Chemistry and Biology of Proteins - F. Haurowitz 1953

Protein Synthesis
Rate of protein synthesis in a living organism

Two main Methods are widely used to determine The rate of protein formation in living organisms. One of these methods is based on plasmapheresis, i.e., reducing plasma protein levels through repeated bleedings (red Blood Cells are usually returned to the animal's Veins, so plasmapheresis noticeably decreases only plasma protein content). In animals subjected to this Procedure, the rate of plasma Protein Synthesis can be determined [29, 30].

These experiments demonstrated that the rate of plasma protein regeneration increases when dogs are fed casein; however, dogs receiving Proteins composed primarily of gelatin or zein show only slight regeneration [31]. This is understandable, as gelatin and zein lack certain Essential Amino Acids (see Table 1). Intraperitoneally administered Hemoglobin is significantly less effective than casein [32] due to the absence of isoleucine in hemoglobin [33]. The maximum amount of Plasma Proteins produced within 24 hours is approximately 1 g per 1 kg of a dog's body weight [34].

When the Liver is removed from animals, the rate of plasma protein regeneration drops sharply, falling to 5% of normal for albumins and 15% for globulins [35, 36]. This led to the Conclusion that plasma protein synthesis occurs mainly in The Liver and that a state of dynamic equilibrium exists between plasma and liver proteins [31]. According to this view, liver Proteins can be formed from plasma proteins and, in turn, converted into plasma proteins. The liver proteins produced during these processes differ from structural proteins, The amount of which does not decrease during plasmapheresis.

If the erythrocytes removed from the body during bleeding are not returned to the animal, the rate of hemoglobin regeneration can be studied simultaneously. Such "double depletion" of the animal, accompanied by the loss of both plasma and erythrocytes, can also be compensated for by administering proteins containing all essential amino acids [37]. As noted above, the maximum amount of plasma proteins produced in a dog's body is about 1 g per day per 1 kg of body weight. The total amount of plasma proteins and hemoglobin produced daily never exceeds 1.5 g per 1 kg of the dog's body weight, with The Nature of the produced proteins depending largely on the Amino Acid Composition of the administered proteins. For example, administering egg proteins to double-depleted dogs induces the formation predominantly of plasma proteins, whereas feeding them beef promotes the formation primarily of hemoglobin [38]. The formation of globin (the protein component of hemoglobin) apparently also takes place in the liver [39].

Protein synthesis occurs not only in the liver but in all other Tissues of the body as well, and it does not cease even in starving animals [40]. Protein synthesis can also be observed in tissue cultures. The type of protein produced in a tissue culture is determined by The properties of the cultured cells and does not depend on the Nature of the protein used to nourish the culture. Thus, for example, when chicken fibroblasts are cultivated in rabbit plasma, specific chicken proteins are formed rather than rabbit proteins. Serological analysis has shown that in this case, alongside chicken fibroblast proteins, chicken plasma proteins are also produced [41]. The synthesis of these proteins presumably occurs from the breakdown products of rabbit proteins. It should be noted, however, that homologous proteins or peptones are utilized by tissue cultures better than proteins or peptones obtained from other animal species [42].

The second method for studying Protein synthesis in living organisms or tissue cultures is based on The Use of isotope-labeled amino acids or proteins.

This method has been used in attempts to determine the half-life of proteins. To this end, animals were administered amino acids labeled with deuterium [43], N¹⁵, or isotopic carbon either parenterally or with food, and the isotope content in their plasma proteins was determined at various time intervals [44, 45].

The interpretation of data obtained by the isotopic method is complicated by the fact that we cannot ascertain whether the isotope-labeled amino acid introduced into the Organism was used only once for the synthesis of a single protein molecule, or whether it repeatedly participated in the synthesis of protein molecules, being released during The breakdown of some proteins and incorporated into others. Experiments with N¹⁵-Glycine, which accounted for this confounding factor, showed that approximately 0.2 g of plasma proteins in humans and about 1 g in rats are produced per 1 kg of body weight within 24 hours [45]. In A number of studies, C¹⁴-Lysine was fed to animals to determine the formation rate of albumins and globulins; it was found that plasma globulins are formed faster than albumins and disappear from the blood faster than albumins [46]. About 10% of plasma proteins are renewed within 24 hours [46]. The renewal rate of Muscle Proteins is significantly lower than that of plasma and liver proteins [47]. Hemoglobin regenerates more slowly than all other proteins, with only 2.5% of this protein being renewed per day [43, 47]. The half-life of hemoglobin is approximately 25–30 days.

