTS IN THE TOTAL MICROSOMAL FRACTION ISOLATED FROM CONTROL RATS BEFORE AND AFTER WASHING THE MEMBRANES WITH 0.3 M SUCROSE CONTAINING 0.1 M SODIUM PYROPHOSPHATE, pH 7.5 After initially assaying the microsomes, 25 mg of microsomal protein was resuspended to a protein concentration of 1 mg/ml in sucrose containing sodium pyrophosphate. This suspension was cen- trifuged at 105,000 xg for 90 minutes to pellet the microsomal membrane. The pellet was then resuspended to a protein concentration of 4.5 mg/ml for assay (18.0 mg of protein was found in the pellet resulting in a recovery of 72% of the original protein). Specific Activity Constituent % Recovery Before After NADPH-cytochrome c reductase* 0.160 0.224 102 NADH-ferricyanide reductase* 4.30 5.61 93 Catalase** 97.0 10.2 7.6 Cytochrome b5*** 0.423 0.658 112 Cytochrome P450*** 0.809 1.28 114 RNA+ 107.0 32.4 21.9 *umoles of receptor reduced/min per mg protein **umoles of H202 consumed/min per mg protein ***nmoles of cytochrome/mg protein +ng of RNA/mg protein 39 lactoperoxidase-catalyzed protein iodination since catalase would interfere with the iodination because H202 is a substrate for both enzymes. The SDS-polyacrylamide gel electrophoresis profiles of rough and smooth microsomes were similar after washing the membranes with sucrose and sodium pyrophosphate. This can be seen in Figure 2 which compares the electrophoresis profiles characteristics of rough, smooth, and total microsomes before and after washing. The major polypeptide constituents for each type of membrane have molecular weights between 40,000 and 60,000 daltons. Unwashed rough microsomes, however, appear to contain several components of molecular weight above 100,000 and below 40,000 which are not present in smooth microsomes. Since Dice and Schimke (113) have reported that the majority of the ribosomal polypeptides have molecular weights between 15,000 and 34,000 on SDS- gels, the ribosomes may be the source of the lower molecular weight components in rough microsomes. After washing, the profiles obtained for rough and smooth microsomes are identical and resemble those obtained for the total microsomal fraction after washing. This suggests that the membranes have similar protein compositions after being washed free of ribosomes and adsorbed proteins. Such a conclu- sion is consistent with several other recent investigations of the protein components of rough and smooth microsomes which have been made using polyacrylamide gel electrophoresis (60,67,114,115). On this basis we concluded that for these studies it did not appear necessary to subfractionate the total microsome fraction if the membranes were first washed with sucrose containing sodium pyrophosphate. 40 .How some on vofiamam we: :«opoum mo m: we on oe coozuom .moceansoa vozmmz mo one mcmom uozoa one came: moceanEoE Boswezas :o :39 mHow mo mum mceom need: one .cofluoom muonpoz use mfiewaoumz on» ca venfiaomeu me any Houucoo m Eoum noumHOmH one: maofiuoenm oneunEoe one mbm ZDHQom oszHHq Amu<>AOQImom .m oHSMAm 41 . «- OOQIN- -"0 009 8| - o v ' O O. -49 8 ’ 8 .3, C) ._ ‘=:::::;_:::=, » O t; 8 ._ m. 000.0” _ _ .. ooo‘on - " 1 Jr- "PN § .. z s. ; ' ’ ‘1' 1 . x . p0 OOS8I- ooggb.’ d» < db “P 1;) f0 8 v Q-<” """ g. 4F g _._:_. 1 — ——- 1“ w- o - :;- - - -_, .0 _, - - .. 8 ~55- -— :7. .. "3., .. , ' .L E OOO'OII -‘~ ,. 000 0" ‘j gs. "\S 7. v.) '0“ -:. 12 uh- —. "x. «F L—— 2 L A T; N I - a - 3 a: 4 g 3 _____. -b d 0098!- oog‘eI _(~’ . JI— .1» y 53 O H 2;) Ji- -1 o 0 <3 0 2 A O x: 5 " -- O s M” «I (5"‘"\51 C2‘<4-;ff;‘” ‘0 " , “ 8 '5‘” ' 47> 1" OOO'Ol|-- _ ‘ ooo'ou -=.-.—_— — t, + '1; ‘" 5 "“‘ ‘ :3 __ '7 '—\; db .3 i f; i manne- oIsmIcI (cu) Figure 2 42 For convenience, the initial experiments conducted to adapt the lactoperoxidase-catalyzed iodination technique for use with rat liver microsomes were performed using the microsomes from PB-pretreated rats. PB-pretreatment causes a proliferation of the liver endoplasmic reticulum (l4) and therefore large amounts of microsomes could be isolated at one time. Lipid Peroxidation During Enzymatic Iodination of Rat Liver Microsomes Phillips and Morrison (90,91) foresaw one potential problem in using the lactoperoxidase-catalyzed iodination technique. It required exposing membranes to H202 which at the time was thought to be involved in the peroxidative breakdown of membrane lipids. Lipid peroxidation has been associated with the increased permeability of membranes including those of red blood cells (116-118) and such subcellular organelles as mitochondria (119-121), microsomes (122), and lysosomes (123,124). Therefore it was thought that this could be a major problem if one were trying to 125I-label only those proteins located on the exterior of a membrane. To solve this problem, Phillips and Morrison suggested that the concentration of H202 to which the membranes are exposed be kept low by use of multiple additions of a small amount of H202 (8 nmole/ml reaction) periodically throughout the course of the iodination. In initial experiments I used a similar technique, adding small aliquots of H202 (5 nmole/ml reaction) to the iodination mixture at one minute intervals over a three minute reaction period. To deter- mine if lipid peroxidation were occurring during the iodination, malon- dialdehyde levels were measured in the microsomes after iodination. Malondialdehyde is a breakdown product commonly used to measure the 43 peroxidation of unsaturated fatty acids (125). As can be seen in Table 111, when iodination of rat liver endoplasmic reticulum was carried out using multiple additions of a small amount of H202, lipid peroxidation still occurred. Iodination in the presence of several agents known to inhibit lipid peroxidation either by chelating iron (EDTA) or acting as antioxidants (dithiothreitol or BHT), indicated that a very low concentration of BHT combined the desirable effects of completely inhibiting lipid peroxidation while doubling the amount of 1251 incorporation into microsomes during the iodination. The mechanism by which inhibition of lipid peroxidation leads to an increase in the level of 125I incorporated is unknown but is currently under investi- gation in Dr. Aust's laboratory. It is now known that H202 actually does not promote the peroxidation of membrane lipids (98) therefore implying that the lipid peroxidation observed in these experiments may be catalyzed enzymatically by lactoperoxidase. Determination of Optimum Hggz:and Lactoperoxidase Concentrations for Maximum 1251 Incorporation into Microsomes Having determined the proper conditions for inhibiting micro- somal lipid peroxidation during an iodination, studies were then conducted to determine the optimum concentrations of H202 and lactoper- oxidase to use during an iodination. The results of these experiments .are shown in Figures 3 and 4. On the basis of these results, iodina- 'tions were carried out using 5 x 10'7 M lactoperoxidase and 5 nmole 11(32/ml aliquot additions of H20 at one minute intervals over a three 2 2 minute reaction period. 