J.A. Rathmacher
Metabolic Technologies Inc., Ames, Iowa, USA
Introduction
The accretion of body proteins is the net result of both the synthesis and breakdown of protein. The dynamic nature of protein metabolism has been known for 60 years thanks to the pioneering work of Schoenheimer and others (Schoenheimer et al., 1939). Using stable isotopes of amino acids, they demonstrated that proteins con- tinually were being broken down and resynthesized. In addition, they reported that different organs have different rates of protein synthesis. The dynamic process by which body proteins are continually synthesized and broken down is protein turnover.
Studies on the growth of body protein stores and the metabolism of protein have been a major area of research. For example, a major reason for this is the dramatic fact that up to 20–25% of the muscle protein can be broken down per day early in the life of humans and farm animals. This rate slows with age to 1–2% day1 in adults.
Rates of synthesis and breakdown are influ- enced not only by age, but by plane of nutrition, stress, disease, hormones, exer- cise and inactivity.
Nomenclature
The nomenclature employed in studying growth and protein metabolism is rela- tively straightforward, but it is a worth- while exercise to define briefly the terms that will be used in this chapter.
● Synthesis. The conversion of amino acids into proteins by the protein synthetic apparatus in the cytoplasm in the cell.
● Breakdown. The proteolysis of polypep- tides by different proteinases within both the cytoplasmic and lysosomal compartments of the cell. The terms breakdown and degradation are used interchangeably.
● Growth. The net accumulation of protein that occurs when the rate of synthesis is greater than the rate of breakdown. The term growth is often used interchangeably with accretion or net synthesis.
● Wasting. The loss of protein that occurs when the rate of breakdown is greater than the rate of synthesis.
● Turnover. A general term that involves both synthesis and breakdown. However,
© CAB International2000. Farm Animal Metabolism and Nutrition
(ed. J.P.F. D’Mello) 25
it is sometimes used to represent break- down. In this chapter, it will be used to describe protein metabolism, which includes both protein synthesis and protein breakdown.
● Fractional synthesis and breakdown.
When quantitating protein synthesis and breakdown, one often expresses the rates as per cent per day. This allows one to compare different organs of different sizes or different muscles of different sizes.
Three intrinsic problems are associ- ated with measuring protein synthesis and breakdown in body tissues. Details of these problems can be found reviewed elsewhere (Bier, 1989). The goal of this chapter will be to describe the methodology used to quantitate indirect whole-body protein turnover and direct measurement of tissue protein synthesis and breakdown.
One of the key problems in studying protein synthesis and degradation is that labelled (radio-labelled or stable isotopes) amino acids which are to measure their incorporation into protein, or conversely, release of amino acids from labelled proteins, and the true enrichment or specific activity of the amino acid or
metabolite are difficult to measure in an experiment. The labelled amino acid has various states in a cell, summarized in Fig.
2.1.
The amino acid can be transported into a free amino acid pool. Once in the cell, it can be transported back out, degraded to other metabolites (depending on the amino acid) or linked to a specific aminoacyl-tRNA. From the tRNA pool, it can be translated into protein by ribosomes (detailed below) and eventually degraded to free amino acids (mechanism described below).
Even though it is known that protein tissue accretion is influenced by both the synthesis and degradation of protein, protein synthesis and degradation are not always measured simultaneously. However, mechanisms controlling protein synthesis and degradation are distinct (Reeds, 1989) and, therefore, can be influenced indepen- dently. Often breakdown is ignored, especially when measuring direct tissue protein turnover. For example, very signifi- cant gains in muscle protein can be increased by decreasing protein break- down, if protein synthesis remains the same. A 10% decrease in the fractional breakdown rate of muscle will result in a
Fig. 2.1. Pathway of uptake, utilization and reutilization of amino acids for protein synthesis and breakdown.
