Contributors to Volume 166 elcitrA numbers are in paten~ following the names of conu'ibutors. snoitailiffA detsil are .tnerruc D. JOHN ABERHART (3, 4), Worcester Foun- MARIA GORDON BUSE (24), stnemtrapeD of dation for Experimental ,ygoloiB -swerhS enicideM and ,yrtsimehcoiB Medical -inU ,yrub sttesuhcassaM 54510 ytisrev of South Carolina, ,notselrahC R. PAUL AFrRn~O (24), Departments of South aniloraC 52492 enicideM and ,yrtsimehcoiB Medical -inU SINED B. NOTXUB (60), noisiviD of raelcuN ytisrev of South Carolina, ,notselrahC Medicine and ,scisyhpoiB Department of South aniloraC 52492 lacigoloidaR ,secneicS ytisrevinU of -ilaC TREBOR H. NELLA (49, 50, 51), noisiviD of fornia Los Angeles Medical ,loohcS Los Hematology, ytisrevinU of odaroloC ,selegnA ainrofilaC 42009 Health secneicS ,retneC ,revneD odaroloC HPESOJ M. OVLAC (27, 57), Department of 26208 Biochemistry and Molecular ,ygoloiB YMMAT K. ICCUNOTNA (31), tnemtrapeD of llenroC ,ytisrevinU Ithaca, New York Immunology and ,ygoloiborciM ytisrevinU 05841 of ,nagihciM Medical ,loohcS Ann ,robrA YNOHTNA G. YESUAC (11), Department of nagihciM 90184 Drug Metabolism, Pfizer Central Re- ZE'EV KARAB (29, 57), Department of -loiB ,hcraes Sandwich, Kent CTI3 9NJ, -nE ,ygo Ben Gurion ytisrevinU of the ,vegeN dnalg Beer Sheva ,50148 Israel DIVAD M. CmPMAN (29), tnemtrapeD of -iB KIM TTELTRAB (11), stnemtrapeD of dlihC ,ygolo Ben noiruG ytisrevinU of the ,vegeN Health and Clinical ,yrtsimehcoiB The Beer Sheva 48 ,501 Israel Medical School, ytisrevinU of eltsacweN DIVAD T. GNAUHC (18, 19), Department of upon Tyne, Newcastle upon Tyne NE2 ,enicideM Cleveland Veterans Medical 4HH, England ,retneC and tnemtrapeD of Medicine and MARK SOGEB (21, 22), tnemtrapeD of -inilC ,yrtsimehcoiB Case nretseW Reserve -inU lac ,yrtsimehcoiB University of ,drofxO ,ytisrev ,dnalevelC Ohio 60144 John Radcliffe Hospital, Oxford 3XO HTENNEK G. COOK (2, 39), Department of ,UD9 England ,yrtsimehcoiB University of eltsacweN L. L. REBmB (37), tnemtrapeD of -simehcoiB upon Tyne, Newcastle upon Tyne NE2 ,yrt nagihciM State ,ytisrevinU East -snaL 4HH, dnalgnE ,gni nagihciM 42884 ARAaRAB E. YEKROC (9), Division of -aiD NIVEK P. KCOLB (24), stnemtrapeD of -ideM betes and ,msilobateM Boston ytisrevinU enic and ,yrtsimehcoiB Medical ytisrevinU School of Medicine, Boston, -uhcassaM of South ,aniloraC ,notselrahC South -raC sttes 81120 anilo 52492 NEAL W. LLENROC (10), yrotarobaL of Me- WERDNA P. BRADFORD (2), Department of msilobat and raluceloM ,ygoloiB NIAAA, ,yrtsimehcoiB University of eltsacweN ,ellivkcoR Maryland 25802 upon Tyne, Newcastle upon Tyne NE2 NIALA-SEUQCAJ COTTING (3), retsecroW 4HH, England noitadnuoF for Experimental ,ygoloiB NOJ REMERB (59), Institute for Medical -oiB ,yrubswerhS sttesuhcassaM 54510 ,yrtsimehc ytisrevinU of ,olsO Oslo ,3 -roN YDOR P. Cox (18), tnemtrapeD of ,enicideM yaw dnalevelC snareteV Medical ,retneC and xi xii CONTRIBUTORS TO VOLUME 166 Case Western Reserve University, Cleve- HTENNEK M. NOSBIG (25, 26), Kimberly H. land, Ohio 44106 Courtwright and Joseph .W Summers ALEMAP L. LLEWORC (7), Department of Nu- Metabolic Disease Center, Baylor -revinU tritional Sciences, College of Agricultural sity Medical Center, Dallas, Texas and Life Sciences, University of