44 TABLE III THE EFFECT OF INHIBITORS OF LIPID PEROXIDATION ON THE FORMATION OF MALONDIALDEHYDE AND INCORPORATION or 1251 INTO MICROSOMES Liver microsomes from a PB-pretreated rat were washed in 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5. They were then resuspended to a concentration of 0.5 mg/ml in 0.1 M Tris-HCl, pH 7.5 (at 25°C) containing 10.6 M K1 (1 uC 12517ml), 5 x 10.7 M lactoper- oxidase, and the additions indicated below. After a two minute equilibration at 25°C, 5 nmole H202/m1 were added at 1 minute intervals over a three minute reaction period. The reaction was terminated by diluation in cold 0.1 M Tris-HCl, pH 7.5 (at 25°C) and centrifugation at 105,000 xg for 90 minutes at 0-4°C. The pelleted microsomes were 125I Incorporation resuspended in 0.05 M Tris-HCl, pH 7.5 (at 25°C). was assayed by removing an aliquot for gamma counting. Malondialdehyde content was assayed as described in the Materials and Methods. Malondialdehyde formation 125I-Incorporation Additions nmoles/mg protein Total cpm/mg protein None 22.0 800,000 40 mM Dithiothreitol 1.3 0 2‘mM EDTA 6.1 860,000 0.0001% BHT 0.0 1,610,000 45 .cofiuohomhoocw H NH ocMEHouov on nouoeonuoomm «EEmm oMmoAEUIemoHosz a co voucsoo onos moaomouowe eopoaaom och .oov-o no mopscue om coo mx ooo.oo~ pa acupawsoaaueoo new m.a mm .Hu:-muae : H.o no“: coausfiue an wouocfiEpop mos :owuomou och .nofiuom cowuooon ousafis oouzu m uo>o mao>uoucfi ouscfie oco um cowuomou HE\NON: mo mucsoso wouooflvcfi on» mcflvvo x9 wouofiuwcfi mm: coflpecfle0fi .mouscwa N pom oomm um coauonzocfinoum uoum< .Hmm wflooo.o one .omonfixouomouoefi 2a-oH x m .AHE\-HmNH on NV He 2 o-oH mcmcflaucoo m.a :a .Hum-maae 2 H.o as He\me m.o mo :owueuucoocoo m o» eoncommsm onoz moEomonoflE wonmmzuouenmmonmouxm Edflvom was omOHQSm NON: mo monem<> osz: HHq OHZH HmNH mo onho mHe>poucfi ounces one no soapoeoh He\NoNx moHoec m maflvuo an wopofluwcfi mm: coaumcfivofi .mouscfle m How oomm no coflumnsucfi-opm aoum< .Hmm waooo.o use .omoeflxopomouooa mo m¢0flumuucoucoo medaaa> .AHE\HmNH u: my H2 2 o-oH meucfimpcoo m.a 2d .Huz-muae 2 2.0 :2 Ha\me m.o mo came -mupcoucoo m on coocoamsmon onoz moEOmopoflE eonmozuopocmmocmonxa esfleom one omonosm monHm<> osz: HHq OHZH HmNH mo onbwH emu ogh HHg mxk mom omzHum<>qoaImom .m opswflm 54 u” 099 EONVOUOSGV 00 J r _ 1 I I’ ..m OOQ‘BI MIGRATION DISTANCE (CM) Figure 5 55 was obtained. This pattern was very reproducible from preparation to preparation of microsomes. From this pattern it appears as if many of the minor protein constituents of microsomes are capable of being 125I-labeled and hence located on the membrane exterior. Also major protein constituents having molecular weights of approximately 50,000 incorporate 125I and therefore are exposed to the membrane's exterior. This 12$I-labeling pattern does not change when the specific activity 125 of I in the iodination mixture is increased (Figure 6) and therefore it reflects the true distribution of 1' into the membrane proteins. 125 Also if I-labeled microsomes are extracted with chloroform:methanol (2:1) to remove lipid and the lipid-depleted microsomal proteins then electrophoresed on SDS-gels, the same protein and 125I-labeling pattern is obtained (Figure 7). This indicates that membrane lipids are not interfering with either pattern. Recovery of 125I from SDS- Polyacrylamide Gels When the recovery of 125I from SDS-gels was first examined, it seemed extremely low. Only 20-25% of the original cpm of 125I applied to the gels were recovered in the fractions obtained from the gel. 125I were found at the top of the gel, this did not Since no Cpm of appear to be a problem resulting from the inability of some of the membrane proteins to enter the gel. Because the gels were being fractionated after being stained with Coomassie blue, it was initially thought that the radio-activity might be lost from the gels during the staining and destaining process. This could not completely account for the low recovery, however, since the recovery was still low (33%) even if the gel was fractionated immediately after electrophoresis. - ;.v 4 . . .1: «cm Figure 6. 56 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROTEIN AND 1251- INCORPORATION PROFILES OBTAINED FROM THE LIVER MICROSOMAL FRACTION FROM A PB-PRETREATED RAT WHEN THE SPECIFIC ACTIVITY OF 1251 IN THE IODINATION REACTION WAS VARIED Membranes were treated as described in the legend to Figure 5. The upper profile is the protein pattern obtained by scanning gels at 550 nm followin Coomassie blue staining. The lower scans are the 25I-incorporation profiles obtained when the membranes were enzymatically iodinated in the presence of 2 HC 125I/ml reaction (A-A), 6 HC 125I/ml reaction (u-u), and 10 uC 125I/ml reaction (-e---e-). 40 ug of protein was applied to each gel. 57 comm. 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OOO'GA 0006i gr I II’ N 0 '5 *r CD (.0 cfimucoo mHHoz House one .ncflououm mo m1 oNHU omH ommuuseou o esopcoouxu-maaHq omNHquaqomahzmommth oz< .mmUumeQOmHHz< 2mm29mm monHuqzoqmmhruao .AH mezmfla 102 103 TABLE VIII NADPH-FERRICYANIDE REDUCTASE ACTIVITIES IN THE IMMUNOPRECIPITATES FORMED BETWEEN ANTIBODY TO BROMELAIN REDUCTASE AND DETERGENT-SOLUBILIZED LIVER MICROSOMAL PROTEINS FROM CONTROL, PB-, AND 3-MC-PRETREATED RATS As described in the Materials and Methods, liver microsomes from control, PB-, and 3-MC-pretreated rats were solubilized in sodium deoxycholate, centrifuged, and antibody was added to the supernatants to form immunoprecipitates. After washing, the immunoprecipitates were suspended in 0.05 M Tris-HCl (pH 7.5 at 25°C) and 10 mM EDTA. A11 fractions were assayed for NADPH—ferricyanide reductase activity. Total nmoles of ferricyanide reduced/min Fraction PB 3-MC Control Microsomes 1.6 0.9 0.8 Sodium deoxycholate- solubilized microsomes 1.6 0.9 0.8 Immunoprecipitate 0.4 0.2 0.2 Supernatant from immunoprecipitation 0.0 0.0 0.0 Figure 18. 104 A COMPARISON OF THE SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROTEIN PROFILES OF THE IMMUNOPRECIPITATES FORMED BETWEEN ANTIBODY PREPARED TO PURIFIED BROMELAIN-SOLUBILIZED NADPH- CYTOCHROME C REDUCTASE AND DETERGENT-SOLUBILIZED LIVER MICROSOMES FROM CONTROL, PB-, AND 3-MC-PRETREATED RATS The scans on the left-hand side of this figure compare the profiles obtained for immunoprecipitates formed between reductase antibody and detergent-solubilized liver micro- somes from control, PB-, and 3-MC—pretreated rats while the scans on the right-hand side compare the profiles obtained when mixtures of these samples were elctro- phoresed on a single gel. Molecular weight markers, based on Rf values, were determined independently for each gel. ABSORBANCE 550nm 105' CONTROL 8 PB CONTROL 2 — 2“ O 8 8 Q I? I -1 I9 I " ‘ I I ' ' T T CONTROL 3 3'~MC 3'MC 2 - 2- O 8 OT . s I _ I | — O). 