differentiation of embryonic stem cells

Preface
Since their isolation over twenty years ago, embryonic stem (ES) cells
have come to play a prominent role in many different fields in biomedical
research. While used extensively for gene targeting studies for the generation
of ‘‘knock-out mice,’’ it is their capacity to differentiate into a wide array of
lineages in culture that has most recently captured the attention of basic
scientists, clinical researchers, and the lay public. The capacity of an ES cell
to differentiate into almost any cell type in a culture dish offers
unprecedented opportunities for studies in lineage commitment and
development, gene function, and cancer. In addition, the development of
human ES cells in 1998 has expanded the potential uses of the in vitro
differentiation approach to include the generat ion of specific cell types for
cell replacement therapy and regenerative medicine.
It has been known for some time that mouse ES cells can differentiate in
culture and generate derivatives of the three primary germ cell layers:
ectoderm, endoderm, and mesoderm. In many of these early studies,
however, differentiation was not well controlled and the cultures often
consisted of mixtures of tissues. Significant advances in the methodologies
of ES cell differentiation have been made in recent years, and it is now
possible to generate relatively pure populations of cells from a number of
different lineages in a reproducible fashion. These new approaches have
enabled investigators to begin to use the system to probe the molecular
events regulating early lineage commitment, as well as to generate
populations appro priate for cell replacement therapy in preclinical models.
With the establishment of human ES cells, the challenge for those in the field
of stem cell and developmental biology is to now translate the information
from the mouse to the human system.
In this volume, we have brought together a comprehensive collection of
the most up-to-date methods for the differentiation of both mouse and
human ES cells into a broad spectrum of lineages. Together with the
differentiation protocols, we have solicited a select set of protocols that

highlight approaches for gene discovery and lineage selection using ES cells.
The chapters are written by leaders in the field from the international
community and provide technical details that will enable the reader to
establish and maintain ES cell cultures and induce their differentiation into a
large number of different lineages.
We hope that this volume on Differentiation of Embryo nic Stem Cells
will find its way into many laboratories and proves to be useful at the bench
xv
for investigators world-wide. We are extremely grateful to the many authors
for their excellent contributions to this volume and their patience in dealing
with publication schedules. Finally, we extend our appreciation to Shirley
Light at Academic Press who organized the assembly of this and hundr eds
of other volumes of Methods in Enzymology over several decades.
PAUL M. WASSARMAN
GORDON M. KELLER
xvi PREFACE
Table of Contents
CONTRIBUTORS TO VOLUME 365 ix
P
REFACE xv
V
OLUMES IN SERIES xvii
Section I. Differentiation of Mouse Embryonic Stem Cells
Early Commitment Steps and Generation of Chimeric Mice
1. Lineage Specific Differentiation of Mouse ES Cells:
Formation and Differentiation of Early Primitive
Ectoderm-like (EPL) Cells
J
OY RATHJEN AND
PETER D. RATHJEN 3

2. Differentiation of F1 Embryonic Stem Cells into
Viable Male and Female Mice by Tetraploid
Embryo Complementation
K
EVIN EGGAN AND
RUDOLF JAENISCH 25
Differentiation to Mesoderm Derivatives: Hematopoietic and Vascular
3. Hematopoietic Commitment of ES Cells in
Culture
M
ARION KENNEDY AND
GORDON M. KELLER 39
4. In Vitro Differentiation of Mouse Embryonic Stem
Cells: Hematopoietic and Vascular Cell Types
S
TUART T. FRASER,
JUN YAMASHITA,
L. M
ARTIN JAKT,
MITSUHIRO OKADA,
MINETARO OGAWA,
SATOMI NISHIKAWA, AND
SHIN-ICHI NISHIKAWA 59
5. In Vitro Differentiation of Mouse Embryonic Stem
Cells to Hematopoietic Cells on an OP9 Stromal
Cell Monolayer
K
ENJI KITAJIMA,
M
AKOTO TANAKA,

JIE ZHENG,
E
IKO SAKAI-OGAWA, AND
TORU NAKANO 72
6. In Vitro Differentiation of Mouse ES Cells:
Hematopoietic and Vascular Development
J
OSEPH B. KEARNEY AND
VICTORIA L. BAUTCH 83
7. In Vitro Differentiation of Mouse ES Cells into
Hematopoietic, Endothelial, and Osteoblastic
Cell Lineages: The Possibility of In Vitro
Organogenesis
M
OTOKAZU TSUNETO,
TOSHIYUKI YAMANE,
HIROMI OKUYAMA,
H
IDETOSHI YAMAZAKI, AND
SHIN-ICHI HAYASHI 98
v
8. Development of Hematopoietic Repopulating
Cells from Embryonic Stem Cells
MICHAEL KYBA,
R
ITA C. R. PERLINGEIRO,
AND GEORGE Q. DALEY 114
9. The In Vitro Differentiation of Mouse Embryonic
Stem Cells into Neutrophils
J

ONATHAN G. LIEBER,
GORDON M. KELLER, AND
G. SCOTT WORTHEN 129
10. Development and Analysis of Megakaryocytes
from Murine Embryonic Stem Cells
K
OJI ETO,
ANDREW L. LEAVITT,
TORU NAKANO, AND
SANFORD J. SHATTIL 142
11. Development of Lymphoid Lineages from
Embryonic Stem Cells In Vitro
S
ARAH K. CHO AND
JUAN CARLOS
ZU
´
N
˜
IGA-PFLU
¨
CKER 158
12. Probing Dendritic Cell Function by Guiding the
Differentiation of Embryonic Stem Cells
P
AUL J. FAIRCHILD,
KATHLEEN F. NOLAN, AND
HERMAN WALDMANN 169
13. Gene Targeting Strategies for the Isolation of
Hematopoietic and Endothelial Precursors from

Differentiated ES Cells
W
EN JIE ZHANG,
YUN SHIN CHUNG,
BILL EADES, AND
KYUNGHEE CHOI 186
14. Establishment of Multipotent Hematopoietic
Progenitor Cell Lines from ES Cells Differentiated
In Vitro
L
EIF CARLSSON,
EWA WANDZIOCH,
P
ERPE
´
TUA PINTO DO O
´
,
AND A
˚
SA KOLTERUD 202
15. Vasculogenesis and Angiogenesis from In Vitro
Differentiation of Mouse Embryonic Stem Cells
O
LIVIER FERAUD,
M
ARIE-HE
´
LE
`

