ì i l I FEATHER KERATI N SYNTHESI S A thesi s submi tted bY (H0NS. DAVID JAl4ES KtMP, B.Sc. ) ' to the Univers'itY of Adelaide' South Australia, for the deçjree of Doctor of PhilosoPhY. DEPARTMENT OF BIOCHElvlISTRY, UNIVERSITY OF ADELAIDE, SOUTH AUSTRAL IA. SEPTEMBER, 1972. To Kath ' for her occasional he'l p and encouragement, and to Andrew and Ben, al though they were of no hel P whatsoever. CONTENTS page j SUMMARY STAT EM E NT iìi ACKNOhJLEDGEMENTS iv ABBREVIATIONS vi CHAPTER I. GENERAL INTRODUCTION A. ]NTRODUCTORY COMMENTS I B. FEATHER STRUCTURE AND DEVELOPMENT I t Structure of Embryonic and Adult Feathers (a) Embryonjc Feathers I (b) Adult Feathers 2 2 Feather Kerati ns 2 (a) Keratins 2 (b) Mol ecul ar Structure of Feather Kerati n 3 (c) Feather Kerati n Protei ns 3 3. Deveì opment of the Embryonì c Chi ck , Feather 5 4. Keratjn Synthesis in the Embryonic Feather 7 c THE GROl^fTH AND DEVELOPMENT OF KERATINOCYTES 9 I Dermo-epi dermal Interact'i ons I (a) The Specìfy'i ng Action of Dermis 9 (b) The Response of EP'idermis l0 (c) The Nature of the Interactjons between Dermi s and EPi dermi s 1l (d ) The Mechani sm of ResPonse of the Epidermjs l3 2 0ther Factors Affecti ng Kerati nocyte Devel opment l6 (a) Hormones l6 (b) Vi tami n A 17 (c) Epi dermal Chal one ì9 (d) Epi dermal Growth Factor 20 (e) Nutri tional Factors 22 D. KERATIN BIOSYNTHESIS 22 't. Control of the 0nset of Kerati n Synthesi s 22 2. DNA Synthesjs and Mitosis 24 3. RNA Synthesis 25 4. Stab'i ì i ty of the Kerati n mRNA 26 5. The Pathway of Cytodi fferenti atj on 27 6. Ki neti cs of Kerati n SYnthesis 28 7. Ini ti ati on of Keratj n SYnthesi s 30 8. Assembìy of Keratj n Subuni ts 3l 9. Post-synthetic Modjfjcation of K.eratjns 32 10. Keratin Synthesis rn vitro 33 E. AIMS OF THE PROJECT 34 CHAPTER 2. IqATERIALS AI{D GENERAL METHODS A. MATER IALS 36 I Tissue 36 2 Enzymes and Protei ns 36 3 Radioactive Compounds 37 4 Fine Chem'icals for Specific Procedures 37 (a) Extraction Reduction and Carboxymethylation of Protei ns 37 ( b) Polyacryl amì de Gel El ectrophoresi s 3B (c) Measurement of Radioactiv'i ty 3B (d ) Col umn Chromatography 39 (e) Antibiotics 39 5. Miscellaneous Chemicals 39 6. Mi scel I aneous Materi al s 40 B. GENERAL METHODS 40 I Amino-Acid Analysis 40 2 Determination of Radioactivity 4l 3 Densitometry of Polyacry'l amide Gels 4l c. NOMENCLATURE 42 CHAPTER 3. CHARACTERIZATION OF THE KERATIN PROTEINS FROM ADULT AND EMBRYONIC FEATHERS AND SCALES. A. INTRODUCTION 43 B. METHODS 44 I Preparation of Tissues 44 2 Preparation of Reduced and S-carboxymethyl ated Protei n Extracts 44 3 Po'lyacrylamide Gel Electrophoresjs 46 4 Preparative Polyacrylam'ide Gel Electrophoresis 47 5 Isoel ectri c Focussi ng 47 6 Peptide Mappìng 47 7 Immunol ogical Techn'i ques 4B 8 Aggregation 4B c RESULTS I . Preparati on of P.educed S-carboxymethyl Protei ns 48 2 Properti es of Reduced S-carboxymethyl Protei ns from Adult and Embryonic Feathers and Scales 50 (a) Solubì1ity of the Proteins in ['Jater 50 (b) Poìyacrylamide Gel Electrophoresis at pH 9.5 50 (c) Polyacryl amide Gel El ectrophoresi s at pH 7 .5 52 (d) Po'lyacry'l amide Gel Electrophoresis at pH 2.7 52 (e) Isoelectric Focussing 53 (f ) Pepti de