"Medicine, Faculty of"@en . "Cellular and Physiological Sciences, Department of"@en . "DSpace"@en . "UBCV"@en . "Otte, Susan Carol"@en . "2010-05-20T04:14:45Z"@en . "1984"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Gel filtration coupled with radioimmunoassay of fractions has\r\ndemonstrated the existence of an 8000 dalton immunoreactive form of GIP\r\n(glucose-dependent insulinotropic polypeptide or gastric inhibitory\r\npolypeptide), which may be a precursor in the biosynthetic pathway. A\r\nmonoclonal antibody to GIP has been shown to have highly suitable\r\ncharacteristics for affinity purification of different species of IR-GIP.\r\nAn enzyme-linked immunosorbent assay (ELISA) was developed for GIP,\r\nemploying the monoclonal antibody and was used for screening fractions for\r\npeptides with the same antigenic determinant i.e. IR-foras of GIP.\r\nClassical strategy used in peptide purification may result in loss of\r\nrelated peptides if they are sensitive to the pH or temperature conditions\r\nused. Tissue from hog duodenal and jejunal mucosa was boiled and extracted\r\ninto acetic acid. Peptides were then adsorbed to alginic acid, eluted with\r\n200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The\r\ndesalted material was adjusted to pH 7.0 with 200mM ammonia and extracted\r\nwith methanol. The methanol insoluble fraction demonstrated the highest\r\ncontent of IR-GIP\u00E2\u0082\u0088\u00E2\u0082\u0080\u00E2\u0082\u0080\u00E2\u0082\u0080\u00E2\u008B\u0085 The overall acidic charge on the larger IR-GIP oUUU\r\nmoiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP\u00E2\u0082\u0085\u00E2\u0082\u0080\u00E2\u0082\u0080\u00E2\u0082\u0080 was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A \u00C2\u00B5Bondapak C\u00E2\u0082\u0081\u00E2\u0082\u0088 column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues."@en . "https://circle.library.ubc.ca/rest/handle/2429/24877?expand=metadata"@en . "PURIFICATION AND PARTIAL CHARACTERIZATION OF A PEPTIDE CROSS REACTING WITH ANTIBODIES TO GASTRIC INHIBITORY POLYPEPTIDE by SUSAN CAROL OTTE B.Sc, The University of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Physiology, Faculty of Medicine) We accept t h i s thesis as conforming ta the required standard EXTERNAL EXAMINER THE UNIVERSITY OF BRITISH COLUMBIA December 1984 (ci w Susan Carol Otte, 1984 AUTHORIZATION In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Univ e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r sc h o l a r l y purposes may be granted by the Head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Physiology The U n i v e r s i t y of B r i t i s h Columbia 2146 Health Sciences M a l l Vancouver, Canada V6T 1W5 ABSTRACT Gel f i l t r a t i o n coupled with radioimmunoassay of f r a c t i o n s has demonstrated the existence of an 8000 dalton immunoreactive form of GIP (glucose-dependent i n s u l i n o t r o p i c polypeptide or g a s t r i c i n h i b i t o r y polypeptide), which may be a precursor i n the biosynthetic pathway. A monoclonal antibody to GIP has been shown to have highly s u i t a b l e c h a r a c t e r i s t i c s f o r a f f i n i t y p u r i f i c a t i o n of d i f f e r e n t species of IR-GIP. An enzyme-linked immunosorbent assay (ELISA) was developed f o r GIP, employing the monoclonal antibody and was used for screening f r a c t i o n s f o r peptides with the same antigenic determinant i . e . IR-foras of GIP. C l a s s i c a l strategy used i n peptide p u r i f i c a t i o n may r e s u l t i n loss of related peptides i f they are s e n s i t i v e to the pH or temperature conditions used. Tissue from hog duodenal and jejunal mucosa was bo i l e d and extracted i n t o a c e t i c a c i d . Peptides were then adsorbed to a l g i n i c a c i d , eluted with 200 mM HC1, p r e c i p i t a t e d with NaCl and desalted on Sephadex G-25. The desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted with methanol. The methanol insol u b l e f r a c t i o n demonstrated the highest content of IR-GIPQ,.,.-.. The o v e r a l l a c i d i c charge on the larger I R - G I P oUUU moiety suggested the p o s s i b i l i t y that i t might not be adsorbed to a l g i n i c a c i d . The monoclonal antibody to porcine G I P ^ Q ^ Q was coupled to cyanogen bromide activated Sepharose -4B. The peptide f r a c t i o n which was not adsorbed to a l g i n i c a c i d was applied to the column and the f r a c t i o n which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further p u r i f i e d to a monocomponent by HPLC. A yBondapak C^g column and a l i n e a r gradient of a c e t o n i t r i l e i n water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), A la (8), Val (5), Met (1), H e (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was i d e n t i f i e d as vali n e using the dansylation method. Cleavage of the molecule with t r y p s i n and separation of the t r y p t i c peptides on HPLC showed 2 peptides with e l u t i o n times s i m i l a r to t r y p t i c peptides of GIP. Ap p l i c a t i o n of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate e n t i t i e s . B i o l o g i c a l a c t i v i t y was assessed i n the i s o l a t e d perfused r a t pancreas, a model used for measurement of the i n s u l i n r e l e a s i n g e f f e c t of GIP. IR-LGIP C did not demonstrate i n s u l i n o t r o p i c a c t i v i t y . It i s u n l i k e l y that t h i s polypeptide i s a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. iv TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES . . . . . . . . . . . . . . v i i i LIST OF FIGURES ix LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . xi STATISTICAL ANALYSIS x i i i ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . xiv INTRODUCTION . . 1 METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 I. PEPTIDE PURIFICATION METHODS 7 A. Af f i n i t y Chromatography 7 1. Rationale 7 2. Activation of Sepharose-Cl-4B 7 3. Ligand purification 8 4. Coupling of purified ligand to activated Sepharose 4B . 9 5. Affinity chromatography column 10 B. Gel F i l t r a t i o n . . . . . . . . . . . . . . . . . . . . . . 10 1. Rationale 10 2. Gel preparation 11 3. Sample application 11 C. Ion-exchange Chromatography 12 1. Rationale 12 2. Gel preparation 12 3. Sample Application 13 D. Reverse Phase High Performance Liquid Chromatography (HPLC)..13 1. Rationale 13 2. HPLC equipment and buffers 13 3. Determination of elution range 14 4. Purification of peptide purifications 14 V I I . PEPTIDE ANALYSIS AND QUANTITATION . 15 A. Enzyme-linked Immunosorbent Assay (ELISA) 15 B. Radioimmunoassay (RIA) 17 1. Rationale 17 2. Gastric I n h i b i t o r y Polypeptide . . 17 a) Iodination of GIP 17 b) Assay buffer 19 c) Antiserum 20 d) Standards 20 e) Controls . 20 f ) Non-specific binding 21 g) Unknown or experimental samples 21 h) Assay protocol 21 i ) Charcoal separation 22 j ) Calculations 22 3. I n s u l i n 23 a) Iodination of i n s u l i n 23 b) Assay buffer 24 c) Antiserum 24 d) Standards 25 e) Controls 25 f ) Non-specific binding 25 g) Unknown or perfusate samples 25 h) Assay protocol 25 i ) Charcoal separation 26 C. Thin Layer Chromatography 26 1. S p e c i f i c colour reactions 27 I I I . PEPTIDE CHEMISTRY . . . . . . 27 Fragments of IR-LGIP C 27 1. Cyanogen bromide cleavage . . . . . . . 27 2. Tryptic digestion of IR-LGIP C 28 3. Amino acid analysis 28 4. I d e n t i f i c a t i o n of N-terminal residue 29 v i IV. PHYSIOLOGY OF PURIFIED PEPTIDE (IR-LGIP C) . . . . . 31 Isolated Perfused Rat Pancreas Bioassay 31 1. Reagents 31 a) Krebs concentrate 31 b) Perfusate . . . . . . . . . . . . . . . . . . . . . . . . 31 c) Side arm infusates 32 2. Procedure . . . . . . . . . . . . . . . . . . 32 a) Sur g i c a l preparation 32 b) Apparatus 33 c) Perfusion 33 APPENDIX TO METHODS - CHEMICAL SOURCES . . . . . . . . 34 RESULTS I. PURIFICATION OF IMMUNOREACTIVE-GIP (IR-LGIP C) MOIETY 36 A. A f f i n i t y Chromatography 36 1. Coupling and p u r i f i c a t i o n of ligand 36 2. Characterization 38 3. Capacity determination and p u r i f i c a t i o n 45 B. Gel F i l t r a t i o n . . . . . . . . . . . . . . . . . . . . . . 45 C. Ion-exchange chromatography 50 D. HPLC . . . . . . . . . . . . . . . . . . . . . . 50 1. Terminology 53 2. I n t r i n s i c Artefacts 54 3. Gradient development 54 4. P u r i f i c a t i o n assessment 58 5. Fr a c t i o n a t i o n of IR-LGIP on HPLC . . . . . . . 58 v i i I I . PEPTIDE QUANTITATION AND IMMUNOREACTIVITY . . . . . . . . . . . 64 A. ELISA 64 1. Antibody d i l u t i o n study 64 2. I n h i b i t i o n study 65 B. Radioimmunoassay 72 C. Thin Layer Chromatography 72 I I I . PEPTIDE CHEMISTRY . . . . . . . . . . . . . . . . . . . . . . . 74 Fragments of IR-LGIP C . . . . . . 74 1. Cyanogen bromide cleavage 74 2. Tryptic digestion 74 3. Amino acid analysis 77 4. I d e n t i f i c a t i o n of N-terminal residue 77 IV. PHYSIOLOGY OF PURIFIED IR-LGIP C . . . . . . . . . . . . . . . 79 Isolated Perfused Rat Pancreas Bioassay 79 1. Control Studies . . . . . . . . . . . . . . . . . . . 79 2. GIP Infusion Studies . . . . . . . . . . 79 3. IR-LGIP C Infusion Studies . . . . . . . . . . . . . . . 80 DISCUSSION . . . . . . . . . . . . . . . 86 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 V l l l LIST OF TABLES Table Page 1 Amino acid composition of IR-LGIP C 78 2 Insulin secretion in response to 80 mg% and 160 mg% glucose: control, with GIP infusion and with IR-LGIP C infusion 80 > i x LIST OF FIGURES Figure Page 1 P u r i f i c a t i o n of GIP monoclonal antibody on DEAE Sephacel 37 2 A f f i n i t y chromatography column showing i t s s p e c i f i c i t y for 1 2 5 I GIP 37 3 E f f e c t of 1 2 5 I GIP i n plasma on binding to the immunosorbent column 40 4 Demonstration of s p e c i f i c i t y of a f f i n i t y chromatography system for GIP using porcine G I P 5 0 0 0 ^1 5 Further c h a r a c t e r i z a t i o n of a f f i n i t y chromatography system with GIP p u r i f i c a t i o n side f r a c t i o n EG I I - I I I FrA . . . . . . . . . . . . . . . . . . . . 42 6 Characterization of a f f i n i t y chromatography system with 1 2 5 I I n s u l i n . . . . . . . 43 7 y c J \ M o t i l i n on a f f i n i t y chromatography column . . . 44 8 Scheme of i n i t i a l steps of GIP p u r i f i c a t i o n 46 9 Capacity determination of immunosorbent column for GIP p u r i f i c a t i o n side f r a c t i o n - a l g i n i c a c i d non-adsorbed material 47 1 0 1 g a l g i n i c a c i d non-adsorbed material on immunosorbent column 48 1 1 Desalting immunoreactive component of 250 mg, 500 mg and 1 g of a l g i n i c acid non-adsorbent material . 49 1 2 Ion exchange chromatography of IR-LGIP using DEAE A-25 Sephadex 51 13 Desalting of ion-exchange chromatography f r a c t i o n s . . . 52 X Figure Page 14 Artefacts and background of HPLC system 55 15 Gradient development on HPLC f o r IR-LGIP . . . . . . . 56 16 Optimum gradient for IR-LGIP on HPLC 57 17 DEAE Fr A and DEAE Fr B on HPLC . . . . . . . . . . . . 59 18 P u r i f i c a t i o n of IR-LGIP on HPLC 60 19 IR-LGIP Fr A run on HPLC . . . . . . . . . . . . . . 61 20 IR-LGIP Fr B run on HPLC 62 21 IR-LGIP C run on HPLC . . . . . . . . . . . . . . . 63 22 IR-LGIP C and GIP on HPLC . 65 23 ELISA ( d i r e c t ) determination of GIP immunoreactivity . 67 24 ELISA ( d i r e c t ) determination of IR-LGIP C immunoreactivity 68 25 I n h i b i t i o n studies using ELISA system with anti-GIP monoclonal antibody (3.65H) 69 26 I n h i b i t i o n studies using ELISA and anti-GIP a n t i s e r a (Go5) on plates with 0.06nM GIP 70 27 I n h i b i t i o n ELISA studies with Gb\"5 on plates coated with 0.06nM IR-LGIP C . 71 28 Immunoreactivity determination using GIP radioimmunoassay 73 29 IR-LGIP C t r y p t i c digest on HPLC 75 30 GIP t r y p t i c digest on HPLC . . . . . . . . . . . . 76 31 Control studies i n i s o l a t e d perfused r a t pancreas . . 83 32 GIP i n f u s i o n studies i n i s o l a t e d perfused r a t pancreas 84 33 IR-LGIP C i n f u s i o n studies i n i s o l a t e d perfused rat pancreas 85 x i LIST OF ABBREVIATIONS AUFS BSA CNBr DEAE Fr A DEAE Fr B DNA cDNA EG I I - I I I Fr A ELISA GIP GIPomas HPLC IR-GIP IR-LGIP IR-LGIP C mRNA NSB PBS PSI A r b i t r a r y Units F u l l Scale (a measurement of spectrophotometry absorbance provided by the HPLC system consistent throughout a l l HPLC traces but not nece s s a r i l y corresponding with actual absorbance). Bovine Serum Albumin Cyanogen Bromide F i r s t peak eluted o f f DEAE A-25 Sephadex i n p u r i f i c a t i o n of IR-LGIP Second peak eluted o f f DEAE A-25 Sephadex i n p u r i f i c a t i o n of IR-LGIP Deoxyribonucleic acid Complementary DNA Side Fra c t i o n from f i n a l p u r i f i c a t i o n of natural porcine GIP. Enzyme Linked Immunosorbent Assay. Gastric Inhibitory Polypeptide (Glucose-dependent Insulinotropic Polypeptide). GIP producing tumour tis s u e High Pressure Liquid Chromatography Immunoreactive GIP Immunoreactive-like GIP Immunoreactive-like GIP (Fraction C of IR-LGIP off HPLC) Messenger r i b o n u c l e i c acid Non-specific binding Phosphate Buffered Saline Pounds per Square Inch x i i RAMIg Rabbit Anti-Mouse Immunoglobulin RT Room temperature RIA Radioimmunoassay SARIg Sheep Anti-Rabbit Immunoglobulin SDS Sodium-Dodecyl Sulphate TFA Trifluoroacetic acid TLC Thin Layer Chromatography x i i i STATISTICAL ANALYSIS Sta t i s t i c a l analyses of results presented in this thesis were performed using the paired Student's t-Test via a Hewlett Packard 41 - C calculator program. xiv ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my supervisor, Dr. John C. Brown. His endless friendship and support have made this possible. I also wish to thank Dr. John Brown for providing the invaluable opportunity to work with his associates and colleagues. 