NE PRANDINI,
AND DANIEL VITTET 214
Other Derivatives of Mesoderm
16. ES Cell Differentiation to the Cardiac Lineage K
ENNETH R. BOHELER 228
17. Bone Nodule Formation via In Vitro
Differentiation of Murine Embryonic Stem Cells
S
ARAH K. BRONSON 241
18. In Vitro Differentiation of Mouse ES Cells: Bone
and Cartilage
J
AN KRAMER,
C
LAUDIA HEGERT, AND
JU
¨
RGEN ROHWEDEL 251
19. Development of Adipocytes from Differentiated
ES Cells
B
RIGITTE WDZIEKONSKI,
P
HI VILLAGEOIS, AND
CHRISTIAN DANI 268
Differentiation to Endoderm Derivatives
20. In Vitro Differentiation of Embryonic Stem Cells
into Hepatocytes
T
AKASHI HAMAZAKI AND

NAOHIRO TERADA 277
vi TABLE OF CONTENTS
21. Differentiation of Mouse Embryonic Stem Cells
into Pancreatic and Hepatic Cells
GABRIELA KANIA,
P
RZEMYSLAW BLYSZCZUK,
J
AROSLAW CZYZ,
ANNE NAVARRETE-SANTOS,
AND ANNA M. WOBUS 287
Differentiation to Ectoderm Derivatives
22. Generating CNS Neurons from Embryonic, Fetal,
and Adult Stem Cells
J
ONG-HOON KIM,
DAVID PANCHISION,
RAJA KITTAPPA, AND
RON MCKAY 303
23. Defined Conditions for Neural Commitment and
Differentiation
Q
I-LONG YING AND
AUSTIN G. SMITH 327
24. Development of Melanocytes from ES Cells T
AKAHIRO KUNISADA,
TOSHIYUKI YAMANE,
H
ITOMI AOKI,
N

AOKO YOSHIMURA,
K
ATSUHIKO ISHIZAKI, AND
TSUTOMU MOTOHASHI 341
Section II. Differentiation of Mouse Embryonic Germ Cells
25. Isolation and Culture of Embryonic Germ Cells MARIA P. DE MIGUEL AND
PETER J. DONOVAN 353
Section III. Gene Discovery by Manipulation of
Mouse Embryonic Stem Cells
26. Gene Trap Mutagenesis in Embryonic Stem Cells WEISHENG V. CHEN AND
PHILIPPE SORIANO 367
27. Gene Trap Vector Screen for Developmental
Genes in Differentiating ES Cells
H
EIDI STUHLMANN 386
28. Gene-Based Chemical Mutagenesis in Mouse
Embryonic Stem Cells
Y
IJING CHEN,
JAY L. VIVIAN, AND
TERRY MAGNUSON 406
Section IV. Differentiation of Monkey and Human
Embryonic Stem Cells
29. Growth and Differentiation of Cynomolgus
Monkey ES Cells
HIROFUMI SUEMORI AND
NORIO NAKATSUJI 419
30. Isolation, Characterization, and Differentiation of
Human Embryonic Stem Cells
M

ARTIN F. PERA,
A
DAM A. FILIPCZYK,
SUSAN M. HAWES, AND
ANDREW L. LASLETT 429
TABLE OF CONTENTS vii
31. Factors Controlling Human Embryonic Stem Cell
Differentiation
MAYA SCHULDINER AND
NISSIM BENVENISTY 446
32. Development of Cardiomyocytes from Human ES
Cells
I
ZHAK KEHAT,
MICHAL AMIT,
AMIRA GEPSTEIN,
IRIT HUBER,
J
OSEPH ITSKOVITZ-ELDOR,
AND LIOR GEPSTEIN 461
A
UTHOR INDEX 475
S
UBJECT INDEX 501
viii TABLE OF CONTENTS
Contributors to Volume 365
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
MICHAL AMIT (32), Department of
Physiology and Biophysics, Bruce Rappa-

port Faculty of Medicine, Technion-Israel
Institute of Technology, 2 Efron Street,
POB 9649, Haifa 31096, Israel
H
ITOMI AOKI (24), Department of Tissue and
Organ Development, Regeneration and
Advanced Medical Science, Gifu University
Graduate School of Medicine, Gifu, 500-
8705, Japan
V
ICTORIA L. BAUTCH (6), Department of
Biology and Program in Genetics, Univer-
sity of North Carolina at Chapel Hill,
CB#3280, Chapel Hill, North Carolina
27599
N
ISSIM BENVENISTY (31), Department of
Genetics, The Silberman Institute of Life
Sciences, The Hebrew University, Jerusa-
lem 91904, Israel
P
RZEMYSLAW BLYSZCZUK (21), In Vitro
Differentiation Group, Institute of Plant
Genetics and Crop Plant Research, Cor-
rensstr. 3, Gatersleben, D-06466, Germany
KENNETH R. BOHELER (16), Molecular Car-
diology Unit, Laboratory of Cardiovascular
Science, National Institute on Aging, NIH
5600 Nathan Shock Drive, Baltimore,
Maryland 21224

S
ARAH K. BRONSON (17), Department of
Cellular & Molecular Physiology, Penn
State College of Medicine, The Milton
S. Hershey Medical Center, H166, 500,
University Drive, Hershey, Pennsylvania
17033-0850
LEIF CARLSSON (14), Umea
˚
Centre for Mole-
cular Medicine, Umea
˚
University, 901 87
Umea
˚
, Sweden
W
EISHENG V. CHEN (26), Program in
Developmental Biology, Division of Basic
Sciences, Fred Hutchinson Cancer Research
Center, 1100 Fairview Avenue N, A2-025,
P.O. Box 19024, Seattle, Washington
98109-1024
Y
IJING CHEN (28), Department of Genetics,
University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599
S
ARAH K. CHO (11), Division of Biological
Sciences, University of California at San