Wiscon- 64257 sin-Madison, Madison, Wisconsin 53706 NATAN GOLLOP (29), Department of Biol- ZAHi DAMUNI (42), Department of ,ygoloiB ogy, Ben Gurion University of the Negev, University of South Carolina, Columbia, Beer Sheva 84 105, Israel South Carolina 29208 GARY W. GOODWIN (23), Indiana Univer- DEAN J. DA~mER (14, 38), Division of Medi- sity, School of Medicine, Department of cal Genetics, Department of Pediatrics, Biochemistry, Indianapolis, Indiana Emory University School of Medicine, At- 46223 lanta, Georgia 30322 ANNAVOIG GRIFFO (30), International Insti- E. JACK DAVIS (59), Indiana University tute of Genetics and Biophysics, 80125 School of Medicine, Department of Bio- Naples, Italy chemistry, Indianapolis, Indiana 46223 DLONRA HAMPEL (33), Department of Bio- OILIRUAM DE FELICE (30), International In- logical Sciences and Chemistry, Northern stitute of Genetics and Biophysics, 80125 Illinois University, DeKalb, Illinois 60115 Naples, Italy, and Istituto di lngegneria MARY E. NETGOLDNAH (32), Department of Chimico-Alimentare, FacolM di Ingegne- Biochemistry and Molecular Biology, J. ria, UniversiM di Salerno, 84100 Baro- Hillis Miller Health Center, University of nissi, Salerno, Italy Florida, Gainesville, Florida 32610 MAILLIW T. E. SDRAWDE (20), AFRC Insti- ALFRED E. HARPER (7, 24), Departments of tute of Food Research, Norwich Labora- Nutritional Sciences and Biochemistry, tory, Norwich NR4 7UA, England University of Wisconsin-Madison, Madi- SONIRAM ELlA (5), MRC Dunn Clinical Nu- son, Wisconsin 53706 trition Center, Cambridge CB2 IQE, Eng- TREBOR A. HARRIS (15, 23), Indiana Uni- land ,ytisrev School of Medicine, Department of Louis J. SASLE (14), Division of Medical -eG Biochemistry, Indianapolis, Indiana netics, Department of Pediatrics, Emory 46223 University School of Medicine, Atlanta, HTENNEK HATTER (48), Department of Bio- Georgia 30322 chemistry and Molecular Biology, The MARK EGATPME (12), Central Research De- University of Oklahoma Health Sciences velopment Department, Experimental Sta- Center, Oklahoma City, Oklahoma tion E328/246A, E. I. du Pont de Nemours 09137 & Company, Wilmington, Delaware EUS C. HEFFELFINGER (38), Department of 19898 Pathology, Baylor School of Medicine, LILLIAN EOYANG (55), Department of Mo- Texas Children's Hospital, Houston, lecular Biology, Division of Biological Texas 77030 Sciences, Albert Einstein College of Medi- ELLEN HILDEnRANDT (60), Department of cine, Bronx, New York 10461 Biochemistry, Saint Judes Childrens Hos- JOSEPH ESPINAL (21, 22), Department of pital Memphis, Tennessee 38101 Clinical Biochemistry, University of Ox- ANTHONY HOBSON-FRoHOCK (5), AFRC ford, John Radcliffe Hospital, Oxford OX3 Institute of Food Research, Norwich Labo- 9DU, England ,yrotar Norwich NR4 7UA, England ALEGNA L. GASKING (5), AFRC Institute of YASUVUKI IKEDA (20, 46, 47), National Food Research, Norwich Laboratory, Nor- Cardiovascular Center, Research Institute, wich NR4 7UA, England Suita City 565, Osaka, Japan CONTRIBUTORS TO VOLUME 166 xiii SUILUJ H. NOSKCAJ (28), Department of Mi- TRAUTS LITWER (14), Division of Medical erobiology, Meharry Medical College, ,sciteneG Department of Pediatrics, Emory Nashville, Tennessee 37208 University School of Medicine, Atlanta, HGALEEHS