ALKALINE PHOSPHATASE

ALKAUNE PHOSPHATASE

Robert B. McComb George N. Bowers, Jr. Hartford Hospital Hartford, Connecticut

and

Solomon Posen Sydney Hospital Sydney, Australia

PLENUM PRESS • NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data

McComb, Robert B 1926-Alkaline phosphatase.

Includes bibliographical references and index. 1. Alkaline phosphatase. I. Bowers, George N., joint author. II. Posen, Solomon,

joint author. III. Title. QP609.A4M32 ISBN-13: 978-1-4613-2972-5 DOl: 10.1007/978-1-4613-2970-1

© 1979 Plenum Press, New York

574.1'925 e-ISBN-13: 978-1-4613-2970-1

Softcover reprint of the hardcover 1 st edition 1979 A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011

All rights reserved

79436

No part of this book 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

To our wives

ALICE, MYRA, and JEAN

ACKNOWLEDGMENTS

Our thanks are due to numerous individuals in the laboratories, offices, and libraries of Hartford and Sydney Hospitals without whom this work could not have been undertaken. We also thank Doctors Donald Moss, A. J. Kirby, Milton Schlesinger, Florence Moog, Claude Petitclerc, Ted Reid, Lawrence Raisz, and Stephen Mistilis, who reviewed sections of the manuscript and offered helpful suggestions.

We are most appreciative of the support of our departments and in particular Dr. R S. Beckett, of Hartford Hospital. One of us (S.P.) was in receipt of grants from the National Health and Medical Research Council of Australia, the N.S.W. State Cancer Council, and the University of Sydney Cancer Research Fund.

vii

Robert B. McComb George N. Bowers, Jr.

Hartford, Connecticut

Solomon Posen Sydney, Australia

FOREWORD

There can be no doubt that alkaline phosphatase is one of the most extensively in­vestigated of all enzymes. This has resulted from the ubiquity of its distribution, and from the ease and sensitivity with which its activity can be measured. Unfortunately, these wide-ranging but often superficial experimental studies have been followed up by intensive and systematic investigations in only a few limited areas of the biochemistry and chemical pathology of alkaline phosphatase. The result has been the accumulation of a scientific literature of intimidating proportions, and the inevitable rediscovery of already­known facts about the enzyme. Scientists are taught early in their careers that, in the words of Sir John Herschel, "Hasty generalization is the bane of science." Nevertheless, moments arrive in all spheres of scientific activity when generalization becomes essential, to codify and to select from the mass of data already accumulated, and to provide starting points for new developments and new lines of investigation. This is especially true in a field such as alkaline phosphatase research, in which very real dangers exist that the seeds of fundamental understanding will be lost amidst an unexamined harvest of empirical observations.

The history of the study of alkaline phosphatase provides several instances when valuable generalizations have emerged. Occasionally, the conclusions drawn on the basis of available evidence were wrong; more frequently, they have stood the test of further experimentation, and always, they have provided new insights into the nature and proper­ties of this enzyme.

Generalizations about changes in the activity of alkaline phosphatase in serum in disease appear surprisingly early in the history of the clinical use of this enzyme test. Thus, the correlation between raised serum alkaline phosphatase activity and pathological or physiological processes characterized by increased osteoblastic activity is to be found in reviews by H. D. Kay and others published around 1930, and this principle has con­tinued to be the basis of interpretations of serum alkaline phosphatase measurements in bone diseases to the present day. Similarly, the changes in serum alkaline phosphatase associated with the main categories of liver disease were apparent shortly after that date. However, the long debate over the tissue of origin of increased activities of alkaline phos­phatase in serum in hepatobiliary disease has only been resolved in recent years.

Another prolonged debate in the field of alkaline phosphatase studies, and one which is amply documented in the following pages, has concerned the question of the differ­ences between, or identity of, the alkaline phosphatases from various human tissues. The resolution of this particular problem has had to await the development of newer techniques

ix

x FOREWORD

of fractionation of protein mixtures and enzyme characterization, and is still incomplete. Difficulties in the experimental study of the intra- and intertissue heterogeneity of alka­line phosphatase in animal cells are related to a large extent to the enzyme's location in the plasma membrane of cells in which it occurs, with the consequent problems of solubi­lization of the enzyme and differing contents of nonprotein components of phosphatase molecules which this entails. Therefore, the active investigations of membrane structure currently being pursued can be expected to throw further light on the molecular nature of the enzyme, even though they may not be primarily directed toward studies of alka­line phosphatase itself.