1. Introduction Protein i s o l a t i o n and p u r i f i c a t i o n involves a sequence of steps which vary i n s p e c i f i c i t y from peptide to peptide but for a p a r t i c u l a r substance should successively r e s u l t i n increased p u r i t y and a c t i v i t y of the material (Lehninger, 1975; Schulz and. Schirmer, 1979). A r i c h source of t i s s u e i s required since i n i t i a l steps are r e l a t i v e l y crude and do not have much resolving power. P r e c i p i t a t i o n of large molecules i n e a r l y stages may through a l t e r a t i o n of various c h a r a c t e r i s t i c s ( e l e c t r o s t a t i c i n t e r a c t i o n , hydrogen bonding, hydrophobic and dispersion forces) produce loss of or mask separation of desired products. For example, the discarded f r a c t i o n s may contain large molecular weight forms which may be precursors or biosynthetic intermediates of the substance being p u r i f i e d . With a good source of material steps may be var i e d to r e t r i e v e masked products. Prohormones are c l a s s i c a l l y considered to be the precursors of the bio a c t i v e moiety, possible i n h i b i t o r s of receptor binding, promoters of transfer to secretory v e s i c l e s and s t a b i l i z e r s of structure (Steiner et a l , 1980). Large molecular weight forms possibly represent a streamlining of structure and function i n the evolutionary process. For example, adr e n o c o r t i c o t r o p i n / l i p o t r o p i n (ACTH/endorphin) i s a multihormone prohormone composed of adrenocorticotropin 1-39, 3 - l i p o t r o p i n , 3-endorphin, c o r t i c o t r o p i n - l i k e intermediate lobe peptide and ot-melanotropin (Mains and Eipper, 1980). Protein biosynthesis e n t a i l s several complex steps which decode the nuclear DNA (gene) to form a b i o l o g i c a l l y active protein (Habener and Potts, 1978; Rubenstein et a l , 1975). B r i e f l y , t r a n s c r i p t i o n of RNA from the DNA 2. template i s followed by post transcriptional modification of RNA to produce messenger RNA (mRNA). mRNA translates i t s message to the cel lular machinery to i n i t i a t e protein synthesis of specific molecules i n the ribosomes of the endoplasmic reticulum. Subsequently, the amino acid assembly is subjected to a possible variety of reactions referred to as post translational modification. At this stage intracel lular proteolyt ic - l ike cleavage of larger moieties to intermediates or bioactive forms and packaging into secretory granules may take place or cleavage may occur following secretion. Glycosylation, phosphorylation and folding of proteins are further examples of processes that may occur at this stage. It i s well documented that the synthesis of secretory proteins frequently involves precursor forms (Habener and Potts, 1978; Judah et a l , 1973; Steiner et a l , 1967; Schechter et a l , 1980) which are simply extensions of the secreted moiety and are cleaved either i n t r a - or extracellularly prior to the active form interacting with i t s specific receptor or target. Common properties of pro-hormones have evolved and provided insight into their role i n the regulatory process of the active hormone both physiologically and pathophysiologically. Generally i t has been established that precursors are N-terminally extended bioactive peptides; that paired basic amino acid residues occur at the site of cleavage between the extension and the active segment and that the mid-region of the extension i s composed of hydrophobic amino acids. Furthermore, the central hydrophobic region may form a 3 -pleated sheet structure with in t r ins ic membrane proteins f a c i l i t a t i n g enzymatic cleavage of the extension, the carboxyl end of the hydrophobic fragment is often composed of amino acid residues with small neutral side chains and f i n a l l y 3. precursor forms show l i t t l e biological activity (Gammeltoft and Gilemann, 1973; Goltzman et a l , 1976; Judah et a l , 1973; Schechter et a l , 1980). It is these similarities of physical properties that enable one to determine i f a large moiety is a precursor, intermediate or simply another peptide. Purification of several precursor hormones has employed techniques that take advantage of common properties between the larger moiety and the bioactive smaller substance. Classical examples of c e l l secreted proteins with a precursor formed prior to eventual release of the active polypeptide are insulin and parathyroid hormone (Goltzman et a l , 1976; Steiner et a l , 1967). For several gastrointestinal hormones - glucagon, somatostatin and gastrin, larger molecular weight forms have been described (Hakanson et a l , 1982; Patzelt et a l , 1979; Patzelt et a l , 1980; Ravazzola and Orci, 1980; Tager and Steiner, 1973). Extraction and purification of these various peptides ut i l i z e d pulse chase experiments, acid-ethanol precipitation, immunoprecipitation and gel f i l t r a t i o n chromatography techniques. Due to the diffuse nature of the gastrointestinal endocrine system i t is very d i f f i c u l t to isolate and purify sufficient amounts of these peptides. Gut hormones are dispersed throughout the gastrointestinal mucosa rather than being localized in identifiable glands. As a result as much as 70 kg of mucosal tissue may be required to obtain 1-2 mg of purified peptide. Since the gut is a diffuse rich source of peptides the existence of peptide secreting tumour tissue has provided a distinct advantage in the isolation of larger molecular weight forms, for example, progastrin and proinsulin (Rehfeld et a l , 1976; Steiner and Oyer, 1966). Rapid and localized proliferation of tumour tissue provided an excellent source of both secreted hormones and related larger molecular weight species. It facilitated extraction, isolation and determination of synthesis of humoral agents. 4. G a s t r i c I n h i b i t o r y Polypeptide or Glucose-dependent Insul i n o t r o p i c Polypeptide (GIP) i s a member of the g a s t r o i n t e s t i n a l endocrine system. Comparable to other peptides located throughout the gut mucosa, GIP may also be derived from a larger r e l a t e d moiety. GIP i s a hormone of 42 amino acids which has been i s o l a t e d and p u r i f i e d from porcine duodeno-jejunal mucosa ( J o r n v a l l et a l , 1981). I n i t i a l studies which led to i t s ultimate p u r i f i c a t i o n and sequencing were based on i t s g a s t r i c a c i d i n h i b i t o r y properties observed i n denervated pouches of the dog stomach (Brown et a l , 1970; Pederson and Brown, 1972). I t was p u r i f i e d from a side f r a c t i o n of impure CCK-PZ preparations. Since these e a r l y studies were performed, i t has been shown that GIP i s devoid of g a s t r i c a c i d i n h i b i t i o n i n the i n t a c t innervated stomach (Soon-Shiong et a l , 1979a; Soon-Shiong et a l , 1979b). The possible explanation for these observed differences was the release of somatostatin i n response to GIP. GIP was shown to stimulate the release of somatostatin from the p a r i e t a l c e l l area of the rat stomach. Somatostatin i s a potent i n h i b i t o r of acid secretion. Vagal stimulation and acetyl c h o l i n e administration i n h i b i t e d the release of somatostatin stimulated by intravenous i n j e c t i o n of GIP (Mcintosh et a l , 1981). The acid i n h i b i t o r y e f f e c t of GIP was not observed i n the i n t a c t , innervated stomach. GIP has an enterogastrone e f f e c t (Kosaka and Lim, 1980) which i s i n d i r e c t . The enterogastrone a c t i o n as a p h y s i o l o g i c a l t e s t i n g of pure natural GIP (1-42) and synthetic preparations has revealed that the peptide and not a contaminant or IR-form has the enterogastrone a c t i v i t y . GIP has another important p h y s i o l o g i c a l r o l e , i t i s i n s u l i n o t r o p i c i n the presence of hyperglycaemia (Pederson and Brown, 1976) and i t has been shown to be a major hormone of the enteroinsular a x i s , i . e . gut regulation 5. of pancreatic endocrine secretion. GIP i s released i n response to two secretagogues: f a t and glucose. Ingestion of f a t or glucose r e s u l t s i n a r i s e i n c i r c u l a t i n g immunoreactive-GIP (IR-GIP) l e v e l s from approximately 200-400 pg/ml to 1000-1500 pg/ml. The onset of t h i s response i s immediate peaking at 90 minutes following f a t and 45 minutes a f t e r glucose intake. The duration of action i s 3h for f a t and 2h for glucose (Brown 1973). Studies by Polak et a l , (1973) using immunohistochemistry have l o c a l i z e d GIP c e l l s i n the duodeno-jejunal regional of the gut and showed that they were the c e l l s formerly considered to be K - c e l l s . Following the p u r i f i c a t i o n of GIP Kuzio et a l , (1974) developed a radioimmunoassay (RIA) f o r measurement of c i r c u l a t i n g immunoreactive-GIP l e v e l s (IR-GIP). To date, c l a s s i c a l p u r i f i c a t i o n methods have been employed i n attempt to e s t a b l i s h the presence of a precursor form of GIP. Tissue and serum extraction u t i l i z i n g p r e c i p i t a t i o n , gel f i l t r a t i o n chromatography and RIA were performed. Lack of described GIPomas (tumour ti s s u e ) necessitated the extraction of normal tis s u e and normal serum following provocative t e s t s . Three d i s t i n c t immunoreactive peaks were i d e n t i f i e d following gel f i l t r a t i o n on Sephadex G*50 (Pharmacia, Sweden). The f i r s t immunoreactive peak eluted at a p o s i t i o n comparable to molecular weight of about 9,000 daltons, the second comparable to 7,500-8000 daltons and the t h i r d peak eluted at a p o s i t i o n equivalent to porcine GIP of molecular weight 5000 daltons. High voltage electrophoresis of the pooled, l y o p h i l i z e d peaks indicated that the la r g e r molecular weight form (7500-8000 daltons) was a c i d i c (Dryburgh, 1977). Attempts were subsequently made to further p u r i f y the larger species by ion exchange using anion exchange chromatography on DEAE A-25 Sephadex (Brown et a l , 1979). This method separated two of the moieties showing they were not a r t i f a c t s of extraction techniques. 6. Another characteristic of precursor peptides i s the presence of antigenic determinants common to the bioactive peptide. In the case of the ACTH/endorphin family of peptides, antibodies directed to the middle region of ACTH 1-39 which were not affected by C-terminal or N-terminal extensions were employed in conjunction with immunoprecipitation, sodium dodecyl (SDS) gel electrophoresis and gel f i l t r a t i o n to isolate and purify the prohormones (Eipper and Mains, 1980; Mains and Eipper, 1980). The larger GIP moiety has demonstrated common immunoreactivity with the bioactive GIP molecule (Brown et a l , 1979; Dryburgh, 1977). Absence of tumour tissue, suitable bioassays and diffuse distribution indicated that an immunological approach was reasonable for attempted purification of the larger molecular weight form. In the course of GIP purification several precipitation steps for removal of large molecules were undertaken and in the process related forms of GIP were possibly eliminated. Recently a monoclonal antibody to GIP was developed (Buchan et a l , 1982) and i t s binding traits suggested i t would be ideally suited as a ligand in a f f i n i t y chromatography. The investigation of the existence of related IR-GIP forms i n GIP purification side fractions is the subject of the research presented in this thesis. Exploitation of common antigenicity u t i l i z i n g an exquisitely selective a f f i n i t y chromatography system is the approach undertaken. The GIP monoclonal antibody designated 3.65H is specific for the mid to the C-terminal portion of the GIP molecule (Buchan et a l , 1982) and since most precursor molecules are N-terminal extensions of the bioactive peptide, binding interference should not occur. Such a ligand should provide a means of selecting not only very specific antigenic substances but most probably larger substances that are N-terminally extended. Whether or not the larger species are precursors requires elucidation of amino acid sequences for confirmation. 7. METHODS I. Peptide P u r i f i c a t i o n Methods A. A f f i n i t y Chromatography 1. Rationale A f f i n i t y chromatography i s a technique which allows p u r i f i c a t i o n of molecules by s p e c i f i c a l l y and r e v e r s i b l y absorbing substances to an immobilized ligand with an exposed complementary binding s i t e . Antibodies are frequently u t i l i z e d as the ligand and those substances with s p e c i f i c antigenic determinants are adsorbed and contaminants separated due to lack of s p e c i f i c i t y . Desorption of bound material i s achieved by lowering pH of eluant, using chaotropic agents or denaturing agents. I t i s a process of weakening the binding between the ligand and s p e c i f i c a l l y bound material. Any agent that weakens non-covalent forces, that i s , e l e c t r o s t a t i c i n t e r a c t i o n s , hydrogen bonding and/or hydrophobic i n t e r a c t i o n s s u c c e s s f u l l y separates the p u r i f i e d substance. 2. A c t i v a t i o n of Sepharose -CL-4B Sepharose -CL- 4B (Pharmacia, Sweden) was selected as the support and i t was activat e d with cyanogen bromide to produce ligand binding s i t e s . Equal volumes of Sepharose -CL-4B (30 ml) and deionized d i s t i l l e d water were mixed together i n an i c e bath f o r 15 min. Cyanogen bromide at a concentration of 250 mg/ml i n dimethylformamide was added dropwise (33 mg/ml beads) over 1 min, i n the fume hood. The pH was maintained between 10.5 and 8. 11.0 f o r 15 min with 4M NaOH. When the pH remained stable with no further a d d i t i o n of NaOH, the reaction was considered complete. The mixture was washed i n a glass funnel with 250 ml c o l d , deionized, d i s t i l l e d water and then with 500ml cold 0.05M borate buffer. 3. Ligand p u r i f i c a t i o n A monoclonal antibody to GIP designated 3.65H was generated i n Balb/c mice using GIP conjugated to keyhole limpet haemocyanin (KLH). The animals were immunized subcutaneously with 10 nmoles of GIP conjugated to KLH i n 50% Freund's complete adjuvant and boosted 30 days l a t e r . The mouse with the best serum response determined by ELISA was selected f o r fusion. Splenocytes were fused with SP/2 myeloma c e l l s as described by Oi and Herzenborg, 1980. Fusion products were cultured and culture supernatants were tested f o r antibody secretion using the ELISA technique ( V o l l e r et a l , 1976). Those i n d i c a t i n g the presence of antibodies to GIP were subcultured and cloned and the c e l l l i n e was grown up as an a s c i t e s i n i r r a d i a t e d outbred mice pre-treated with 2, 3, 10, 14 - tetramethylpentadecane. Ascites f l u i d (20 ml) was f i l t e r e d through Whatman #4 paper (Whatman, England) and the f i l t r a t e adjusted to pH 8.0 with 0.2M a c e t i c a c i d . The s o l u t i o n was applied to an anion exchange column, DEAE Sephacel (Pharmacia, Sweden). The column (2.5 x 10 cm) was previously e q u i l i b r a t e d with 0.050M Tris-hydrochloride buffer pH 7.4 and eluted with a stepwise i o n i c strength gradient (0-0.2M NaCl i n T r i s b u f f e r ) . Two peaks were eluted, the f i r s t eluted with e q u i l i b r a t i n g buffer and the second with 0.2M NaCl. Each f r a c t i o n was pooled and p a r t i a l l y p u r i f i e d by ammonium 9. sulphate[(NH^^SO^] precipitation. Saturated ammonium sulphate was added to each fraction to a f i n a l concentration of 50%. The precipitates were centrifuged for 15 min at 4\u00C2\u00B0C at 11,000 rpm using a Sorvall RC-5B centrifuge (Dupont Instruments, Conn). The sedimented fractions were reconstituted in 0.05M sodium borate buffer pH 8.0 and dialyzed against 2 1 of sodium borate pH 8.0 for 60h. At every stage of purification, elution from DEAE Sephacel ammonium sulphate precipitation, centrifugation and after dialysis fractions were assayed for immunoreactivity employing the ELISA method. Fraction II consistently showed most immunoreactive activity. 4. Coupling of purified ligand to activated Sepharose 4B An i n i t i a l absorbance reading at 280nm was taken on the ligand. An equal volume of activated beads and monoclonal antibody to GIP 3.65H (5-10mg/ml) in 0.05M borate buffer was mixed overnight at 4\u00C2\u00B0C. The mixture was then f i l t e r e d on a buchner funnel and the absorbance of the f i l t r a t e measured at 280nm to estimate the degree of coupling. The coupled gel was washed with 0.01M Tris buffer pH 8.0 and resuspended in 25ml of 0.16M ethanolamine to block unreacted active groups on the gel matrix. Stock ethanolamine was diluted with 0.05M borate buffer to a concentration of 0.16M and adjusted to pH 8.0 with 6M hydrochloric acid. Ethanolamine treated coupled gel was l e f t for 2h at 4\u00C2\u00B0C and subsequently washed on a buchner funnel with 0.01M Tris buffer. Beads were stored at 4\u00C2\u00B0C in Tris buffer with 0.2% sodium azide. 10. 