M. A. SENOJ (2), Department of Georgia 30322 Biochemistry, University of Newcastle YERFFOEG YESEVIL (1, 5, 36), AFRC Insti- upon Tyne, Newcastle upon Tyne NE2 tute of Food Research, Norwich Labora- 4HH, England tory, Norwich NR4 7UA, England Rvo KzDo (35), Department of Biochemis- RETEP N. EWOL (43), Department of Molec- try, Wakayama Medical College, Wa- ular Sciences, Wellcome Research Labora- kayama 640, Japan tories, Beckenham, Kent BR33BS, Eng- LEAHCIM S. GREBLIK (32), Department of land Biochemistry and Molecular Biology, J. AICIRTAP LUND (1, 36), Metabolic Research Hillis Miller Health Center, University of Laboratory, Nujffield Department of Clini- Florida, Gainesville, Florida 32610 cal Medicine, Radcliffe Infirmary, Oxford M. TODD KIN~ (10), Laboratory of Metabo- OX2 6HE, England lism and Molecular Biology, NIAAA, RUN ADLOOAM (12), Central Research De- Rockville, Maryland 20852 velopment Department, Experimental Sta- GUNTER B. WAHLHOK (52, 53, 54), Depart- tion E328/246A, E. I. du Pont de Nemours ment of Biochemistry, Purdue ,ytisrevinU & Company, Wilmington, Delaware West Lafayette, Indiana 47907 19898 J. FRED ESUOHLOK (50, Division of Hema- LAVRO A. REMAM (6, 8), McGill University tology, University of Colorado Health Biomedical Mass Spectrometry Unit, Sciences Center, Denver, Colorado 80262 Montreal, Quebec, Canada H3A IA3 T. K. ALEPROK (34), Department of Bio- TREBOR H. MILLER (7), ROSS Laboratories, chemistry, University of Turku, SF-20500 Columbus, Ohio 43216 Turku, Finland NIBOR J. YHCAVOK (49), Division of Hema- ENAJ A. YREMOOTNOM (6, 8), McGill Uni- versity Biomedical Mass Spectrometry tology, University of Colorado Health ,tinU Montreal, Quebec, Canada H3A 1A3 Sciences Center, Denver, Colorado 80262 AHTRAM J. KUNTZ (15), Department of Bio- NNELG E. EROMITROM (58), Department of chemistry, Indiana University School of Physiology, The Milton S. Hershey Medi- Medicine, Indianapolis, Indiana 46223 cal Center, The Pennsylvania State Uni- ,ytisrev Hershey, Pennsylvania 17033 ENIMRAC T. LAGO (30), International Insti- tute of Genetics and Biophysics, via .G ELREM S. NOSLO (60), Department of Bio- Marconi ,01 80125 Naples, Italy chemistry, University of Texas Health Science Center, San Antonio, Texas 77284 DAVID S. LAPOINTE (60), Department of Biochemistry, University of Texas Health ELAD L. REDNEXO (31), Department of Bio- Science Center, San Antonio, Texas 77284 logical Chemistry, University of Michigan, TREBOR A. ASSORAL (13), Central Research Ann Arbor, Michigan 48109 and Development Department, E. I. du TARUN B. PATEL (60), Department of Phar- Pont de Nemours & Company, Wilming- macology, University of Tennessee Center ton. Delaware 19898 for the Health Sciences, Memphis, Tennes- EEH-GNUS OHC EEL (59), Hyo-Sung -revinU see 38163 sity, Department of Food Science and Nu- PHILIP A. PATSTON (22), Department of trition, Taegu, Korea Clinical Biochemistry, University of Ox- ALINAD ORUAMIL (30), International Insti- ford, John Radcliffe Hospital, Oxford OX3 tute of Genetics and Biophysics, via .G 9DU, England Marconi ,01 80125 Naples, Italy HPLAR NOTXAP (23, 4 l), Department of Bio- xiv CONTRIBUTORS TO VOLUME 166 logical Sciences, Texas Technical -revinU DLANOR NOSPMIS (15), Sandoz