Diego, 9500 Gilman Drive, La Jolla,
California 92093
K
YUNGHEE CHOI (13), Department of Pathol-
ogy & Immunology, Washington University
School of Medicine, 660 S. Euclid Avenue,
Box 8118, St. Louis, Missouri 63110
Y
UN SHIN CHUNG (13), Department of
Pathology & Immunology, Washington
University School of Medicine, 660 S.
Euclid Avenue, Box 8118, St. Louis,
Missouri 63110
J
AROSLAW CZYZ (21), In Vitro Differentiation
Group, Institute of Plant Genetics and Crop
Plant Research Corrensstr. 3, Gatersleben,
D-06466, Germany
G
EORGE Q. DALEY (8), Whitehead Institute
for Biomedical Research, Harvard Medical
School, Cambridge, Massachusetts 02142-
1479
C
HRISTIAN DANI (19), Centre de Biochimie,
Institut de Recherches Signalisation, Biolo-
gie du Developpement et Cancer, Universite
´
de Nice-Sophia Antipolis, UMR 6543
CNRS, Parc Valrose, Nice cedex 2, 06108

France
M
ARIA P. DE MIGUEL (25), Department of
Microbiology, Kimmel Cancer Center,
Thomas Jefferson University, Philadelphia,
Pennsylvania 19107
ix
PETER J. DONOVAN (25), Kimmel Cancer
Center, Thomas Jefferson University, Phil-
adelphia, Pennsylvania 19107
B
ILL EADES (13), Department of Pathology &
Immunology, Washington University School
of Medicine, 660 S. Euclid Avenue, Box
8118, St. Louis, Missouri 63110
K
EVIN EGGAN (2), Whitehead Institute for
Biomedical Research, 9 Cambridge Center,
Cambridge, Massachusetts 02142
K
OJI ETO (10), Division of Vascular Biology,
Department of Cell Biology, Scripps
Research Institute, 10550 North Torrey
Pines Road, VB-5, La Jolla, California
92037
P
AUL J. FAIRCHILD (12), Sir William Dunn
School of Pathology, University of Oxford,
South Parks Road, Oxford, OX1 3RE, UK
O

LIVIER FERAUD (15), Laboratoire Developpe-
ment et Vieillissement de L’endothelium,
EMI INSERM 0219, DRDC/DVE, CEA
Grenoble, 17 rue des martyrs, 38054
Grenoble cedex 09, France
A
DAM A. FILIPCZYK (30), Monash Institute of
Reproduction and Development, Monash
University, 246 Clayton Road, Clayton,
Victoria, 3168, Australia
S
TUART T. FRASER (4), Laboratory of
Molecular Mouse Genetics, Institute for
Toxicology, Johannes Gutenberg-Univer-
sity, Obere Zahlbacher Strasse 67, Mainz
55131, Germany
L
IOR GEPSTEIN (32), Cardiovascular Research
Laboratory, Bruce Rappaport Faculty of
Medicine, Technion-Israel Institute of Tech-
nology, 2 Efron Street, POB 9649, Haifa
31096, Israel
A
MIRA GEPSTEIN (32), Cardiovascular
Research Laboratory, Bruce Rappaport
Faculty of Medicine, Technion-Israel Insti-
tute of Technology, 2 Efron Street, POB
9649, Haifa 31096, Israel
T
AKASHI HAMAZAKI (20), Department of

Pathology University of Florida College of
Medicine, P.O. Box 100275, Gainesville,
Florida 32610 0275
S
USAN M. HAWES (30) Monash Institute of
Reproduction and Development, Monash
University, 246 Clayton Road, Clayton,
Victoria, 3168, Australia
S
HIN-ICHI HAYASHI (7), Division of Immunol-
ogy, Department of Molecular and Cellular
Biology, School of Life Science, Faculty of
Medicine, Tottori University, 86 Nishi-
Machi, Yonago, Tottori 683-8503, Japan
C
LAUDIA HEGERT (18), Department of Med-
ical Molecular Biology, Medical University
of Lu
¨
beck, Ratzeburger Allee 160, Lu
¨
beck,
D-23538, Germany
I
RIT HUBER (32), Cardiovascular Research
Laboratory, Department of Biophysics and
Physiology, The Bruce Rappaport Faculty
of Medicine, Technion-Israel Institute of
Technology, 2 Efron Street, POB 9649,
Haifa 31096, Israel

K
ATSUHIKO ISHIZAKI (24), Department of
Tissue and Organ Development, Regenera-
tion and Advanced Medical Science, Gifu
University Graduate School of Medicine,
Gifu 500-8705, Japan
J
OSEPH ITSKOVITZ-ELDOR (32), Department
of Physiology and Biophysics, Bruce
Rappaport Faculty of Medicine, Technion-
Israel Institute of Technology, 2 Efron
Street, POB 9649, Haifa 31096, Israel
R
UDOLF JAENISCH (2), Whitehead Institute for
Biomedical Research, 9 Cambridge Center,
Cambridge, Massachusetts 02142
L. M
ARTIN JAKT (4), RIKEN Centre for
Developmental Biology, 2-2-3 Minatojima-
minamimachi, Chuo-ku, Kobe, Japan
650-0047
G
ABRIELA KANIA (21), In Vitro Differentiation
Group, Institute of Plant Genetics and Crop
Plant Research, Corrensstr. 3, Gatersleben,
D-06466, Germany
J
OSEPH B. KEARNEY (6), Program in Genetics
and Molecular Biology, University of North
x CONTRIBUTORS