5. A f f i n i t y chromatography column The Sepharose -CL- 4B coupled with anti-GIP monoclonal antibody (3.65H) was poured i n t o a column 1.5 x 10 cm and e q u i l i b r a t e d at room temperature for several hours with 0.05M T r i s buffer pH 7.4. M a t e r i a l to be p u r i f i e d , a l g i n i c a c i d non-adsorbed f r a c t i o n , was dissolved i n s t a r t i n g b uffer, centrifuged to remove p r e c i p i t a t e and the pH of the supernatant adjusted to 5.25 with 0.1M ammonia. Four m i l l i l i t r e f r a c t i o n s of the eluant were c o l l e c t e d and monitored at 280nm through a 1cm l i g h t path i n a Pye Unicam spectrophotometer Sp8-100 (Canlab, Canada). Those substances which did not adsorb to the ligand eluted i n the T r i s buffer (0.05M pH 7.4) and the material bound to ligand was eluted with 0.1M HC1. The respective f r a c t i o n s were pooled and rotary evaporated on a Biichi Rotavapor R110 (Brinkman, New York) and subsequently desalted on Sephadex G25. The a f f i n i t y chromatography column was characterized before use, for i t s immunoreactive \u00E2\u0080\u00A2 V \u00E2\u0080\u00A2 1 2 5T rTT> 1 2 5T T T 1 2 5T \u00C2\u00AB \u00C2\u00AB. \u00E2\u0080\u00A2 1 \u00E2\u0080\u00A2 s p e c i f i c i t y , by passing I GIP, I I n s u l i n , I M o t i l i n , porcine GIP and GIP p u r i f i c a t i o n side f r a c t i o n s through the column. B. Gel f i l t r a t i o n 1. Rationale The g e l support acts as a molecular seive and separation depends on the a b i l i t y of molecules to enter the stationary gel or remain i n the mobile phase outside the g e l . Large molecules do not enter the gel pores but pass between them, whereas, smaller molecules are retarded i n movement by the degree of entry i n the g e l . Peptides are, therefore eluted i n decreasing molecular s i z e . 11. 2. Gel preparation Sephadex G-25 (Pharmacia, Sweden) was the bed support used in this study. The appropriate weight of gel was suspended in excess buffer (0.2M acetic acid) at room temperature and l e f t for several hours or overnight. The fines were decanted and the gel was de-aerated under vacuum for 30 minutes. The column (0.9 x 100 cm) was mounted vertically and approximately 10 cm of buffer was poured in before pouring the slurry of gel into the closed column in one pass. I n i t i a l packing of the column occurred under gravity t i l l the gel reservoir could be removed, then the buffer reservoir was connected and column packing was completed under hydrostatic pressure. A f i l t e r disc (Whatman 3MM paper) was placed on top of gel to stabilize gel-liquid interface. The column was equilibrated overnight. Between runs the column was stored i n buffer containing 0.01% sodium azide as a preservative. 3. Sample application The buffer above the gel was removed and the sample dissolved in a small volume (0.5-1.0 ml) of buffer was applied and allowed to settle into the gel. A volume of buffer comparable to that used to dissolve the sample was run i n after the sample. Buffer was then placed above the gel and the column connected to a buffer reservoir. Fractions of 1.5ml were collected at a flow rate of 18 ml/h and monitored by measuring absorbance at 280nm in a spectrophotometer. Peaks were pooled, lyophilized and subjected to further purification. Elution of the salt peak was recorded with a type CDM 2d conductivity meter (Radiometer, Copenhagen). 12. C. Ion-exchange Chromatography 1. Rationale Ion exchange chromatography is a technique used to separate molecules according to charge. The gel support carries either positive (anion exchanger) or negative (cation exchanger) charges. Peptides bind to the support with varying a f f i n i t y depending on their net charge and are eluted by increasing the ionic strength of the solvent. Cation exchange separates substances in order of alkalinity and anion exchange separates substances i n order of increasing acidity. 2. Gel preparation An anion exchanger, DEAE A-25 Sephadex (Pharmacia, Sweden) was the chosen gel and since i t was purchased in salt form no pre-cycling in acid and a l k a l i was required before equilibration. The required weight of gel was swollen i n starting buffer (0.05M ammonium acetate, pH 6.0) for 2 days at room temperature. The supernatant was decanted several times and replaced with fresh buffer during this period. After swelling the gel was washed extensively on a Buchner funnel with starting buffer and resuspended in the same as a slurry, and poured into a vertically supported column (0.9 x 10 cm). The gel was permitted to settle by gravity then connected to a buffer reservoir and allowed to settle further under hydrostatic pressure as described for gel f i l t r a t i o n . The column was equilibrated for several hours at room temperature, equilibration was considered complete when inflow and outflow matched in pH and conductivity. Between runs the column was stored in 1.0M acetic acid with 0.01% sodium azide to prevent deterioration. Reswelling was not necessary between runs but adequate equilibration was important. 13. 3. Sample a p p l i c a t i o n The sample was dissolved i n 2 ml s t a r t i n g buffer (0.05M ammonium acetate pH 6.0), and applied to top of the g e l following removal of excess buffer. When the sample s e t t l e d into the matrix the buffer r e s e r v o i r was connected and 1.5 ml f r a c t i o n s were c o l l e c t e d at a flow rate of 12 ml/h. Eluant was monitored as described for gel f i l t r a t i o n . A stepwise gradient of increasing i o n i c strength was used i n t h i s system, strongly adsorbed material was removed with 1.0M a c e t i c a c i d . D. Reverse Phase High Performance L i q u i d Chromatography - HPLC 1. Rationale HPLC (reverse phase) i s a competitive process, based on p o l a r i t y , between adsorbed substances and solvent for s i t e s on the support. HPLC provides rapid separation of peptides due to fast flow rates that can be attained i n such a system. 2. HPLC equipment and buffers Samples were applied to a uBondapak C-18 column (3.9 x 300 mm) using a Waters U6K i n j e c t i o n system (Waters Associates, Inc., Mississauga). The column matrix was s i l i c a with attached hydrocarbon chains - eighteen carbon atoms i n length. Outflow passed through a Waters Model 450 Variable Wavelength Detector. A Hewlett Packard Integrator 3380A (Hewlett Packard, C a l i f . ) p l o t t e d the absorbance p r o f i l e s . Water and non-polar solvent a c e t o n i t r i l e (CH^CN) each with 0.1% t r i f l u o r o a c e t i c a c i d were separately de-aerated under vacuum for 10 min and 14. then placed i n reservoirs reserved for HPLC solvents. Gradients of increasing polarity were mixed by regulated control of pumping speed of two pumps (H20 - Pump A, Waters Model M-45; CH-jCN-Pump B, Waters Model 6000A). A constant solvent flow rate was maintained although solvent composition varied. 3. Determination of elution range The Waters U6K injection system for sample application ensured that samples were applied to the mixed solvents just prior to the point that the flow entered the column. I n i t i a l l y a broad linear gradient 0-70% acetonitrile over 10 min was employed to ascertain the acetonitrile elution concentration at which peptides eluted. This information aided in the development of a purification strategy. When the acetonitrile concentration range for a particular peptide had been determined gradient duration was altered to obtain optimum resolution (Dahl, 1983). 4. Purification of peptide preparations Once conditions of resolution had been established purification of the 'peaks' could be carried out. For analytical purposes 10-20 ,yg of peptide were applied, whereas, for preparative or purification studies, approximately 500 yg aliquots of samples were used. The sensitivity for analytical applications was generally set at 0.1 Arbitrary Units Full Scale (AUFS) but for purification of 500 jig of material this was changed to 0.4 AUFS so that the detection limit was not exceeded. Spectrophometric detection occurred simultaneously with column outflow which permitted peak 15. c o l l e c t i o n to coincide with i t s recording. Injection of 500 yg al i q u o t s were repeated several times and the desired f r a c t i o n s were pooled and a c e t o n i t r i l e removed by rotary evaporation. For t h i s step a Buchii Rotavapor R110 was used. The evaporated material was taken up i n 0.1M ac e t i c a c i d and l y o p h i l i z e d on an Edwards L y o p h i l i z e r (Edwards, England). Rechromatography of the p u r i f i e d material on HPLC was undertaken to check the p u r i t y of the preparation. The y i e l d of a p a r t i c u l a r protein was calculated as the amount of protein present i n the appropriate f r a c t i o n divided by the t o t a l amount of p r e p u r i f i c a t i o n protein. Desalting on Sephadex G-25 and subsequent r e l y o p h i l i z a t i o n produced f r a c t i o n s from which more accurate y i e l d s could be calculated as stated above. I I . Peptide Analysis and Quantitation A. Enzyme-linked Immunosorbent Assay (ELISA) The ELISA technique i s a s o l i d phase assay which provides a r e l a t i v e l y quick, s e n s i t i v e and isotope free method of quantifying antibodies and determining immunoreactivity of p a r t i c u l a r peptides ( V o l l e r et a l , 1976). Ninety-six w e l l Falcon 3912 m i c r o t i t r e plates (Becton Dickinson, C a l i f . ) were coated with e i t h e r GIP or IR-LGIP C at concentrations of 0.06 nM i n carbonate buffer pH 9.6 (0.02% NaN^, 0.01 M Na 2C0 3, 0.03 M NaHC0 3). One hundred m i c r o l i t r e aliquots were used. The plates were incubated overnight at 4\u00C2\u00B0C. Aft e r washing with PBS-Tween (phosphate buffered s a l i n e with 0.5% Tween-20) various concentrations of GIP monoclonal antibody (3.65H) or GIP antiserum (Gb5) were applied i n 0.1 ml al i q u o t s and plates were incubated f o r 1.5h at room temperature. Plates were then washed and 16. the developing a l k a l i n e phosphatase l a b e l l e d rabbit anti-mouse immunoglobulin (RAMIg) or sheep a n t i - r a b b i t immunoglobulin (SARIg) was applied i n 0.1 ml a l i q u o t s at d i l u t i o n s of 1:3200 and 1:2500 re s p e c t i v e l y . The a l k a l i n e phosphatase l a b e l l e d RAMIg and SARIg were produced by Dr. Levy (Dept. of Microbiology, UBC). A f t e r incubation of the second antibodies (RAMIg and SARIg) for 1.5h at 22\u00C2\u00B0C the plates were again washed with PBS-Tween. One hundred m i c r o l i t r e a l i q u o t s of the enzyme substrate s o l u t i o n (Sigma 104-105) at a concentration of 5mg i n 5ml 10% diethanolamine buffer were then added. The reaction was permitted to continue f o r 30-120 min at room temperature and the colour development was monitored at 405nm using a MR 580 M i c r o e l i s a reader (Dynatech Laboratories, V i r g i n i a ) . The immunological c r o s s - r e a c t i v i t y of the IR-LGIP C peptide was assessed by i n h i b i t i o n t e s t s using the ELISA system. Varying concentrations of GIP and IR-LGIP C were incubated with either the monoclonal antibody 3.65H or p o l y c l o n a l antibody GS5 at h a l f the f i n a l d i l u t i o n to be used (5 g/ml or 1:2500 resp e c t i v e l y ) for 2h at 22\u00C2\u00B0C. Subsequently 0.1ml a l i q u o t s of the mixture were transferred i n t r i p l i c a t e to plates previously coated overnight with GIP or IR-LGIP C (0.006nmoles/well). The plates were incubated f o r 1.5h at room temperature then washed with PBS-Tween and further incubated with RAMIg (1:3200) or SARIg (1:2500) for 1.5h. The plates were then washed again and developed with the enzyme substate s o l u t i o n for 30-120 min at R.T. and absorbance read at 405 nm. A l l d i l u t i o n s and incubations were c a r r i e d out i n PBS-Tween pH7.4 unless otherwise stated. Similar tests were performed with normal mouse serum and normal rabb i t serum to act as non-specific binding determinants. 17. B. Radioimmunoassay 1. Rationale Radioimmunoassay i s a technique based on a competitive binding p r i n c i p l e . Constant concentrations of antibody and r a d i o l a b e l l e d antigen are used but varying concentrations of unlabelled antigen are introduced to create competition between the two antigens ( l a b e l l e d and unlabelled) f o r the antibody. Increasing concentrations of unlabelled antigen (standards) are included to produce displacement of r a d i o l a b e l l e d antigen and to provide a comparative measurement f or the samples. The method can be used to give s e n s i t i v e determinations of peptide c i r c u l a t i n g l e v e l s . To achieve t h i s a r a d i o l a b e l l e d peptide with a high r a t i o of isotope to polypeptide (high \" s p e c i f i c a c t i v i t y \" ) i s necessary. The incorporation of radioactive iodine 125 ( I) into tyrosine residues, i s the common method used. Secondly, an antigen s p e c i f i c and high a f f i n i t y antibody i s required. 2. Gastric Inh i b i t o r y Polypeptide a) Iodination of GIP Phosphate buffer was used throughout t h i s procedure and was prepared by t i t r a t i n g 1 1 of 0.4M Na^PO^ with 0.4 M NaH 2P0 4- H 20 to pH7.5. The following solutions were prepared f o r use i n the io d i n a t i o n procedure: 18. ( i ) 5.0 mg Chloramine-T i n 10 ml of 0.4M phosphate buffer pH 7.5. ( i i ) 20 mg sodium metabisulphite i n 10 ml of 0.4M phosphate buffer pH 7.5. ( i i i ) GIP (porcine) 15 yg f r e s h l y weighed on a Cahn microbalance i n t o a s i l i c o n i z e d glass test tube (12 x 75 mm) and dissolved i n 20 y l 0.4M phosphate buffer pH 7.5. The reagents were then mixed together i n the r e a c t i o n vessel containing GIP according to the following protocol: lmCi N a 1 2 5 I i n 10 y l NaOH so l u t i o n (pH 7-11) 20 y l porcine GIP (15 yg) i n 0.4 M phosphate buffer, pH 7.5 5 y l chloramine-T (2.5 yg) 15 second exposure with gentle mixing 10 y l sodium metasulphite (20 yg) with gentle mixing 125 P u r i f i c a t i o n of the I-GIP was by g e l f i l t r a t i o n on a G-25 Sephadex column (0.9 x 10 cm) eluted with 0.2M a c e t i c a c i d with 0.5% Bovine Serum Albumin (BSA) and 2.0% t r a s y l o l ( p r o t e o l y t i c enzyme i n h i b i t o r ) . The column was previously e q u i l i b r a t e d with t h i s eluant. The i o d i n a t i o n mixture was applied to the column and eluted at a flow rate of 24 ml/h. Four hundred m i c r o l i t r e f r a c t i o n s were c o l l e c t e d and 10 y l al iq uo ts counted for 0.1 min. The column p r o f i l e was plotted - counts/10 yl/O.lmin against f r a c t i o n 125 number. Two peaks were obtained, the f i r s t representing I GIP and the 125 second free I. Samples across the f i r s t peak were d i l u t e d to 5000 cpm/100 y l (that deemed optimal for RIA as developed by Kuzio et a l , 1974) 19. and added to 900 y l assay diluent (0.04M P0 4 pH 6.5 1.5% t r a s y l o l and 5% charcoal extracted plasma). Charcoal separation was c a r r i e d out by addition of 200 y l of dextran coated charcoal (1.25% carbon decolourizing N o r i t , 0.25% dextran T-70 i n 0.04M phosphate buffer pH 6.5 with 5% charcoal extracted plasma) and subsequent c e n t r i f u g a t i o n at 3000 rpm, 4\u00C2\u00B0C for 30 min. The supernatant was decanted and both i t and the charcoal p e l l e t were counted using a 1285 y-counter (Searle Instruments, I l l i n o i s ) . The fr a c t i o n s with best adsorption to charcoal were pooled and d i l u t e d 1:1 with eluant and a c i d ethanol (1500 ml ethanol; 500 ml H^O; 30ml concentrated HC1) to a f i n a l concentration of 2.5 x 10 cpm/100 y l and stored at -20\u00C2\u00B0C. The s p e c i f i c a c t i v i t y of the l a b e l was not r o u t i n e l y calculated since i t c o n s i s t e n t l y showed a s p e c i f i c a c t i v i t y of approximately lOOmCi/mg. Fresh iodinations were performed every 4-6 weeks. b) Assay buffer The diluent buffer used i n the GIP RIA was 0.04M phosphate buffer pH 6.5 made by t i t r a t i n g 0.4MNaH2P04 H 20 with 0.4M Na 2HP0 4 to pH 6.5 and d i l u t i n g 1:10 for the assay. The buffer contained 5% charcoal extracted plasma prepared as follows: 1) Outdated bloodbank plasma was f i l t e r e d through sharkskin paper, then mixed with 1% Carbon Decolourizing Norit f o r l h . 