Alexander Scott PhD thesis

146 Pages·2017·8.39 MB·English

A STUDY OF VERTEBRATE CORTICOTROPHINS AND
RELATED PEPTIDES

Alexander P. Scott

A Thesis Submitted for the Degree of PhD
at the
University of St Andrews

1974
Full metadata for this item is available in
St Andrews Research Repository
at:
http://research-repository.st-andrews.ac.uk/

Please use this identifier to cite or link to this item:
http://hdl.handle.net/10023/14878

This item is protected by original copyright
UNIVERSITY ON ST. ANDREW’ PI i. ■' 1CGT D
A STUDY OP VERTEBRATE CORTICOTROPHINS
AND RELATED PEPTIDES
Alexander P. Scott
Department of Zoology, St. Andrews University
and the Department of Chemical Pathology,
St. Bartholomew’s Hospital, London.
A thesis submitted to the University of
St. Andrew’s for the degree of
Doctor* of Philosophy
ProQuest Number: 10171216
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
uest.
ProQuest 10171216
Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author.
All rights reserved.
This work is protected against unauthorized copying under Title 17, United States Code
Microform Edition © ProQuest LLC.
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106- 1346
? 7
1 declare that this thesis has been composed by myself and
the work of which it is a record has been done by myself. It has
not been accepted in any previous application for a higher degree
The date of admission as a research student and candidate for a
Ph.I)., was October 1st, 1968.
A. P. Scott.
I declare that the conditions of the Ordinance and Regulations
(Ph.l).) have been fulfilled.
Supervisor
ACKNOWLEDG ;' ENTS
I wish, to express my sincere gratitude to Professor
J. Landon, Dr, J. Ratcliff's and Dr. G.M. Besser of St.
Bartholomew’s Hospital for their encouragement and
guidance throughout this study, and to Dr. D. Scott
who acted as my supervisor in St. Andrew’s. Thanks
are also due to Dr. B. Elliott for the opportunity to
collaborate with Dr. P.J. Lowry, Mr. C. McMartin and
Mr. Hugh Bennett at CIBA Laboratories.
I gratefully acknowledge the help of many others
who helped me in the course of this study, including
Dr. L. Rees, for the organ culture of rat pituitaries,
Dr. B. Smith for advice and assistance with irarauno-
staining, Dr. B. Evennett for the dissection of lamprey
pituitaries and Dr. H. Slack for the use of the
facilities at the Glasgow University Field Station on
Loch Lomond.
I am indebted to the Science Research Council for
their support from 1st October, 1968 - 30th September,
1971, and to the Tenovus Institute, Cardiff, for the
award of my current grant.
ABBREVIATIONS
ACTH - adrenocorticotrophic hormone
MSH - melanocyte stimulating hormone
LPH - lipotrophic hormone
CLIP - corticotrophin-like intermediate lobe peptide
Asp - aspartic acid ( Asn - asparagine , Asx - either
Asp or Asn )
Thr - threonine
Ser - serine
Glu - glutamic acid (Gin - glutamine , Glx - either
Glu or Gin )
Pro - proline
Gly - glycine
Ala - alanine
Cys - cystine
Val - valine
Met - methionine
Leu - leucine
He - isoleucine
Tyr - tyrosine
Ph e - ph. e ny 1 a 1 an i n e
Lys - Lysine.
Ilis - histidine
Arg - arginine
Try - t ryp t o ph an
IIC1 - hydrochloric acid
HAc - acetic acid
TEA - trifluoroacetic acid
MeqNAc - trimethylamine acetate
I - iodine
gram
io'3e
rag - milligram
-1 ~“6
Jig - micro,-gram 10 g
1 o-9g
ng - nanogram
, „-12
PC - picogram 10 g
M - molar
N - normal
mM - millimolar
nM - nanomolar
cm - centimetre
1 O"\ietre
rnm - millimetre
_o
nm ~ nanometre 10 "metre
ml - millilitre 10 ’litre
, -6
hl - inicrolitre 10 litre
I.S - international standard
I.U - international units
1 0“^U
mU - milliunits
- „ —
(lU - microunits 10 u
v/v - volume/volume
w/v - weight/volume
IV - Intravenous
S.E. - standard error
S.D. - standard deviation
sec - second
min - minute
h - hour
/ - percentage
° - temperature in degrees centigrade
rpni - revolutions per minute
cps - counts per second
CONTENTS
ABSTRACT . 1
INTRODUCTION
SECTION 1 : THE VERTEBRATE PITUITARY..................................................................3
SECTION 2 : ACTH AND RELATED PEPTIDES IN MAMMALS...................................7
a) Definition and structure of ACTH. 7
b) Definition and structure of MSII. 10
c) Other ACTH-like peptides. 14
d) Biol ogical and immunological properties of ACTII and MSH.16
e) Localisation of ACTH in the mammalian pituitary. 21
f) Localisation of MSII in the mammalian pituitary. 23
SECTION 3 : ACTII AND RELATED PEPTIDES IN NON-MAMMAL J AN
VERTEBRATES.............................................................................................26
a) The vertebrate pituitary-adrenal axis and ACTH. 26
b) Evidence for related pituitary hormones. 28
c) Localisation of corticotrophic and melanotrophic
activity in vertebrate pituitaries. 29
SECTION 4 : PROBLEMS CONCERNING THE EVOLUTION OF THE
ACTII-LIKE PEPTIDES..................................................................... 31
METHODS
SECTION 1 : PEPTIDE EXTRACTION AND ISOLATION PROCEDURES...................33
a) Treatment of pituitaries. 33
b) Extraction of ACTH and related peptides from tissue. 33
c) Purification of ACTH-like peptides. 36
d) Chemical analysis of peptides. 37
SECTION 2 : ASSAY PROCEDURES 41
a) Bioassay. 41
b) Radioimmunoassay of ACTH. 45
SECTION 5 : IMMUNOHISTOLOGICAI; AND ORGAN CULTURE PROCEDURES.....51
a) Immunofluorescent histology. 51
b) Organ culture. 51
RESULTS AND PRELIMINARY DISCUSSION
SECTION 1 : CHARACTERISATION OF METHODS............... 55
a) Radioimmunoassay. 55
b) Extraction. 58
SECTION 2 : COMPARATIVE STUDIES...................................... ......64
a) Immunological characterisation of vertebrate
corticotrophins. 64
b) Pituitary content of corticotrophin in several
vertebrate species. 70
SECTION 5 : STUDIES ON THE LOCALISATION OF ACTH IN THE
RAT PITUITARY....................................................................................................... 77
a) Comparison of immunoactive ACTH concentrations in
separated pituitary lobes. 77
b) Organ culture of rat pituitaries. 80
c) Immunofluoroscent staining of rat pituitaries. 85
SECTION 4 : PURIFICATION AND CHARACTERISATION OF ACTH-LIKE
PEPTIDES IN RAT , PIG AND MAN.......................................................85
a) Rat. 85
b) Pig. 92
c) Human tumour. 99
d) Partial sequencing studies. 102
e) Comparison of C-terminal peptides with known
corticotrophins. 105