Research In- sity, Lubbock, Texas 79409 stitute, Sandoz Pharmaceutical Corpora- DRAHCIR N. MAHREP (43), Department of tion, East Hanover, New Jersey 07936 Biochemistry, University of Cambridge, NHOJ R. HCTAKOS (44, 45, 48), Department Cambridge CB2 1QW, England of Biochemistry and Molecular Biology, AROLF H. PETTIT (40), Clayton Foundation The University of Oklahoma Health Biochemical Institute, The University of Sciences Center, Oklahoma City, Okla- Texas at Austin, Austin, Texas 78712 homa 73190 A. REETA POSt) (58), Department of Bio- NIETSY~~ DLOVEDYPS (59), Institute for chemistry, College of Veterinary Medicine, Medical Biochemistry, University of ,olsO Hdmeentie ,75 SF-00550, Helsinki ,55 Oslo ,3 Norway Finland YLLAS P. RELBATS (49, 50, 51), Division of J. LEAHCIM NOTSOP (16, 17), Laboratory of Hematology, University of Colorado Biochemistry, National Heart, Lung, and Health Sciences Center, Denver, Colorado Blood Institute, National Institutes of 80262 Health, Bethesda, Maryland 20892 P. J. SEKYS (45), Department of Haematol- PrIILlP J. RANDLE (21, 22), Department of ogy, Flinders Medical Centre, Bedford Clinical Biochemistry, University of Ox- Park, South Australia 5042, Australia ford, John Radcliffe Hospital, Oxford OX3 KAY TANAKA (20, 46, 47), Yale University 9DU, England School of Medicine, Department of RETSEL J. REED (40, 42), Clayton Founda- Human Genetics, New Haven, Connecticut tion Biochemical Institute and Department 06510 of Chemistry, The University of Texas at DRAHCaR TRITZ (33), Department of -goloiB Austin, Austin, Texas 78712 ical Sciences and Chemistry, Northern Illi- LUAP D. SSIER (10), MilliGen ,noitaroproC nois University, DeKalb, Illinois 60115 Bedford, Massachusetts 0371O WERD E. NAV KVD (56), Central Research EzIo RICCA (30), International Institute of and Development, Experimental Station Genetics and Biophysics, 80125 Naples, E328, E. L du Pont de Nemours & Com- Italy, and IABBAM Consiglio Nazionale pany, Wilmington, Delaware 19898 della Ricerche, 80147 Ponticelli, Naples, TINA K. NAV DYK (13), Central Research Italy and Development, Experimental Station E328, E. L du Pont de Nemours & Com- P. J. N1RUOBAS (37), Department of Bio- chemistry, Michigan State University, pany, Wilmington, Delaware 19898 East Lansing, Michigan 48824 NAVRAP P. KCAMYAW (60), Center for Dis- NHOJ V. SSOLHCS (12, 56, 57), Central Re- ease Control, Atlanta, Georgia 30033 search and Development, Experimental NEHPETS J. NAMAEY (2, 39), Department of Station E328, E. L du Pont de Nemours & Biochemistry, University of Newcastle Company, Wilmington, Delaware 19898 upon Tyne, Newcastle upon Tyne NE2 EILLIL L. SELRAES (27), Department of Biol- 4HH, England ogy, University of North Carolina, Chapel IEB ZHANG (23), Indiana University, School Hill, North Carolina 27599-3280 of Medicine, Department of Biochemistry, PILIHP M. NAMREVLIS (55), Department of Indianapolis, Indiana 46223 Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medi- cine, Bronx, New York 10461 Preface Branched-chain amino acids were first discussed in Methods ni Enzy- ygolom in 1970 in a section of Volume XVII: Metabolism of Amino Acids and Amines, Part A. That section dealt primarily with biosynthetic enzymes of branched-chain amino acid