Carolina at Chapel Hill CB#3280, Chapel
Hill, North Carolina 27599
I
ZHAK KEHAT (32), Cardiovascular Research
Laboratory, Bruce Rappaport Faculty of
Medicine, Technion-Israel Institute of Tech-
nology, 2 Efron Street, POB 9649 Haifa
31096, Israel
G
ORDON M. KELLER (3,9), Carl C. Icahn
Center for Gene Therapy and Molecular
Medicine, Mount Sinai School of Medicine,
1425 Madison Avenue, Box 1496, New York,
New York 10029-6574
M
ARION KENNEDY (3), Carl C. Icahn Institute
for Gene Therapy and Molecular Medicine,
Mount Sinai School of Medicine, 1425
Madison Avenue, Box 1496, New York,
New York 10029-6574
J
ONG-HOON KIM (22), Laboratory of Mole-
cular Biology, NINDS, National Institutes
of Health, Bethesda, Maryland 20892-4092
K
ENJI KITAJIMA (5), Department of Molecular
Cell Biology, Research Institute for
Microbial Diseases, Osaka University, 3-1
Yamadaoka, Suita Osaka, Osaka 565-0871,
Japan

R
AJA KITTAPPA (22), Laboratory of Molecular
Biology, NINDS, National Institutes of
Health, Bethesda, Maryland 20892-4092
A
˚
SA KOLTERUD (14), Umea
˚
Centre for Mole-
cular Medicine, Umea
˚
University, 901 87
Umea
˚
, Sweden
J
AN KRAMER (18), Department of Internal
Medicine I, University of Lu
¨
beck, Lu
¨
beck,
D-23538, Germany
T
AKAHIRO KUNISADA (24), Department of
Tissue and Organ Development, Regenera-
tion and Advanced Medical Science, Gifu
University Graduate School of Medicine,
Gifu 500-8705, Japan
M

ICHAEL KYBA (8), Center for Developmental
Biology, UT Southwestern Medical Center,
Dallas, Texas 75390-9133
A
NDREW L. LASLETT (30), Monash Institute of
Reproduction and Development, Monash
University, 246 Clayton Road, Clayton,
Victoria, 3168, Australia
A
NDREW L. LEAVITT (10), Departments of
Laboratory Medicine and Internal Medi-
cine, University of California at San Fran-
cisco, San Francisco, California 94143
J
ONATHAN G. LIEBER (9), Department of
Medicine, National Jewish Medical and
Research Center, 1400 Jackson Street,
Denver, Colorado 80206
T
ERRY MAGNUSON (28), Department of
Genetics, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina
27599
R
ON MCKAY (22), Laboratory of Molecular
Biology, NINDS, National Institutes of
Health, Bethesda, Maryland 20892-4092
T
SUTOMU MOTOHASHI (24), Department of
Tissue and Organ Development, Regenera-

tion and Advanced Medical Science, Gifu
University Graduate School of Medicine,
Gifu 500-8705, Japan
T
ORU NAKANO (5,10), Department of Mole-
cular Cell Biology, Research Institute for
Microbial Diseases, Osaka University, 3-1
Yamadaoka, Suita Osaka, Osaka 565-0871,
Japan
N
ORIO NAKATSUJI (29), Department of Devel-
opment and Differentiation, Institute for
Frontier Medical Sciences, Kyoto Univer-
sity, Sakyo-ku, Kyoto, 606-8507, Japan
A
NNE NAVARRETE-SANTOS (21), Department
of Anatomy and Cell Biology, Martin
Luther University, Halle-Wittenberg, Halle
(Saale), D-06108, Germany
S
ATOMI NISHIKAWA (4), RIKEN Centre for
Developmental Biology, 2-2-3 Minatojima-
minamimachi, Chuo-ku, Kobe 650-0047,
Japan
S
HIN-ICHI NISHIKAWA (4), RIKEN Centre for
Developmental Biology, 2-2-3 Minatojima-
minamimachi, Chuo-ku, Kobe, Japan
650-0047
CONTRIBUTORS xi

KATHLEEN F. NOLAN (12), Sir William Dunn
School of Pathology, University of Oxford,
South Parks Road, Oxford, OX1 3RE, UK
M
INETARO OGAWA (4), Department of Cell
Differentiation, Institute of Molecular
Embryology and Genetics, Kumamoto Uni-
versity, Japan
M
ITSUHIRO OKADA (4), Department of Cell
Differentitation, Institute of Molecular
Embryology and Genetics, Kumamoto Uni-
versity, Kumamoto, Japan 650-0047
H
IROMI OKUYAMA (7), Biotechnology
Research Laboratories, Takara Bio Inc.,
Otsu, Siga 520-2193, Japan
D
AVID PANCHISION (22), Laboratory of Mole-
cular Biology, NINDS, National Institutes
of Health, Bethesda, Maryland 20892-4092
M
ARTIN F. PERA (30), Monash Institute of
Reproduction and Development, Monash
University, 246 Clayton Road, Clayton,
Victoria 3168, Australia
R
ITA C. R. PERLINGEIRO (8), ViaCell Inc, 26
Landsdowne St, Cambridge, MA 2139
P

ERPE
´
TUA PINTO DO O
´
(14), Umea
˚
Centre
for Molecular Medicine, Umea
˚
University,
901 87 Umea
˚
, Sweden
M
ARIE-HE
´
LE
`
NE PRANDINI (15), Laboratoire
Developpement et Vieillissement de L’en-
dothelium, EMI INSERM 0219, DRDC/
DVE, CEA Grenoble, 17 rue des martyrs,
38054 Grenoble cedex 09, France
J
OY RATHJEN (1), Department of Molecular
Biosciences and ARC Special Research
Centre for the Molecular Genetics of Deve-
lopment, Adelaide University, Molecular
Life Sciences/335, Adelaide, 5005, Australia
P

ETER D. RATHJEN (1), Department of Mole-
cular Biosciences and ARC Special
Research Centre for the Molecular Genetics
of Development, Adelaide University, Mole-
cular Life Sciences/335, Adelaide, 5005,
Australia
J
U
¨
RGEN ROHWEDEL (18). Department of
Medical Molecular Biology, Medical
University of Lu
¨
beck, Ratzeburger Allee
160, Lu
¨
beck, D-23538, Germany
E
IKO SAKAI-OGAWA (5), Department of Mole-
cular Cell Biology, Research Institute for
Microbial Diseases, Osaka University, 3-1
Yamadaoka, Suita Osaka, Osaka 565-0871,
Japan
M
AYA SCHULDINER (31), Department of
Genetics, The Silberman Institute of Life
Sciences, The Hebrew University, Jerusa-
lem 91904, Israel
S
ANFORD J. SHATTIL (10), Division of Vascular