2) Mixture was centrifuged at 4\u00C2\u00B0C, 10,000 rpm for 30 min. 3) Supernatant was f i l t e r e d , aliquoted and stored at -20\u00C2\u00B0C. 20. c) Antiserum The antiserum used was Go 5 (28/4/75). It was raised in rabbits against GIP (porcine) conjugated to BSA by the carbodiimide method and injected emulsified 1:1 with Freund's complete adjuvant. The antiserum was stored, lyophilized i n 200 VI aliquots or frozen i n 2ml (1:10 dilution) 3 aliquots. The f i n a l dilution used in the RIA was 1:30 x 10 which produced 25-30% zero binding. The antiserum at this dilution detected IR-GIP concentrations within the range of 0.125 to 4.0ng/ml. Levels found to be below 0.125ng/ml were considered to be non-detecable that is less than 0.125ng/ml and those levels above 4 ng/ml were recorded as >4.0 ng/ml. d) Standards Porcine GIP was weighed on a Cahn -25 microbalance (Cahn Instruments, Calif.) and dissolved i n 0.2M acetic acid with 0.5% BSA and 2.0% trasylol at a concentration of 1 yg/100 y l lyophilized and stored at -20\u00C2\u00B0C. Dilution of an aliquot to 12.5ml with assay diluent gave 80 ng/ml. A 1:10 dilution of this yielded 8ng/ml and serial dilutions with diluent buffer of this concentration provided standards of 4, 2, 1, 0.5, 0.25 and 0.125 ng/ml. e) Controls Standardized porcine GIP control tubes were included in every assay. Controls were prepared by dissolving gut extract In phosphate buffer (0.04M), pH 6.5 containing 20,000 KIU trasylol per 100ml and 0.5% plasma to a concentration of 1000 pg/ml. The controls were stored at -20\u00C2\u00B0C i n 1.5 ml plastic Eppendorf micro test tubes. Control tubes were included at two points in each assay following the standard curve and at the end of the assay. In 10 ar b i t r a r i l y chosen assays, the i n i t i a l control value was 116 + 7 pg/100 yl (mean + SD) and the f i n a l control was 121 + 19 pg/100,yl. Any assay i n which controls deviated more than one SD from those values was invalidated. f) Non-specific binding Non-specific binding (NSB) of radioligand in absence of antiserum was checked throughout the assay with antiserum being replaced by diluent buffer. The NSB tubes preceded the standard curve, control tubes and each series of unknowns. The latter two NSB tubes contained 100 y l of control and unknown respectively. NSB for standard curves and controls ranged from 3-10% depending on age of the tracer and was generally 2-3% higher for unknowns. g) Unknown or Experimental Samples Samples were kept at -20\u00C2\u00B0C prior to assay and then thawed for measurement. After aliquots were taken for assay, samples were discarded because of the labile nature of the peptide. h) Assay protocol 100 y l sample standard or control 125 100 y l I GIP containing approximately 5000 cpm 100 y l antiserum at f i n a l 3 dilution of 1:30x10 fi n a l volume was made up to 1.0 ml with diluent buffer. 22. Assays were performed on serum samples in trip l i c a t e at 4\u00C2\u00B0C in 12x75mm siliconized tubes and incubated for 48h at 4\u00C2\u00B0C. Tubes containing 100 yl of 125 I-GIP were stored with the assay, for the measurement of total counts. i ) Charcoal separation On the day of separation of bound and free radioactivity 0.5% dextran T-70 was dissolved in 100 ml 0.04M Phosphate pH 6.5, 1.25% charcoal and 5% charcoal extracted plasma were added and stirred for lh at 4\u00C2\u00B0C. Two hundred microlitre aliquots of this mixture were added to a l l but total count tubes, vortexed, let stand for 10 min at 4\u00C2\u00B0C and centrifuged at 3000 rpm for 40 min. The supernatant containing antibody bound radioactivity was discarded and charcoal pellet containing unbound radioactivity was measured in a Searle 1285ycounter. j) Calculations Percent bound was determined employing the following formula: %B =Total counts - Free (pellet) Total Counts Standard curves were plotted on logarithmic standard concentration, standard curve by finding equivalent bound. - Total ~ Free (NSB pellet) Total Counts semilog paper with %B versus Control and unknowns were read off standard concentration for particular % 23. 3. Insulin a) Iodination of insulin Phosphate buffer (0.4M, pH 7.5) as described for GIP iodination was used as stock solution. In preparation for iodination the following solutions were made: (i) 40 mg Chloramine-T in 10 ml 0.2M phosphate buffer, pH 7.5. ( i i ) 24 mg sodium metabisulphite in 10ml 0.2M phosphate buffer, pH 7.5. ( i i i ) 100 mg potassium iodide in 10ml 0.2M phosphate buffer pH 7.5-. (iv) Insulin (porcine) freshly weighed (5yg) on a Cahn microbalance, into 12x75mm test tube and dissolved in 5 yl 0.01N HC1 plus 5 y l 0.2m phosphate buffer, pH 7.5. The reagents were mixed together in a 12x75mm glass test tube recording to the following protocol: lmCi Na 1 2 5I in 10 yl NaOH solution (pH7-ll) 10 yl porcine insulin (5y g) in 0.2M phosphate 25 y l chloramine-T (100 yg) 10 second exposure with gentle vortex mixing 100 y l sodium metabisulphite (240 yg) 45 sec exposure with gentle vortex mixing 50 y l potassium iodide (500 yg) Purification of I insulin was by adsorption onto lOmg of microfine s i l i c a (QUSO) in a total volume of 2ml 0.04M PO^ pH 7.5. The mixture was vortex-mixed and centrifuged i n a table top centrifuge. The supernatant containing free iodine was discarded and the pellet washed three times with d i s t i l l e d water to ensure removal of free iodine. The iodinated insulin was removed from the pellet by washing with 3ml acid ethanol. The acid ethanol supernatant was stored at -20\u00C2\u00B0C. Incorporation of iodine into the peptide was calculated on the basis of the following formula: % Incorporation = Acid ethanol supernatant + Pellet 125 Total I (supernatant,1,2,& 3 and pellet) As for GIP, specific activity of label was not assessed with each iodination since incorporation was generally of the order of 75% which represents a specific activity of 150 mCi/mg. Iodinations were done monthly. b) Assay buffer The stock buffer was 0.4M P0^ pH 7.5 made as described i n the section on GIP iodination. It was diluted 1:10 for the assay and 5% charcoal extracted plasma was added. Charcoal extraction of plasma has been previously described. c) Antiserum An antiserum GP01 raised in guinea pigs by Dr. R.A. Pederson was u t i l i z e d . The antibody was raised against unconjugated porcine insulin in Freund's complete adjuvant. Aliquots (1ml) were stored frozen at 1:5000 dilution and used in the assay at a f i n a l dilution of 1:10^ giving 40-55% zero binding. The antiserum detected rat insulin over a range of 5 \"yU/ml to the 160 yU/ml. 25. d) Standards Rat insulin obtained from Novo Pharmaceuticals (Denmark) was weighed on a Cahn microbalance and diluted i n assay buffer to a concentration of 160 UU/ml. Serial dilutions were made at the time of assay to the following concentrations: 160, 80, 40, 20, 10, and 5yU/ml. e) Controls Controls were prepared from effluent of isolated perfused rat pancreas studies which had been subject to perfusion with 300 mg% glucose and lOmM arginine and assayed for insulin content. The effluent was diluted to give a concentration of 40 y U/ml and 1.5ml aliquots were stored frozed in plastic Eppendorf micro-test tubes at -20\u00C2\u00B0C. Controls were included in the assay as per GIP assay and deviation from the mean of 41 U/ml (n=14) by greater than 10 yU/ml resulted in invalidation of assay results. f) Non-specific binding tubes These were the same as for the GIP RIA. g) Unknowns or perfusate samples These were frozen at -20\u00C2\u00B0C and thawed for the assay. Following removal of aliquots for the assay the samples were refrozen and stored at -20\u00C2\u00B0C. h) Assay protocol Samples were assayed in duplicate except for the standard curve which was routinely done in tr i p l i c a t e . Non-siliconized glass tubes (12x75mm) tubes were used and the assay was set up at 4\u00C2\u00B0C. A disequilibrium protocol was used: 26. 100 y l standard, sample, or control 100 y l of antiserum at a fi n a l dilution of 1:10^ buffer to 900 y l was added to each tube 24h incubation at 4\u00C2\u00B0C 125 100 y l I-insulin containing approximately 10,000 cpm 24h incubation at 4\u00C2\u00B0C. i) Separation This was the same as described for the GIP RIA except no plasma was included i n the charcoal mixture and the dextran T-70 concentration was 0.5% (w/v) and charcoal concentration 5% (w/v). Tubes were vortexed, permitted to s i t 10 min at 4\u00C2\u00B0C and centrifuged at 3000 rpm for 30 min. The amount of insulin in each sample was determined as reported for the GIP RIA. C. Thin Layer Chromatography Thin layer chromatography, a type of adsorption chromatography was employed as a means of assessing peptide purity. Twenty-five to f i f t y micrograms of peptide were applied in 5-10 yl d i s t i l l e d deionised water using a disposable micro pipette (DADE, Florida) onto s i l i c a gel coated plates ( S i l i c a r , TLC-7GF Mallinckrodt, Missouri). The peptides were applied as a discrete spot (l-2mm diameter) 4cm from the bottom of the plate and permitted to air dry between successive applications. The plate was developed at room temperature i n a sealed tank 22x22x9 cm (Dessago, Heidelberg, West Germany) with a solvent mixture of n-butanol, acetic acid, pyridine and d i s t i l l e d water i n a ratio of 5:1:3.4:5. The plate was placed in a fume hood and dried overnight at 22\u00C2\u00B0C. Detection of peptides was achieved by spraying the plate with 0.5% ninhydrin reagent in acetone with acetate i n 34% glacial acetic acid solution v/v. The RF value was determined by dividing the distance travelled by the distance from the origin to the solvent front. Purity of a particular substance was arbitrarily determined by the discreteness of the developed spot and i t s appearance relative to monocomponent GIP, a known pure substance. 1. Specific Colour Reactions The amino acid residue, tryptophan was detected qualitatively by the Ehrlich reagent (Smith, 1960). The reagent, 10% p-Dimethylaminobenzaldehyde in concentrated hydrochloric acid (w/v) added to acetone in a ratio of 1:4 (v/v), was sprayed on thin layer chromatography plates after developing with ninhydrin. A characteristic purple colour reaction was observed i f tryptophan was present in the peptide. III. Peptide Chemistry Fragments of IR-LGIP C 1. Cyanogen bromide cleavage Cyanogen bromide (CNBr) cleaves a peptide on the C-terminal side of the methionine residue (Gross et a l , 1961; Gross et a l , 1962). The reaction was performed at R.T. overnight i n a sealed flask wrapped in f o i l to exclude light. CNBr was dissolved in 90% formic acid at a concentration of 250 mg/ml (w/v) and the peptide was dissolved in 90% formic acid at a concentration of 5mg/ml (w/v). The CNBr solution was added to the peptide to give a peptide: CNBr ratio of 1:20 (v/v). At the end of the reaction 28. time the flask contents were diluted 1:20 with d i s t i l l e d water, rotary evaporated and lyophilized. The unseparated cleaved material and uncleaved peptide were examined by dansylation. 2. Tryptic digestion of IR-LGIP C Trypsin cleaves a peptide molecule between the C-terminal of lysine or arginine residue and the amide group of the adjacent residue. Enzymatic cleavage of the peptide was carried out in 1% NH^ HCO^ at a peptide concentration of 0.2% (w/v) and an enzyme substrate ratio of 1:50 (w/v). The TPCK treated trypsin obtained from Worthington Biochemical was added every 2h and the reaction was allowed to proceed at RT for 6h from the f i r s t addition of enzyme. The reaction mixture was lyophilized, the residue was then taken up i n d i s t i l l e d water, boiled for 6 min. to inactivate the enzyme, centrifuged and the decanted supernatant relyophilized. Cleaved peptides were subsequently compared to GIP on HPLC. 3. Amino Acid Analysis Amino acid compositions of peptides were determined following acid hydrolysis u t i l i z i n g a Dionex MSB/SS amino acid analyzer k i t (Sunnyvale, California). Twenty to thirty nanomoles of peptide were hydrolyzed in 6NHC1 in vacuo at 110\u00C2\u00B0C for 22h. The HC1 was removed in a high vacuum and the peptide was dissolved in analyzer sample buffer pH 2.0 (Femto-buffer system 2 Durrum, Calif.) at a concentration of lnmol/20 1. The sample was then applied to a cation exchange column (resin DC-5A Durrum, Calif.) and eluted with three buffers at a flow rate of 12 ml/h at pressures of 1400-1600 p.s.i. Amino acids were detected using a ninhydrin reagent and passed through a spectrophotometer (Model 56, Glenco, Texas) at 590nm. Peak areas were recorded and integrated using a Hewlett Packard 3390A instrument 29. (Hewlett Packard, California). The identification and quantification of amino acids were based on comparison with 5nmol aliquots of amino acid standards (Pierce, I l l i n o i s ) . 4. Identification of N-terminal residue The N-terminal residue of peptides were identified using the dansylation technique described by Gray (1967) and modified by Burton and Hartley (1970). The peptide was tagged with a fluorescent molecule, 1-dimethylaminonaphtalene -5-sulphonyl-Cl, (dansyl chloride) and hydrolyzed. The reaction causes the terminal amino group to displace chlorine and to produce a fluorescent residue. Approximately 10 nmoles of peptide was transferred to a glass pyrex tube (4x30 mm) and 2 pi 0.1M NaHCO^ was added, centrifuged and lyophilized. Two and one-half microlitres each of d i s t i l l e d , deionized water and dansyl chloride (2.5mg/ml in acetone) was added and the mixture centrifuged, covered with parafilm and incubated for l h at 37\u00C2\u00B0C. The contents were again lyophilized and taken up in 20 yl 6NHC1. The tube was drawn out, evacuated under nitrogen, heat sealed and incubated for 20h at 110\u00C2\u00B0C. When acid hydrolysis was complete the tube was cooled, centrifuged, opened and dried under vacuum over sodium hydroxide pellets. Thin layer chromatography was carried out on dansylated material. The peptide was dissolved i n 2.5 yl aqueous pyridine. Approximately 0.5.yl was 2 spotted on each side of a 5cm polyamide plate (Cheng Chin Trading Co., Taipei, Taiwan). A standard solution (0.5 yl) combining dansyl derivatives of phenylanine, serine, isoleucine, arginine, proline, glycine, and glutamic acid, 1 ymole of each amino acid/ml in acetone: 0.1M acetic acid (3:2 v/v) was spotted on one side only. The plate was subjected to ascending chromatography i n two dimensions in the appropriate solvent systems. 30. Dimension Solvent 1 I Water: 90% formic acid 200 : 3 (v/v) Woods and Wang (1967). 2 II Benzene: glacial acetic acid. 9 : 1 (v/v) Woods and Wang (1967). 