lr ßf rþs J Þ I THE RAT PANCREATI C MICROSOIITE ENZYI"ÍE )' i RELEASE PHENOMENON 't{ I i I { A Thesis Submitted for the Degree of Doctor of PhilosoPhY at the UniversitY of Adelaide t I by LINDA MARIE TABE, B.Sc. (Hons.) Department of BiochemistrY University of Adelaide South Australia October L982 CONTENTS Page No. (i) STATEIVIENT ( ACKNOWLEDGE}ÍENTS ii) ( SUMMARY iii) CHAPTER 1 GENERAL INTRODUCTION r.1 Introduction I L.2 Transfer of toxin molecules across membranes 2 1.3 Vectorial transfer of proteins into mitochondria 5 r.4 Vectorial transfer of proteins into other organelles I2 r.5 Theoretical mechanisms of "biosynthetic transfer" of proteins across membranes 16 1.6 Translocation of newly-synthesized proteins across the plasma membrane of prokarYotes 24 L.7 Translocation of newly-synthesized proteins across the ER membrane of eukaryotes 34 1.8 Translocation mechanisms: a sunìmary 49 1.9 The pancreatic microsome enzyme 53 release Phenomenon 55 r. 10 Aim of the investigation CHAPTER 2 MATERIALS AND I4ETHODS 2.I Materials 57 2.2 Methods 59 Page No CHAPTER 3 RESULTS PROTEASE-SENSITIVE A}IYLASE RELEASE FROM MICROSO¡MS INVOLVES THE PASSAGE OF PROTEIN ACROSS AN APPARENTLY INTACT MEMBRANE 3.1 Introduction 72 3-2 Release of amylase from microsomes 73 3.3 Protection of intra-microsomal amylase from proteolysis 76 3.4 Protection of intra-microsomal amylase from iodination 79 3.5 Inhibition of amylase release by proteolysis of the microsomes 83 3.6 Summary and Discussion 85 CHAPTER 4 RESULTS THE EFFECTS OF INHTBITORS OF SIGNAL PEPTIDASE ACTIVITY ON THE RELEASE OF AMYLASE FROM RÀT PANCREATI C MÏCROSOIVIES 4.L Introduction B8 4.2 The effects of inhibitors of eukaryotic signal peptidase activity on amylase release from rat pancreatic microsomes.. 90 4.2.1 Inhibitors of the signal peptidases of dog pancreatic microsomes and of ascites membranes 90 4.2.2 Inhibitors of DOC-solubilized signal peptidase from dog pancreatic microsomes 91 4.2.3 Inhibitors of OBG-solubilized signal peptidase from dog pancreatic microsomes 93 4.3 The effects of inhibitors of Pro- karyotic signat peptidase activity on amylase release from rat pancreatic microsomes 94 4.4 The molecular form of intra- microsomal amYlase 96 4.5 Summary and Discussion 99 CHAPTER 5 RESULTS INHIBITION OF THE PANCREATIC IVIICROSOME ENZYI4E RELEASE PHENOMENON BY DENATURED PROTEINS 5.1 Introduction 105 5.2 Amylase release from rat pancreatic microsomes in the Presence of ovalbumin r06 5.3 Amylase release from rat pancreatic mi-crosomes in the Presence of other Proteins .. .. 107 5-4 Summary and Discussion r09 CHAPTER 6 RESULTS ON THE RAT THE EFFECTS OF NEM AND SALT WASHING PANCREATIC MICROSOME ENZYME RELEASE PHENOMENON 6.1 Introduction 115 6.2 Protease and high salt treatment of rat pancreatic microsomes LI7 6.3 KCl-extraction of rat Pancreatic microsomes L2L 6-4 Protease- or NEM-treatment of salt extract L24 6.5 Inhibition of amylase release from rat pancreatic microsomes bY NEM L26 6-6 Summary and Discussion 130 Page No. CHAPTER 7 RESULTS BETWEEN STUDIES ON A POSSIBLE RELATIONSHIP RELEASE RIBOSOME DISINTEGRA TION AND AMYLASE FROM RAT PANCREA TIC MICROSOI4ES r35 7.L Introduction 7.2 Correlative evidence for a relation- ship between amylase release from rat pancreatic microsomes and the disintegration of membrane-bounél 138 ribosomes 138 7 .2.I Introduction 7.2.2 Comparative rates of amylase release and microsomal membrane degranulation r39 7.2.3 The effects of changes in incubation temPerature on amYlase release and microsomal 140 membrane degranulation 7.2.4 The effects of inhibitors on amylase release and microsomal membrane degranulation 143 7 3 Removal of ribosomes from rat pancreatic microsomal membranes L44 L44 7.3 . I Introduction 11 .2 Degranulation of microsomes 145 with PuromYcin/KCL 7.4 The sensitivitY of enzYme release, r50 to inhibitors r53 7.5 SummarY and Discussion CHAPTER 8 CONCLUDING DISCUSSION 8.r The rat pancreatic microsome enzyme L62 release phenomenon Page No. 8.2 The characteristics of amylase release ]-62 8.3 Suggestions regarding the relevance of the release phenomenon to secretion 166 8.4 Conclusion 175 ABBREVIATIONS BIBLTOGRAPHY (i) STAÎEMENT This thesis contains no material which has been accepted for the award of any other degree or diploma. The studies $/ere carried out in the DepartmenÈ of Biochemistry, University of Adelaide, under the supervision of Professor W.H. Elliott and Dr. B.K. May. The experiments were performed only by myself and to the best of my knowledge this thesis contains no material previously published or written by another person except where due reference is made in the text. (r,rnoa TABE) t. ( ii) I ACKNOWLEDGEMENTS ,{ Þ I am grateful to Professor W.H. Elliott for the I of working in the Biochemistry Department, I opportunity