As experiments with labeled glycine have shown, the formation of embryonic proteins occurs at a rate significantly higher than that of adult proteins. However, it remains unclear whether the high growth rate of the embryo is determined by the rapid synthesis of proteins [49] or by their slowed breakdown [48]. The highest rate of protein formation is observed in the intestinal mucosa and pancreatic tissue. This is not surprising, considering that these Organs secrete large amounts of protein [50].

A number of researchers have found that labeled Amino acids can also be incorporated into the proteins of organ slices or homogenates. For instance, the isotopic sulfur S³⁵, which is part of cystine or Methionine, is rapidly detected in the proteins of liver slices. However, it remains unknown whether true protein synthesis occurs in these cases, since the majority of labeled amino acids can be cleaved off using reducing agents. It is highly probable that some of the Sulfur-Containing Amino Acids incorporated into the proteins of slices or homogenates are bound within them by Disulfide Bonds that are cleaved by reducing agents [51].

The incorporation of C¹⁴-glycine into the proteins of the intestinal mucosa is inhibited by azide and can also be suppressed by tissue maceration. Protein synthesis in this tissue is apparently coupled with an oxidative process catalyzed by Enzymes whose activity is closely linked to a specific Cell Structure [52]. The incorporation of Alanine labeled with C¹⁴ in the carboxyl group into liver slices is likewise an aerobic process [53]. Liver slices are capable of incorporating even C¹⁴O₂ into their proteins [54] and converting C¹⁴-glycine into C¹⁴-Serine [55]. All this indicates that protein synthesis in liver slices and other tissues does not simply amount to the formation of peptide bonds between amino acids, but is a more complex reaction. According to some authors, labeled Amino acids are not incorporated directly into proteins, but first combine with a peptide, which should therefore be regarded as a precursor of the resulting protein [56].

Radioactive isotopes have also been used to elucidate The Mechanism of thyroglobulin formation. It was found that radioiodine administered to an animal accumulates exclusively in The Thyroid Gland and that thyroglobulin is synthesized solely in this organ. All other organs contain only traces of iodine, the presence of which is most likely due to iodine diffusion into the tissues [57]. If the thyroid gland is removed from an animal, thyroxine begins to be produced in the liver and intestines [58], which can also be detected using radioactive iodine.

Although experiments using isotope-labeled substances as tracers have provided much valuable information regarding protein synthesis, fundamental problems in this field still remain unresolved. Based on these experiments, it is impossible to determine whether continuous self-renewal of proteins occurs via the synthesis and subsequent breakdown of individual protein molecules, or whether it is due to the fact that each of these molecules, without completely disintegrating, constantly exchanges its individual constituent parts. Such exchange can be achieved, for example, by the temporary opening of peptide bonds and the incorporation of an amino acid between the ends of the opened chains. Immunological methods have been used to resolve this problem. As already noted in Chapter XIV, Antibodies are located in the serum γ-globulin fraction. If antibody formation is induced in a rabbit by immunizing it with an antigen, the newly formed immune γ-globulins can be differentiated from the γ-globulins present prior to immunization by their ability to precipitate the corresponding antigen. For instance, injection of pneumococcal polysaccharide SIII leads to the formation of SIII antibodies in the globulin fraction of immune serum. If N¹⁵-glycine is administered to the experimental rabbits In addition to the antigen, the labeled amino acid is detected in the antibodies after a short period [59], indicating that it has been incorporated into the newly formed protein.

In another series of studies aimed at resolving whether amino acid incorporation into protein occurs via the opening and closing of its peptide chains, the following experiments were set up. Serum from another rabbit, previously immunized against polysaccharide SI, was injected into a non-immunized rabbit. Thus, SI antibodies were transferred into the blood of this non-immunized rabbit. At the same time, an injection of N¹⁵-glycine was administered to it. It turned out that N¹⁵-glycine was not incorporated into the transferred SI antibodies in these cases [59].

Based on these data, one might conclude that the renewal of serum protein molecules occurs not As a result of the brief opening of peptide chains and the insertion of new amino acids at the rupture site, but through the complete breakdown of individual protein molecules followed by the formation of new protein particles. However, if C¹⁴-leucine was administered to the experimental rabbits instead of N¹⁵-glycine, the incorporation of the labeled amino acid into SI antibodies was observed [60]. These latter experiments with the isotope carbon-labeled amino acid appear more convincing than those with the isotope nitrogen-labeled amino acid, because labeled nitrogen can be cleaved off and exchanged as a result of deamination and Transamination processes. Nevertheless, it is difficult to assume that antibody protein molecules can undergo continuous renewal of their amino acid composition while simultaneously preserving their antibody properties unchanged. These properties are most likely due to the fact that the surface geometry of the antibody molecule is complementary to the shape of the antigen's determining group. It is hard to imagine how this complementary shape could be maintained if the antibody molecule continuously exchanged its constituent amino acids for those of the surrounding environment.