2 III Hexane: n-butanol:glacial acetic acid 3 : 3 : 1 (v/v) Crowshaw et a l (1967) 2 IV .1% Ammonia: Ethanol 9 : 1 (v/v) After running in solvents I and II the plates were examined under ultraviolet light and dansylated residues identified. Dansyl serine, dansyl threonine, dansyl glutamic acid, dansyl aspartic acid, dansyl glycine and dansyl alanine may only be differentiated after running in solvent III. Solvent IV was used to separate arginine, histidine and X -lysine. If lysine or tyrosine was present at any position in the peptide \u00C2\u00A3 -dansyl lysine or 0-dansyl tyrosine were seen. If lysine or tyrosine was the N-terminal residue bis-dansyl-lysine or bis-dansyl-tyrosine must also be present. For identification of proline as N-terminal residue hydrolysis must be restricted to 4h. IV. Physiology of Purified Peptide Isolated Perfused Rat Pancreas Bioassay 1. Reagents a) Krebs concentrate 285 ml 154 mM KC1 243 ml 102.7 mM CaCl 2 78 ml 154 mM MgS04 7H20 97 ml 154 mM KH\u00E2\u0080\u009EP0 2 4 stored at 4\u00C2\u00B0C. b) Perfusate Dextran (Clinical grade) and BSA (RIA grade) in a concentration 3% and 0.2% respectively were dissolved in 0.9% saline overnight at 4\u00C2\u00B0C. the day of the experiment Krebs concentrate, NaCl and NaHCO^ and glucose were added to give the f i n a l concentrations of: 4.4mM KC1 2.5mM CaCl 2 1.5mM KH2P04 2.5mM NaHC03 120mM NaCl 0.2% BSA 3% Dextran Glucose as desired (80mg% or 160mg%) 32. c) Side arm infusates Substances delivered to the pancreas via side arm infusions were dissolved in perfusate such that 0.206ml/min delivery rate mixed with perfusion rate of 4ml/min gave the appropriate concentration of infusate (2ng/ml for GIP and 20 ng/ml for IR-LGIP C). 2. Procedure Surgical preparation The method used was a modification of that described by Grodsky et a l (1963). Male Wistar rats weighing 200-300g fasted overnight were anaesthetized with sodium pentobarbital (60mg/kg) injected intraperitoneally. A midline abdominal incision from pelvis to sternum was made i n i t i a l l y and descending colon including i t s ar t e r i a l blood supply was doubly ligated and cut. A drainage tube was inserted into the small intestine just inferior to the level of the pancreas. The mesenteric vessels were doubly ligated distal to the drainage tube and severed between ligatures. The colon was doubly-ligated immediately orad to the caecum, cut and the entire intestinal segment between drainage and caecum removed. The oesophagus and associated blood vessels were doubly ligated and sectioned. Stomach removal was accomplished after single ligatures were placed around vessels of the antrum, greater curvature and pylorus, sections were made on the gastric side of the ties. Single ligatures were tied around spleen vessels and sectioning was done on the spleen side of ligatures. The right kidney and associated vessels were tied with a single ligature whereas the le f t renal gland and blood supply were double ligated and severed between ties to ensure adequate exposure of aorta. A single ligature was placed orad to 33. where the coeliac artery branched from the aorta. A double ligature was placed below the superior mesenteric branch of the aorta and adjacent to the renal artery stump. The aorta was cannulated with polyethylene tubing (PE160) to the level of the superior mesenteric artery. One to one and half m i l l i l i t r e s of heparinized saline was then injected into the aorta. The animal was hemi-sected at the diaphragm and the upper half of the carcass discarded. The portal vein was cannulated with polyethylene tubing (PE 90) to collect effluent. b) Apparatus Perfusate was pumped through the isolated pancreas via a peristaltic pump at 4ml/min. Pressure was monitored and maintained between 40 and 60 mmHg. Two separate servo controlled temperature regulators maintained isolated gland and perfusate at 37\u00C2\u00B0C. Perfusate pH was maintained at 7.4 by gassing with 95% 0^ 5% CO^ saturated with water vapour. Side arm infusions into the ar t e r i a l cannula were achieved by u t i l i z i n g a variable-speed infusion pump (Harvard Apparatus Co., Mass.). Effluent was collected after one pass through the pancreas using an automated fraction collector (LKB, Sweden). c) Perfusion Two parallel recirculating perfusate systems one containing perfusate with 80mg% glucose and the second with glucose at a concentration of I60mg% glucose were presented to the pancreas in a square wave manner. I n i t i a l l y the 80mg% glucose solution passed through the pancreas for a 10 min equilibration period and subsequent 5 minute experimental period. At the end of 5 min the two systems were interchanged at the level of the bubble trap and the gland was subjected to a glucose concentration of 160mg% for 40 min. Side arm infusions were presented to the pancreas during this time from 11 minutes to 30 minutes inclusive. 34. APPENDIX TO METHODS - Chemical Sources Chemical Grade * Source Acetic a c i d Acetone A c e t o n i t r i l e ( f o r HPLC) Ammonium acetate Ammonium bicarbonate Ammonium hydroxide Ammonium sulphate [(NH 4) 2S0 4] 2 Aminoethanol (ethanolamine) Benzene Bovine Serum Albumin Cadium acetate C a C l 2 Carbon Decolourizing Norit Chloramine -T Concentrated HC1 Cyanogen bromide Dansyl amino acids standards Dextran Dextran T-70 Diethanolamine Dimethylaminobenzaldehyde (para) l-Dimethylaminonapthalene-5-sulphonyl Chloride (dansyl chloride) Dimethylformamide Ethanol l-Ethyl-3(3-dimethyl-amino-propyl) Carbodiimide, HC1 Freund's Complete Adjuvant Formic Acid G l a c i a l A c e t i c Acid Glucose (Dextrose) Heparin Hexane H 20 (for HPLC) I n s u l i n (porcine) I n s u l i n (rat) KC1 Keyhole Limpet Hemocyanin KH 2P0 4 KI MgCl 2 6H20 MgS04 7H20 n-Butanol Na 2C0 3 N a i 2 5 I i n NaOH NaHCOo A r i s t a r Omnisolve Analar A r i s t a r RIA A r i s t a r C l i n i c a l 99% Laboratory Omnisolve IMS-30 BDH Fisher BDH BDH MCB BDH Fisher Sigma Fisher Sigma Fisher Fisher Fisher Eastman BDH Eastman Calbiochem-Behring Sigma Pharmacia Baker Fisher Pierce Fisher Liquour Control Board Calbiochem-Behring Difco Fisher Fisher F i s h e r Fisher Fisher BDH Novo Novo Baker Calbiochem Behring Baker Fisher Fisher Fisher Fisher MCB Amersham Baker 35. Chemical Grade * Na2HP04 NaH2PC>4 H20 NaOH Ninhydrin Pyridine Phosphatase Substrate 104 -QUSO microfine s i l i c a G 32 Sodium Azide Practical Sodium Borate -Sodium Chloride Sodium Metabisulfite 2,3,10,14 - tetramethylpentadecane -Trasylol (10,000 KlU/ml) Trifluoroacetic acid Sequanal Tris-HCl Trypsin-TPCK Tween-20 Source Fisher Fisher Fisher Pierce Fisher Sigma Philadelphia Quartz Baker Baker Fisher Fisher Aldrich Miles Pierce Sigma Worthington Sigma * Where unspecified, chemicals were of standard reagent grade purity. 36. RESULTS I. Purification of Immunoreactive-GIP (IR-LGIP C) Moiety A. Aff i n i t y Chromatography 1. Coupling and Purification of Ligand The ligand used i n the a f f i n i t y chromatography system employed in this study was a monoclonal antibody to GIP designated, 3.65H (Buchan et a l , 1982). The antibody was harvested from ascites i n irradiated outbred mice pretreated with 2,3,10,14-tetramethylpentadecane. Prior to coupling to the a f f i n i t y chromatography maitrix the ascites f l u i d was purified by passing i t through an anion exchange column (2.5x10cm) of DEAE Sephacel (Pharmacia). Twenty m i l l i l i t r e s of ascites f l u i d were applied to the column and 6 ml fractions were collected at a flow rate of 96 ml/h. The column was eluted with a stepwise gradient of 0-0.2M NaCl in 50 mM Tris HC1 buffer pH 7.4. Two peaks were obtained, the f i r s t eluted in 50 mM Tris HC1 buffer and the second i n 50 mM Tris HC1 buffer with 0.2 M NaCl (figure 1). Each peak was pooled and subjected to ammonium sulphate precipitation, centrifugation and dialysis. Each fraction was assayed for immunoreactivity using the ELISA technique and fraction II consistently showed the greatest a f f i n i t y for GIP. Fraction II was coupled to the a f f i n i t y chromatography support, Sepharose-CL-4B, as described in the methods (section I, A4). 50mM TRIS-HCI pH 7.4 50mM TRIS-HCI pH 7.4 40 \u00E2\u0080\u00A2 with 2 0 0 m M N a C I o 00 CM LU o < 00 DC o CO 00 < 3.0 . 2.0 -1.0 . 0 8 16 24 32 FRACTION NUMBER FIGURE 1 P u r i f i c a t i o n of GIP monoclonal antibody, 3.65H. Twenty m i l l i l i t r e s of a s c i t e s f l u i d was applied to DEAE Sephacel anion exchange column (2.5 x 10 cm). The column was eluted with a stepwise gradient of 0-0.2M NaCl i n 50mM Tris-HCL pH 7.4. Two peaks eluted, the f i r s t with 50 mM Tris-HCl and the second with 0.2M NaCl i n 50mM T r i s - H C l . The second f r a c t i o n demonstrated the greatest a f f i n i t y for GIP when assayed i n the ELISA and was used for coupling to the a f f i n i t y chromatography support. 2. Characterization I n i t i a l l y , to separate immunoreactive-like GIP peptides from side fractions of GIP purification an a f f i n i t y chromatography system was used. The column support, cyanogen bromide activated Sepharose - CL - 4B, was coupled with the monoclonal antibody to G I P ^ Q Q Q . Specificity for G I P was 6 125 determined by respectively passing 2 ml (1x10 cpm) I-GIP (figure 2), 2 ml (lxlO^cpm) 1 2 ^ I - I n s u l i n (figure 6) 2 ml (1x10^ cpm) 125 I-Motilin (figure 7), 5 mg GIP purification side fraction EG II-III FrA (Brown et a l , 1970) (figure 5), and 0.5 g porcine G I P 5 0 0 Q (figure 4) through the column. Iodinated GIP was applied i n 50 mM Tris HC1 pH 7.4 and in 2 ml plasma respectively, (figure 3); the other peptides were applied in 50 mM Tris HC1 pH 7.4. Material unbound to the ligand was washed off the column with 50 mM Tris HC1 pH 7.4 and substances binding to 3.65H were desorbed with 100 mM HCL. Eluant was collected in 2.0 ml fractions at a flow rate of 120 ml/h. One hundred microlitre aliquots of iodinated substances were counted for 1 min in a gamma counter (1285 Searle Instrument). Absorbance of GIP side fraction EG II-III FrA and porcine G I P , - Q Q Q was read at 280 nm through a 1 cm light path (figures 4 and 5). Column profiles (figures 2-7) indicate the specificity of the ligand for GIP. Application of GIP (iodinated and non-iodinated) showed most of the 125 peptide binding to the ligand (figures 2-4). Application of GIP in plasma (figure 3) demonstrated that plasma components did not affect or hinder specificity. The iodinated GIP peak eluting in the wash probably was composed of fragments of labelled GIP with no antigenicity for the ligand (figure 2 and 3). The elution profile of EG II-III FrA confirmed i t s 39. FIGURE 2 Characterization of Immunosorbent system with 1 Z 3 I GIP. Two m i l l i l i t r e s of 1 2 5 I GIP (lx l O 6 cpm) in 50mM Tris-HCl pH 7.4 were applied to the a f f i n i t y chromatography column with GIP monoclonal antibody, 3.65H, as the ligand. The elution profile illustrates the preferential binding of 125T GIP to the ligand. Material unbound to the ligand was washed off the column with 50mM Tris HC1 pH 7.4 and fractions with a f f i n i t y for GIP monoclonal antibody were eluted with lOOmM HC1. 40. 5 0 m M TRIS pH 7.4 100mM HCI CO \u00E2\u0080\u00A2 O 150 _ 100 50 \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 I 1 2 5 GIP in TRIS B U F F E R 125 \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 I GIP in P L A S M A / V v . 8 16 24 32 FRACTION NUMBER FIGURE 3 Comparison of 1 2 5 I GIP (lx l O 6 cpm) in 2 ml 50mM Tris-HCl (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) and in 2 ml plasma (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2) on immunosorbent column with GIP monoclonal antibody 3.65H as the ligand. The peaks eluted with 100 mM HCI demonstrate the a f f i n i t y of 1 2 5 I GIP for the ligand and show that plasma does not interfere with the ligand specificity and binding capacity. 41. 50mM TRIS pH 7.4 100mM HCI 0.2 r o oo CM UJ o z < 00 DC O 00 CO < 0.1 0 / 8 16 24 32 FRACTION NUMBER \u00E2\u0080\u00A2 \ 40 FIGURE 4 GIP (0.5 mg) applied to af f i n i t y chromatography column with GIP monoclonal antibody, 3.65H, as the ligand. Specificity of ligand for GIP molecule i s shown in above profile. The percentage of applied material which did not bind to the ligand was probably contaminants present in the peptide that lack antigenic determinants for the ligand. 42. 50mM TRIS pH 7.4 100mM HCI LU O z < CD GC O CO CO < 0.4 ^ o 0.3 oo CM 0.2 0.1 / / \ 1 m \ 20 40 60 FRACTION NUMBER FIGURE 5 E l u t i o n p r o f i l e of GIP p u r i f i c a t i o n side f r a c t i o n (EG I I - I I I Fr A) on immunosorbent column with GIP monoclonal antibody, 3.65H, as the ligand. M a t erial not adsorbed to the ligand was eluted with 50 mM T r i s HCI. The peak eluted with 100 mM HCI shows the component of EG I I - I I I FrA with a f f i n i t y for the GIP monoclonal antibody. 43. 200 50mM TRIS pH 7.4 100mM HCI CO 150 100 _ x CL d 50 . ^. L J 8 16 24 32 FRACTION NUMBER FIGURE 6 125 6 Two m i l l i l i t r e s I I n s u l i n (1x10 cpm) i n 50mM Tris-HCl pH 7.4 applied to a f f i n i t y chromatography column with GIP monoclonal antibody as the ligand. 125 I I n s u l i n demonstrated no a f f i n i t y f o r the ligand. The el u t i o n p r o f i l e shows a l l material eluted i n the wash with 50mM T r i s HCI. No material was eluted following a p p l i c a t i o n of lOOmM HCI 125 ^ 44, 5 0 m M TRIS pH 7.4 100mM HCI 100 . ~ 75 co o Q. o 50 25 ML \u00E2\u0080\u00A2 \ !\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 k \u00E2\u0080\u00A2 k \u00E2\u0080\u00A2 8 16 24 32 FRACTION NUMBER profile of 2ml i z - ) l Motilin ( l x l O 6 cpm) on immunosorbent column. Specificity of ligand for GIP was confirmed. No l 2 5 i Motilin bound to the ligand. A l l material was eluted in wash phase with 50mM Tris HCI and no fraction was desorbed with lOOmM HCI. impurity and showed that a significant amount of IR-GIP was also present in this fraction (figure 5). Iodinated insulin and iodinated motilin demonstrated the reverse profile compared to GIP, that i s , a l l the peptide eluted i n the wash (figure 6 and 7). 3. Capacity Determination and Purification Having established the specificity of the immunosorbent system a side fraction of GIP purification - material not adsorbed to alginic acid (figure 8) was applied to column. Varying amounts of this material were dissolved in 10 ml of washing buffer (Tris HCI) and pH was adjusted to 5.25 with 0.1 M ammonia to ensure complete solubility. Aliquots of the starting material, 0.25, 0.50 or 1.0 g were applied to the column and eluted as described for column characterization. Elution profiles (figure 9) indicate the capacity of the system for this starting material to be approximately 1 g. Absorbance increased proportionately with each weight applied. There was an indication that 1 g of material saturated the binding sites. In figure 10 a typical purification profile of this material is illustrated. Repetitive applications of 1 g of material not adsorbed to alginic acid were carried out as described above. The fractions i n the peaks were pooled and rotary evaporated to dryness. B. Gel F i l t r a t i o n A Sephadex G-25 column (0.9 x 100cm) was used to desalt the fractions pooled from a f f i n i t y chromatography. Both bound and unbound material from the immunosorbent system were desalted but i t was the bound fraction that was used for further purification. Each fraction following rotary S U M M A R Y O F T I S S U E E X T R A C T I O N P R O C E D U R E 46, H E A T C O A G U L A T E D H O G D U O D E N O J E J U N A L M U C O S A A C E T I C A C I D A C E T I C A C I D E X T R A C T i N O T A D S O R B E D T O A L G I N I C A C I D A L G I N I C A C I D A D S O R P T I O N N A C I P R E C I P I T A T E C O N T A I N I N G S N , C C K - P Z A N D G I P A C T I V I T Y | S E P H A D E X G 2 5 F R A C T I O N I C O N T A I N I N G I R - G I P ( H I G H M . W T . ) 0 . 0 4 M A M M O N I U M A C E T A T E P H 7 . 0 N E U T R A L I N S O L U B L E F R A C T I O N M E T H A N O L S O L U B L E F R A C T I O N N E U T R A L S O L U B L E F R A C T I O N M E T H A N O L M E T H A N O L I N S O L U B L E F R A C T I O N FIGURE 8 Flow chart of GIP purification from hog duodeno-jejunal mucosa. The side fraction not adsorbed to alginic acid was used as starting material. 