'T I I I I I I j I PB PB 8 3m 2 "I 8 2.. Q 0.2 § I m I\ I _ _ I I“ Luv“) \_____, L...” - -1, I I I I 2 4 6 8 MIGRATION DISTANCE (CIW N— .b-I CD 00 Iigurc 18 106 immunoprecipitate contains three protein constituents, one of molecular weight 79,000, one of molecular weight 52,000, and the other of mole- cular weight 25,000. The constituents of molecular weights 52,000 and 25,000 have previously been shown to be derived from the antibody. It appears from this experiment, that the "native" reductases from each type of microsomes have identical molecular weights. Even in mixing experiments in which combinations of two different immunoprecipitates were electrophoresed on a single gel, no difference could be detected in the molecular weights of the "native" reductases (Figure 18, right column of scans). Discussion From the results presented in this chapter it can be concluded that the "native" NADPH-cytochrome c reductase enzymes present in the liver microsomes from control, PB-, and 3-MC—pretreated rats appear to be identical both on the basis of their antigenic similarity and their apparent molecular weights on SDS-polyacrylamide gels. This conclusion concurs with those from other studies in which the trypsin—solubilized enzymes from liver microsomes of control and PB-pretreated rats have been shown to be immunologically identical and behave similarly during chromatography on Sephadex G-100 or DEAE-celluclose (58). Furthermore, other investigators have demonstrated that antibody to trypsin- solubilized reductase from PB—pretreated rats inhibits the reductase activity to the same degree in the liver microsomes from control, PB-, or 3-MC-pretreated rats (53,58), again suggesting that the reductases are identical. The one anomalous finding, however, is that by Lu e£_213 (73) in which it has been shown that "native" NADPH-cytochrome c 107 reductases partially purified from detergentfsolubilized PB- and 3-MC- microsomal proteins appear to have a role in determining substrate specificity in reconstituted microsomal drug hydroxylation systems. This would not be expected if the reductase from the two types of microsomes were identical. Since the reductase preparations used in those studies were not pure, however, the observations made by Lu g£_§13 (73) may have resulted from a contaminant in the preparations. Alternatively, the ”native" reductases in the three different types of microsomes may differ in some very subtle manner which cannot be detected by studying their immunochemical or physical properties. Clearly, it will be necessary to isolate the "native" reductase from the three types of microsomes to differentiate between these possibili- ties. It appears by SDS-polyacrylamide gel electrophoresis analysis that "native" NADPH-cytochrome c reductase has a molecular weight slightly larger than its proteolytically solubilized form. This suggests a structure for the "native" enzyme which is very similar to those already described by Strittmatter and co-workers for two other microsomal enzymes, NADH-cytochrome bS reductase (33) and cytochrome b5 (34). These enzymes are amphipathic proteins-—that is, they consist of a single polypeptide chain containing a large hydrophilic segment which contains the active site of the enzyme and is exposed to the cytoplasm of the cell and a smaller hydrophobic "tail" segment which interacts with the phospholipids of the microsomal membrane. Proteases cleave the hydrophilic portion of these proteins from the membrane while leaving behind the hydrophobic tail. If this is also the case with NADPH-cytochrome c reductase, this enzyme would appear to have a 108 tail of approximately 70 amino acids on the basis of the 8,000 dalton molecular weight difference of the native and proteolytically solubi- lized enzymes, determined in this study by SDS-gel electrophoresis. In actuality, the molecular weight difference may be greater, since Spatz and Strittmatter (33) have reported that SDS-gel e1ectr0phoresis gives a 20% underestimation of the molecular weight of the amphipathic NADH-cytochrome bS reductase. They attribute this disparity to the ability of the hydrophobic "tail" of this protein to bind extra SDS, increasing its mobility during electrophoresis and therefore decreasing its apparent molecular weight. If this is also the case with NADPH- cytochrome c reductase, the actual molecular weight of the "native" protein would be larger than 79,000. The results of the studies presented in this chapter are con- sistent with the view that a single form of the enzyme NADPH-cytochrome c reductase is present in the microsomes from control, PB-, and 3-MC- pretreated rats. This suggests that the different hydroxylation activities found in these microsomes are not the result of multiple mixed-function oxidase electron transport chains. Therefore multi- plicity must lie entirely within the oxygen- and substrate-binding component of the chain, cytochrome P450. In the next two chapters, the role of this cytochrome is further investigated. CHAPTER THREE MULTIPLICITY OF CYTOCHROME P450 HEMOPROTEINS IN RAT LIVER MICROSOMES Abstract Cytochrome P450 is thought to be a major microsomal protein constituent having a molecular weight of approximately 50,000. There- fore the existence of multiple forms of this cytochrome in rat liver microsomes and their differential inducibility by PB- and 3—MC- pretreatment of rats could be investigated by SDS-polyacrylamide gel electrophoresis. Using this technique the 50,000 molecular weight protein components of the liver microsomes from control and PB- or 3-MC-pretreated rats were compared. It was observed that 3-MC- pretreatment induced a protein of slightly higher molecular weight than 50,000 (53,000) while PB induced a protein(s) of slightly lower molecular weight. The induced proteins co-purified with cytochrome P fractions prepared from the three types of microsomes. A method 450 was developed by which benzidine and H202 could be used to stain for the peroxidase activity of cytochrome P450 hemoproteins on SDS-gels. Three hemoproteins were observed in rat liver microsomes using this technique and they have molecular weights of 53,000, 50,000, and 45,000. 