The substrate specificity of alkaline phosphatase has been the subject of particularly interesting changes of view during 50 years of study. Although early investigators recog­nized the almost unrestricted specificity of the enzyme, the view subsequently acquired currency that alkaline phosphatase does not hydrolyze inorganic pyrophosphate, or other pyrophosphate esters. However, recent investigations have shown that nonspecific alka­line orthophosphatases are almost invariably pyrophosphatases also, when allowed to act on pyrophosphates under suitable conditions. This represents a return to the views of the earliest investigators, such as Robert Robison. In this particular instance, therefore, a widely accepted generalization about alkaline phosphatase has proved to be less well founded than the earlier concept which it replaced.

In some of the areas of phosphatase research mentioned above, progress has un­doubtedly been hindered by the rapid, uncritical accumulation of apparently random observations. It seems unlikely that the earlier literature on alkaline phosphatase will ever again be reexamined on the scale that has been attempted in this monograph and placed in its modern context by authors who are themselves active contributors to alkaline­phosphatase research. This volume therefore presents a consolidated view of our current knowledge of this ubiquitous and enigmatic enzyme, and one which, while clarifying the significance of past discoveries, will serve as a starting point for future investigations.

D. W. Moss, M.A., M.Sc., Ph.D., D.Sc.

Professor of Clinical Enzymology Royal Postgraduate Medical School University of London, and Honorary Biochemist to Hammersmith Hospital London, England

CONTENTS

1 Introduction, General Aims, and Scope

2 Historical Background 2.1. Early Studies of Phosphorus Metabolism . . . . . . . . . . . . . . . . . 5 2.2. Hydrolysis of Phosphate Esters ......................... 5 2.3. Identification of Phosphatases as a Separate Group

of Enzymes .......................................... 6 2.4. Nomenclature ........................................ 7 2.5. Robert Robison and His Collaborators .................. 8 2.6. Early Experiments Concerning Solubilization and

Purification .......................................... 9 2.7. Properties of Alkaline Phosphatases ..................... 9 2.8. Phosphatase Assays ................................... 10 2.9. Reaction Mechanisms ................................. 13 2.10. Microbiological Studies ................................ 13 2.11. Early Clinical Applications ............................. 14 2.12. Histochemistry ....................................... 14 2.13. Procedures for the Separation of Alkaline Phosphatase

Isoenzymes ........................................... 15 2.14. Function ............................................. 16 References ................................................ 16

3 Distribution in Nature 3.1. Introduction ......................................... 27 3.2. Plants ............................................... 28 3.3. Algae ............................................... 29 3.4. Slime Molds ......................................... 29 3.5. Bacteria ............................................. 30 3.6. Fungi ............................................... 32 3.7. Protozoa ............................................ 32 3.8. Cnidaria (= Coelenterates) and Ctenophora ............... 34 3.9. Platyhelminthes....................................... 35

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xii CONTENTS

3.10. Nemertina ........................................... 40 3.11. Phylum Aschelminthes: Class Nematoda ................. 40 3.12. Acanthocephala ...................................... 41 3.13. Annelids ............................................. 41 3.14. Molluscs ............................................. 43 3.15. Arthropoda .......................................... 45 3.16. Phoronida ........................................... 49 3.17. Echinodermata ....................................... 49 3.18. Vertebrates........................................... 51 3.19. Vertebrates: Individual "Systems" ....................... 54 References ................................................ 112

4 Purification 4.1. Introduction ......................................... 153 4.2. Preliminary Treatment of Living Organisms to Increase

Yield ................................................ 153 4.3. Preparation and Storage of Starting Material ............. 156 4.4. Disruption of Cells .................................... 156 4.5. Subcellular Fractionation as a Preparative Step ........... 158 4.6. Solubilization of the Enzyme ........................... 158 4.7. Removal of Water and Unwanted Reagents .............. 163 4.8. Fractionation by Column Techniques .................... 166 4.9. Other Methods of Fractionation ........................ 173 4.10. Assessment of the Progress of Purification ............... 175 4.11. Crystallization ........................................ 177 4.12. Final Product ........................................ 177 4.13. Comparison of States of Purity of Different Alkaline