t and for the guidance and friendly encouragement that he and Dr. B.K. May have given me throughout the course of i i my studies. I am also grateful to all the people in the ! to Mike Calder I department who contributed verbal input; i for helping me with the electron microscopy; to Jenny Rosey for her help with the diagrams; and to Lucy and Terror Thommo for their buffer-making and their friendship. I thank Richard for listening; Annie for so many lovely dinners; and the many and varied Lurliners' (both permanent, and part-time) , for a happy and stimulating I fii atmosphere at home. ål fl I am very grateful to Jan Hemming for doing an excellent job of typing the manuscript. I would also like to say a special thank you to our cheerful, efficient departmental secretaries Jane, June and caroline who, despite being perpetually over-v/orked, were always happy to help with bits and Pieces. I dedicate this thesis to Dad, Mum and Ferne for their unwavering confidence, support and thoughtfulness. During the course of this work I was supported by a Commonwealth Postgraduate Research Award. ( iii) .t¡ SUMMARY È^. THE RAT PANCREATIC MICROSOì4E ENZY¡{E RELEASE PHENOMENON It has been re¡rorted that active secretory enzymes are released into the suspending medium when rat pan- creatic microsomes are incubated at 37oC j-n cation-depleted medium. The enzyme release process, which seemed to involve the transfer of fully-formed proteins across intact microsomal membranes, was inhibited by incubation of the vesicles in the Presence of active proteases (Pearce et aL., 1978). The work described in this thesís was directed towards defining the possible relationship between the pancreatic microsome enzyme release phenomenon and the physiological process of secretory protein trans- location across the ER membrane in uiuo. I. It was demonstrated that intra-microsomal amylase was protected from attack by ad.ded subtilisin, although amylase released from the vesicles by incubation at 37oC was readily degraded by the protease. SimiIarly, intra- microsomal amylase was inaccessible to membrane surface- specific labelling with r25T, whereas released, extra- vesicular enzyme was strongly labelled- Brief incubation of microsomes in the presence of a protease was found to retard the subsequent release of amylase from the vesicles when they \¡rere incubated at 37oC, after their removal from contact with the hydrolase. These results supported the previous Suggestions (Pearce et aL., Ig78), that the enzyme release phenomenon involved the passage of secretory proteins across intact microsomal ( iv) ,l I .l't membranes in a manner dependent on the integrity of a Þ- membrane-associated Prote in. ¡ frOm rat pancreatic microsOmes Iâ/aS ,1, 2. Amylase release t inhibited by several protease inhibitors which have been reported to inhibit the activity of the signal peptidase ) I enzyme. Inhibitors which did not affect signal peptidase effect on amylase release' I had no inhibitory I 3. The amylase release process was blocked by denatured ovalbumin and denatured or native BSA, but not by other mature, processed secretory proteins, or by the denatured or native forms of a cytoplasmic protein. The denatured form of catalaser âD enzyme which in uíuo, is transported across the peroxisomal membrane without proteolytic ! "J processirg, also inhibited the release of amylase from fl rat pancreatic microsomes. 4. The treatment of rat microsomes with protease and/or high salt was found to inhibit the amylase release phenomenon. This inhibition $/as partly reversed by the re-addition of the salt extract to the salt-washed membranes. The active constituent of the extract could be destroyed either by proteolysis or by treatment with NEM. 5. The release of amylase from rat pancreatic microsomes u¡as found to be synchronized, with the disintegration of the ribosomes in the vesicle suspensions, durinq incubation at 37oC in the absence of tutg2+. An increase in the sensitivi-ty of the amylase release process to various inhibitors correlated temporally with the complete disappearance of the membrane-bound ribosomes. (v) 6. The similarities between the experimentally- þ determined properties of the rat pancreatic microsome ri enzyme release phenomenon, and the published properties t' proteins ER uioo of the transport of secretory into the in fi are discussed in the light of current theories of trans- membrane protein translocation. I I CHAPTER 1 GENERAL INTRODUCTTON 1 1-l i' ì -. l.r INTRODUCTION Biologicalmembranesarethevitaltyimportant barriers which separate specialized intra-ceIIuIar compartmentsandmaintaintheintegrityofliving cells in the face of hostile external environments. These comPlex structures are permeable to a selected few of the molecules on either side