3-MC appeared to induce the 53,000 dalton hemoprotein while PB induced the 45,000 dalton hemoprotein. These hemoprotein were also 109 110 present in partially purified fractions of this cytochrome from the three types of microsomes. These results suggest that multiple cytochrome P450 hemoproteins are present in rat liver microsomes. The spatial position in the microsomal membrane of the proteins induced by PB and 3-MC was then investigated by combining the tech- niques of SDS-polyacrylamide gel electrophoresis and lactoperoxidase- catalyzed protein iodination. The liver microsomes isolated from rats pretreated with these compounds incorporate more 125I into proteins of 50,000 molecular weight than do control microsomes. This suggests that the proteins induced by PB and 3-MC may be inserted onto the exterior of the microsomal membrane. Introduction For several years, investigations conducted using whole micro- somal suspensions have suggested that different spectral forms of cytochrome P450 appear to be present in the liver microsomes isolated from control and PB- or 3-MC-pretreated rats. For example, in 1966 Imai and Sato (151) observed that a reduced difference spectrum of the cytochrome P in control microsomes, assayed in the presence of 450 the ligand, ethyl isocyanide, exhibited two absorption maxima, one at 430 nm and the other at 455 nm. Subsequently Sladek and Mannering (152) reported that PB-pretreatment of animals caused an identical increase in the levels of both of these peaks while 3-MC-pretreatment only increased the level of the 455 nm peak. These results were interpreted t: suggest that two forms of cytochrome P450 existed in microsomes and showed differential inductibility by PB and 3-MC. The 111 next year this idea was further substantiated when Alvares 33 31. (153) showed that the reduced CO-difference spectra of microsomes from control and PB- or 3-MC-pretreated rats also exhibited different absorbance maxima. The cytochrome assayed in the liver microsomes from control and PB-induced rats had an absorbance maximum at 450 nm while the maximum for the cytochrome in the microsomes from 3-MC- pretreated animals was at 448 nm. (Hence the cytochrome present in the liver microsomes from control and PB-pretreated rats is called cytochrome P450, while that present in the microsomes from 3-MC- pretreated rats is termed cytochrome P448.) Comparisons of substrate- binding spectra (154) and absolute spectra (66) of the cytochromes present in the liver microsomes from control and PB- or 3-MC-pretreated rats also suggested the presence of two forms of this cytochrome in these microsomes. Evidence other than spectral data has also supported this hypothesis. Thus, Levin and Kuntzman (155) reported a biphasic decay in the degradation of cytochrome P450 heme in microsomes, suggesting that two pools of this cytochrome exist in microsomes. They further noted that administration of PB increased the level of the component responsible for the fast turnover of heme while 3—MC- pretreatment induced the level of the component with the slow heme turnover. In the past few years, more refined studies using detergent— solubilized microsomal fractions have also provided evidence which suggests that multiple forms of cytochrome P450 may exist in rat liver microsomes. For example, Lu and co-workers (73,74) have partially purified cytochrome P 0 fractions from detergent-solubilized liver 45 microsomes isolated from control and PB- and 3—MC—pretreated rats and 112 shown that each cytochrome P450 fraction appears to have different substrate specificities when assayed in a solubilized reconstituted microsomal hydroxylation system. Those hydroxylation activities normally induced in liver microsomes by PB-pretreatment of animals appeared to be catalyzed most effectively by the cytochrome fraction prepared from microsomes isolated from PB-pretreated rats. Similarly, those activities normally induced in liver microsomes by pretreatment of animals with 3-MC, were most effectively catalyzed by a cytochrome P448 fraction isolated from microsomes prepared from a rat pretreated with this compound. In addition, they discovered that cytochrome P450 fractions prepared from control microsomes appeared to have a different substrate specificity than the cytochrome fractions isolated from either the liver microsomes of 3-MC- or PB-pretreated rats. Thus on the basis of these studies using cytochrome P450-enriched fractions from the liver microsomes of control and PB- or 3-MC-pretreated rats, it was suggested that not only did multiple catalytic forms of cyto- chrome P450 appear to exist in microsomes, but there appeared to be three forms of this cytochrome rather than two, as had been suggested by earlier work. The existence of three forms of cytochrome P 50 was also 4 suggested by the results of another type of fractionation study conducted on detergent-solubilized microsomal proteins by Comai and Gaylor (156). In this study, protease-treated microsomes were solubilized with sodium deoxycholate and then subjected to DEAE- cellulose chromatography. This procedure resulted in the separation of three fractions of microsomal proteins which appeared to contain spectrally distinct forms of cytochrome P450. The three forms of this 113 cytochrome were distinguished on the basis of their binding constants fer cyanide and octylamine, as assayed by difference spectroscOpy in the presence of these ligands. PB administration to rats was shown to induce one form while 3-MC-pretreatment induced a second. The third form could also be induced preferentially by ethyl alcohol. Thus, these observations too suggested that multiple forms of cytochrome P450 may exist in microsomes, but because the three spectrally distinct forms of this cytochrome observed in this study were separated from protease-treated microsomes, the possibility also existed that they may have resulted from proteolytic degradation of a single form of the cytochrome. In any case, it is becoming an increasingly popular view that multiple forms of cytochrome P450 may be present in rat liver micro- somes. All evidence for this proposal, however, has been obtained indirectly, because methods are not yet available to purify the cyto- chrome(s) free from other microsomal proteins. Thus, in actuality, little is known about the mechanism by which the apparently different spectral and catalytic forms of this cytochrome result in microsomes. It may be that different cytochrome P hemoproteins are present in 450 microsomes and are preferentially induced by pretreatment of animals with various lipophilic compounds. Alternatively, only one cytochrome P hemoprotein may be present in microsomes and the spectral and 450 catalytic properties of this hemoprotein may then be modified by other membrane constituents directly associated with the hemoprotein (i.e., perhaps a "regulatory" polypeptide or phospholipid?). In the studies presented in this chapter, the technique of SDS-polyacrylamide gel electrophoresis was used to further study this question. 