Cell membrane Extracellular pool
Free IC amino acids
tRNA amino acids
Protein amino acids
Protein Intracellular pool
AA AA AA-tRNA
Protein breakdown and AA reutilization α-OXO-derivative
CO2 Etc.
23% increase in the protein accretion rate, whereas a 10% increase in the fractional synthesis rate will result in an 11%
increase in the protein accretion rate. One could theorize even greater increases in growth if synthesis and degradation were both affected.
Protein synthesis and breakdown It is evident that both synthesis and break- down of proteins are necessary to evaluate the regulation of protein turnover. A better understanding of these processes is needed before we proceed. A brief overview of the mechanisms involved is presented.
Protein synthesis
Protein synthesis (translation) requires the coordination of >100 macromolecules working together. They include DNA, mRNA, tRNA, rRNA, activating enzymes and protein factors. Information encoded in DNA is transcribed into the RNA molecules, which are responsible for the synthesis of the individual protein. The formation of single-stranded mRNA occurs in the nucleus and is called tran- scription. The mRNA is transported to the cytosol, where it associates with ribo- somes, and the translation of the mRNA sequence into an amino acid sequence occurs. There are three phases of pro- tein synthesis: initiation, elongation and termination.
Initiation occurs when the mRNA and ribosome bind. The elongation cycle proceeds with aminoacyl-tRNA (tRNA molecules bound to specific amino acids) assembling on a specific codon on the mRNA. Many ribosomes can attach to a single mRNA and translate a protein.
Synthesis is terminated when a stop codon is encountered. A newly synthesized protein may undergo post-translational modifications before it can become a func- tional protein. The protein synthetic process is probably regulated in two ways which can affect the rate of protein synthesis measured: the amount of RNA and the rate of translation to form protein.
Protein breakdown
Once a protein is synthesized, it is sub- ject to breakdown. The mechanism of protein breakdown involves the hydroly- sis of an intact protein to amino acids.
Protein breakdown is selective, and spe- cific proteins degrade within the cell at widely different rates. There are two gen- eral mechanisms involved in breakdown, lysosomal and non-lysosomal systems.
The lysosomal system is characterized by the following: (i) it is located in lyso- somes at pH 3–5 and includes the cathep- tic peptidases (cathepsins B, D, H and L);
(ii) it is involved in degradation of endo- cytosed proteins; and (iii) it is involved in bulk degradation of some endogenous proteins. It is unclear how such a degra- dation system can produce different half- lives for different proteins.
A second system is the error-eliminat- ing system which includes peptidases located in the cell cytoplasm. This system is specific for proteins containing errors of translation (abnormal), short-lived pro- teins, long-lived proteins and membrane proteins, and it requires ATP. This system is the ubiquitin–proteasome pathway reviewed by Mitch and Goldberg (1996).
Proteins are degraded by this pathway when ubiquitin binds to the protein. It is accomplished by three enzymes: (i) the E1 enzyme activates ubiquitin in an ATP- requiring reaction; (ii) activated ubiquitin is transferred to E2 carrier protein; and (iii) this is transferred to the protein, catalysed by the E3 enzyme. This process is repeated to form a ubiquitin chain. The ubiquitin- conjugated proteins are recognized by the proteasome and degraded within the proteasome by multiple proteolytic sites.
The peptides are released and degraded in the cytoplasm.
Another cytoplasmic system is the calpain system consisting of two iso- enzymes, µ- and m-calpain. This system is regulated by Ca2+-binding, autoproteolytic modification, and its inhibitor, calpastatin (Emori et al., 1987). It has been hypothe- sized that the calpain system is involved in the rate-limiting step of myofibrillar protein breakdown (Reeds, 1989). The calpains are
candidates for the disassembly of the myofibril into filaments. The filament proteins are then broken down in the cyto- plasm by other proteolytic enzymes.
The remainder of this chapter will deal with methodology used to measure protein synthesis and degradation. Discussion will be divided into two categories: (i) indirect and direct measurements of whole-body