Protein Deposition in Animals. Proceedings of Previous Easter Schools in Agricultural Science

304 Pages·1980·5.078 MB·English

by  P.J. Buttery and D.B. Lindsay (Auth.)

Published Proceedings of Previous Easter Schools in Agricultural Science
SOIL ZOOLOGY*
Edited by D.K.McE. Kevan (Butterworths, London, 1955)
THE GROWTH OF LEAVES*
Edited by F.L. Milthorpe (Butterworths, London, 1956)
CONTROL OF THE PLANT ENVIRONMENT*
Edited by J.P. Hudson (Butterworths, London, 1957)
NUTRITION OF THE LEGUMES*
Edited by E.G. Hallsworth (Butterworths, London, 1958)
THE MEASUREMENT OF GRASSLAND PRODUCTIVITY*
Edited by J.D. Ivins (Butterworths, London, 1959)
DIGESTIVE PHYSIOLOGY AND NUTRITION OF THE RUMINANT*
Edited by D. Lewis (Butterworths, London, 1960)
NUTRITION OF PIGS AND POULTRY*
Edited by J.T. Morgan and D. Lewis (Butterworths, London, 1961)
ANTIBIOTICS IN AGRICULTURE*
Edited by M. Woodbine (Butterworths, London, 1962)
THE GROWTH OF THE POTATO*
Edited by J.D. Ivins and F.L. Milthorpe (Butterworths, London, 1963)
EXPERIMENTAL PEDOLOGY*
Edited by E.G. Hallsworth and D.V. Crawford (Butterworths, London, 1964)
THE GROWTH OF CEREALS AND GRASSES*
Edited by F.L. Milthorpe and J.D. Ivins (Butterworths, London, 1965)
REPRODUCTION IN THE FEMALE MAMMAL*
Edited by G.E. Lamming and E.C. Amoroso (Butterworths, London, 1967)
GROWTH AND DEVELOPMENT OF MAMMALS*
Edited by G.A. Lodge and G.E. Lamming (Butterworths, London, 1968)
ROOT GROWTH*
Edited by W.J. Whittington (Butterworths, London, 1968)
PROTEINS AS HUMAN FOOD*
Edited by R.A. Lawrie (Butterworths, London, 1970)
LACTATION*
Edited by I.R. Falconer (Butterworths, London, 1971)
PIG PRODUCTION
Edited by D.J.A. Cole (Butterworths, London, 1972)
SEED ECOLOGY*
Edited by W. Heydecker (Butterworths, London, 1973)
HEAT LOSS FROM ANIMALS AND MAN: ASSESSMENT AND CONTROL
Edited by J.L. Monteith and L.E. Mount (Butterworths, London, 1974)
MEAT*
Edited by D.J.A. Cole and R.A. Lawrie (Butterworths, London, 1975)
PRINCIPLES OF CATTLE PRODUCTION*
Edited by Henry Swan and W.H. Broster (Butterworths, London, 1976)
LIGHT AND PLANT DEVELOPMENT
Edited by H. Smith (Butterworths, London, 1976)
PLANT PROTEINS
Edited by G. Norton (Butterworths, London, 1977)
ANTIBIOTICS AND ANTIBIOSIS IN AGRICULTURE
Edited by M. Woodbine (Butterworths, London, 1977)
CONTROL OF OVULATION
Edited by D.B. Crighton, N.B. Haynes, G.R. Foxcroft and G.E. Lamming (Butterworths,
London, 1978)
POLYSACCHARIDES IN FOOD
Edited by J.M.V. Blanshard and J.R. Mitchell (Butterworths, London, 1979)
SEED PRODUCTION
Edited by P.D. Hebblethwaite (Butterworths, London, 1980)
* These titles are now out of print
Protein Deposition
in Animals
P.J. BUTTERY, BSc, PhD
Department of Applied Biochemistry and Nutrition
University of Nottingham School of Agriculture
D.B. LINDSAY, MA, DPhil
ARC Institute of Animal Physiology
Babraham, Cambridge
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First published 1980
© The several contributors named in the list of contents, 1980
ISBN 0 408 10676 X
British Library Cataloguing in Publication Data
Easter School in Agricultural Science, 29th
University of Nottingham, 1980
Protein deposition in animals.
