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The Hexokinase System of the Erythrocyte

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The Hexokinase System of the Erythrocyte THE HEXOKINASE SYSTEM OF THE ERYTHROCYTE by Charles Prévost A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science. Department of Biochemistry, Mc Gill University, Montreal. September 1961 i ABSTRACT The writer has developed an as say for estimation of optimal hexokinase activity in stroma-free hemolyzate, and shawn that hexokinase remains stable and active for at least 30 days in human erythrocytes preserved in CD or ACD at 4 ° C. Addition of ADP or DPN to the assay medium could increase the enzyme 1s activity; nicotinamide or alloxan depressed it. It is suggested that failure of glycolytic activity during preservation is not therefore due to the formerly supposed lability of hexokinase, but to inhibition of hexokinase attributable to falling pH and ATP, and conditions favouring glucose-6-P accumulation such as decrease of phosphofructokinase, DPN and ADP. Pyruvate produced in cells during the first 15 days of storage tended to be expelled into the external medium. Cyclic adenylate was utilised by the human red cell, as evidenced by conversion of its ribose moiety into lactic acid with concomitant esterification of inorganic phosphate into the stable phosphate fraction. ii AC KNOWLEDGE MEN TS I am most grateful to Dr. O. F. Denstedt for his sympathetic guidance and understanding throughout the course of this investigation. The kindness and spirit of my colleagues are greatly appreciated and I wish to express my thanks to Mrs. Marina van Ermengen, Miss Anne Hemphill and Miss Arlene Maximchuk for assistance in some of the experimenta, and to Miss Maximchuk for proofreading the manuscript. Many thanks to my friend Samuel Refetoff for completing the colored diagrams in the final copies. I am most indebted and thankful to my wife, Francine, for assistance in experimenta which ran into the night and for her meticulous care in typing the thesis. Grateful acknowledgement is made also for financial assistance from a grant to Professer O. F. Denstedt from the Defence Research Board of Canada (Grant Number 9350-01). iii PREFACE The work presented here is part of a larger programme of research on the preservation of blood under the direction of Dr. O. F. Denstedt. The main object was to examine the validity of a current hypothesis that the key factor in the metabolic failure of the erythrocyte during preservation in the cold (4"C} is the progressive inactivation of hexokinase. In order to understand how the loss of cell viability cornes about during the preservation of blood, it is helpful to review the properties of the hexokinase system and also per'tinent aspects of the structure of the erythrocyte and properties of the membrane in relation to the metabolic activity of the cell. lV TABLE OF CONTENTS ABSTRACT. • • • . i ACKNOWLEDGEMENTS .• . ii PREFACE • • • • iii LIST OF TABLES • .vi LIST OF FIGURES • . • vii LIST OF ABBREVIATIONS. viii INTRODUCTION. 1 ERYTHROCYTE COMPOSITION AND STRUCTURE 1 PERMEABILITY . 4 Glucose. 6 Sodium and potassium 8 Inorganic phosphate • 13 METABOLISM OF THE ERYTHROCYTE. 15 Hexokinase 17 Phosphoglucoisomerase . 20 Phosphofructokinase . 21 Enzymes of the metabolism of triose phosphates 21 Enzymes connected with the metabolism of high energy phosphate compounds 23 Other enzymes 26 Metabolism of red cells during preservation at 4°C. 29 EX PERIMENT AL 36 I. MA TE RIALS AND REAGENT5. 36 A. Substrates 36 B. Buffers 36 C. Enzymes . 37 II. METHODS 38 A. Procedure for collection and preservation of blood 38 B. Analytical procedures • 39 Glucose, 39 Pyruvic acid 40 Lac tic acid. 40 Pentose. 40 Phosphate fractions • 40 Hemoglobin 41 Hematocrit. 41 v C. General method of taking samples for analysis 41 D. Electrophoretic separation of nucleotides 42 E. Assay of hexokinase activity . 43 III. EXPERIMENTAL RESULTS • 44 A. Optimum conditions for hexokinase activity 44 1. The influence of hydrogen ion concentration . 44 2. Influence of added ATP and ADP on glucose utilisation and lac tic acid production • 46 3, The influence of other glycolytic enzymes and the optimal concentration of DPN • 51 4. Inhibition of glucose utilisation and lactic acid production by nicotinamide and alloxan 55 B. The activity of hexokinase during preservation of blood at 4°C 58 C. Influence of added 3 1, 5'-cyclic adenylic acid on the metabolic activity of blood during storage in ACD at 4°C. 73 DISCUSSION AND CONCLUSIONS 83 SUMMAR Y . 94 CLAIMS TO ORIGINALITY • 96 BIBLIOGRAPHY. 97 Vl LIST OF TABLES I. The influence of ATP and ADP on hexokinase activity 47 II. Hexokinase activity in the SFH from two specimens of blood during storage in CD and ACD respectively at 4°C • 60 III. Changes in pH, pyruvic acid, glucose and lactic acid concen­ tration in two specimens of blood during storage in CD and ACD respectively at 4°C . 