Mapp'i ng 54 (g) Immunodiffusion 54 (h) Aggregation 55 (i ) Amino Acid Composjtions 55 3 Isolation of Rachis Protein Fractions by Preparat'ive Pol yac ryl ami de Ge I El ec trophores i s 56 4 Nature of the "Water-Insoluble" Proteins of Embryon'ic Feather 57 (a) Identification of the y-Prorei ns and the "Fast Band" 57 (b) Isolation of y-Proteìns by DEAE-Cellulose Chromatography 58 5. Embryonic Feather-Sheath Proteins 59 D. DISCUSSION 60 1 Heterogenei ty of Feather Protei ns 60 2 Di fferences i n the Protei ns of Feathers and Scales 6l 3 Microheterogeneity and Relatjonship to Kerati n Structure 6l (a) ß-Proteins 6l (b) o¿- and y-Proteins 62 4. Embryonic Feather Sheath proteins 64 5. S'ignificance in Relation to Deve'lopment 64 CHAPTER 4. N.TERMINAL SEQUENCES OF EMBRYONIC FEATHER PROTEINS. A I NTRO D IICTION 67 B METHODS 67 I Proteolytic D'i gestions 68 (a) Pronase Procedure 6B (b) 0ther Enzymes 6B 2 Deprote'i njzat'ion of Proteo'lytic Digests 69 3 Isol ati on of Bl ocked Pepti des by Dowex-50 Chromatography 69 4 Chromatography on SePhadex G-l 0 69 5 Chromatography on Dowex-l 70 6 Chromatography on SePhadex G-50 70 7 Hi gh Voì tage Paper El ectrophoresi s 71 8 N-terminal Anaìysis 71 c RESULTS 72 I Isolation of the N-Termi nal Di pePtide 72 (a) Isolation by the Pronase Procedure 72 (i) Fract'ionation of the Dowex-50 eluate 72 (ii) Nature of the fracti ons i sol ated by Sephadex G-l 0 and Dowex-l chromatography 72 (b) Isol atj on by the Chymotryps i n/Carboxy- peptidase-A Procedure 73 (c) Isolation of th eN -terminal D'i pept'ide from Puri fi ed F rac tions of Embryonic Feather Prote'i n s 74 2. Isolation of N-Terminal Trìpeptides from Unfracti onated and Fracti onated Embryoni c Feather Protei ns 75 3 Isolation of N-Terminal Pgptides After Digestion with Elastase 76 4 Isolatjon of N-Term'inal Tryptic Peptjdes 77 (a) Tryps'i n/Cargoxypept'idase B Procedure 77 ( b) Sephadex G-50 Procedure 77 (i ) Identification of Sephadex G-5q fractions contai ning N-terminal trypt'ic PePt'ides 77 (ii) Isolation of N-termjnal trYPtic pept'ides 78 (iii) Mol ecul ar wei g ht of N-termi nal tryptic pepti d es 79 D. DISCUSS ION E. APPENDIX I FIBRILLATION OF A TRYPTIC PEPTIDE FRACTION FROM EMBRYONIC FEATHER PROTE I NS 83 CHAPTER 5. KERATIN SYNTHESIS DURTNG DEVELOPMENT OF THE TMBRYONIC FEATHER A INTRODUCTION 86 B METHODS 87 Prepara ti on of Embryo n i c Fea thers B7 2 Extraction of Feather Proteins 87 j 8B 3 Qua n ti ta ti ve Poi yacry'l amì de Gel El ec tro phores s l4C-Leucí 4 Incorporatl'on of ne by Feather Ti s sue 90 5 Poìyacrylamide Gel Electrophoresis of Radioacti ve Protei ns 90 3H-thymidine 3 6 Incorporation of and H-Uridi ne by 12-day Feathers 9l 7 . tl ectron Mi croscoPY 92 B. El ectron-mi croscopi c Au toradi ography 93 C. RESULTS Changes i n Total Protei n Content Duri ng Feather Devel oPment 93 2 Determinatjon of Keratin Content Duri ng Feather Deveìopment by Quantitative Polyacrylam'ide Gel Electrophoresis 94 (a) Polyacrylamide Gel Electrophoresis at pH 9.5 94 (b) Po'lyacrylamjde Gel Electrophoresis at pH 7 .5 96 (c) Polyacry'l amide Gel El ectrophoresis at pH 2.7 96 (d ) Quanti tati on of Protei n Bands on Pol yacry'l am'ide Gel s 97 (e) Changes in Total Keratin