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Aspects of the Metabolic Role and Biosynthesis of Carnitine

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Aspects of the Metabolic Role and Biosynthesis of Carnitine WAITE INSTITUTE LrÌltì,qRY ASPECTS OF THE METABOLIC ROLE AND BIOSYNTHESIS OF CARNITINE A Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy by NICK DIMITRI C0STA, B.Ag.Sc. (Hons) Department of Agricultural Biochem'istry and Soiì Science I'laite Agri cul tural Research Insti tute The University of Adelaide South Australia January 1977 give ï consent to this copy of my thesis, when d.eposited. in the Universit¡r Library, being available f or loan and. otocopying. Date .. J P.'. Signe d, L ACKNOWLEDGEMENTS I acknowledge with deepest gratitude the advice, insight and Christian frienclship given throughout the course of this work by my supervisor, Dr. Alan M. Snoswell, Reader in Agricuìtural Biochemistry. My thanks go to Mr. G.D. Henderson, Mr. R.C. Fishlock, Mr. D.t,J. Pethick and Ms. L.C. Read for heìp ìn collecting samples and preparation of hepatocytes from rats and sheep. The help of Dr. G.H. Mclntosh in the surgical preparation of'lactating ewes and Dr. J. Mclean ìn providing non-lactating and lactating cows and in the surgical removal of liver biopsy samples is gratefuììy acknow'ledged. Professor D.J.D. Nicholas, Ms. J.0. Peavy and the late Mrs. M. Pickford are also thanked for their encouragement and he1p. Many thanks also go to Mr. J. Magarey for competent maintenance of the sheep and goats used in this work and to Mr. B. Palk for the preparation of the figures presented in this work. Ms. P. Falloon is thanked for her perseverance and competence in typing this thesis. My love and thanks go to my w'ife, Robyn, who gave me encouragement and inspiration when I needed it, drìve when I needed it, and love, warmth and friendship a'lways. The financial support of the Australjan hlool Corporation Postgraduate Award is gratefuìly acknowledged. ÅlL DECLARAT I ON I hereby declare that the work presented in this thesis has been performed by myseìf, except Where otherwise stated in the text, and that it has not been submitted in any previous application for a degree. NI TRI COSTA ,UrL PUBLICATIONS Part of the work presented in this thesis has been reported elsewhere: N.D. Costa and A.M. Snoswell Acety'lcar.nitine hydro'lysis in I iver. ?noe. Autt. BLochen. Soc. (1974) 7, 36. N.D. Costa and A.M. Snoswell Conversion of acetyl-CoA to acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase. Pnoc. Au.tt. Bíochen. Soc. (1975) B, 51. A.M. Snoswell, G.H. Mclntosh and N.D. Costa Endogenous acetate production by the livér in lactating ewes. Pnoe. Au¿t. Biochen, Soc. (1975) 8, 5.l . N.D. Costa and A.M. Snoswell Enzymic hydroìysis of acety'lcarnitine in liver from rats, sheep and colvs. B.Loeh¿n. J. (.l975) 152, l6l-166. N.D. Costa and A.M. Snoswell Acetyl-Coenzyme A hydrolase, an artifact? ßíochem. J. (1975) lSZ, 167 -172. N.D. Costa, G.H. Mclntosh and A.M. Snoswell Production of endogenous acetate by the liver in lactating ewes. Au,st. J. ßioX-. Sei. (1976) 29, 33-42. .LV N.D. Costa and A.M. Snoswell Biosynthesis of carnitine in hepatocytes isolated'from rats and sheep. Pnoc. Atut. Bíochem. Soe. (1976) 9, 35. N.D. Costa, J.G. Mclean and A.M. Snoswell 0n the role of carnitine and acetylcarnitine in the liver of I actati ng and non-l actati ng co\^,s . Submi tted for publ i cati on. v PREFACE Abbreviations approved by the Biochemical Journal (.l976) for use without exp'lanation are used as such throughout this thesis. Chemical compounds, their Sources and degrees of purity are described in the text. The fol'lowing enzymes are referred to by name only: Acetare thiokinase EC 6. 2.1 .1 Acetyl-CoA hydrolase EC 3. 1 .2.1 Acetyì-CoA synthetase EC 6. 2.1 .1 Adenylate kinase EC 2.7.4.3 Alanjne dehydrogenase EC l.4.l.l 4-aminobutyric acìd - glutamate transaminase EC 2.6.1 .19 'lyase ATP-ci trate EC 4.1.3.8 Carboxyì ic ester hydrolase EC 3.t.l.l Carni ti ne acetyì transferase EC 2.3.1.7 Carni ti ne pa'l mi toy'l trans ferase EC 2.3.1.a Catal ase EC 1.il.1.6 Choì i nesterase EC 3.1.t.8 Citrate synthase EC 4.1.3.7 Gì ucose-6- phos phatase E0 3.r.3.e Gl utamate decarboxyì ase EC 4..l .l .l s Gl utamate dehydrogenase EC t . 4.1.2 L-glyceroì -3-phosphate dehydrogenase EC 1.1.1.8 .