47. 8 16 24 32 FRACTION NUMBER FIGURE 9 The a f f i n i t y chromatography system u t i l i z i n g a monoclonal antibody to GIP designated 3.65H as the ligand was subjected to various amounts of starting material to assess column capacity. The elution profile of 250mg (O O), 500mg (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) and l.Og (\u00E2\u0080\u00A2\u00E2\u0080\u0094 - 1 ) of starting material i s illustrated here. Peptides within the starting material with no a f f i n i t y for the ligand were washed off the column with Tris HCI and that component specific for the ligand was desorbed with lOOmM HCI. A positive correlation between the weight applied and absorbance of bound fraction was observed. An upper limit of lg of starting material was noted. 48. o co CM UJ o < CD DC O CO CQ < 0.05M TRIS 2.0 _| 1.5 _ 0.1M HCI 1.0 . 0.5 m \ 8 16 24 FRACTION NUMBER 32 FIGURE 10 A t y p i c a l e l u t i o n p r o f i l e u t i l i z i n g immunosorbent chromatography to i n i t i a l l y separate an immunoreactive GIP component from l g of s t a r t i n g material (material not adsorbed to a l g i n i c a c i d ) . The immunoreactive-like GIP (IR-LGIP) component was desorbed with HCI and subjected to further p u r i f i c a t i o n . 49. s I CvJ UJ O z < I 00 o C O CO i < 1.0 ^ 0.8 0.6 0.4 0.2 0 J I I L J 8 16 24 32 F R A C T I O N N U M B E R FIGURE 11 Desalting on Sephadex G-25 column (0.9 x 100 cm) Following immunosorbent separation of 250mg, 500mg, and lg of starting material, the respective desorbed bound compound was desalted on Sephadex G-25. The open circles (O O) labelled \"a\" show desalting of ligand-bound fraction in 250mg of starting material. The closed circles (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) labelled \"b\" represent the same in 500mg and the closed squares (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) labelled 'c\" illu s t r a t e the same in l g of starting material. The respective recoveries were 4.9mg, 7.5mg and 11.3mg. 50. evaporation was dissolved i n the smallest possible volume of 0.2 M acetic acid (1-1.5 ml), applied to the gel f i l t r a t i o n system and eluted with 0.2 M acetic acid. Fractions of 1.5 ml were collected at a flow rate of 18 ml/h. and absorbance monitored at 280 nm through 1 cm light path. In conjunction with capacity determination of the immunosorbent system, the respective bound components were desalted. Figure 11 illustrates the desalting profiles of 250 mg, 500 mg and 1 g. The recovery of desalted bound material was found to be 4.9 mg, 7.5 mg and 11.3 mg respectively. Repetitive desalting of the bound component obtained from immunosorbent separation of 1 g starting material (material not adsorbed to alginic acid) yielded a consistent recovery of approximately 10 mg. This fraction was designated immunoreactive-like GIP (IR-LGIP). C. Ion-exchange Chromatography Further purification of IR-LGIP was attempted u t i l i z i n g ion exchange chromatography. Due to the acidic properties of the starting material (Dryburgh, 1977) an anion exchanger, DEAE A-25 Sephadex, was employed. IR-LGIP (25 mg) i n 2 ml ammonium acetate pH 6.0 was applied to a column of DEAE A-25 Sephadex (0.9x5 cm) and eluted with a stepwise gradient of 0.05 M ammonium acetate pH 6.0 and 1.0 mM acetic acid. Eluant was collected in 1.5 ml fractions at a flow rate of 12 ml/h and absorbance detected at 280 nm through 1 cm light path. The chromatogram i s shown in figure 12. Fractions designated DEAE FrA and DEAE FrB were pooled and lyophilized and desalted on Sephadex G-25 (figure 13). Recovery was 13.0 mg and 1.0 mg respectively. Each fraction was applied to HPLC (see Results section 1(D), part 4, figure 17). The peak DEAE FrA contained more hydrophobic material than DEAE FrB o CO CvJ UJ o 2.0 1.6 -1.2 50mM AMMONIUM ACETATE 1.0M ACETIC ACID < 0.8 m DC O co 0 4 pH 6.0 \DEAE FrA \ DEAE FrB 8 16 24 32 40 FRACTION NUMBER FIGURE 12 IR-LGIP (25mg) on anion exchange column, DEAE A-25 Sephadex (0.9 x 5 cm) The f i r s t peak eluted with 50mM ammonium acetate was designated DEAE FrA and the second peak eluted with 1.0M Acetic acid was designated DEAE FrB. 52. 8 16 24 32 FRACTION NUMBER FIGURE 13 Desalting on Sephadex G-25 of respective f r a c t i o n s (DEAE FrA and DEAE FrB) separated by anion exchange chromatography of IR-LGIP. Closed squares (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2) l a b e l l e d \"a\" shows the desalting of DEAE FrA and (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) l a b e l l e d \"b\" gives the desalting p r o f i l e of DEAE FrB. The recoveries were 13.0 mg and 1.0 mg r e s p e c t i v e l y . 53. which was predominantly hydrophilic. Anion exchange chromatography did not enhance purification of the hydrophobic components of the immunoreactive fraction (IR-LGIP) extracted from a f f i n i t y chromatography. Immunoreactivity of each fraction obtained from ion-exchange was measured using the ELISA technique. Indirect inhibition studies and direct dilution studies were carried out. DEAE FrA and DEAE FrB showed a response comparable to IR-LGIP. D. HPLC 1. ^Terminology HPLC figures shown in the results section have certain common features unless stated otherwise which are summarized below:-(i) figures show absorbance (225 nm) versus time ( i i ) sensitivity of detector i s 0.1 AUFS (Arbitrary Units Ful l Scale) ( i i i ) solvent flow rate is 2 ml/min (iv) solvent pressure is 2900-3500 PSI (v) acetonitrile gradients were linear and ran for 10 minutes. With each figure a description of the programmed solvent gradient i s provided, for example: \"35-45% CH^CN, 10 minutes\" would describe a linear acetonitrile gradient ranging from 35% to 45%. 54. 2. Intrinsic Artefacts Several inherent artefacts exist in the HPLC system used here. Injection which occurs simultaneously with the start of the gradient results in a consistent delay interval between programmed gradient beginning and i t s detection by the spectrophotometer. During this time the peptide i s exposed to a constant acetonitrile concentration, the same concentration at which the gradient begins. Acetonitrile has a greater absorbance than water at 225 nm and a linear gradient of acetonitrile results in a rising baseline. The baseline i s not completely linear due to different compressibilities of the two solvents. A slight concave effect occurs in the gradient background which is produced when a lower operating pressure pumps a high concentration of acetonitrile solvent mixture. Figure 14 illustrates these consistently produced artefacts. 3. Gradient Development As described in the methods section a suitable gradient system for each particular peptide must be developed. Figures 15 and 16 illu s t r a t e the establishment of a high resolution gradient system for IR-LGIP. I n i t i a l l y a broad gradient of 25-55% CH^ CN, 5 min was used and 3 peptides within the fraction of IR-LGIP eluted (figure 15). Narrowing the gradient further improved the resolution (figure 16). Due to the hydrophobicity of IR-LGIP a linear gradient of 35-45% CH^ CN was found to be optimum. Increasing the time to 10 min between i n i t i a l and f i n a l acetonitrile concentrations produced better separation of Fraction \"B\" and Fraction \"C\" without broadening the peaks excessively (figure 16). FIGURE 14 HPLC artefacts and background 1. injection marker spike 2. injection artefact 3. lag time between gradient beginning and i t s detection by the spectrophotometer 4. start of gradient 5. end of gradient gradient: 35-45% CH3CN, 10 min. FIGURE 15 Development of optimum gradient system for IR-LGIP. -20 yg IR-LGIP - 25-55% C H 3 C N , 5 min. FIGURE 16 Development of optimum gradient system for IR-LGIP - 20 yg IR-LGIP - 35-45% CH3CN, 10 min. 58. 4. Purification Assessment At each stage of separation the immunoreactive-like GIP was applied to the HPLC column, that i s , following desalting (IR-LGIP) and ion exchange chromatography (DEAE FrA and DEAE FrB). In a typical application 20 yg of each fraction i n 20 y l HPLC water with 0.1% TFA was injected onto the y Bondapak C18 column and run with a broad gradient of 25-55% CH^CN, 5 or 10 min. Ion exchange chromatography was successful in separation of the hydrophilic component of IR-LGIP but did not improve separation of hydrophobic components (Figure 16 and 17). ELISA measurement of immunoreactivity demonstrated no difference between ion exchange fractions and IR-LGIP. HPLC was subsequently used to further purify IR-LGIP. 5. Fractionation of IR-LGIP on HPLC In a typical separation 500 yg of IR-LGIP was applied to a y Bondapak C18 column. Three fractions eluted with a linear gradient of acetonitrile (35-45% CH-jCN, 10 minutes). The peaks were labelled Fraction A, Fraction B and Fraction C (figure 18). Each peak was pooled, acetonitrile removed by rotary evaporation and lyophilized from 0.1 M acetic acid. The lyophilized fractions were reconstituted i n HPLC water with 0.1% TFA and rerun on the HPLC column. Each respective fraction eluted as a single component with a retention time comparable to that obtained in i t s i n i t i a l separation. The respective retention times were: Fraction 'A' 1.89 + 0.16 min. (n=10), Fraction 'B' 11.8 + 0.26 min. (n=10), and Fraction *C' 12.54 + 0.26 min. (n=10) (figures 19-21). 5 10 ! TIME (MIN) | FIGURE 17 Assessment of purity of IR-LGIP post anion exchange chromatography The upper panel shows the elution profile of 20 yg DEAE FrA on HPLC. The lower panel illustrates the elution pattern of 50 yg DEAE FrB. DEAE FrB contained more hydrophilic material indicated as \"A\" and DEAE FrA possessed more hydrophobic components shown as \"B\". 35-45% CH3CH, 5 minutes. 60. 0 5 10 15 TIME (MIN) FIGURE 18 Purification of IR-LGIP - 500 ug IR-LGIP - 35-45% CH3CN, 10 min. Three peptides eluted in this system and were designated FrA, FrB, and FrC (IR-LGIP C). FIGURE 19 FrA separated from IR-LGIP using HPLC -20 ug FrA - 35-45%, 10 min FIGURE 20 FrB separated from IR-LGIP using HPLC - 20 yg FrB - 35-45% CH3CN, 10 min 0.10 _ co u. LU o z < GO GC o CO 00 < 0.05 _ 5 1 0 T I M E ( M I N ) j 1 5 FIGURE 21 F r C ( IR -LG IP C) s e p a r a t e d f rom IR-LGIP u s i n g HPLC - 12 yg IR-LGIP C - 35-45% CH 3 CN, 10 min 64. Immunoreactivity of each HPLC separated fraction was determined using the ELISA. Fraction C demonstrated the greatest a f f i n i t y for 3.65H and thin layer chromatography analyses of these fractions revealed Fraction C to be the most pure. The major peak, designated IR-LGIP C, obtained after pooling 16 applications of 500 yg of IR-LGIP yielded 3.24 mg 69% of total recovery of 4.7 mg (Fraction A,B,C). This material was reconstituted and run on HPLC with a linear gradient of acetonitrile (25-45% CH^ CN, 10 minutes) and compared with GIP in the same solvent system. The gradient was broadened to include an elution range for GIP. IR-LGIP C is less hydrophilic than GIP and when the equimolar amounts of each peptide were applied simultaneously i t was confirmed that the two substances were separate entities (figure 22). II. Peptide Quantitation and Immunoreactivity A. ELISA 1. Antibody Dilution Study An ELISA system was used to confirm the immunoreactivity of the purified IR-LGIP (IR-LGIP C). Microtitre plates (Falcon 3912) were coated overnight at 4\u00C2\u00B0C with GIP and IR-LGIP C in equimolar concentrations (0.06 nM). Monoclonal anti-mouse GIP antibody (3.65H) and anti-rabbit GIP antiserum (G05) were applied for 1.5h at R.T. at dilutions ranging from 1:300 to l:30xl0 3 (30 ug/ml to 3 ng/ml) for 3.65H and 1:300 - l:10xl0 3 for G05. The second antibodies RAMIg and SARIg coupled with alkaline phosphatase were applied to appropriate wells at concentrations of 1:3200 and 1:2500 respectively. The reaction was then developed with alkaline phosphatase substrate and monitored at 405nm on a Dynatech microelisa reader. i 5 10 15 i | T I M E (MIN) FIGURE 22 Equimolar amounts of natural porcine GIP and IR-LGIP C on HPLC Panel A shows the e l u t i o n p r o f i l e of lnmole GIP Panel B shows the e l u t i o n p r o f i l e of lnmole IR-LGIP C Panel C shows the e l u t i o n p r o f i l e of lnmole GIP plus lnmole IR-LGIP C. gradient: 25-45% CH3CN, 10 min. 66. Comparable a f f i n i t i e s for G05 were noted for both GIP and IR-LGIP C. GIP showed decreasing a f f i n i t y for 3.65H with increasing antibody dilution. IR-LGIP C did not bind to 3.65H (figure 23 and 24). 2. Inhibition Study To determine i f lack of binding of IR-LGIP C to 3.65H was due to i t s antigenic site and plate binding site being the same, inhibition studies were performed using the ELISA technique. The plates were coated with 0.06 nM GIP or IR-LGIP C. Concentrations of GIP and IR-LGIP C ranging from 0.1 -50 g/ml were preincubated with 3.65H antibody at half the f i n a l dilution to be used (5 yg/ml). The same was done with G05 antiserum at half the desired f i n a l dilution of 1:2500. Following 2h incubation at 22\u00C2\u00B0C 100 yl aliquots of each mixture were applied i n t r i p l i c a t e . Each antibody (uninhibited) at the desired f i n a l concentration was also applied. The reaction was then developed with alkaline phosphatase labelled RAMIg at 1:3200 for 3.65H incubated material and with alkaline phosphatase labelled SARIg at 1:2500 for Gb5 incubations. Response was detected at 405 nm in a microelisa reader (Dynatech). Results are illustrated in figures 25, 26 and 27. IR-LGIP C coated plates produced no binding or maximum inhibition when GIP or IR-LGIP C preincubated with 3.65H was applied (figure 25A). On GIP coated plates both peptides showed a positive correlation between the degree of inhibition and preincubation antigen concentration. GIP was more remarkable in the varying degree of inhibition than IR-LGIP C (figure 25B) . Whether the plate was coated with IR-LGIP C or GIP, preincubation with Gb5 produced comparable inhibition for both antigens i n a l l studies (figure 26 and 27). 67. I T ) O UJ o z < CO cc o CO DQ < \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 RABBIT \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 MOUSE MOAB 0.3 3.0 30 ANTIBODY DILN. (TO ) FIGURE 23 Confirmation of immunoreactivity employing ELISA technique on microtitre plates coated with 0.06nM GIP. Closed squares (\u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2) show the decreasing (GIP) antigen a f f i n i t y for increasing monoclonal antibody (Mouse MoAB) dilutions. Open squares (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2) show the decreasing antigen a f f i n i t y for increasing polyclonal antiserum (RABBIT) dilutions. 68. 