62 IV. Partition of glucose and lactic acid between cells and plasma in two specimens of blood during storage at 4°C 65 V. Changes in the concentration of phosphate fractions in two specimens of blood during storage in CD and ACD respectively at 4 oc 66 VI. Partition of inorganic phosphate between cells and plasma in two specimens of blood during storage at 4 oC 69 VII. Partition of pyruvic acid between cells and plasma in two specimens of blood during storage at 4 oC 71 VIII. Changes in pH and ribose concentration in blood during storage in ACD at 4°C to which nucleoside was added on the 25th day 74 IX. Influence of added nucleoside on pyruvic acid concentration in blood during storage at 4°C, and partition of pyruvic acid between cells and plasma in the inosine-containing sample . 77 X. Influence of added nucleoside on the concentration of inorganic and stable phosphate, and partition of inorganic phosphate between the cells and the plasma during storage at 4°C. 79 vii LIST OF FIGURES 1 . The glycolytic and pentose metaholic pathways in the red cell . 16 2. The influence of the hydrogen ion concentration on hexokinase activity • 45 3. The influence of ATP and ADP on hexokinase activity 48 4. Nucleotide composition of the SFH during hexokinase assay 50 5. The influence of added SFH and DPN on the activity of yeast hexokinase • 53 6. The influence of nicotinamide on the metabolic behaviour of intact red cells and SFH • 56 7. The influence of alloxan on the metabolic behaviour of intact cells and SFH . 57 8. Hexokinase activity of SFH from blood during storage at 4°C . 61 9. Changes in pH, glucose and lactic acid concentration during preservation of blood at 4°C 63 10. Changes in the phosphate fractions during storage of blood at 4°C 67 11, Partition of inorganic phosphate between cells and plasma during storage of blood at 4 oC 70 12.. Changes in the concentration of pyruvic acid and its partition between tells and plasma during storage of blood at 4°C 72 13. The influence of added 3 1, 5'-cyclic adenylic acid or inosine on the utilisation of ribose by blood during storage in ACD at 4°C 75 14. {a) Theillfluen'ce ofadded 3',5'-cyclic adenylic acid or inosine on the pyruvic acid concentration during storage of blood in ACD at 4°C 78 (b) The influence of added inosine on the partition of pyruVié. acld between cells and plasma during storage of blood in ACD at 4 oC 78 15. The influence of added 3 1, 5 1 -cyclic adenylic a cid or inosine on the concentration of inorganic and stable phosphate of blood during storage in ACD at 4" C . 80 1 16. The influence of added 3 , 5 '-cyclic adenylic ac id or inosine on the partition of inorganic phosphate between cells and plasma during storage of blood in ACD at 4 oC 81 viii LIST OF ABBREVIATIONS AMP, ADP and A TP adenosine mono-, di-, and triphosphate CD! ACD citrate-dextrose medium, acid citrate-dextrose medium. Suffixes +CA or + 1 indicate the 1 1 medium to which had been added 3 , 5 -cyclic adenylic acid or inosine respectively. 1, 3-DPG, 2, 3-DPG 1, 3- and 2, 3-diphosphoglyceric acid DPN, DPNH diphosphopyridine nucleotide and reduced DPN GTP, UTP g:uanosine triphosphate and uridine triphosphate G-6-P glucose-6-phosphate Hb h;emoglobin IMP, ITP inosine mono- and triphospha.te molar, millimolar concentration inorganic phosphate, labile phosphate, and stable phosphate SFH stroma-free hemolyzate TCA trichloroacetic acid TPN, TPNH triphosphopyridine nucleotide and reduced TPN w/v, w/w weight/volume, and weight/weight 1 Cyclic AMP 3 , 5 1-cyclic adenylic acid INTRODUCTION ERYTHROCYTE COMPOSITION AND STRUCTURE Of the cells that make up a complex organism such as the animal body, the erythrocyte plays a peculiarly important role, since it carries oxygen from the lunga to every cell in the body and on its return journey to the lunga picks up and transports carbon dioxide. Its hemoglobin also is one of the most important buffers in the regulation of the hydrogen ion concentration of the blood and the tissùes. Knowledge of the detailed structure of the erythrocyte still is incomplete. The red cell of the mammal is a nonnucleated biconcave disk with a diameter of about O. 008 millimeter (8Jl) and a thickness around O. 0005 millimeter (O. S)l). It is a highly specialized cell which in the process of its maturation loses most of its organelles.- nucleus, mitochondria, microsomes, etc. , and becomes filled with a respiratory pigment, hemoglobin. The cellular membrane comprises 3 o/o of the cell mate rial; the rest of the cell is made up of hemoglobin 32o/o and water 65o/o (1 ).
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