Amino acid pancreatic ribonuclease sequence

Merrifield used this procedure to prepare a number of peptides. For example, he synthesized the nonapeptide bradykinin in 68% yield in only eight days, a remarkable feat at the time.The biological activity of the synthetic peptide was identical with that of the natural peptide. Merrifield was ultimately able to automate all the steps in his technique for solid-phase peptide synthesis and demonstrated its power by using a homemade machine to prepare bovine pancreatic ribonuclease, an enzyme that contains 124 amino acids. This synthesis proceeded in 17% overall yield and required 369 chemical reactions and 11,931 individual operations The synthetic ribonuclease had a specific activity that was 13-24% that of the native enzyme. The lower activity of the synthetic enzyme can probably be attributed to the fact that each coupling step did not proceed with 100% efficiency, so some polypeptides lacking one or more individual amino acids in the sequence were also produced. Because of their close similarity to ribonuclease, it was not possible to separate these polypeptides from the synthetic enzyme. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

Identify the signal peptide and family signature sequences of pig pancreatic ribonuclease with the given amino acid sequence ... 

This process is routinely automated in commercially available machines. Solutions of all of the protected amino acids required are stored in separate containers and a programmed sequence of coupling and deprotection leads rapidly to the complete peptide in days rather than the years needed for solution chemistry. The most dramatic illustration of this came with the publication of a heroic traditional synthesis of bovine pancreatic ribonuclease A (an enzyme with 124 amino acids) by Hirschmann, side-by-side with one by Merrifield using functionalized polystyrene as we have described. The traditional method required 22 co-workers, while the Merrifield method needed only one. 

Prepn. 3, 9 (1953). Bovine pancreatic RNase A is a single-chain peptide of 124 amino add residues. Primary structure C. H. W. Hits et al J. Biol. Chem. 235, 633 (I960). Amino acid sequence D. H. Spackman et al.. Ibid. 648. Pictorial representation of entire structure Stein, Moore, Sci. Am. 204, no. 2, 81-92 (Feb. 1961). Ribonuclease from plant leaves has slightly different characteristics Markham, Strominger, Biochem. J. 64, 46p (1956). Can be obtained as a by-product of microbial erythromydn production Japan, pat. 263>38( 63) (to Shionogi). Chemical synthesis of materials possessing partial RNase enzyme activity Gutte,... 

All glycoforms show the same amino acid sequence but differ in the location, number or sequence of attached glycan, and importantly their bioactivities. For example, bovine pancreatic ribonuclease (RNase) occurs naturally as a mixture of the unglycosylated protein (RNase A) and five glycoforms (RNase B, Man 5-9), in which 5-9 mannose residues are attached to the di-A-acetylchitobiose core. These glycoforms differ in their enzyme activities, i.e. decreasing activities in the order of RNase A > RNase ManO = RNase Manl > RNase Man5 = RNase B (Rudd et al, 1994). 

Pancreatic ribonuclease. Ribonuclease (MW 13,600), with known amino-acid sequence and tertiary structure, is quite heat stable and frequently a great nuisance, because of its ubiquitous presence and Its stability. It attacks only single-stranded RNA and cleaves at the 5 -side of pyrimidine nucleotides (see Fig. 3.8). The reaction, therefore, yields oligonucleotides with -Yp, I.e., with the 3 -phosphate group on the terminal pyrimidine nucleoside. Its reaction mechanism is well known and closely follows that of alkaline hydrolysis, i.e., the intermediate is a pyrimidine nucleoside-2, 3 -cycllc phosphate. 

Some 30 years ago Christian Anfinsens groundbreaking work on the refolding of bovine pancreatic ribonuclease conclusively demonstrated that the information necessary to specify the three-dimensional structure of a folded protein in general is completely specified in the protein s amino acid sequence. Experimentally, the thermodynamic hypothesis of protein folding is now well supported. It states that the native... 

Ribonudeases. Pancreatic ribonuclease has already been mentioned in the chapter on proteins. Its molecular weight is not very high (13,000), and its amino acid sequence has been unraveled. As an enzyme it has one Temarkable property It is relatively heat stable solutions may briefly be warmed up to 80°. 

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