Biology, Department of Cell Biology, The
Scripps Research Institute, 10550 North
Torrey Pines Road, VB-5, La Jolla,
California 92037
A
USTIN G. SMITH (23), Institute for Stem Cell
Research, University of Edinburgh, King’s
Buildings, West Mains Road, Edinburgh,
Scotland EH9 3JQ, UK
P
HILIPPE SORIANO (26), Program in Develop-
mental Biology, Division of Basic Sciences,
Fred Hutchinson Cancer Research Center,
1100 Fairview Avenue N, A2-025, P.O. Box
19024, Seattle, Washington 98109-1024
H
EIDI STUHLMANN (27), Department of Cell
Biology, Division of Vascular Biology,
Scripps Research Institute, Mail CVN-26,
10550 North Torrey Pines Road, La Jolla,
California 92037
H
IROFUMI SUEMORI (29), Stem Cell Research
Center, Institute for Frontier Medical
Sciences, Kyoto University, Sakyo-ku,
Kyoto, 606-8507, Japan
M
AKOTO TANAKA (5), Department of Mole-
cular Cell Biology, Research Institute for
Microbial Diseases, Osaka University, 3-1

Yamadaoka, Suita Osaka, Osaka 565-0871
Japan
N
AOHIRO TERADA (20), Department of
Pathology, University of Florida College
of Medicine, P.O. Box 100275, Gainesville,
Florida 32610-0275
M
OTOKAZU TSUNETO (7), Division of Immu-
nology, Department of Molecular and
xii CONTRIBUTORS
Cellular Biology, School of Life Science,
Faculty of Medicine, Tottori University, 86
Nishi-Machi, Yonago, Tottori 683-8503,
Japan
P
HI VILLAGEOIS (19), Centre de Biochimie,
Institut de Recherches Signalisation,
Biologie du Developpement et Cancer,
Universite
´
de Nice-Sophia Antipolis, UMR
6543 CNRS, Parc Valrose, Nice cedex 2,
06108 France
D
ANIEL VITTET (15), Laboratoire Developpe-
ment et Vieillissement de L’endothelium,
EMI INSERM 0219, DRDC/DVE, CEA
Grenoble, 17 rue des martyrs, 38054 Gre-
noble cedex 09, France

J
AY L. VIVIAN (28), Department of Genetics,
University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599
H
ERMAN WALDMANN (12), Sir William Dunn
School of Pathology, University of Oxford,
South Parks Road, Oxford, OX1 3RE, UK
E
WA WANDZIOCH (14), Umea
˚
Centre for
Molecular Medicine, Umea
˚
University, 901
87 Umea
˚
, Sweden
B
RIGITTE WDZIEKONSKI (19), Centre de Bio-
chimie, Institut de Recherches Signalisation,
Biologie du Developpement et Cancer,
Universite
´
de Nice-Sophia Antipolis, UMR
6543 CNRS, Parc Valrose, Nice cedex 2,
06108 France
A
NNA M. WOBUS (21), In Vitro Differentiation
Group, Institute of Plant Genetics and Crop

Plant Research, Corrensstr. 3, Gatersleben,
D-06466, Germany
G. S
COTT WORTHEN (9), Department of
Medicine, National Jewish Medical and
Research Center, 1400 Jackson Street,
Denver, Colorado 80206
T
OSHIYUKI YAMANE (7,24), Department of
Pathology, Stanford University School of
Medicine, B259 Beckman Center, Stanford,
California 94305
J
UN YAMASHITA (4), RIKEN Centre for
Developmental Biology, 2-2-3 Minatojima-
minamimachi, Chuo-ku, Kobe 650-0047,
Japan
H
IDETOSHI YAMAZAKI (7), Division of Regen-
erative Medicine and Therapeutics, Depart-
ment of Genetic Medicine and Regenerative
Therapeutics, Institute of Regenerative
Medicine and Biofunction, Tottori Univer-
sity Graduate School of Medical Science,
and Division of Immunology, Department of
Molecular and Cellular Biology, School of
Life Science, Faculty of Medicine, Tottori
University, 86 Nishi-Machi, Yonago, Tot-
tori 683-8503, Japan
Q

I-LONG YING (23), Institute for Stem Cell
Research, University of Edinburgh, King’s
Buildings, West Mains Road, Edinburgh,
Scotland, EH9 3JQ, UK
N
AOKO YOSHIMURA (24), Department of
Tissue and Organ Development, Regenera-
tion and Advanced Medical Science, Gifu
University Graduate School of Medicine,
Gifu 500-8705, Japan
W
EN JIE ZHANG (13), Department of Pathol-
ogy & Immunology, Washington University
School of Medicine, 660 S. Euclid Avenue,
Box 8118, St. Louis, Missouri 63110
J
IE ZHENG (5), Department of Molecular Cell
Biology, Research Institute for Microbial
Diseases, Osaka University, 3-1 Yama-
daoka, Suita Osaka, Osaka 565-0871,
Japan
J
UAN CARLOS ZU
´
N
˜
IGA-PFLU
¨
CKER (11),
Department of Immunology, University of

Toronto, Toronto, Ontario M5S 1A8,
Canada
CONTRIBUTORS xiii
METHODS IN ENZYMOLOGY
VOLUME I. Preparation and Assay of Enzymes
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME II. Preparation and Assay of Enzymes
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME III. Preparation and Assay of Substrates
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME IV. Special Techniques for the Enzymologist
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME V. Preparation and Assay of Enzymes
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VI. Preparation and Assay of Enzymes (Continued)
Preparation and Assay of Substrates
Special Techniques
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VII. Cumulative Subject Index
Edited by S
IDNEY P. COLOWICK AND NATHAN O. KAPLAN
VOLUME VIII. Complex Carbohydrates
Edited by E
LIZABETH F. NEUFELD AND VICTOR GINSBURG