1. Proteins in animal nutrition - Congresses
2. Protein metabolism - Congresses
I. Title II. Buttery, Ρ J HI. Lindsay, D Β
636.089'2'398 SF98.P7 80-49869
ISBN 0-408-10676-X
Typeset by Scribe Design, Gillingham, Kent
Printed by Billing & Sons Ltd, London & Guildford
PREFACE
The 29th Easter School of the University of Nottingham, of which this book is
the proceedings, discussed the factors controlling protein deposition in farm
animals. The aim of the meeting was to mount a forum in which biochemists and
physiologists could discuss with colleagues associated with the more practical
aspects of animal production, the factors which influence protein production by
animals. The book starts by discussing some fundamental aspects of protein syn-
thesis and is followed by a consideration of the molecular control of protein break-
down. Two chapters then consider the measurement of whole-body protein
metabolism and the integration of the metabolism of individual organs with the
rest of the animal.
Two 'tissues', the muscle and the fetus, are singled out for detailed discussion
in subsequent chapters, while another chapter attempts to describe the synthesis
of egg proteins but shows clearly that much more work is required in this area.
The factors which influence overall nitrogen retention by the animal are studied,
as are the energy costs of protein deposition. Hormonal influences on protein
deposition are considered from three different angles: first, a detailed discussion
of hormone action; secondly, the way of manipulating growth with anabolic
agents; and thirdly, the implications from a health point of view of current
practice in the use of these anabolic agents. Two chapters, one on poultry and
the other on ruminants, are concerned with predicting rates of protein deposi-
tion. The book ends by considering protein metabolism in a cold-blooded animal,
the fish.
The meeting clearly indicated that there were numerous areas where addition-
al work is required. The speakers were particularly asked to speculate on future
developments in the area and much of this speculation appears in their papers.
The organizers would like to thank the speakers, the chairmen of sessions,
and indeed all the staff of the University of Nottingham for their efforts in
making the 29th Easter School a success. Particular mention should be made
of Mrs Shirley Bruce and her staff who did so much to keep the organization
of the conference running smoothly. In addition, the financial contributions by
the concerns mentioned elsewhere were most gratefully received since they
enabled the costs of the meeting to the delegates to be kept at a reasonable level.
P.J. BUTTERY
D.B. LINDSAY
ACKNOWLEDGEMENTS
The organizers wish to thank the staff at the University of Nottingham, the
speakers and the chairmen (P.J. Buttery, D.J.A. Cole, Professor G.E. Lamming
and Professor G.A. Lodge), who all contributed to the success of the meeting.
The assistance of the following organizations is also gratefully acknowledged
since without their help the meeting would not have taken place:
BOCM Silcock Ltd
BP Nutrition (UK) Ltd
Colborn Group Ltd
Imperial Chemical Industries Ltd
Pauls and Whites Foods Ltd
Pedigree Petfoods
Roussel Uclaf
Rumenco Ltd
Sun Valley Feed
Unilever Research Laboratory
1
MECHANISM AND REGULATION OF PROTEIN
BIOSYNTHESIS IN EUKARYOTIC CELLS
VIRGINIA M. PAIN
Department of Human Nutrition, London School of Hygiene and
Tropical Medicine
and
MICHAEL J. CLEMENS
Department of Biochemistry, St George's Hospital Medical School, London
Summary
Protein biosynthesis comprises a series of complex processes involving three kinds of
RNA and a large number of proteins. Messenger-RNA (mRNA) carries in its nucleo-
tide sequence a code determining the order of insertion of amino acids into the poly-
peptide chain. Ribosomal-RNA, in combination with about 70 proteins, is formed
into an organelle, the ribosome, which provides the correct structural alignment for
the other protein synthetic components. Transfer-RNA (tRNA) exists as a number of
different species, each specific for a particular amino acid, and is involved in activating
and binding successive amino acids to the ribosome in the order directed by the
structure of messenger-RNA (also bound to ribosomes). Protein factors involved in
protein synthesis include many which function enzymically and others which have
more structural significance.
The overall process, referred to as translation, is divided into three stages: (1)
Initiation. A ribosome and a molecule of a specific initiator tRNA (Met-tRNAf) bind
to a particular site on the mRNA at the beginning of the coding sequence. Another
aminoacyl-tRNA is then able to bind, and synthesis of the first peptide bond takes
place. (2) Elongation. The ribosome moves relative to the messenger-RNA and a
polypeptide chain is elaborated from amino acids in a specific sequence directed by
the order of nucleotides in the messenger-RNA. (3) Termination. The ribosome
reaches the end of the coding sequence on the messenger-RNA and is released together
with the completed protein chain. In most tissues in vivo each molecule of messenger-
RNA is translated simultaneously by several ribosomes, the entire structure being
termed a polyribosome or polysome.