0.3 3.0 30 ANTIBODY DILN. (10 ) FIGURE 24 Confirmation of immunoreactivity employing direct ELISA studies on microtitre plates coated with 0.06nM IR-GIP C. Closed squares (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2) indicate IR-LGIP C has no a f f i n i t y for mouse anti-GIP monoclonal antibody (Mouse MOAB) over dilutions ranging from 0.3 x 10^ to 30xl0 3. Open squares (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) show decreasing antigen (IR-LGIP C) a f f i n i t y for increasing dilutions of rabbit anti-GIP polyclonal antiserum (RABBIT). o LU o z < DQ DC O CO CD 0.4 0.2 0 1.2 0.8 0.4 0 . . . I R - L G I P C . - . G I P B \ _L \u00E2\u0080\u00A2 L 0.1 1.0 10 ANTIGEN CONC. CUG/ML) 100 FIGURE 25 ELISA inhibition studies using 3.65H at a f i n a l concentration of 5 P g/ml. In panel A (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) shows the inhibition of binding produced when GIP at concentrations ranging from 0-50yg/ml was preincubated with 3.65H for 2h at R.T. before application to the plate. (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) shows the inhibition of binding produced when IR-LGIP C was preincubated as described for GIP before application to the plate. The plate was coated with 0.06nM IR-LGIP C. In panel B, the same preincubation mixtures as described in panel A for either GIP (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2) or IR-LGIP (\u00E2\u0080\u00A2\u00E2\u0080\u0094-0) were applied to a plate coated with 0.06nM GIP. 70. FIGURE 26 ELISA inhibition study using antiserum Go5 on microtitre plate coated with 0.06nM GIP. Absorbance versus antigen concentrations preincubated with Go5 at a f i n a l concentration of 1:2500. (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) shows increased inhibition of antibody binding to GIP coated on the plate as preincubation concentration of GIP rises. Preincubation of IR-LGIP C with Gb5 produces parallel binding inhibition seen with GIP (\u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2) . 71 . FIGURE 27 ELISA i n h i b i t i o n studies with GS5 (1:2500, f i n a l d i l u t i o n ) on m i c r o t i t r e plates coated with 0.06nM IR-LGIP C. Absorbance versus preincubation antigen concentration. (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) shows that as preincubation concentration of GIP increases i n h i b i t i o n of binding of Gb5 increases. The same e f f e c t i s observed when IR \u00E2\u0080\u0094LGIP C i s preincubcited with GQ5 (\u00E2\u0080\u00A2\u00E2\u0080\u0094\u00E2\u0080\u00A2\u00E2\u0080\u00A2). 72. B. Radioimmunoassay 3 Radioimmunoassay for GIP employing Gb5 at 1:30x10 was used to compare IR-LGIP, DEAE FrA, DEAE FrB, HPLC FrA, HPLC FrB and IR-LGIP C with a GIP standard curve. The standard concentration range was as described in the methods section. The various fractions were included in the assay at concentrations ranging from 2 to 16 ng/ml. The standard curve i s shown in figure 28 with a profile of IR-LGIP C, the only fraction to demonstrate crossreactivity. The degree of crossreactivity for IR-LGIP C was found to be approximately 7.5%. C. Thin Layer Chromatography Thin layer chromatography (TLC) was used to compare the purification stages of alginic acid non-adsorbed material to monocomponent GIP. Monocomponent GIP as described by Kwauk (1982) develops as a single ninhydrin spot with an RF value of 0.66 on TLC. When IR-LGIP was applied two major but not discrete components developed with Rf values of 0.66 and 0.60 respectively. IR-LGIP C revealed a strong major spot with a Rf value of 0.60 but there was a degree of contamination s t i l l present. Overstaining with Ehrlich's reagent to detect the presence of tryptophan produced a positive reaction for a l l components of IR-LGIP and IR-LGIP C but the response was not as strong as that observed for GIP. 73 . O CD 4 0 ]\u00E2\u0080\u00A2 3 0 2 0 10 0 \u00E2\u0080\u00A2 O-\ o, \u00E2\u0080\u00A2 \ \u00E2\u0080\u00A2 o \ \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 GIP O I R - L G I P X I \ \u00E2\u0080\u00A2 _L _L 0.1 1.0 10.0 P E P T I D E C O N C . ( N G / M L ) FIGURE 28 Radioimmunoassay measurement of IR-LGIP C Standard curve f o r GIP RIA i s shown as % bound against peptide concentration. (\u00E2\u0080\u00A2 \u00E2\u0080\u00A2) shows GIP standards ranging i n concentration from 0-4 ng/ml. Standards are incubated f o r 48h at 4\u00C2\u00B0C with l 2 5 l GIP (5000 cpm) and Go5 antiserum at f i n a l d i l u t i o n o f l : 3 0 x l 0 3 . (O O ) i l l u s t r a t e s the binding curve of IR-LGIP C i n the RIA. The peptide was used at concentrations ranging from 2-16ng/ml. 74. III. Peptide Chemistry Fragments of IR-LGIP C 1. Cyanogen Bromide Cleavage of IR-LGIP C To provide additional structural information, cyanogen bromide cleaved material was subjected to dansylation. If structural hindrance blocked the N-terminus the combination of these two techniques would elucidate a possible terminal amino acid residue. It would also reveal the residue on the C-terminal side of methionine. IR-LGIP C (1.0 mg) was cleaved with cyanogen bromide and an aliquot (approximately 10 nmoles) was reacted with dansyl chloride. Subsequent thin layer chromatography on polyamide plates revealed phenylalanine and valine amino acid residues. 2. Tryptic Digestion GIP and IR-LGIP C were reacted with trypsin and analyzed on HPLC. Ten micrograms of each peptide were applied to a HPLC column and eluted with a linear gradient of 5-70% CH^CN, 30 min. Four major peaks were indicated (figure 29). Additional peaks reveal possible minor cleavage of the major peaks. Tryptic digest of GIP (figure 30) revealed 6 major peaks and 2 definite smaller peaks. Homology between the two tryptic digests existed for 2 peaks and are indicatedby \ i n figures 29 and 30. TIME (MIN) FIGURE 29 Trypsin-cleaved IR-LGIP C on HPLC 10 yg IR-LGIP C tryptic digest in 10 y l HPLC H20 with 0.1% TFA 5 - 70% CH3CN, 30 min. \r denotes homology with GIP tryptic peptides. C O u. 0.1 l O CM CM L D o z < CD DC O C O C D < 0 15 TIME (MIN) 25 FIGURE 30 Trypsin-cleaved GIP on HPLC 10 yg GIP tryptic digest in 10 y l HPLC H20 with 0.1% TFA 5 - 70% CH3CN 30 min. \r denotes peptides with similar retention times to IR-LGIP C tryptic peptides. 3. Amino Acid Composition of IR-LGIP C Amino acid analysis was performed on IR-LGIP C. The results expressed as mole ratios of each amino acid are shown in table 1. The presence of tryptophan was demonstrated qualitatively using Ehrlich reagent and thin layer chromatography (see Results Section II-C). 4. Determination of N-Terminal Residue Dansylation of IR-LGIP C, subsequent hydrolysis and polyamide chromatography revealed valine as the N-terminal residue. This technique also confirmed the presence of lysine and tyrosine in the peptide, that i s , only \u00C2\u00A3 -lysine and 0-tyrosine were detected. No bis-lysine or bis-tyrosine was observed ruling out these amino acids as the N-terminus. Table i Amino Acid Composition Mole Ratios Amino Acid A B C Asp/ASN 6.1 5.9 6 Thr 2.7 2.4 2 Ser 3.6 2.9 3 Glu/Gln 3.3 3.0 3 Pro 3.1 3.1 3 Gly 4.0 4.0 4 Ala 7.9 7.9 8 Val* 5.0 5.0 5 Met** 0.74 0.7 1 l i e 0.4 0.4 0 Leu 7.4 7.4 7 Tyr 1.2 1.0 1 Phe 3.2 3.0 3 His 4.7 4.4 4 Lys 4.7 4.7 5 Arg 2.0 3.6 3 Trp + + 1 A Amino acid Composition of IR-LGIPC B Mean of 10 analyses C Probable Composition ** Methionine sulphoxides and sulphone present + Tryptophan present as determined by Ehrlich reagent * Assigned mole integer of 5.0. 79. IV. Isolated Perfused Rat Pancreas Experiments Insulin Release 1. Control Study Following a 10 min equilibration period, the preparation was perfused with 80 mg/% D-glucose for 5 min. For the remaining 40 min the pancreas was perfused with 160 mg/% D-glucose. The perfusate was delivered at a rate of 4 ml/min and effluent was collected every min. Effluent fractions were stored at -20\u00C2\u00B0C prior to assaying for insulin. An immediate significant increase in insulin output was observed upon delivery of perfusate with 160 mg % (8.9mM) glucose. The i n i t i a l increase was rapid and transient followed by a plateau of elevated insulin secretion compared with 80 mg % (4.4mM) glucose perfusion. Results expressed as mean + S.E. are tabulated in Table 2 and shown in figure 31. 2. GIP Infusion GIP was presented to the isolated perfused rat pancreas, superimposed on control conditions via a side arm infusion. GIP was dissolved in perfusate with 160 mg % glucose and delivered at a concentration of 2 ng/ml in a 10 ml plastic syringe using a Harvard infusion pump at speed 6 (0.206 ml/min) for 20 min. Determinations of insulin release using the insulin radioimmunoassay are tabulated i n Table 2 and shown in figure 32. When GIP was infused a significant increase in insulin output was observed within 1 min of the start of infusion and returned to pre GIP infusion levels within 3 min of cessation of infusion. 80. 3. IR-LGIP C Infusion IR-LGIP C was presented at a concentration of 20 ng/ml to the isolated perfused rat pancreas. The delivery of this peptide was as described for the GIP infusion. Insulin release was detected using the insulin radioimmunoassay and results expressed as mean + S.E. are expressed in Table 2 and figure 33. No change in insulin output compared to control conditions was observed. 81. Insulin secretion in response to 80 mg/dl and 160mg/dl glucose with 2ng/ml GIP and 20ng/ml IR-LGIP C Minutes following the equilibration period Mean IRI (x) output ( U/min) n=7 min. 1-5: 80mg/dl glucose min. 6-45: 160mg/dl glucose Standard error of the mean for Column B (SEM) x IRI ( U/min) n=6 min. 1- 5: 80mg/dl glucose min. 6-45: 80mg/dl glucose min. 11-30: 2ng/ml GIP SEM for Column D *denotes significant (p 0.05) difference between columns B and D x IRI ( U/min) n=6 min. 1-5: 80 mg/dl glucose min. 6-45: 160mg/dl glucose min. 11-30: 20 ng/ml IR-LGIP C SEM for column G. Table 2 A. B. C. D. E. F. G. H. 1 53 20 30 12 34 10 2 52 13 63 17 32 10 3 62 15 49 12 47 25 4 54 17 55 11 25 5 5 40 9 32 7 25 11 6 132 48 51 16 44 8 7 332 80 241 89 60 14 8 256 66 324 124 197 66 9 223 67 204 51 122 42 10 201 83 129 42 114 27 11 160 71 140 22 90 22 12 132 29 290 54 A 75 18 13 121 23 312 80 * 93 16 14 100 19 420 99 * 80 9 15 106 17 584 207 * 121 19 16 109 21 323 107 115 24 17 122 26 239 46 A 99 18 18 145 25 296 87 115 24 19 125 29 344 85 * 85 14 20 152 35 370 80 * 104 7 21 144 29 380 95 A 122 28 22 132 27 425 67 A 120 25 23 121 24 399 85 A 114 10 24 136 25 377 62 A 121 17 25 135 26 346 41 A 109 29 26 140 18 276 36 A 132 21 27 134 29 354 38 A 135 24 28 156 34 321 40 A 130 25 29 130 30 346 38 A 134 27 30 144 33 415 55 A 144 30 31 151 37 281 40 139 21 32 125 24 288 32 A 127 29 33 152 36 327 54 A 133 27 34 144 35 221 37 135 24 35 143 32 261 56 130 24 36 164 48 244 63 130 15 37 182 45 327 134 117 16 38 149 43 276 96 107 16 39 133 49 148 32 189 85 40 166 48 127 38 127 38 41 180 86 125 22 115 24 42 163 58 123 28 168 39 43 162 62 141 20 73 23 44 171 54 185 41 152 33 45 143 40 132 27 129 23 83. 4.4mM 8.9mM G L U C O S E 400 l r 0 I i i i i i i i i i 5 15 25 35 45 TIME (MIN) FIGURE 31 Isolated perfused rat pancreas control study (n=7) The pancreas was perfused with 4.4mM glucose for 5 min and 8.9mM glucose for the remaining 40 min. In th i s f i g u r e and figures 32 and 33 the pancreas was eq u i l i b r a t e d for 10 min with perfusate with 4.4mM glucose. E f f l u e n t from t h i s period was discarded. I n s u l i n output expressed as IRI yU/min i s shown plotted against time i n minutes. 4.4mM 8.9mM G L U C O S E 600 r-400 _ 3. 200 . 2 . 0 N G/ML PORC INE GIP J 1 'AT TT 1 l y T T 15 25 TIME (MIN) 35 45 FIGURE 32 The e f f e c t o f 2ng/ml GIP on IRI r e s p o n s e i n t he i s o l a t e d p e r f u s e d r a t p a n c r e a s (n=6). 8 5 . 20ng/ml IR-LGIP \"C\" 300 4 4 m M 8 ' 9 m M G L U C 0 S E 5 15 25 35 45 TIME (MIN) FIGURE 33 The effect of 20ng/ml IR-LGIP C on insul in output in the isolated perfused rat pancreas (n=6). 86. Discussion Precursor proteins have been described for many biologically active peptide hormones (Lazure et a l , 1982). Classically gel f i l t r a t i o n of tissue or serum extracts has been used to identify the precursor moiety (Steiner and Oyer 1966, Eipper and Mains 1980, Potts et a l . , 1980, Rehfeld et a l . , 1976 and Gozes et a l . , 1983). Identification of the larger molecular weight fractions has been determined using radioimmunoassays established for the respective, active, smaller moieties. Biosynthesis of active peptides from pro-forms has been performed using pulse chase experiments (Eipper and Mains, 1980; Potts et a l . , 1980, Steiner et a l . , 1967). The advent of recombinant DNA technology has provided further invaluable means of monitoring synthesis and elucidation of the sequence of precursors (Itoh et a l . , 1983 and Noda et a l . , 1982). Many gastrointestinal hormones have also been found to be derived from a larger molecular form (Hakanson et a l . , 1982, Ravazzola and Orci, 1980; Patzelt et a l . , 1980). Gastric Inhibitory Polypeptide or Glucose-dependent Insulinotropic Polypeptide (GIP), a hormone of the diffuse endocrine system is most probably generated from a larger precursor peptide. Dryburgh (1977) showed the heterogeneity of GIP in serum and tissue. Controversy regarding circulating IR-GIP levels determined by radioimmunoassay (RIA) has also been reported (Brown, 1982). The variation may be a result of measurement of pro-forms, biosynthetic intermediates, degradation products, or even unrelated peptides. Isolation of definite precursor molecules could help c l a r i f y these observed discrepancies. 87. The sparcity of cells of the diffuse endocrine system precludes the use of recombined DNA technology in the isolation of precursors. This technology has only been of use when peptide-secreting tumours have been available. Common immunoreactivity, however, is an exploitable property of peptides that can be used to extract related peptides. Generally antisera used in RIA are heterogeneous and have numerous antibodies to several antigenic determinants, a factor which contributes to inconsistency in reported circulating levels of a particular peptide. Conventional antisera frequently crossreact with substances classified as families of peptides e.g. the glucagon family which includes: glucagon, VIP, GIP, PHI and secretin. These peptides have several homologous regions to which an antiserum may bind and demonstrate cross reactivity. Hybridoma technology used for monoclonal antibody generation allows screening for one antigenic determinant and therefore demonstration of shared immunoreactivity only i f a peptide has that particular amino acid sequence. It i s possible that the epitope may be common to a family of peptides but screening and cloning can be designed to select for either a specific epitope or an epitope shared by many family members. Selection of appropriate peptides or fragments for screening from areas of greatest or least homology can result in production of antibodies with exquisite sensitivity or antibodies which crossreact with other family members. Monoclonal antibodies possess highly specific binding properties and when used in af f i n i t y chromatography prove very selective agents for isolation of cross reacting peptides (Duffy and Kurosky, 1982). Immunoadsorbents produced with monoclonal antibodies eliminate the need for involved precipitation, chromatographic separation and RIA methods of 88. extraction and detection. The use of monoclonal antibodies in development of a f f i n i t y chromatography systems significantly reduces the number of extractable substances with common antigenicity. A monoclonal antibody to GIP was generated (Buchan et a l . , 1982) and i t was coupled to an insoluble support for a f f i n i t y chromatography. The system was carefully characterized and shown to be highly specific for GIP. 125 Components of I GIP that eluted in the wash phase were possibly fragments of iodinated material lacking antigenic determinants (figure 2 and 3). The unbound fraction of natural porcine GIP may simply have represented non-specific binding and was not the minor contaminant described by Jornvall et a l . (1981) which lacks the N-terminal residues NR^-Tyr-Ala (figure 4). Clearly the immunoadsorbent was specific for GIP and only had a f f i n i t y for peptides with common antigenicity which may be elongated or shortened forms of the GIP molecule (figures 2 - 7 ) . Mouse anti-GIP monoclonal antibody is specific for an epitope in the region 14-42 of the GIP molecule (Buchan et a l . , 1982). If a peptide has an N-terminal extension, a property more often