Phosphatase Preparations .............................. 181 4.14. Conclusion........................................... 183 References ................................................ 183

5 Structural Features 5.1. Introduction ......................................... 189 5.2. Amino Acid Composition .............................. 189 5.3. Amino Acid Sequences ................................ 191 5.4. Substitution of Amino Acids ........................... 193 5.5. Metals............................................... 194 5.6. Carbohydrates........................................ 203 5.7. Inorganic Phosphate .................................. 205 5.8. Interconversion of Molecular Species .................... 206 5.9. Molecular Weights .................................... 212 5.10. Chemical and Physical Modification of Alkaline

Phosphatases ......................................... 213 5.11. Physical and Chemical Probes of Internal Structures ...... 217 5.12. Summary ............................................ 221 References ................................................ 221

CONTENTS xiii

6 Reaction Mechanisms 6.1. Introduction ......................................... 229 6.2. Compounds of Phosphorus ............................ 229 6.3. Nonenzymatic Hydrolysis .............................. 234 6.4. Substrates of Alkaline Phosphatases ..................... 239 6.5. Steps of the Alkaline-Phosphatase-Catalyzed Reaction ..... 244 6.6. Rate-Limiting Factors ................................. 253 6.7. Hydrolysis and Transphosphorylation ................... 256 6.8. Ionic Strength and Alkaline Phosphatase Kinetics ......... 263 6.9. Metals and Alkaline Phosphatase Kinetics ............... 264 6.10. Effect of Hydrogen Ion Concentration:

Why "Alkaline" Phosphatase? .......................... 267 6.11. Inhibition of Alkaline Phosphatases ..................... 268 6.12. Some Unresolved Problems Concerning Reaction

Mechanisms .......................................... 275 References ................................................ 277

7 Measurement of Alkaline Phosphatase Activity 7.1. Introduction ......................................... 289 7.2. Enzyme Sample ...................................... 292 7.3. Substrates ........................................... 305 7.4. pH "Optimum" ....................................... 318 7.5. Buffers .............................................. 323 7.6. Other Components in the Reaction Mixture .............. 329 7.7. Inhibitors ............................................ 332 7.8. Reaction Temperature ................................. 334 7.9. Sample and Reaction Volumes; Reaction Containers ...... 338 7.10. Time Course of the Alkaline Phosphatase Reaction ....... 339 7.11. Reaction Products and Their Measurement............... 341 7.12. Analytical Variability.................................. 345 7.l3. Measurement of Alkaline Phosphatase Activity Based on

Nonoptical Determinations............................. 350 7.14. Methods of Expressing Activity......................... 351 7.15. Measurement of Alkaline Phosphatase Concentration by

Immunological Techniques ............................. 354 References ................................................ 355

8 Isoenzymes 8.1. Introduction ......................................... 373 8.2. Methods Available for the Separation and Identification

of Alkaline Phosphatase Isoenzymes ..................... 377 8.3. Bacterial Alkaline Phosphatases ........................ 417 8.4. Fungi ............................................... 423 8.5. Slime Molds ......................................... 425 8.6. Cnidaria............................................. 425

xiv CONTENTS

8.7. Platyhelminthes....................................... 425 8.8. Nemertina ........................................... 426 8.9. Acanthocephala ...................................... 426 8.10. Annelids............................................. 426 8.11. Molluscs ............................................. 426 8.12. Arthropods .......................................... 427 8.13. Echinoderms ......................................... 429 8.14. Isoenzymes within Vertebrate Tissues .................... 429 8.15. Isoenzymes in Blood Plasma ........................... 465 8.16. Conclusions .......................................... 486 References ................................................ 487