The overall rate of protein synthesis in eukaryotic cells and tissues can be regu-
lated at two levels: (a) by the number of ribosomes available in the tissue, which
determines the maximum rate of protein synthesis possible, and (b) by the activity
of the ribosomes, i.e. the rate of protein synthesis per ribosome in the tissue. The
activity of ribosomes appears, in most cases studied to date, to be regulated at the
level of initiation of protein synthesis. Experiments which have led to this conclusion
and possible mechanisms by which initiation may be regulated will be discussed.
Technical difficulties have limited progress in this area of investigation with many
animal tissues. Data are therefore presented which have been obtained with a model
system of nutritional control (amino acid regulation of protein synthesis in Ehrlich
ascites tumour cells in tissue culture), which can be subjected to more detailed
analysis at the molecular level. Analogies will be drawn, where possible, with results
obtained in various laboratories using normal tissues such as muscle and liver.
1
2 Mechanism and regulation of protein biosynthesis in eukaryotic cells
Introduction
During the 1970s we witnessed a very rapid development of knowledge of the
mechanism of protein biosynthesis in animal cells. Even now our understanding
of many details of the various chemical interactions is far from complete. It is
apparent that, while the overall mechanism of protein synthesis in eukaryotes
is analogous to that operating in bacteria, there are several stages which are
considerably more complicated. Earlier reviews were concerned mainly with the
bacterial process, but several recent articles have been devoted partly (Mazumder
and Szer, 1977) or wholly (Pain, 1978; Revel and Groner, 1978; Pain and
Clemens, 1980) to describing protein synthesis in eukaryotes.
In parallel with the characterization of the mechanism of protein biosynthesis,
there have been developments in our understanding of the regulation of this
process. Improved methods of measurement of rates of protein synthesis in vivo
(see Chapter 3) permit the identification of situations in which physiological
regulation occurs, and pinpoint fruitful areas for investigation at the subcellular
level. At present, however, most of our knowledge of regulatory mechanisms is
derived from model systems, e.g. reticulocytes and tumour cells in culture, in
which very pronounced variations in the protein synthetic rate can be induced
by the investigator. It is hoped that studies in these systems will provide informa-
tion on potential sites of control of protein synthesis which will be applicable
to tissues of normal animals.
In this chapter, the present knowledge of the mechanism of protein biosyn-
thesis in animal cells is summarized, followed by a discussion of recent studies
aimed at increasing our understanding of how the rate of this process may be
controlled at the molecular level.
Mechanism of protein synthesis
TYPES OF RNA
Protein biosynthesis comprises a series of complex processes involving three
kinds of RNA and a large number of proteins. The order of insertion of amino
acids into a polypeptide chain is directed by the sequence of nucleotide bases
in messenger-RNA(mRNA), which is transcribed from DNA in the nucleoplasms
region of the nucleus. Each amino acid is specified by a codon of three bases in
the mRNA. Recent studies have revealed that most eukaryotic mRNAs contain
certain characteristic features in addition to the coding sequence (Figure 1.1).
At the 5' end they carry a 'cap' structure, i.e. a base-methylated guanosine
residue joined 5'—5' to the next nucleotide by a trisophate linkage (see reviews
by Rottman, 1976; Shatkin, 1976; Filipowicz, 1978; Revel and Groner, 1978;
Pain and Clemens, 1980). The function of the cap is not yet certain but this
structure may well play a role in regulating the initiation of protein synthesis
(Filipowicz, 1978). Next to the 'cap' is an untranslated sequence, the length of
which differs between species of mRNA, followed by the coding region which
commences with the codon-specifying methionine (AUG). At the 3' end of the
coding sequence is another untranslated region, and finally a segment of poly-
adenylic acid residues, again untranslated. The presence of this poly(A) tract
VΜ. Pain and M.J. Clemens 3
has been utilized extensively by biochemists in devising procedures for extract-
ing mRNA from cells and tissues, but its physiological function is still obscure
(Brawerman, 1976; Revel and Groner, 1978; Pain and Clemens, 1980). Some
studies in cell-free systems suggest a direct role of poly(A) in regulating the rate
of protein synthesis, but there are also indications that it may be involved in
controlling the turnover of mRNA (e.g. Huez etal, 1975).
