Table Of ContentCYTOCHROMES P-450 AND h5
Structure, Function, and Interaction
ADV ANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
Nathan Back State University of New York at Buffalo
N. R. Di Luzio Tulane University School of Medicine
Bernard Halpern College de France and Institute of Immuno·Biology
Ephraim Katchalski The Weizmann Institute of Science
David Kritchevsky Wistar Institute
Abel Lajtha New York State Research Institute for Neurochemistry and Drug Addiction
Rodolfo Paoletti University of Milan
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Volume 54
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Edited by Laurence W. Hedlund, John M. Franz, and
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CONCANAVALIN A
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Volume 56
BIOCHEMICAL PHARMACOLOGY OF ETHANOL
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Volume 58
CYTOCHROMES P·450 and b5 : Structure, Function, and Interaction
Edited by David Y. Cooper, Otto Rosenthal, Robert Snyder,
and Charlotte Witmer • 1975
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ALCOHOL INTOXICATION AND WITHDRAWAL: Experimental Studies II
Edited by Milton M. Gross • 1975
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Edited by Cesare Sirtori, Giorgio Ricci, and Sergio Gorini • 1975
CYTOCHROMES P-450 AND hs
Structure, Function, and Interaction
Edited by
David Y. Cooper and Otto Rosenthal
Harrison Department of Surgical Research
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania
and
Robert Snyder and Charlotte Witmer
Department of Pharmacology
Thomas Jefferson University
Philadelphia, Pennsylvania
PLENUMPRESS • NEWYORKANDLONDON
Library of Congress Cataloging in Publication Data
Philadelphia Conference on Heme Protein P450, 2d, 1974.
Cytochromes P·450 and b5.
(Advances in experimental medicine and biology; v.58)
Includes bibliographical references and index.
1. Cytochrome P-450-Congresses. 2. Cytochrome b5-Congresses. I. Cooper,
David Y. II. Title. III. Series. [DNLM: 1. Cytochrome P-450-Congresses. 2.
Cytochromes-Congresses. WI AD559 v. 58 1974 / QU135 P544 1974]
QP671.C83P48 1974 574.8'74 75-15625
ISBN-13: 978-1-4615-9028-6 e-ISBN-13: 978-1-4615-9026-2
DOl: 10.1007/978-1-4615-9026-2
Proceedings of the Second Philadelphia Conference on Heme Protein P-450,
held at the Thomas Jefferson University, Philadelphia, Pennsylvania,
April 5-6, 1974
© 1975 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1975
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N.Y. 10011
United Kingdom edition published by Plenum Press, London
A Division of Plenum Publishing Company, Ltd.
Davis House (4th floor), 8 Scrubs Lane, Harlesden, London, NWIO 6SE, England
All rights reserved
No part of this hook may be reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
Preface
P-450 has in common with our nation that it can call Phila-
delphia its hometown. Yet there are differences, too. The
U.S.A. was born and named in this city. P-450 was first recog-
nized here -- an odd CO-combining pigment without family or func-
tion. Japanese workers identified it as an unusual member of the
cytochrome family. Finally, in Philadelphia, its biological func-
tion was established and its growth to a global power initiated.
Since discovery of its function as terminal oxidase of the
21-steroid hydroxylase system of adrenocortical microsomes, P-450
has proved to play the same role in a wide variety of other mixed
function oxidase systems involved in biosynthesis and catabolism
of specific cell or body components as well as in the metabolism
of foreign substances entering organisms. P-450-like oxygenating
enzymes appear to be fundamental cellular constituents in most
forms of aerobic organisms. Activation of molecular oxygen and
incorporation of one of its atoms into organic compounds as cata-
lyzed by P-450 enzymes are reactions of vital importance not only
for biosynthesis and degradation of steroid hormones necessary for
sustaining life, but also for metabolic activation or inactivation
of foreign agents such as drugs, food preservatives and additives,
insecticides and carcinogens. Moreover, P-450 linked enzyme sys-
tems can either be induced or suppressed by these agents with
significant biological consequences.