VOLUME IX. Carbohydrate Metabolism
Edited by W
ILLIS A. W OOD
VOLUME X. Oxidation and Phosphorylation
Edited by R
ONALD W. ESTABROOK AND MAYNARD E. PULLMAN
VOLUME XI. Enzyme Structure
Edited by C. H. W. H
IRS
VOLUME XII. Nucleic Acids (Parts A and B)
Edited by L
AWRENCE GROSSMAN AND KIVIE MOLDAVE
xvii
VOLUME XIII. Citric Acid Cycle
Edited by J. M. LOWENSTEIN
VOLUME XIV. Lipids
Edited by J. M. LOWENSTEIN
VOLUME XV. Steroids and Terpenoids
Edited by R
AYMOND B. CLAYTON
VOLUME XVI. Fast Reactions
Edited by K
ENNETH KUSTIN
VOLUME XVII. Metabolism of Amino Acids and Amines
(Parts A and B)
Edited by H
ERBERT TABOR AND CELIA WHITE TABOR
VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)
Edited by D
ONALD B. MCCORMICK, AND LEMUEL D. WRIGHT

VOLUME XIX. Proteolytic Enzymes
Edited by G
ERTRUDE E. PERLMANN AND LASZLO LORAND
VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)
Edited by K
IVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME XXI. Nucleic Acids (Part D)
Edited by L
AWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME XXII. Enzyme Purification and Related Techniques
Edited by W
ILLIAM B. JAKOBY
VOLUME XXIII. Photosynthesis (Part A)
Edited by A
NTHONY SAN PIETRO
VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)
Edited by A
NTHONY SAN PIETRO
VOLUME XXV. Enzyme Structure (Part B)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
VOLUME XXVI. Enzyme Structure (Part C)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
xviii METHODS IN ENZYMOLOGY
VOLUME XXVII. Enzyme Structure (Part D)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME XXVIII. Complex Carbohydrates (Part B)
Edited by VICTOR GINSBURG
VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by L
AWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)
Edited by K
IVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME XXXI. Biomembranes (Part A)
Edited by S
IDNEY FLEISCHER AND LESTER PACKER
VOLUME XXXII. Biomembranes (Part B)
Edited by S
IDNEY FLEISCHER AND LESTER PACKER
VOLUME XXXIII. Cumulative Subject Index Volumes I–XXX
Edited by M
ARTHA G. DENNIS AND EDWARD A. DENNIS
VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)
Edited by W
ILLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XXXV. Lipids (Part B)
Edited by J
OHN M. LOWENSTEIN
VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)
Edited by B
ERT W. O’MALLEY AND JOEL G. HARDMAN
VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)
Edited by B
ERT W. O’MALLEY AND JOEL G. HARDMAN
VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)
Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY
VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and
Organ Systems)

Edited by J
OEL G. H ARDMAN AND BERT W. O’MALLEY
VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)
Edited by B
ERT W. O’MALLEY AND JOEL G. HARDMAN
METHODS IN ENZYMOLOGY xix
VOLUME XLI. Carbohydrate Metabolism (Part B)
Edited by W. A. WOOD
VOLUME XLII. Carbohydrate Metabolism (Part C)
Edited by W. A. WOOD
VOLUME XLIII. Antibiotics
Edited by J
OHN H. HASH
VOLUME XLIV. Immobilized Enzymes
Edited by K
LAUS MOSBACH
VOLUME XLV Proteolytic Enzymes (Part B)
Edited by L
ASZLO LORAND
VOLUME XLV I. Affinity Labeling
Edited by W
ILLIAM B. JAKOBY AND MEIR WILCHEK
VOLUME XLV II. Enzyme Structure (Part E)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
VOLUME XLV III. Enzyme Structure (Part F)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
VOLUME XLIX. Enzyme Structure (Part G)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF

VOLUME L. Complex Carbohydrates (Part C)
Edited by V
ICTOR GINSBURG
VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism
Edited by P
ATRICIA A. HOFFEE AND MARY ELLEN JONES
VOLUME LII. Biomembranes (Part C: Biological Oxidations)
Edited by S
IDNEY FLEISCHER AND LESTER PACKER
VOLUME LIII. Biomembranes (Part D: Biological Oxidations)
Edited by S
IDNEY FLEISCHER AND LESTER PACKER
VOLUME LIV. Biomembranes (Part E: Biological Oxidations)
Edited by S
IDNEY FLEISCHER AND LESTER PACKER
xx METHODS IN ENZYMOLOGY
VOLUME LV. Biomembranes (Part F: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LVI. Biomembranes (Part G: Bioenergetics)
Edited by SIDNEY FLEISCHER AND LESTER PACKER
VOLUME LVII. Bioluminescence and Chemiluminescence
Edited by M
ARLENE A. DELUCA
VOLUME LVIII. Cell Culture
Edited by W
ILLIAM B. JAKOBY AND IRA PASTAN
VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)
Edited by K
IVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)

Edited by K
IVIE MOLDAVE AND LAWRENCE GROSSMAN
VOLUME 61. Enzyme Structure (Part H)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
VOLUME 62. Vitamins and Coenzymes (Part D)
Edited by D
ONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and
Inhibitor Methods)
Edited by D
ANIEL L. PURICH
VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and
Complex Enzy me Systems)
Edited by D
ANIEL L. PURICH
VOLUME 65. Nucleic Acids (Part I)
Edited by L
AWRENCE GROSSMAN AND KIVIE MOLDAVE
VOLUME 66. Vitamins and Coenzymes (Part E)
Edited by D
ONALD B. MCCORMICK AND LEMUEL D. WRIGHT
VOLUME 67. Vitamins and Coenzymes (Part F)
Edited by D
ONALD B. MCCORMICK AND LEMUEL D. WRIGHT
METHODS IN ENZYMOLOGY xxi
VOLUME 68. Recombinant DNA
Edited by RAY WU
VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)
Edited by ANTHONY SAN PIETRO