114 Since it has been suggested by others that cytochrome P450 has a molecular weight of approximately 50,000 (61,66,67) and comprises 5 to 20% of the total microsomal protein (67,68), it was reasoned that the induction of this protein should be observable on SDS-gels of total microsomal protein. Furthermore, if multiple cytochrome P 50 hemo- 4 proteins exist in microsomes and PB and 3-MC induce different hemo- proteins, one should observe the induction of different proteins on SDS-gels run of liver microsomal proteins after rats are pretreated with these compounds. The results of the studies presented in this chapter demonstrate that PB and 3-MC do indeed induce different major microsomal proteins which are observable in the 50,000 molecular weight region of SDS-gels. These induced proteins are shown to co-purify with cytochrome P450, suggesting their association with this cytochrome. Also they appear to be hemoproteins on the basis of their ability to be stained in SDS-gels with benzidine and H202. Furthermore, because the induction of major microsomal polypeptides after PB- and 3-MC- pretreatment of animals could be observed in SDS-polyacrylamide gels, it was reasoned that it might be possible to determine the position of these induced proteins in the microsomal membrane. Such knowledge would, in turn, allow a better understanding of how the proteins induced by PB- and 3-MC-pretreatment are incorporated into a pre- existing membrane. This information could be obtained by using the technique of lactoperoxidase-catalyzed protein iodination, developed 125I-label those proteins exposed to the exterior in chapter one, to of the liver microsomal membranes isolated from control and PB- or 3-MC-pretreated rats. If the 50,000 molecular weight proteins induced by PB- and 3-MC-pretreatment of rats are located on the exterior of 115 the membrane, more 125I should be incorporated into proteins of this molecular weight in the liver microsomes from P8- and 3-MC-pretreated rats than in the microsomes from control animals. Materials and Methods Materials Male Sprague—Dawley rats weighing between 75-100 g were obtained from Spartan Research Animals, Inc., Haslett, Michigan. Sodium cholate was obtained from the Schwarz-Mann Division of Becton Dickinson and Company, Orangeburg, New York. Benzidine-HCl was obtained from Merck and Company, Inc., Rahway, New Jersey. Purified Cytochrome P450 (cam) from Pseudomonas putida was the gift of Drs. Karl Dus and I. C. Gunsalus of the University of Illinois, Urbana, Illinois. The sources of other reagents have been listed in chapters one and two. Drug Pretreatment of Animals The procedure for drug pretreatment of rats with PB and 3-MC was described in chapter two. Isolation of Microsomal Membranes The isolation and storage procedure used for microsomal membranes was described in chapter one. In some cases, isolated membranes were washed with 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5, also as described in that chapter. 116 Partial Purificiation of Cytochrome P450 Fractions From Liver Microsomes of Control and’PB- or 3-MC-Pretreated Rats The isolation procedure described by Levin et_al: (66) was followed. Unless otherwise stated all procedures were carried out at 0-4°C. Liver microsomes, which had been stored in 0.05 M Tris-HCl, pH 7.5 containing 50% glycerol, were suspended to a protein concentra- tion of 1-2 mg/ml in 1.15% KCl containing 10 mM EDTA, pH 7.5 and centrifuged at 105,000 x g for 90 minutes. The resulting microsomal pellets were resuspended to a protein concentration of 30-40 mg/ml in 0.25 M sucrose. Each 800-1000 mg of microsomal protein was then mixed with 14 m1 of glycerol, 7 ml of 1 M potassium phosphate, pH 7.7, 0.7 ml of 0.1 M dithiothreitol, and 0.7 m1 of 0.1 M EDTA, pH 7.5 and this mixture was then diluted to 61 ml with 0.25 M sucrose. The mixture was sonicated using four, 15 second sonication bursts at the number 4 setting on a Branson Model S-125 Sonifier (4 ma, maximum power output). During sonication the temperature of the mixture was not allowed to rise above 8°C. Eight to 10 ml of 10% sodium cholate was then added, with stirring, to the mixture bringing the final concentration to 1 mg cholate per mg of protein. The mixture was then stirred on ice for an additional 20 minutes and centrifuged at 105,000 x g for 90 minutes. After centrifugation, the supernatant was fractionated with ammonium sulfate. These fractionations were performed by slowly adding solid ammonium sulfate to the saturation desired, stirring the mixture an additional 20 minutes, and then centrifuging at 27,000 x g (15K in refrigerated Sorvall RC 2-B centrifuge, SS34 head) for 20 minutes to pellet the insoluble material. Solid ammonium sulfate was added to 40% saturation (0.224 g/ml) and then to 50% saturation (0.058 g/ml 117 additional ammonium sulfate). The material pelleting between 40 to 50% saturation was resuspended to approximately 13 ml in 0.05 M potassium phosphate buffer, pH 7.7 and centrifuged at 160,000 x g for 60 minutes to remove insoluble material. The supernatant fraction was then dialyzed overnight against 2 liters of 0.02 M potassium phosphate buffer, pH 7.7 containing 20% glycerol, 10.4 M dithiothreitol, 10.4 M EDTA, and 0.1% sodium cholate. After dialysis, the material was centrifuged at 27,000 x g for 20 minutes and the supernatant (usually between 10-20 mg protein/ml) was diluted to 3-4 mg protein/ml with 0.02 M potassium phosphate buffer, pH 7.7 containing 20% glycerol, 10'4 M dithiothreitol, 10'4 M EDTA, and 0.2% sodium cholate. Solid ammonium sulfate was added to 43% saturation (0.25 g/ml) and then to 50% saturation (0.042 g/ml additional ammonium sulfate). The resulting precipitate from 43-50% saturation was dissolved in 2-4 ml of 0.005 M potassium phosphate, pH 7.7 containing 20% glycerol, 10'4 M dithio- threitol, 10"4 M EDTA, and 0.1% sodium cholate. It was dialyzed overnight against this same buffer. The dialyzed sample was then centrifuged at 27,000 x g for 20 minutes and the supernatant was divided into small aliquots which were frozen under N2 in sealed vials. The final protein concentration usually ranged between 3-8 mg/ml. 1% SDS-Polyacrylamide Gel Electrophoresis The procedure described in chapter one was used for studies employing this electrophoresis technique. 