| 3- hydroxybutyrate dehydrogenas e EC .1 ..l .30 Lactate dehydrogenase EC I .1 .1 .27 Monoamine oxidase EC 1.4.3.4 Phos photrans acetyì ase EC 2.3. I .8 V,L Protein methylase III EC 2..l .1.25 Rotenone-insensitive NADH-cytochrome c reductase EC 1.6.99.3 Succinate dehydrogenase EC 1.3.99.'l Succinic semialdehyde dehydrogenase EC ì.2.1.16 4-trimethylaminobutyric acid hydroxylase EC l.l4.lt;l v/.L TABLE OF CONTENTS Page Aet¿nowlødgwent's 'L Oøc.t-a.tulion ,1,.L Publica.tion's iii ?nø$aeø v TabLe o{ CoYttønt's vü suMll,{ARy xví PART I: UTILIZATION OF ACETYLCARNITINE IN ANIMAL TISSUES t I Introducti on ì t.l General Introduction I 1.2 Production and utilization of 0-acetyl-L-carnitine 2 1.3 The relationship between carnitine acety'ltransferase and acetyl-CoA hydroìase in the utilization of acetyl- CoA 6 2 Enzymic hydroìysis of 0-acetyl-L-carnitine in liver from rats, sheep and cows 7 2.1 Matei^ials and Methods 7 2.1.1 Animals ... 7 2.1.2 Alloxan-diabetic animals ... 7 2.1.3 Tissue preparations and homogenates 7 2.1 .4 Ti ssue fracti onati on B 2.1.5 Mitochondrial fractionation I 2.1 .6 Instrumentati on 9 2.1.7 Enzyme assays 9 2.1 .7 .1 Carni ti ne acetyl transferase 9 2.1 .7 .2 Enzymi c ut'i I i zati on of 0-acetyl -L- carn i ti ne t0 2.1.7.3 Rotenone-insensitive NADH-cytochrome c reductase l0 2.1 .7 .4 Adenyl ate ki nase It V/,Å/L Page 2.1.7 .5 Succinate dehydrogenase ll 2.1.7 .6 Gìutamate dehydrogenase ll 2.1.7 .7 Lactate dehydrogenase il 2.1.7 .B Gl ucose-6-phosphatase 12 2.1.8 Metabol ite assays . 12 2.1 .9 Preparation of bromoacetyl -L-cårnitine 12 2.I .10 Chemi cal s t3 2 .2 Resuì ts l3 2.2.1 Enzymic util ization of 0-acety'l-L-carnitine by normai sheep I i ver homogenates l3 2.2 .2 Enzymic hydrolysis of acetyìcarnitine by homogenates of normal rat, sheep and cow liver t4 2.2.3 Enzymic specificity of acetylcarnitine hydroìysis ... l4 2 2.4 Substrate specificity of acetyìcarnitine hydrol ase t5 2.2.5 Michaelis-Menton constants for acetylcarnitine hydro'lase from rat and sheep liver t5 2.2.6 Intraceìlular localization of acetylcarnitine hydrolase in rat liver 16 2.2.7 Tissue distribution of acetylcarnitine hydrolysis in rat and sheep 17 2.2.8 Effect of various physiological states on enzymic hydroìysis of acety'l-L-carnitine by homogenates of rat and sheep ìiver 17 3 Carnitine-dependent transfer of acetyl groups from acetyl-CoA across the inner mitochondrial membrane of liver from rats and sheep t7 3.t Materials and methods 17 3.ì.1 Animals ... l7 3.1.2 Tissue preparat'ions IB 3. I .3 Homogenates l8 3..l.4 Subcellular fractions l8 ix Page 3.t.5 Enzyme assays l9 3.t.5.1 Carnitine acetyltra.nsferase t9 3.1.5.2 Acetylcarnitine hydrolase t9 3.1.5.3 Acetyl-CoA hydrolase t9 3.1.5.4 Rotenone-insensitive NADH-cytochrome c reductase 20 3.1.5.5 Adenylate klnase 20 3.1.5.ó Succinate dehydrogenase 20 3.1.5.7 Glutamate dehydrogenase 20 3.1 .6 Metabol ite assays . 21 3.1.7 Chemicals ... 21 3 .2 Resul ts 21 3.2.l Effect of stereoisomers of carn'itine on acetyì-CoA .hydrolase activ'ity in normal and Sephadex G-25 treated homogenates of rat and sheep liver 2\ 3.2.2 Spectrophotometric assay of acetyl-CoA hydrolase and carn i t'ine acetyl transferase 'in normal and Sephadex-treated homogenates from rat liver 22 3.2"3 The Michaelis-Menton kinetics of acetyl-CoA hydroìase and carnitine acetyìtransferase reactions in rat liver homogenates before and after treatment wìth Sephadex G-25 23 3.2.4 Acetyl -CoA hydrolys'is i n rat I i ver mi tochondri a and in inner and outer mitochondrial membrane fracti ons 24 4. Discussion 25 PART II: ASPECTS OF ACETATE METABOLISM IN THE RUMINANTS 32 l. Introduction 32 1.1 General Introduction 32 1.2 Significance of endogenous acetate production in the rumi nant 33 1.3 Origins of endogenous acetate productìon in the ruminant 35 x Page t.4 Precursors of endogenous acetate in the ruminant 37 .l.5 Enzymic product'ion of acetate in the ruminant 39 1.6 Acetate metabolism by the mammary gland 42 1.7 Objectivesof tþis study 43 2 A role for carnitine in enzymic acetate product'ion in liver from dry and ìactating cows 44 2.1 Materials and methods 44 2.1.1 Animals ... 44 2.1.2 Tissue preparation and homogenates 44 2.1.3 Enz¡rme assays 45 2.1.3.1 Carnitine acetyltransferase 45 2.1.3.2 Acetylcarnitine hydrolase 45 2.1.4 Metabol ite assays .
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