Content During Feather Deve'l opment 98 3 Determi nati on of the Rates of Kerat'i n Synthesis Durj ng Feather Devel oPment bY Incorporation of l4c-Leucine 99 j 4 El ectron Mi croscopt'c 0bservati ons on Kerat n Synthesis During Feather Development 100 (a) 0ccurrence of Kerat'in Fi bri I s i n 12-15- .l00 Day Feather Cel I s (b) 3H-lhym'i di ne Autoradi ograPhY 100 (c) 3H-Uridine Autoradiography l0l D. DISCUSSION .l03 l Changes in Protein Content and Synthesjs 2 Ki neti cs of Keratj n SYnthesi s I05 3 Eìectron-Mjcroscopic 0bservations on Feather Keratìn SYnthesjs 107 CHAPTER 6 . KERAT IN SYNTHES I S IN A CELL-FREE SYSTEM FROM THE EI'IBRYONIC FEATHER A. INTRODUCTION ll0 B. METHODS lll ì. Incorporation of Radioactive Precursors into Proteins in the LYsate SYstem llr 2. Preparatj on of Reduced and Carboxymethyl ated Radi oacti ve Protej ns from the Lysate Supernatanfs lll 3. Polyacryl amide Gel Ej ectrophoresi s 112 4. DEAE-Cel I ul ose ChromatograPhY 112 5. Sephadex G-l 00 ChromatograPhY 112 6. N-termi nal AnalYsis 112 7. Determination of the Specific Activity of 3H-Seri ne i n the Protei ns and N-Termi nal Dipept'ide ll3 B. Preparatjon of "De-acetylated SH-keratin" il3 9. Preparation of Nascent Po'lypeptide Chains ll4 t0. Fracti onatj on of Nascent Cha i n Preparati ons on Sephadex G'25 ll4 lt. Fractionation of Nascent Chajn Preparatjons on Sephadex G-50 ll5 12. Fractj onati on of Nascent Chai n Preparati ons on Sephadex G-l 0 ll5 I 3.. Fracti onatj on of Nascent Chai n Preparati ons on Dowex- 50 115 14. Hi gh Vo'l tage Paper El ectrophoresi s at pH 1.9 il6 15. N-termi nal Ana lys i s by the Dansyl -Chl ori de Procedure 1.l6 C. RESULTS il7 l Characteri zati on of the Protei ns Synthesi zed in'14-Day Lysates 117 (a) Polyacrylam'ide Gel Electrophoresis 117 (i) Polyacrylamide Gel Electrophores'i s at PH 7 .s il7 (ii) Poìyacry'l amide Gel Electrophoresjs at PH 2.7 ilB (b) DEAE-cel lulose Chromatography llB (c ) Sephadex G-l 00 Chromatography 119 2 Comparison of the Proteins Synthesjzed ìn I 4-Day Lysates wi th Protei ns Synthesi zed i n Lysates from Feathers of 0ther Ages 120 3 Isol ati on of the N-termi nal D'i peptjde from the Proteins SYnthesized in I 4-Day Lysates 121 (a) Isol a ti on of the N-Termi nal Dipept'ide by th e Pro nase Proc edure 121 (b) Isol atjon of the N-termjnal Dipeptide by the Chymotryps'i n /Car boxypepti da se A Procedure 122 'Released (c) Cal cul ati on of the Number of Chains Per PolYsome which were Ini tiated i n the Lysate 123 4 Studies on the Mechanism of N-Acetylatjon of Keratj n Protei ns j n 1 4-Day Feather Lysate 125 {a ) Characterj zation of Protei ns Label I ed b.y Incubat j on,of the LYsate i n the Presence of I +C-Acetyl -CoA 125 (b) Isolation of the N-Term'i nal Dipeptide from - Protei ns Label I ed i n the Presence of l4C-Acetyl CoA 126 (c) Effects of "de-acet.ylated" Keratin on Incorporation of I 4C-Acetate i nto the N-terminal DipePt'ide 127 5 Attempts to Determ'i ne the Nature of the Initiator of Keratin Synthes j s Us'ing 1 4-Day Lysates 128 3H-serine (a ) Incorporati on of into Nascent Cha'i ns in'14-DaY Feather Lysates 129 3H-Nethi (b) Incorporatj on of oni ne i nto Nascent Chains in 14-Day Feather Lysates l3l D.