118 0.1% SDS-Polyacrylamide Gel Electrophoresis For 0.1% SDS—polyacrylamide gel electrophoresis, 10 cm gels were prepared according to the method of Fairbanks g£_a1: (106), except the SDS concentration in the gels and in the electrophoresis buffer was lowered to 0.1%. (N233; under these conditions the gels polym- erize in 10—15 minutes, much faster than in the presence of 1% SDS.) Microsome samples were suspended to a protein concentration of 6 mg/ml in 1% 808 containing % sucrose, 10 mM Tris—HCl, pH 8.0 (at 25°C), 1 mM EDTA, and 10 ug/ml pyronin B tracking dye. Partially purified cytochrome P450 fractions were suspended to a protein concentration of 3 mg/ml and the sample was only made 0.5% with respect to SDS. The samples were not boiled prior to electrophoresis. They were instead immediately applied to pre-electrophoresed gels and electrophoresis was performed in the dark at 5°C using an electrophoresis apparatus with a cooling jacket. A voltage gradient of 5 V/cm was used (2 ma/tube). Electrophoresis took about 8 hours under these conditions. After electrophoresis the gels were either stained for protein with Coomassie blue, as described in chapter one, or for peroxidase activity with benzidine and H202 (157). Molecular weights were determined as described in chapter one for the 1% SDS-polyacrylamide gel electro— phoresis sytem. Dithiothreitol (40 mM) was included in the samples containing molecular weight markers. Use of Benzidine and Hzngto Stain for cytochrome P450 Hemoppoteins Immediately after 0.1% SDS-polyacrylamide gel e1ectr0phoresis, the gels were placed in 0.02 M Tris-HCl, pH 7.5 (at 25°C) containing 119 50% methanol for 30 minutes to lower the SDS concentration within the gels. The gels were then placed in 0.25 M sodium acetate, pH 4.7 containing 0.25% benzidine, 25% methanol, and 0.75% H202 (157). This solution was made fresh just prior to use and was prepared by dis- solving the required amount of benzidine-HCl in methanol and then adding the other reagents. H202 was added to the solution last. Since benzidine is carcinogenic, all staining procedures were carried out using rubber gloves and in the hood. After placing the gels in the staining solution, color development took approximately 15 minutes. Because the gels became opaque during the staining procedure and the stain was not stable for long periods of time, the gels could not be spectrophotometrically scanned and had to be photographed immediately after staining. Lactoperoxidase-Catalyzed Protein Iodination This procedure was performed as described in chapter one. The 125 iodination mixture contained 2 uC I'lml. Enzyme Assays Cytochrome P and P420 concentrations were assayed as 450 described by Imai and Sato (62) assuming values of 91 and -11 cm- mM"1 for extinction coefficients between 450 nm and 490 nm for P450 1 1 and P 0’ respectively, and -41 and 110 cm' mM'1 between 420 nm and 42 490 nm for P and P420, respectively. A computer program was used 450 in making these calculations. 120 Results Cpmparison of the 1% SDS-Polyacrylamide Gel Electrophoresis Protein Patterns of the Rat Liver Microsomes from Control andTPB- or 3-MC-Pretreated Rats The 1% SDS-polyacrylamide gel electrophoresis protein patterns of the rat liver microsomes from control and PB- or 3-MC-pretreated rats are compared in Figure 19. It can be seen that pretreatment of animals with either PB or 3-MC induces major microsomal protein constituents which migrate in the 50,000 molecular weight region of the gels. Furthermore, the two compounds do not induce the same proteins in this region. 3-MC appears to induce a component of slightly higher molecular weight than 50,000 while PB induces components of slightly lower molecular weight. Since pretreatment of animals with the two compounds does not appear to induce protein constituents having molecular weights below 40,000, these proteins can be electrophoresed off the gel to better resolve the protein constituents having molecular weights of approximately 50,000 (Figure 20). By comparison, the Rf values of all the proteins resolvable in this region of the gels, 6 different proteins can be observed among the three types of microsomes. To establish that 6 different microsomal proteins are indeed resolvable in this region of the gel, a mixture of the proteins from the liver microsomes of 3-MC- and PB-pretreated rats was electrophoresed in a single gel. In Figure 21 it can be seen that 6 proteins were visualized in this experiment and their apparent molecular weights are presented in Table IX. From Figure 20, at least four of the proteins are common to all three types of microsomes and these have been labeled proteins 1, 2, 4, and 6. Protein 3 is induced by 3-MC-pretreatment of animals. Figure 19. 121 1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROTEIN PROFILES OF THE RAT LIVER MICROSOMES FROM CONTROL AND PB- OR S-MC- PRETREATED RATS Microsomal membranes were isolated by differential centrifugation and washed with 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5 prior to electro- phoresis. As an index of induction by PB and 3-MC, the cytochrome P450 (448) levels (nmoles/mg microsomal pro- tein) in the three types of microsomes were: control, 1.2; PB, 3.4; and 3-MC, 1.7. Between 40—45 ug of micro— somal protein was applied to each gel. The protein banding patterns were visualized by staining with Coomassie blue and scanning the gels at 550 nm using a Gilford Spectrophotometer. The arrows indicate the positions on the gels of the protein(s) induced by 3-MC and PB. 1'22 000.00 000.0: Ill; 000.09 .12 c fie i ‘I.....I..I'I'IIIIIIII...'-'IIII ..... .‘I... u..! A... L. fiI'IIIIIU'. IIIIIIIIIIIIIIIICI A L. 18 v...'.'l..'..II:I:'I" IIIIIIIIIIIIIIIIII I IV #4 w .I.'..I.3 "' IIIIIIIIIIIIIIIIIIIIIIIIIIII Z-II- -II— » b d _ 2 ' Eco-.3 m02<¢¢0an< MIéRAtI ow msmwéa 6 4 Figure 19 Figure 20. 123* 1% SDS—POLYACRYLAMIDE GEL ELECTROPHORESIS PROFILES OF THE PROTEIN CONSTITUENTS HAVING MOLECULAR WEIGHWS LARGER THAN 40,000 WHICH ARE PRESENT IN THE RAT LIVER MICROSOMES FROM CONTROL AND PB-, AND S-MC-PRETREATED RATS Microsomal membranes were prepared as described in Figure 19. Electrophoresis was performed approximately 5-1/2 hours such that components having molecular weights lower than 40,000 would migrate off the gel. The protein patterns were visualized by staining with Coomassie blue and scanning the gels at 550 nm. Between 50-60 ug of protein was applied to each gel. 124 21, Wt 4 o 8 o o O " o o o o o’ O. 0.| o’ 93 9.3 8 26¢ 2‘. me 34 I6 I” C 4 5 2 n 6 ' I I ' I I2 I ‘II Ikqugfkmfi~lvwbfi\fivJM\«J : - . : ¢ = 3 B . MIeIiA'nofi amines cM Figure 20 Figure 20. 18$~ 1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROFILES OF THE PROTEIN CONSTITUENTS HAVING MOLECULAR WEIGHTS LARGER THAN 40,000 WHICH ARE PRESENT IN THE RAT LIVER MICROSOMES FROM CONTROL AND PB-, AND 3-MC-PRETREATED RATS Microsomal membranes were prepared as described in Figure 19. Electrophoresis was performed approximately 5-1/2 hours such that components having molecular weights lower than 40,000 would migrate off the gel. The protein patterns were visualized by staining with Coomassie blue and scanning the gels at 550 nm. Between 50-60 ug of protein was applied to each gel. 124 2., CKINTHC". CD 4- C) C5 C2 E; I C5 2 g m 2 6 ., ZI’ 3Ilc 34 In ‘2 6 4 2‘ PI 5 3 E 4 a 3 é 3 ‘3 . MIGRATION DISTANCE 0M Figure 20 Figure 21. 