VOLUME 70. Immunochemical Techniques (Part A)
Edited by H
ELEN VAN VUNAKIS AND JOHN J. LANGONE
VOLUME 71. Lipids (Part C)
Edited by J
OHN M. LOWENSTEIN
VOLUME 72. Lipids (Part D)
Edited by J
OHN M. LOWENSTEIN
VOLUME 73. Immunochemical Techniques (Part B)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 74. Immunochemical Techniques (Part C)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII,
XXXIV–LX
Edited by E
DWARD A. DENNIS AND MARTHA G. DENNIS
VOLUME 76. Hemoglobins
Edited by E
RALDO ANTONINI,LUIGI ROSSI-BERNARDI, AND
EMILIA CHIANCONE
VOLUME 77. Detoxication and Drug Metabolism
Edited by W
ILLIAM B. JAKOBY
VOLUME 78. Interferons (Part A)
Edited by S
IDNEY PESTKA
VOLUME 79. Interferons (Part B)

Edited by S
IDNEY PESTKA
VOLUME 80. Proteolytic Enzymes (Part C)
Edited by L
ASZLO LORAND
xxii METHODS IN ENZYMOLOGY
VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple
Membranes, I)
Edited by L
ESTER PACKER
VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular
Matrix)
Edited by L
EON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN
VOLUME 83. Complex Carbohydrates (Part D)
Edited by V
ICTOR GINSBURG
VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile
Apparatus and the Cytoskeleton)
Edited by D
IXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM
VOLUME 86. Prostaglandins and Arachidonate Metabolites
Edited by W
ILLIAM E. M. LANDS AND WILLIAM L. SMITH
VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates,
Stereochemistry, and Rate Studies)
Edited by D

ANIEL L. PURICH
VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple
Membranes, II)
Edited by L
ESTER PACKER
VOLUME 89. Carbohydrate Metabolism (Part D)
Edited by W
ILLIS A. W OOD
VOLUME 90. Carbohydrate Metabolism (Part E)
Edited by WILLIS A. WOOD
VOLUME 91. Enzyme Structure (Part I)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies
and General Immunoassay Methods)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
METHODS IN ENZYMOLOGY xxiii
VOLUME 93. Immunochemical Techniques (Part F: Conventional
Antibodies, Fc Receptors, and Cytotoxicity)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 94. Polyamines
Edited by H
ERBERT TABOR AND CELIA WHITE TABOR
VOLUME 95. Cumulative Subject Index Volumes 61–74, 76–80
Edited by E
DWARD A. DENNIS AND MARTHA G. DENNIS
VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and
Targeting (General Methods; Eukaryotes)]

Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and
Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and
Recycling)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 99. Hormone Action (Part F: Protein Kinases)
Edited by J
ACKIE D. CORBIN AND JOEL G. HARDMAN
VOLUME 100. Recombinant DNA (Part B)
Edited by R
AY WU,LAWRENCE GROSSMAN, AND KIVIE MOLDAVE
VOLUME 101. Recombinant DNA (Part C)
Edited by R
AY WU,LAWRENCE GROSSMAN, AND KIVIE MOLDAVE
VOLUME 102. Hormone Action (Part G: Calmodulin and
Calcium-Binding Proteins)
Edited by A
NTHONY R. MEANS AND BERT W. O’MALLEY
VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)
Edited by P. M
ICHAEL CONN
VOLUME 104. Enzyme Purification and Related Techniques
(Part C)
Edited by W
ILLIAM B. JAKOBY

xxiv METHODS IN ENZYMOLOGY
VOLUME 105. Oxygen Radicals in Biological Systems
Edited by LESTER PACKER
VOLUME 106. Posttranslational Modifications (Part A)
Edited by FINN WOLD AND KIVIE MOLDAVE
VOLUME 107. Posttranslational Modifications (Part B)
Edited by F
INN WOLD AND KIVIE MOLDAVE
VOLUME 108. Immunochemical Techniques (Part G: Separation and
Characterization of Lymphoid Cells)
Edited by G
IOVANNI DI SABATO,JOHN J. LANGONE, AND HELEN VAN
VUNAKIS
VOLUME 109. Hormone Action (Part I: Peptide Hormones)
Edited by L
UTZ BIRNBAUMER AND BERT W. O’MALLEY
VOLUME 110. Steroids and Isoprenoids (Part A)
Edited by J
OHN H. LAW AND HANS C. RILLING
VOLUME 111. Steroids and Isoprenoids (Part B)
Edited by J
OHN H. LAW AND HANS C. RILLING
VOLUME 112. Drug and Enzyme Targeting (Part A)
Edited by K
ENNETH J. WIDDER AND RALPH GREEN
VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds
Edited by A
LTON MEISTER
VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)
Edited by H

AROLD W. WYCKOFF,C.H.W.HIRS, AND SERGE N. TIMASHE FF
VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B)
Edited by H
AROLD W. WYCKOFF,C.H.W.HIRS, AND SERGE N. TIMASHE FF
VOLUME 116. Immunochemical Techniques (Part H: Effectors and
Mediators of Lymphoid Cell Functions)
Edited by G
IOVANNI DI SABATO,JOHN J. LANGONE, AND HELEN VAN
VUNAKIS
VOLUME 117. Enzyme Structure (Part J)
Edited by C. H. W. H
IRS AND SERGE N. TIMASHEFF
METHODS IN ENZYMOLOGY xxv
VOLUME 118. Plant Molecular Biology
Edited by ARTHUR WEISSBACH AND HERBERT WEISSBACH
VOLUME 119. Interferons (Part C)
Edited by SIDNEY PESTKA
VOLUME 120. Cumulative Subject Index Volumes 81–94, 96–101
V
OLUME 121. Immunochemical Techniques (Part I. Hybridoma Technology
and Monoclonal Antibodies)
Edited by J
OHN J. LANGONE AND HELEN VAN VUNAKIS
VOLUME 122. Vitamins and Coenzymes (Part G)
Edited by F
RANK CHYTIL AND DONALD B. MCCORMICK
VOLUME 123. Vitamins and Coenzymes (Part H)
Edited by F
RANK CHYTIL AND DONALD B. MCCORMICK
VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P. M
ICHAEL CONN
VOLUME 125. Biomembranes (Part M: Transport in Bacteria,
Mitochondria, and Chloroplasts: General Approaches and Transport
Systems)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria,
and Chloroplasts: Protonmotive Force)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and
Translocation)
Edited by L
ESTER PACKER
VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Struct ure, and
Molecular Biology)
Edited by J
ERE P. SEGREST AND JOHN J. ALBERS
VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology,
and Metabolism)
Edited by J
OHN J. ALBERS AND JERE P. SEGREST
xxvi METHODS IN ENZYMOLOGY
VOLUME 130 Enzyme Structure (Part K)
Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF
VOLUME 131. Enzyme Structure (Part L)
Edited by CHWHIRS AND SERGE N. TIMASHEFF
VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and
Cell-Mediated Cytotoxicity)