Both the wide variety of reactions catalyzed by P-450 and
the versatility of these enzyme systems have attracted increasing
numbers of investigators to this area. Environmentalists, ento-
mologists, nutritional biochemists have joined endocrinologists,
biochemists, biophysicists, biochemical pharmacologists and toxi-
cologists who originally explored this field. The number of in-
vestigators has proliferated so rapidly that the earlier students
of P-450 are little acquainted with the new entries in the field.
v
vi PREFACE
This is the second time we have taken advantage of the geo-
graphical location of Philadelphia to get together the people
interested in P-4S0. Both meetings have been successful, not
only scientifically, but socially, also. Therefore, if there
are any profits from this current publication, we plan to set up
a fund to form a society to perpetuate these Philadelphia Con-
ferences, so that the people working with P-4S0 can meet, exchange
ideas, stimulate new research and enjoy themselves. Such con-
ferences could be held in Philadelphia -- or anywhere else in
the world.
The organizers of this symposium wish to express their
thanks to the following: Dean Robert C. Baldridge and the
College of Graduate Studies for both financial support and use of
the facilities of Thomas Jefferson University; Mr. G. Frederick
Roll whose kind donation, along with that of the Smith Kline and
French Laboratory, helped defray expenses. We also wish to
specially acknowledge the invaluable contribution of Dr. Heinz
Schleyer as Consulting Editor and the editorial work of Mrs.
Beatrice G. Novack, without which publication of this volume
would not have been possible.
David Y. Cooper
Otto Rosenthal
Robert Snyder
Charlotte Witmer
Contents
Official Greetings. xiii
Robert C. Baldridge
Session I - Chairman, John Schenkman
Part 1
Partial Purification and Separation of Multiple Forms
of Cytochrome P-450 and Cytochrome P-448 from
Rat Liver Microsomes. 1
Levin, W., Lu, A. Y. H., Ryan, D., West, S.
Kuntzman, R. and Conney, A. H.
Biochemical Characterization of Highly Purified Cytochrome
P-450 and Other Components of the Mixed Function
Oxidase System of Liver Microsomal Membranes. . 25
Coon, M. J., van der Hoeven, T. A., Haugen, D. A.,
Guengerich, F. P., Vermilion, J. L. and Ballou, D. P.
Immunochemical and Compositional Comparison of Cytochromes
P-4S0 am of Pseudomonas put ida and P-4S0LM of
Pheno£arbital-Induced Rabbit Liver Microsomes.. 47
Dus, K., Litchfield, W. J., Miguel, A. G., van der
Hoeven, T. A., Haugen, D. A., Dean, W. L. and Coon,
W. L.
Immunochemical and Functional Similarities and Differences
among Iron-Sulfur Proteins Involved in Mammalian
Steroidogenesis. . . . . . . . . • . . . . . • .. 55
Baron, J.
General Discussion. 73
Peisach, Coon, Jerina, Schenkman, Levin, Peterson,
Ballou, Kamin, Baron, Masters.
vii
viii CONTENTS
Part 2
Comparison of the Induction Course, Biophysical Chemical
Interactions and Photochemical Action Spectra of
Phenobarbi tal·· and 3-Methylchol anthrene- Induced
Hepatic Microsomal P-450. . . . . . . . . . . . 81
Cooper, D. Y., Schleyer, H., Thomas, J. H., Vars,
H. M. and Rosenthal, O.
Cytochrome P-450 in the Activation and Inactivation of
Carcinogens. . . . . . . . . . . . . . . . . . 103
Greim, H., Czygan, P., Garro, A. J., Hutterer, F.,
Schaffner, F. and Popper, H.
Effect of Cyclic AMP on the Phenobarbital Induced Increase
in Cytochrome P-450 and Hypertrophy of the Endo-
plasmic Reticulum of the Rat Liver. . . . . 117
Hutterer, F., Dressler, K., Greim, H., Czygan, P.,
Schaffner, F. and Popper, H.
Evidence for the Activation of 3-Methylcholanthrene as a
Carcinogen In Vivo and as a Mutagen In Vitro
by P -450 from Inbred Strains of Mice. . . . . 127
l
Nebert, D. W. and Felton, J. S.
Increased Translation as a Result of Elevated Initiation
Factor Activity after Administration of 3-
Methylcholanthrene. . . . . . . . . . . . 151
Bresnick, E., Hopkinson, J. and Prichard, P. M.
General Discussion. . . . . . . . . . . . . . . . 165
Holtzman, Hutterer, Coon, Cooper, Schenkman, Jerina,
Peisach, Remmer, Schleyer, Cinti, Nebert, Bresnick,
Narasimhulu, Orrenius.