pathways in prokaryotes. Al- though at that time the catabolic pathways for these amino acids had been quite well outlined, few of the catabolic enzymes had been purified. Recently, there has been a renaissance in research on several aspects of branched-chain amino acid metabolism which amply justifies a complete volume on this subject. The structure and function of branched-chain keto acid dehydrogenase has attracted the attention of several investigators, in part because of the relationship of this enzyme complex to other keto acid dehydrogenase multienzyme complexes, and in part because of the role of this complex in maple syrup urine disease. There are a number of human genetic diseases associated with the metabolism of branched-chain amino acids which has led to the development of sensitive methods, principally high- performance liquid chromatography, for detecting metabolites of these pathways in serum. The pattern of metabolites helps localize the genetic lesion which can then be identified by specific enzyme assays. New enzyme assays have been developed, both for recently described en- zymes, such as the branched-chain keto acid dehydrogenase, and well- known enzymes, such as acetolactate synthase. Several enzymes, both catalytic and biosynthetic, have been purified and are described in this volume. We believe this volume will be useful to investigators of branched-chain amino acid metabolism, to those contemplating entering the field, and to those interested in the genetic diseases affecting branched-chain amino acid metabolism. TREBOR A. HARRIS JOHN R. HCTAKOS XV METHODS IN ENZYMOLOGY EDITED BY Sidney P. Colowick and Nathan O. Kaplan VANDERBILT UNIVERSITY DEPARTMENT OF CHEMISTRY SCHOOL OF MEDICINE UNIVERSITY OF CALIFORNIA NASHVILLE, TENNESSEE AT SAN DIEGO LA JOLLA, CALIFORNIA .I Preparation and Assay of Enzymes .II Preparation and Assay of Enzymes III. Preparation and Assay of setartsbuS .VI Special Techniques for the Enzymologist .V Preparation and Assay of Enzymes .IV Preparation and Assay of Enzymes )deunitnoC( Preparation and Assay of setartsbuS laicepS Techniques VII. Cumulative Subject Index iivx METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF Sidney P. Colowick and Nathan O. Kaplan EMULOV VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG EMULOV IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD EMULOV X. Oxidation and Phosphorylation Edited by RONALD W. KOORBATSE DNA MAYNARD E. PULLMAN EMULOV XI. Enzyme Structure Edited by C. H. W. HIRS EMULOV XII. Nucleic Acids (Parts A and B) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE EMULOV XIII. Citric Acid Cycle Edited by J. M. NIETSNEWOL EMULOV XIV. Lipids Edited by J. M. NIETSNEWOL EMULOV XV. Steroids and Terpenoids Edited by DNOMYAR B. NOTYALC EMULOV XVI. Fast Reactions Edited by HTENNEK NITSUK EMULOV XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR xix XX METHODS IN ENZYMOLOGY EMULOV XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DLANOD B. KCIMROCCM DNA LEMUEL D. WRIGHT EMULOV XIX. Proteolytic Enzymes Edited yb EDURTREG E. NNAMLREP DNA OLZSAL DNAROL EMULOV XX. Nucleic Acids and Protein Synthesis (Part C) Edited by IOVlE EVADLOM DNA ECNERWAL NAMSSORG EMULOV XXI. Nucleic Acids (Part D) Edited by