125 1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PATTERN OF THE PROTEIN CONSTITUENTS HAVING MOLECULAR WEIGHTS GREATER THAN 40,000 WHICH ARE PRESENT IN A MIXTURE OF THE LIVER MICROSOMES FROM 3—MC— AND PB-PRETREATED RATS Microsomal membranes were prepared as described in the legend to Figure 19. Electophoresis was performed approximately 5-1/2 hours such that components having mole- cular weights below 40,000 migrated off the gel. Approxi- mately 30 ug of protein from each type of microsome was applied to the gel. After electrophoresis the gel was stained with Coomassie blue and scanned at 550 nm. 126 3MC& PB 4 ii (3:755‘L ES . 3 6 ' ' I g W g I 0.25- : I g g g 1 1 1 % .LJ 2 4 e ' a MIGRATION DISTANCE (CM) Figure 21 127 TABLE IX THE MOLECULAR WEIGHTS OF THE MAJOR PROTEIN CONSTITUENTS OF RAT LIVER MICROSOMES The molecular weights of the major protein constitutents of rat liver microsomes were calculated as described by Weber and Osborn (108) from a standard curve prepared using the following proteins as mole— cular weight standards: B-galactosidase (130,000), bovine serum albumin (68,000), catalase (60,000), alcohol dehydrogenase (37,000), and carbonic anhydrase (29,000). The values reported for proteins 1, 2, 4, and 6 are the average and standard deviations from 9 determi- nations. The values for bands 3 and 5 are the averages and standard deviations from 3 determinations. Protein Molecular Weight 1 61,000 :_1,000 2 58,000 : 1,000 3 53,000 :_l,000 4 50,000 :_l,000 5 47,000 :_2,000 6 45,000 :_1,000 128 This component is probably also present in the liver microsomes from control and PB-pretreated animals but at levels at which it cannot be resolved from component 4. Proteins 2 and 6 may also be induced by 3-MC but to a much lesser extent than protein 3. The induction pattern after PB-pretreatment is more complex. It appears as if proteins 4, 5, and 6 are induced by pretreatment with this compound but since these proteins cannot be completely separated from one another during electrophoresis, this cannot be easily quantitated. Protein 5 appears to be induced to the greatest extent. This protein too is probably also present in the liver microsomes from control and 3-MC- pretreated rats but at levels at which it cannot be resolved from proteins 4 or 6. 1% SDS-Polyacrylamide Gel ElectrOphoresis Protein Profiles of the Liver Microsomes from Control and PB- or 3-MC-Pretreated' Rats and the Cytochrome P450 Fractions Purified from the Microsomes If the liver microsomal proteins induced by PB- or 3-MC- pretreatment of rats are associated with cytochrome P they should 450’ co-purify with this cytochrome. To further explore this possibility, this cytochrome was partially purified from the liver microsomes of control and PB- or 3-MC-pretreated rats. The purification procedure used was similar to that described by Levin E£.El: (66). Their purification procedure consisted of two ammonium sulfate fractionations of sodium cholate-solubilized microsomes and a calcium phosphate gel extraction step. Since the calcium phosphate gel extraction did not appear to greatly increase the specific activity of the cytochrome and had a low yield, it was omitted from these studies. Table X summarizes 129 TABLE X PARTIAL PURIFICATION OF CYTOCHROME R450 448) FROM THE LIVER MICROSOMES OF CONTROL AND B- OR 3-MC-PRETREATED RATS The purification procedure of Levin £3.31: (66) was followed, however the final calcium phosphate gel extraction was omitted. Cytochrome P and P4 concentrations were assayed and 450 (448) 20 calculated as described in the Materials and Methods. The values in parentheses are those reported by Levin §£_31: (66). Specific Specific Specific Activity Activity Activity Total P450 448) P420 CytOChrome FOId Sample nmoleImg nmole/mg nmole/mg Purification Control Washed microsomes 1.0 0.2 1.2 (0.8) . . 2nd Ammonium Sulfate 2.2 0.6 2 8 (2.0) 2.4 (2.6) (43-50%) 3-MC Washed microsomes 1.6 0.1 1.7 (1.4) . . 2nd Ammonium Sulfate 3.5 0.5 4 0 (3.7) 2.4 (2.6) (43—50%) BE Washed microsomes 3.1 0.3 3.4 (1.8) . . 2nd Ammonium Sulfate 4.5 1.3 5.8 (4.4) 1.7 (2.4) (43-50%) 130 the specific activities of the cytochrome fractions purified from the liver microsomes isolated from control and PB- or 3-MC-pretreated rats. The final specific activities and fold purifications were similar to those reported by Levin g£_a13 (66). Thus if the 50,000 molecular weight proteins induced by PB and 3-MC are associated with cytochrome P450, these proteins should also appear in SDS-gels run of these partially purified cytochrome fractions. Figure 22 compares the 1% SDS-polyacrylamide gel electrophoresis patterns of the proteins present in each partially purified cytochrome fraction with those present in the microsomes from which they were prepared. The major proteins in the cytochrome P450 preparations migrate in the 50,000 molecular weight region of the gels and, again, to best visualize components in this region of the gel, these samples were electrophoresed longer, allowing all components with molecular weights lower than 40,000 to migrate off the end of the gel (Figure 23). Using this procedure, four major proteins can be resolved among the three types of P450. Again this can be seen most clearly by electro- phoresing cytochrome fractions purified from both the liver microsomes of 3-MC- and PB-pretreated rats on a single gel (Figure 24). From Figure 23, protein 3 (53,000 daltons) which was induced in rat liver microsomes by pretreatment of rats with 3-MC is still present in the partially purified cytochrome fraction prepared from these microsomes. And, those proteins which appeared to be induced in the microsomes after pretreatment of rats with P8 also co—purify with the cytochrome P450 preparation from these microsomes. But even though it appeared from these studies that PB and 3-MC did induce different major microsomal proteins of molecular Figure 22. 131 1% SDS—POLYACRYLAMIDE GEL ELECTROPHORESIS PROTEIN PROFILES OF RAT LIVER MICROSOMES FROM CONTROL AND PB- OR 3-MC- PRETREATED RATS AND THE CYTOCHROME P450 FRACTIONS PURIFIED FROM THE MICROSOMES Cytochrome P450 fractions were partially purified from rat liver microsomes as described in the Materials and Methods. The microsomal samples had been washed with 1.15% KCl containing 10 mM EDTA, pH 7.5. The cytochrome P450 levels in each sample are indicated in the upper right hand corner of the scan. Between 35—40 ug of total microsomal protein and 20-25 ug of protein from partially purified cytochrome P450 fractions were applied to a gel. The gels were stained with Coomassie blue and scanned at 550 nm. 132 20" 10"“. 