ascribed to precursor proteins than C-terminal extension, binding to the anti-GIP monoclonal antibody was unlikely to be affected. Purification of natural GIP was performed using classical precipitation steps with organic solvents and neutral salts under conditions of controlled pH and temperature (figure 8). Related peptides sensitive to these conditions may have been lost in the purification procedures (Mutt, 1976). An immunoreactive-like GIP (IR-GIP) described by Dryburgh (1977) vras isolated using.the conditions described (figure 8) and was shown to demonstrate an overall acidic charge when applied to high voltage 89. electrophoresis. Alginic acid adsorption used in purification of GIP eliminates most acidic peptides (figure 8). Material not adsorbed to alginic acid was found to contain GIP-like immunoreactive fraction when applied to the immunoadsorbent system described (figure 10). Purification of this immunoreactive-like GIP (IR-GIP) material was undertaken to determine i t s status and structure. I n i t i a l l y ion exchange chromatography using the anion exchanger, DEAE A-25 Sephadex was performed and two fractions were separated from IR-LGIP (figure 12). When these fractions were examined on thin layer chromatography (TLC) no discernible difference was noted between the ion exchange fractions and IR-LGIP. HPLC did, however, indicate that anion exchange chromatography significantly separated a hydrophilic component of IR-LGIP (figure 17). The f i r s t fraction eluted from DEAE A-25 sephadex with 0.05M ammonium acetate had less hydrophilic material but contained the same hydrophobic components that IR-LGIP demonstrated on HPLC (Figure 16 and 17). The ELISA technique used to assay IR-LGIP, DEAE Fr A and DEAE Fr B showed a l l these substances to have comparable immunoreactivity. Based on these observations i t was realized that ion exchange chromatography did not markedly improve separation of the hydrophobic components of IR-LGIP. The superior resolution of HPLC and i t s a b i l i t y to separate the hydrophilic and hydrophobic fractions made i t the method of choice for further purification of IR-LGIP. The rationale for isolating the most polar fraction obtained on HPLC designated IR-LGIP C, was that i t demonstrated the greatest degree of purity on TLC and demonstrated most significant immunoreactivity when measured i n the ELISA. 90. Development of an assay for IR-LGIP C was attempted following HPLC purification. The use of solid phase ELISA methodology has facilitated peptide measurement. It i s a rapid method done in small volumes without use of radioisotopes (Voller et a l . , 1976). Heterogeneous anti-GIP antisera (Go 5) showed comparable a f f i n i t y for GIP and IR-LGIP C in both direct and indirect ELISA experiments using plates coated with either antigen (figures 23, 24, 26 and 27). Indirect inhibition studies using the ELISA were necessary however for assaying IR-LGIP C with the GIP monoclonal antibody, 3.65H. The lack of a f f i n i t y of 3.65H for IR-LGIP C in direct ELISA studies (figure 23) was possibly due to properties of solid phase assays. Steric hindrance of antigen may occur in solid phase and prevent antibody-antigen interaction. Liquid phase conditions used in inhibition studies may f a c i l i t a t e antigen-antibody binding. Indirect ELISA studies performed with preincubation mixtures of IR-LGIP C or GIP and 3.65H on plates coated with IR-LGIP C failed to confirm this possibility (figure 25A). The antigen-antibody interaction took place in liquid phase but as antigen concentration decreased excess antibody did not recognize the antigen (IR-LGIP C) used to coat the plate. If assay conditions were the reason for binding differences, displacement of IR-LGIP C would be expected to occur in RIA (liquid phase) when either Go 5 (figure 28) or 3.65H was used and not to take place in direct ELISA studies (solid phase) with Go 5 (figure 23). The heterogeneity of the antibodies to different antigenic determinants present in the antiserum (GS5) limits the possibility that a l l sites would be sterically hindered in solid phase. Direct studies with Gb5 on plates coated with either IR-LGIP C or GIP showed that solid phase hindrance of 91. antigenic determinants was not a factor (figures 23 and 24). In the RIA using antiserum Gb'5, GIP and IR-LGIP C did not demonstrate comparable displacement curves as found in ELISA studies with Gb5. Solid phase assay appeared to be a better system when Gb5 was used. Neither GIP nor IR-LGIP C showed displacement in RIA when 3.65 H was used. This could be attributed 125 to the remarkable avidity of 3.65H for I GIP, i.e. no amount of GIP or cross reacting peptide can displace with the iodinated material once bound to 3.65H. The conclusion reached from these observations was that the nature of the assay was not the determining factor for variation in af f i n i t y of IR-LGIP C for 3.65H seen on the immunoadsorbent column and in the ELISA (figures 10, 23 and 25A). A more probable explanation for the difference was that the region of IR-LGIP C which adheres to the ELISA microtitre plates and i t s antigenic determinant for binding to 3.65H was the same. If so, only inhibition studies using plates coated with GIP would be able to confirm the immunoreactivity of IR-LGIP C for 3.65H. This was found to be the case (figure 25B). Clearly 3.65H is very specific and IR-LGIP C does not possess a region capable of adhering to plastic different from i t s antigenic determinant for anti-GIP monoclonal antibody (3.65H) (figure 23). This may be a result of tertiary structure formation where folding of the molecule has masked an alternate suitable site for adhering to the microtitre plate. Historically, identification of a biological activity has been followed by peptide extraction, isolation, purification and f i n a l l y composition, and sequence determination. In order to learn the structural properties of a peptide, amino acid analyses are generally performed on the intact peptide, enzymatic digestion products and chemical cleavage fractions. The traditional strategy for sequencing peptides involved stepwise Edman N-terminal degradation of the molecule and chromatographic identification of residues (Doolitle, R.F. 1982). The current methodology of determining composition and peptide sequence analyses employs both traditional degradations in combination with new, sensitive and sophisticated techniques (Mutt, 1983 and Wittman-Liebold, 1982). The latter include HPLC and nucleotide sequencing. Progress in this f i e l d has permitted analysis of nanomolar quantities of peptide and eliminated the need for massive tissue sources to obtain sufficient material for enzymatic digestion, hydrolysis and sequence analyses. Certain properties have been attributed to precursor proteins and composition analyses should reveal i f the status of proform can be assigned to a peptide. These traits are not without exception and deviation may occur. However, certain common denominators do exist between a precursor and the active fragment. A core of common amino acid residues should be present in both i f a larger molecular weight species i s a proform. Enzymatic digestions and chemical cleavage should reveal common fragments. Chemical and enzymatic cleavage sites should reveal certain residues and the postulated cleavage site of the biologically active fragment at double base residues should be apparent with trypsin digestion. Shared immunoreactivity with the active peptide and lack of biological activity are other characteristics frequently observed in precursor hormones (Eipper and Mains, 1980). If structural analysis procedures reveal some or a l l of these properties i t could be speculated that a particular peptide has precursor status. 93. IR-LGIP C, when subjected to acid hydrolysis, did not show a core of amino acids common to GIP. In particular the ratio of isoleucine: leucine (0:7) did not concur with that found in GIP (4:2). Further composition information was obtained using the dansylation technique for N-terminal amino acid residue identification. Valine was found to be the N-terminal amino acid residue. This technique confirmed the presence of lysine and tyrosine in the molecule. N-terminal amino acid analyses on IR-LGIP C cleaved with cyanogen bromide indicated the presence not only of the valine residue but also a phenylalanine residue. It could be deduced that phenylalanine is located on the C-terminal side of methionine, the site of cyanogen bromide cleavage. This would also exclude this peptide as a precursor because the GIP sequence is Met-Asp. Erhlich reagent used with TLC confirmed the presence of tryptophan in IR-LGIP C. Amino acid analysis of IR-LGIP C revealed approximately 60 amino acid residues in the molecule confirming work of Dryburgh (1977) which estimated the larger moiety of IR-GIP to have a molecular weight of 8000 daltons. HPLC analysis of purified IR-LGIP C showed that i t was a separate entity from GIP, each peptide eluted with different retention times (figure 22). Tryptic digestion of each peptide (IR-LGIP C and IR-GIP) was performed and the products when applied to HPLC demonstrated common retention times for only 2 tryptic peptides, (figures 29 and 30). Most precursors are described primarily as N-terminal extensions of the secreted moiety and occasionally as C-terminal extended forms (Mutt, 1983). If IR-LGIP C were an extension of GIP i t would be expected that most of tryptic peptides would be identical. Additional fragments would also occur i f an extension had the appropriate cleavage sites, arginine and lysine residues. Classical strategy of peptide purification employs a bioassay at each stage to ensure that fractions exhibiting the activity are isolated further and unnecessary steps are avoided. The gastrointestinal tract is the site of many polypeptides and proteolytic enzymes. The diffuse nature of the endocrine system of the GI tract and the multiple bioactivities of the peptides limits the value of assays based on biological activity to monitor crude extracts. Synergistic, antagonistic or degradation product activity in crude extracts may complicate the interpretation of bioassays. To assess the biological function of a gastrointestinal peptide i n i t i a l purification followed by bioassay permits a more definitive description of activity. To date, precursor proteins have been described as devoid of biological activity. The isolated perfused rat pancreas bioassay developed by Pederson et a l (1976) i s an in vitro model for determining insulinotropic action of GIP. GIP has been shown to potentiate insulin release in a glucose dependent manner in rat, man and dog (Pederson et a l . 1976, Anderson et a l . , 1978 and Kwauk, 1982). Using the isolated perfused rat pancreas the biological activity of IR-LGIP C was investigated. Infusion of IR-LGIP C was compared with natural porcine GIP, but the dose was 10 fold greater. GIP was infused at a concentration of 2 ng/ml over 20 min and in keeping with the lack of or minimal activity attributed to precursors, IR-LGIP C was infused at 20 ng/ml. No potentiation of insulin release was detected. IRI secretion was comparable to control studies when IR-LGIP C was infused. In summary i t can be stated that IR-LGIP C has a molecular weight of approximately 8000 daltons, i t crossreacts with anti-GIP antisera and anti-GIP monoclonal antibody and i t demonstrates no insulinotropic 95. activity. The composition and tryptic digest show that GIP is not part of IR-LGIP C structure. It is not a pro-form of GIP but simply another gut peptide with common immunoreactivity. Locating the sequence homology of IR-LGIP C and GIP either by isolating and performing amino acid analyses on common tryptic peptides or sequence analysis of the entire molecule and comparing i t to GIP w i l l elucidate the antigenic determinant for 3.65H. To resolve the identity of IR-LGIP C i t would be expedient to employ microsequence analysis techniques to determine at least a partial sequence. Subsequent computer screening for existence of comparable sequences can greatly f a c i l i t a t e the direction of further investigation. Examining the physiology of the peptide would be redundant i f the peptide i s a known entity. If sequence analysis indicates a new gastrointestinal peptide has been isolated, function can be screened employing a number of bioassays. For example, gut motility in isolated ileum strips (Bakich et a l . , 1984); somatostatin secretion using isolated perfused stomach preparation (Mcintosh et a l , 1981); acid secretion using an anesthetized rat model (Goto and Debas, 1982); exocrine and endocrine pancreatic function employing isolated perfused pancreas (Pederson and Brown, 1976) lipoprotein lipase activity u t i l i z i n g isolated fat cells (Dahl, 1983) and g a l l bladder contractility using guinea pig bioassay (Ljunberg, 1964) can be ascertained. Generating monoclonal antibodies to the peptide would be an invaluable tool for determining cellular localization and providing further information about function, that i s , neural or endocrine. Whether the sequence shows the peptide to be known or unknown, the dilemma of pro-GIP s t i l l exists. DNA technology i s one approach that could be used to isolate the nucleotide sequence of a proform. The nucleotide sequence of the proform could be obtained using a cDNA probe of GIP to extract i t s mRNA. Chromosome walking techniques could then be employed to isolate an extension of GIP mRNA which might represent the nucleotide sequence of a precursor (Mutt, 1983). Since exceptions in proform properties exist, caution is required. For example, there is not a definitely established rule for the cleavage site of active peptides from either N-terminal extensions or C-terminal extensions. It is conceivable that isolated mRNA may have nucleotides for a peptide that is not a precursor of the active hormone. IR-LGIP C purified in this study may be a factor which does interfere with measurement of circulating IR-GIP levels. Certain RIA antibodies may crossreact with IR-LGIP C more than others and cause the discrepancy in levels observed in various assays. The function of IR-LGIP C may be that of a modulator of GIP action or i t may be a circulating degradation product of a structural protein or enzyme product of c e l l lysis that occurred during sampling. Isolation of the proform of GIP is necessary not only to help elaborate the biosynthesis and regulation of GIP but also, because i t may be responsible for the equivocal results reported concerning the circulating levels of GIP in pathophysiological situations (Brown & Otte, 1979). 97. 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Proc Nat Acad Sci USA 70: 2321-2325 Voller A, Bidwell D, Bartlett A (1976) Microplate enzyme immunoassays for the immunodiagnosis of v i r a l infections. In: Rose, N. and Freedman, H (eds) Manual of Cli n i c a l Immunology. American Society for Microbiology, Washington p 506-512 Wittman-Liebold B (1982) An evaluation of the current status of protein sequencing. In: Elzinga M (ed) Methods in Protein Sequence Analysis. Humana Press Clifton, New Jersey pp. 27-63 Woods KR, Wang KT (1967) Separation of dansyl-amino acids by poly-acrylamide layer chromatography. Biochim Biophys Acta 133: 369-371 3780/31A "@en . "Thesis/Dissertation"@en . "10.14288/1.0096202"@en . "eng"@en . "Physiology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Purification and partial characterization of a peptide cross reacting with antibodies to gastric inhibitory polypeptide"@en . "Text"@en . "http://hdl.handle.net/2429/24877"@en .