Edited by G
IOVANNI DI SABATO AND JOHANNES EVERSE
VOLUME 133. Bioluminescence and Chemiluminescence (Part B)
Edited by M
ARLENE DELUCA AND WILLIAM D. MCELROY
VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile
Apparatus and the Cytoskeleton)
Edited by R
ICHARD B. VALLEE
VOLUME 135. Immobilized Enzymes and Cells (Part B)
Edited by K
LAUS MOSBACH
VOLUME 136. Immobilized Enzymes and Cells (Part C)
Edited by K
LAUS MOSBACH
VOLUME 137. Immobilized Enzymes and Cells (Part D)
Edited by K
LAUS MOSBACH
VOLUME 138. Complex Carbohydrates (Part E)
Edited by V
ICTOR GINSBURG
VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-
Binding Protei ns)
Edited by A
NTHONY R. MEANS AND P. MICHAEL CONN
VOLUME 140. Cumulative Subject Index Volumes 102–119, 121–134
V
OLUME 141. Cellular Regulators (Part B: Calcium and Lipids)
Edited by P. M
ICHAEL CONN AND ANTHONY R. MEANS

VOLUME 142. Metabolism of Aromatic Amino Acids and Amines
Edited by S
EYMOUR KAUFMAN
VOLUME 143. Sulfur and Sulfur Amino Acids
Edited by W
ILLIAM B. JAKOBY AND OWEN GRIFFITH
METHODS IN ENZYMOLOGY xxvii
VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular
Matrix)
Edited by L
EON W. CUNNINGHAM
VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular
Matrix)
Edited by L
EON W. CUNNINGHAM
VOLUME 146. Peptide Growth Factors (Part A)
Edited by D
AVID BARNES AND DAVID A. SIRBASKU
VOLUME 147. Peptide Growth Factors (Part B)
Edited by D
AVID BARNES AND DAVID A. SIRBASKU
VOLUME 148. Plant Cell Membranes
Edited by L
ESTER PACKER AND ROLAND DOUCE
VOLUME 149. Drug and Enzyme Targeting (Part B)
Edited by R
ALPH GREEN AND KENNETH J. WIDDER
VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of
B and T Cell Functions and Lymphoid Cell Receptors)
Edited by G

IOVANNI DI SABATO
VOLUME 151. Molecular Genetics of Mammalian Cells
Edited by M
ICHAEL M. GOTTESMAN
VOLUME 152. Guide to Molecular Cloning Techniques
Edited by S
HELBY L. BERGER AND ALAN R. KIMMEL
VOLUME 153. Recombinant DNA (Part D)
Edited by R
AY WU AND LAWRENCE GROSSMAN
VOLUME 154. Recombinant DNA (Part E)
Edited by RAY WU AND LAWRENCE GROSSMAN
VOLUME 155. Recombinant DNA (Part F)
Edited by R
AY WU
VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related
Transport: The Na, K-Pump)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
xxviii METHODS IN ENZYMOLOGY
VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related
Transport: Calcium, Proton, and Potassium Pumps)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 158. Metalloproteins (Part A)
Edited by J
AMES F. RIORDAN AND BERT L. VALLEE
VOLUME 159. Initiation and Termination of Cyclic Nucleotide
Action
Edited by J

ACKIE D. CORBIN AND ROGER A. JOHNSON
VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose)
Edited by W
ILLIS A. W OOD AND SCOTT T. KELLOGG
VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin)
Edited by W
ILLIS A. W OOD AND SCOTT T. KELLOGG
VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and
Inflammation)
Edited by G
IOVANNI DI SABATO
VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and
Inflammation)
Edited by G
IOVANNI DI SABATO
VOLUME 164. Ribosomes
Edited by H
ARRY F. NOLLER,JR., AND KIVIE MOLDAVE
VOLUME 165. Microbial Toxins: Tools for Enzymology
Edited by S
IDNEY HARSHMAN
VOLUME 166. Branched-Chain Amino Acids
Edited by R
OBERT HARRIS AND JOHN R. SOKATCH
VOLUME 167. Cyanobacteria
Edited by L
ESTER PACKER AND ALEXANDER N. GLAZER
VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides)
Edited by P. M
ICHAEL CONN

VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A)
Edited by J
ACEK HAWIGER
METHODS IN ENZYMOLOGY xxix
VOLUME 170. Nucleosomes
Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG
VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model
Membranes)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and
Characterization)
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport:
Eukaryotic (Nonepithelial) Cells]
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 174. Biomembranes [Part U: Cellular and Subcellul ar Transport:
Eukaryotic (Nonepithelial) Cells]
Edited by S
IDNEY FLEISCHER AND BECCA FLEISCHER
VOLUME 175. Cumulative Subject Index Volumes 135–139, 141–167
V
OLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques
and Dynamics)
Edited by N
ORMAN J. OPPENHEIMER AND THOMAS L. JAMES
VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and
Mechanism)

Edited by N
ORMAN J. OPPENHEIMER AND THOMAS L. JAMES
VOLUME 178. Antibodies, Antigens, and Molecular Mimicry
Edited by J
OHN J. LANGONE
VOLUME 179. Complex Carbohydrates (Part F)
Edited by V
ICTOR GINSBURG
VOLUME 180. RNA Processing (Part A: General Methods)
Edited by J
AMES E. DAHLBERG AND JOHN N. ABELSON
VOLUME 181. RNA Processing (Part B: Specific Methods)
Edited by J
AMES E. DAHLBERG AND JOHN N. ABELSON
xxx METHODS IN ENZYMOLOGY