8 P450 28anle 0. l.2nmo|e/mg o In L00 CONTROL 8 | o IQ 05» 8 °—° I o' (n t *s s’ I I I I If. I I I I I I I I I [On l.7nmol e An 9 3" I 4.0nmole/mg g M [00 I 6 ' II I sue ». I osI w W 0.5“ K I I 4 I I I I I I I I I I I I I I I I 2.0" 4 II I I I I5 I I .5 I 3.4nmoIe/mg ‘ . 5.8nmoIM'nq I | ‘ IoI II [OI n I I I 0.50 I I K I 4% 4I I I I I I I ‘ ' ‘ I I I I * ‘7 2 4 6 8 Figure 22 6 moimow panacea cu Figure 23. 133 1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROFILES OF THE PROTEIN CONSTITUENTS HAVING MOLECULAR WEIGHTS LARGER THAN 40,000 WHICH ARE PRESENT IN THE RAT LIVER MICROSOMES FROM CONTROL AND PB- OR 3-MC—PRETREATED RATS AND IN THE CYTO— CHROME P450 FRACTIONS PURIFIED FROM THE MICROSOMES Samples of rat liver microsomes (washed with 1.15% KCl containing 10 mM EDTA, pH 7.5) and partially purified cytochrome P450 fractions were electrophoresed for approximately S-l/2 hours such that all protein consti- tuents having molecular weights lower than 40,000 migrated off the gels. Between 50-60 ug of total microsomal protein and 30-40 ug of protein from the cytochrome fractions were applied to a gel. The cytochrome P450 level in the various samples is indicated in the upper left hand corner of the gel scan. Gels were stained with Coomassie blue and scanned at 550 nm. 134 '5" TOTAL If» P450 l.2nmole/mg 2 8nmole/hng 4 ‘0‘ CONTROL I 8 . O O O 8 C5 I 2 I 8 6 O o °° I o’ 0.50 O, . IV 5., O 2 W V/\ I5‘ 2 “ 6 E 5 L0 3 4.0nmole/mg L0 I.7nmole/Ing ‘4 LL] [01> ‘Ufl‘ IO‘I U 2 3MC Z P < H 6 E V I o 050 I I 0.5.» I 8 I ewo> och .Ha N one: moesao> :ofiuomnm may .meozpoz one mamwuoumz on» cw wonfiuomov coon o>mn mouseooonm Heucoefiuomxo och omzuHmzmn omv HHq mmb 20mm 20H90U awhmP < m0 rmm""°99qv asuonoaa OIAo-Hdovw o—o—e .m/sqomu 9a awoaHDOIAD p... Irv/septa: 03% awoawoouo‘ I 0 9 0 ID .9. N _ _. O '2 ‘8 FRACTION NUMBER I f I T Q' '0 N - I'll/5w NIBLOHd Figure 30 174 dalton component originally present in the cytochrome P450-enriched fraction used for proteolysis (Figure 31). Because this protein was prepared by proteolysis, it may have been partially degraded and could not be directly correlated with this protein component of the cyto- chrome P450 preparation, however. Electrophoresis of the cytochrome P420 fractions from the Sephadex G-100 column on 0.1% SDS-gels for benzidine-staining indicated that the major component of these fractions was a hemoprotein and appeared to migrate in nearly the same position on these gels as hemoprotein 3, the major hemoprotein found in PB-microsomes (Figure 32). Therefore it seemed, on the basis of such analyses, that a method had been found which could be used to isolate hemoprotein 3 from PB-microsomes in a form suitable for antibody production. Furthermore, because this hemoprotein gave a cytochrome P420 spectrum when assayed by reduced, CO-difference spectroscopy, it appeared to be an altered form of cytochrome P450. One incongruency in the isolation procedure, however, resulted from trying to correlate the purity of the hemoprotein with cytochrome P420 specific activity data. Thus while it appeared, by SDS-gel electrophoresis, that a pure hemoprotein had been isolated, the maximum specific activities of cytochrome P420 attained by this procedure ranged from S to 9 nmole/mg protein (20 nmole/mg would be expected for a pure protein). Several explanations can be offered for this, however. One is that during proteolysis the protein is altered in such a fashion that the extinction coefficients normally used for calculating cytochrome P420 concentrations are no longer valid. A second is that heme dissociated from the cytochrome P450 hemoprotein during the isolation and this, in fact, is probably very true since Figure 31. 175 1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS PROTEIN PROFILES OF THE CYTOCHROME P450-ENRICHED FRACTION BEFORE TRYPSIN TREATMENT AND THE CYTOCHROME P420 PREPARATION RESULTING FROM TRYPSIN DIGESTION AND SEPHADEX G-lOO COLUMN CHROMATOGRAPHY The upper scan is of the cytochrome P450-enriched preparation used for proteolysis while the lower scan is of the cytochrome P420 preparation after Sephadex G-100 column chromatography. The samples were prepared in the presence of 40 mM dithiothreitol. After electrophoresis the gels were stained with Coomassie blue and scanned at 550 nm. 24 ug of prbtein was applied to the upper gel while 15 ug was applied to the lower. ABSORBANCE 55O nm 0.5 . Figure 31 176 0 50,000 45000 I I I 4SOOO‘ I ‘ 2 4 6 8 MIGRATION DISTANCE (CM) Figure 32. 177 0.1% SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS HEMOPROTEIN PROFILES OF THE CYTOCHROME P450-ENRICHED FRACTION BEFORE TRYPSIN TREATMENT AND THE CYTOCHROME P420 PREPARATION RESULTING FROM TRYPSIN DIGESTION AND SEPHADEX G-lOO COLUMN CHROMATOGRAPHY The gel on the left is of the cytochrome P450-enriched fraction before proteolysis while the gel on the right is of the cytochrome P420 preparation obtained by column chromatography. The hemoprotein profiles were visualized by staining the gels with benzidine and H202 as described in the Materials and Methods. 40 ug of each sample was applied to the gels. Figure 32 179 heme is known to dissociate from cytochrome P420 very easily (162,163). Finally, a third explanation is suggested by the observation that the hemoprotein is eluted from the Sephadex G-100 column in the form of a high molecular weight aggregate. Thus it is possible that this aggre- gate contains, in addition to hemoprotein 3, small hydrophobic peptides which undoubtedly result when membrane proteins are proteolytically degraded. Such peptides could be of such small molecular weights that they could not be observed on SDS-gels. To further explore this third possibility, the hemoprotein 3 preparation was solubilized in SDS and chromatographed on Sephadex G-200 also in the presence of this deter- gent. The results of such an experiment are shown in Figure 33. It can be seen that two peaks of material which absorb at 280 nm eluted from the column during this procedure. When the first peak was analzyed by 1% SDS-gel electrophoresis, it contained the 45,000 dalton protein but when the second peak of material was similarly analyzed, no proteins could be detected on the gel. Because the results of this experiment confirmed the possibility that the cytochrome P420 prepa- ration might also contain small peptides, in addition to hemOprotein 3, which are not detectable by SDS-gel electrophoresis, initial attempts at preparing antibody to this hemoprotein employed protein which had been further purified by SDS-column chromatography on Sephadex G-200. In this form the protein was not a good antigen, however, and eventually antibody was prepared by injecting the entire hemoprotein 3 aggregate into rabbits. 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