Session II: Chairman, Daniel Nebert
Part 1
Optical and EPR Studies of Partially Purified Rabbit
Liver Cytochrome P-450. .. .... 175
Witmer, C., Nehls, P., Krauss, P., Remmer, H. and
Snyder, R.
Studies on the Spin State of 3-Methylcholanthrene-Induced
Cytochrome P-450 from Rat Liver. . . . . . . . .. 189
Stern, J. 0., Peisach, E., Peisach, J., Blumberg,
W. E., Lu, A. Y. H., West, S., Ryan, D. and Levin, W.
CONTENTS ix
An Analysis of the Optical Titrations of the 430 and
455 nm Chromophores of Ethyl Isocyanide
Complexes of Mammalian Hepatic Cytochrome P-450 203
Peisach, J.
Implications of Ligand Modified Spectra of Cytochrome
P-450 Associated with Pregnenolone Synthesis
in Mitochondria from Corpus Luteum ..... . 213
Uzgiris, V. I., McIntosh, E. N., Graves, T. and
Salhanick, H. A.
General Discussion. . 229
Mannering, Witmer, Lu, Snyder, Levin, Nebert,
Holtzman, Remmer, Vore, Narasimhulu, Stern,
Kamin, Peisach, Schenkman, Hildebrandt,Coon
Part 2
Studies on the Interaction of Water with Microsomal
Cytochrome P-450. . . . . . . . . . . . . . 239
Holtzman, J. L.
Drug Metabolism in Isolated Rat Liver Cells . . . . . . 251
Grundin, R., Moldeus, P., Vadi, H., Orrenius, S.,
von Bahr, C., B~ckstrom, D. and Ehrenberg, A.
Role of Phospholipids in Adrenocortical Microsomal
Hydroxylation Reactions: Activation of Lipid-
Depleted Microsomal Preparations by Non-Ionic
Detergents. . . . . . . . . . . . . . . . . . . 271
Narasimhulu, S.
On the Structure of Putidaredoxin and Cytochrome P-450 ,
cam
and Their Mode of Interaction 287
Dus, K.
Metabolic Control of Cytochrome P-450 . 311
cam
Peterson, J. A. and Mock, D. M.
General Discussion. . 325
Stern, Peterson, Dus, Peisach, Bresnick, Orrenius,
Holtzman, Remmer, Cinti, Thurman, Nebert,
Narasimhulu, Coon, Lu, Jerina
x CONTENTS
Session III - Chairman, Charlotte Witmer
Relationship between Microsomal Hydroxylase and Glucuronyl-
transferase. . . . . . . . . . . . . . . . 335
Remmer, H., Bock, K. W. and Rexer, B.
A Possible Role of Copper in the Regulation of Heme
Biosynthesis through Ferrochelatase. . . . . 343
Wagner, G. S. and Tephly, T. R.
Mixed Function Oxidation and Intermediary Metabolism:
Metabolic Interdependence in the Liver. 355
Thurman, R. G., Marazzo, D. P. and Scholz, R.
Dissociation of Microsomal Ethanol Oxidation from
Cytochrome P-450 Catalyzed Drug Metabolism. 369
Vatsis, K. P. and Schulman, M. P.
General Discussion. . . . . . . .. .......... 383
van der Hoeven, Remmer, Mannering, Thurman, Coon,
Vatsis, Orrenius, Schenkman, Kamin, Bresnick, Wagner,
Hildebrandt, Thurman.
Session IV Chairman, Otto Rosenthal
Interaction between Microsomal Electron Transfer
Pathways. .. .......... . 387
Schenkman, J. B. and Jansson, I.
Role of Cytochrome b in the NADH Synergism of NADPH-
5
Dependent Reactions of the Cytochrome P-450 ..... 405
Mannering, G. J.
The Role of Cytochrome b in Cytochrome P-450 Enzymes .... 435
5
Sasame, H. A., Thorgeirsson, S. S., Mitchell, J. R.
and Gillette, J. R.
Role of Cytochrome b in NADPH- and NADH-Dependent
5
Hydroxylation by the Reconstituted Cytochrome
P-450- or P-448-Containing System. . . . 447
Lu, A. Y. H., Levin, W., West, S. B., Vore, M.,
Ryan, D., Kuntzman, R. and Conney, A. H.
The Role of Cytochrome b in Mixed Function Oxidations:
5
Effect on Microsomal Binding of the Hemoprotein
on Hepatic N-Demethylations ...... ' ....... 467
Cinti, D. L. and Ozols, J.
