Hemoglobin amino acid incorporation

Some phases of the synthesis of hemoglobin have been studied in vitro and in vivo in both animal and human subjects by various investigators [(G9, Hll, M4, N13) and others]. Labeled amino acids, i.e., leucine- C, phenylalanine- C, and leucine- H, are used for the study of amino acid incorporation. The procedure consists of obtaining either human or animal mRNA, reticulocyte ribosomes from the study case, and radioactive amino acids. The three are combined and incubated for... 

Hll. Heywood, J. D., Karon, M., and Weissman, S., Amino acids Incorporation into o and 8 chains of hemoglobin by normal and thalassemic reticulocytes. Science 146, 530-531 (1964). 

In the second phase of these investigations on protein synthesis, an attempt was made to study the process in vitro. At first, intact cells had to be used. Amino acid incorporation was studied in liver slices amylase, lipase, and ribonuclease synthesis was investigated in slices of pigeon pancreas and the incorporation of glycine into reticulocyte hemoglobin was measured. The first of the above systems was exploited with exceptional success in Zamecnik s laboratory [138]. 

We have already seen that the amino acid incorporated is very firmly bound in the ribonucleoprotein particles, that it cannot be washed out by nonisotopic amino acid, that most of it is not at the ends of chains and that after partial hydrolysis of the microsomal protein, labeled peptides can be isolated. And it has also been sbown S26, S 6) that after an in vitro incubation in the usual way there appears on liver microsomes a hi y labeled substance which has the immimolo cal characteristics of serum albumin, and on those of reticuloc3rtes 128), a substance which seems indistinguishable from hemoglobin, except that it is not soluble. 

In fact, the malaria parasite has a limited capacity for de novo amino acids synthesis, and its survival is dependent upon hemoglobin proteolysis. The amino acids derived from the hydrolysis of globins are incorporated into the parasite s proteins and appear to be available for energy metabolism. This digestion of hemoglobin releases heme, which is... 

Does this mischarged tRNA recognize the codon for cysteine or for alanine The answer came when the tRNA was added to a cell-free protein-synthesizing system. The template was a random copolymer of U and G in the ratio of 5 1, which normally incorporates cysteine (encoded by UGU) but not alanine (encoded by GCN). However, alanine was incorporated into a polypeptide when Ala-tHNA ys was added to the incubation mixture. The same result was obtained when mRNA for hemoglobin served as the template and [i CJalanyl-tRNACys was used as the mischarged aminoacyl-tRNA. The only radioactive tryptic peptide produced was one that normally contained cysteine but not alanine. Thus, the amino acid in aminoacyl-tRNA does not play a role in selecting a codon. 

When such an amino acid is incorporated into a protein, the local chemical environment in the protein may affect the pK of a group and thus alter its charge. Such changes can be very important in the function of proteins (e.g., the Bohr effect in hemoglobin depends on structural changes that affect the pK of histidine... 

Progress has been made in demonstrating synthesis of specific proteins by cell-free systems, e.g., on the synthesis of cytochrome c by isolated rat liver mitochondria (S3, cf. 383) and on the synthesis of serum albumin by the isolated microsome fraction of rat liver 34, cf. 335,336). Campbell et al. 34) concluded that while specific serum albumin is, indeed, synthesized on the ribonucleoprotein particle fraction of the microsomes, it is not readily released in soluble form. In other words, the isolated microsomes have lost their ability to promote substrate turnover. The same is true for the hemoglobin-synthesizing RNP particles from rabbit reticulocytes 35, cf. 138). Ogata and associates 36) have essentially confirmed the results of Campbell et al. 34) on the synthesis of serum albumin by liver microsomes they have also studied the relative effect of both stimulatory and inhibitory factors on the incorporation of different amino acids into total ribonucleoprotein and into serum albumin, and showed that the requirements for the two processes were generally the same it may be noted, however, that pretreatment of the pH 5 enzymes with ribonuclease, which caused a 95% inhibition of the ineorporation into ribonucleoprotein, inhibited the corresponding incorporation into serum albumin by only 55%. 

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