Untranslated Untranslated
5, J Cap J sequence | Coding sequence ,sequence Poly(A)| t
7-methylguanine Base
Figure 1.1 Generalized structure of messenger-RNA. The majority of eukaryotic mRNAs
contain the five regions shown (see text). The relative lengths of the different parts of the
molecule are not drawn to scale. The expanded portion shows the structure of the 5'—
terminal cap
Amino acid
attachment site
Anticodon
Figure 1.2 Characteristic structure of transfer-RNA, showing the position of the anticodon
and the site for attachment of the specific amino acid
The genetic information represented by the sequence of bases in mRNA is
decoded by transfer-RNA (tRNA) and converted into sequences of amino acids
in proteins. The structure of tRNA (Figure 1.2) includes three features which
enable it to carry out this function: (1) an anticodon triplet of bases for
complementary binding to the codon in mRNA specifying a particular amino
4 Mechanism and regulation of protein biosynthesis in eukaryotic cells
acid, (2) a site for attachment of that same amino acid at the 3' terminus, and
(3) a recognition site for the specific aminoacyl-tRNA synthetase enzyme which
catalyses the binding of the amino acid. The formation of aminoacyl-tRNA
involves the hydrolysis of ATP to AMP; hence, the preparation of each molecule
of amino acid for protein synthesis requires the expenditure of two high-energy
bonds.
The third type of RNA involved in protein synthesis is ribosomal-RNA
(rRNA), which is synthesized in the nucleolus and becomes incorporated,
together with about 70 proteins, into the ribosomes. Ribosomes consist of two
subunits, termed by their sedimentation behaviour on sucrose-density gradients
as 40S and 60S. The nature and possible roles of the protein components of
ribosomes have been the subject of much recent investigation (Wool and Stoffler,
1974), and there is some evidence, as yet inconclusive, linking changes in protein
synthetic activity of ribosomes with covalent modifications, e.g. phosphoryla-
tion, of certain ribosomal proteins.
THE RIBOSOME CYCLE
Figure 1.3 gives an overall scheme of the process of protein synthesis, often
referred to as translation. This can be divided into three stages:
(1) Initiation. A ribosome and a molecule of a specific initiator tRNA (Met-
tRNAf) bind to a particular site on the mRNA at the beginning of the coding
sequence.
m Direction of translation
Initiation Polysome Elongation Termination
δ
Figure 1.3 Diagram of the ribosome cycle, showing the incorporation of native ribosomal
subunits into polysomes (peptide chain initiation), the transit of the ribosomes along the
mRNA as the nascent polypeptide chains are extended (elongation), and the release of the
ribosomes and completed protein (termination). (From Pain, 1978)
V. Μ. Pain and Μ J. Clemens 5
(2) Elongation. The ribosome moves relative to the mRNA and a polypeptide
chain is elaborated from amino acids in a specific sequence directed by the
genetic information encoded in the order of bases in the mRNA.
(3) Termination. The ribosome reaches the end of the coding sequence on the
mRNA and is released together with the completed protein chain. The
ribosome is then available for reattachment on the same or another molecule
of mRNA. If it is not required immediately for another round of protein
synthesis, however, it may remain in the cytoplasm, in an 'idling pool' of
monomeric ribosomes unattached to mRNA.
In most tissues in vivo, as shown in Figure 1.3, each molecule of mRNA is trans-
lated simultaneously by several ribosomes. The entire structure is called a
polyribosome or polysome.
The three stages of protein synthesis will now be described in more detail.
The text concentrates mostly on the process of initiation, since this has been
the subject of most dramatic expansion of knowledge in recent years and is
thought to be an important site of regulation of overall rates of protein synthesis
(see page 8).
Figure 1.4 Expansion of the region of Figure 1.3, showing the initiation of protein syn-
thesis. The diagram shows: (1) the dissociation of a monomeric (80S, i.e. 40S+60S subunits)
ribosome into native 4OS and 60S subunits. (2) the presence of extra proteins (initiation
factors) on the native 40S subunits. (3) the formation of a complex between 40S subunits
and the initiator tRNA (Met-tRNAf). (4) the binding of this complex to mRNA. The anti-
codon of Met-tRNAf recognizes the initiation codon, AUG, on mRNA. (5) Addition of the
60S subunit and release of the initiation factors associated with the 40S subunit