9 Clinical Utilization of Alkaline Phosphatase Measurements 9.1. Introduction ......................................... 525 9.2. Reference Values ...................................... 527 9.3. Associations with Human Disorders ..................... 549 9.4. Skin and Subcutaneous Tissues ......................... 550 9.5. Breast ............................................... 553 9.6. Hemopoietic and Lymphatic Systems .................... 555 9.7. Bone ................................................ 570 9.8. Joints ............................................... 596 9.9. Muscle .............................................. 599 9.10. Adipose, Fibrous, and Other Connective Tissues .......... 601 9.11. Respiratory System ................................... 602 9.12. Cardiovascular System ................................ 608 9.13. Digestive System ...................................... 610 9.14. Liver and Biliary Tract ................................ 620 9.15. Pancreas............................................. 658 9.16. Urinary System ....................................... 660 9.17. Male Reproductive System............................. 665 9.18. Female Reproductive System ........................... 667 9.19. Endocrine System..................................... 674 9.20. Nervous System ...................................... 681 9.21. Brief Aids for Resolving Unexpected Results ............. 684 9.22. Summary ............................................ 703 References ...............................•................ 704

10 Utilization of Alkaline Phosphatase Measurements in Veterinary Practice 10.1. Introduction ........................................ 787 10.2. Dogs ............................................... 788 10.3. Cats................................................ 796 10.4. Horses ............................................. 798 10.5. Cattle .............................................. 799

CONTENTS xv

10.6. Sheep .............................................. 804 10.7. Pigs................................................ 806 10.8. Goats .............................................. 808 10.9. Rats ............................................... 809 10.10. Mice ............................................... 814 10.11. Guinea Pigs......................................... 817 10.12. Hamsters ........................................... 817 10.13. Rabbits............................................. 817 10.14. Nonhuman Primates ................................. 818 10.15. Miscellaneous Eutherian Mammals..................... 826 10.16. Marsupials .......................................... 828 10.17. Birds ............................................... 828 10.18. Amphibians, Reptiles, and Fish ........................ 834 References ................................................ 835

11 Industrial and Other Uses 11.1. Use in the Dairy Industry .............................. 851 11.2. Use in the Identification and Classification of

Microorganisms ...................................... 853 11.3. Use as an Analytical Reagent ........................... 854 11.4. Industrial Uses ....................................... 856 11.5. Use in the Assessment of Soil and Water Samples ......... 856 11.6. Use in the Measurement of Plasma Volume .............. 857 11.7. Use in the Detection of Fecal Matter.................... 858 11.8. Use in Enzyme-Linked Immunosorbent Assays

(ELISA) ............................................. 859 11.9. Use in Biological Assays ............................... 860 References ................................................ 860

12 Physiological Functions 12.1. Introduction ........................................ 865 12.2. Enzymatic Activity as a Clue to Function ............... 865 12.3. Distribution in Nature as a Clue to Function ............ 868 12.4. Embryonic Development as a Clue to Function .......... 870 12.5. Transport of Specific Substances as a Possible Function. . . 872 12.6. Hydrolysis of Intracellular Metabolites as a Possible

Function ............................................ 878 12.7. Role in Phagocytosis ................................. 881 12.8. Role in Calcification Mechanisms ...................... 882 12.9. Induction of Alkaline Phosphatases .................... 883 12.10. Denervation Experiments ............................. 890 12.11. Role in the Development of Cancer .................... 890 12.12. Conclusions Regarding the Physiological Functions of

Alkaline Phosphatases ................................ 891 References ................................................ 891

xvi CONTENTS

Appendix: Procedures A.l. Introduction .......................................... 903 A.2. Manual Reference Method: Continuous Spectrophotometric

Measurement of Total Alkaline Phosphatase Activity in Human Serum ........................................ 903

A.3. Manual Sampling Method: Measurement of Total Alkaline Phosphatase Activity in Human Serum...... ....... . . ... . 907

A.4. Automated Continuous-Flow Method: Measurement of Total Alkaline Phosphatase Activity in Human Serum ..... 909

A.5. Automated Continuous-Flow Micromethod: Automated Fluorometric Method for Alkaline Phosphatase Microassay ........................................... 911

A.6. Leukocyte Staining Method: Cytochemical Method for Straining Granulocytic Leukocytes for Leukocyte Alkaline Phosphatase Activity (LAPA) ........................... 913

References ................................................ 915

Index .................................................... 917

ALKALINE PHOSPHATASE