ECNERWAL NAMSSORG DNA KIVIE EVADLOM EMULOV XXII. Enzyme Purification and Related Techniques Edited yb MAILLIW B. YBOKAJ EMULOV XXIII. Photosynthesis (Part A) Edited by YNOHTNA NAS ORTEIP EMULOV XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by YNOHTNA NAS ORTEIP EMULOV XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVI. Enzyme Structure (Part C) Edited yb C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS DNA EGRES N. FFEHSAMIT EMULOV XXVIII. Complex Carbohydrates (Part B) Edited by ROTCIV GRUBSNIG EMULOV XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by ECNERWAL NAMSSORG DNA KIVIE EVADLOM EMULOV XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE EVADLOM DNA ECNERWAL NAMSSORG EMULOV XXXI. Biomembranes (Part A) Edited by YENDIS REHCSIELF DNA RETSEL REKCAP METHODS IN ENZYMOLOGY iXX EMULOV XXXII. Biomembranes (Part B) Edited by YENDIS REHCSIELF DNA RETSEL REKCAP EMULOV XXXIII. Cumulative Subject Index Volumes I-XXX Edited by AHTRAM G. SINNED DNA DRAWDE A. SINNED EMULOV XXXIV. Affinity Techniques (Enzyme Purification: Part )B Edited by MAILLIW .B YBOKAJ DNA MEIR KEHCLIW EMULOV XXXV. Lipids (Part )B Edited by NHOJ M. NIETSNEWOL EMULOV XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by TREB W. YELLAM'O DNA LEOJ G. NAMDRAH EMULOV XXXVII. Hormone Action (Part :B Peptide Hormones) Edited by TREB W. YELLAM'O DNA LEOJ G. NAMDRAH EMULOV XXXVIII. Hormone Action (Part :C Cyclic Nucleotides) Edited by LEOJ G. NAMDRAH DNA TREB W. YELLAM'O EMULOV XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by LEOJ G. NAMDRAH DNA TREB W. YELLAM'O EMULOV XL. Hormone Action (Part :E Nuclear Structure and Function) Edited by TREB W. YELLAM'O DNA LEOJ G. NAMDRAH EMULOV XLI. Carbohydrate Metabolism (Part )B Edited by W. A. DOOW EMULOV XLII. Carbohydrate Metabolism (Part )C Edited by W. A. DOOW EMULOV XLIII. Antibiotics Edited by NHOJ H. HSAH EMULOV XLIV. Immobilized Enzymes Edited by SUALK HCABSOM EMULOV XLV. Proteolytic Enzymes (Part )B Edited by OLZSAL DNAROL xxii METHODS IN ENZYMOLOGY EMULOV XLVI. Affinity Labeling Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK EMULOV XLVII. Enzyme Structure (Part E) Edited by .C H. .W SRIH DNA EGRES N. FFEHSAMIT EMULOV XLVIII. Enzyme Structure (Part F) Edited yb C. H. W. SRIH DNA EGRES N. FFEHSAMIT EMULOV XLIX. Enzyme Structure (Part G) Edited by .C H. .W SRIH DNA EGRES N. FFEHSAMIT EMULOV L. Complex Carbohydrates (Part C) Edited yb ROTCIV GRUBSNIG EMULOV LI. Purine and Pyrimidine Nucleotide Metabolism Edited by PATRICIA A. HOFFEE AND MARY NELLE SENOJ EMULOV LII. Biomembranes (Part :C Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER EMULOV LIII. Biomembranes (Part D: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER EMULOV LIV. Biomembranes (Part :E Biological Oxidations) Edited by YENDIS REHCSIELF DNA RETSEL REKCAP EMULOV LV. Biomembranes (Part F: Bioenergetics) Edited yb YENDIS REHCSIELF DNA RETSEL REKCAP EMULOV LVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEY FLEISCHER AND RETSEL PACKER EMULOV LVII. Bioluminescence and Chemiluminescence Edited by MARLENE A. DELUCA EMULOV LVIII. Cell Culture Edited by WILLIAM B. JAKOBY AND IRA PASTAN EMULOV LIX. Nucleic Acids and Protein Synthesis (Part G) Edited yb KIVIE MOLDAVE AND LAWRENCE GROSSMAN