Amino-acid sequence

The amino acid sequence of the human erythrocyte glucose transporter was deduced from the nucleotide sequence of a cDNA clone in 1985 [106]. Polyclonal antibodies raised against the protein were used to screen a Xgtl I cDNA library prepared from the human hepatocellular carcinoma cell line HepG2. (Like many other transformed 

The amino acid sequence of the di-heme CCP from P. denitrificans has been determined by chemical methods [9]. The sequence shows 60% similarity to the P. aeruginosa CCP. 

Fiilop et al. [10] have shown that the heme of the P. aeruginosa CCP N-terminal domain is coordinated by the proximal histidine 55 (69 in P. denitrificans CCP) and 

2 Three-dimensional structure of P. aeruginosa CCP and proposal for the P. denitrificans three-dimensional structure 

CCPs from P. aeruginosa and P. denitrificans catalyze similar reactions, where the electron donors are either cytochrome C551 or azurin. Eq. (6.1) represents the P. aeruginosa CCP-catalyzed reaction. Ellfolk and Soininen purified P. aeruginosa CCP for the first time in 1970 [12]. P. aeruginosa CCP has been extensively used as a model for di-heme peroxidases. 

C55o(Fe ) + H2O2 -I- 2H+ - 2cyt.C5so(Fe ) -b 2H2O 

Long-acting insulin analogues. The more extensive alteration of amino acids in insulin glargine changes the electric charge of the molecule. At pH 4 of the injectate, it is dissolved however, at the pH of tissue it is poorly water-soluble and precipitates. Resolubilization and diffusion into the bloodstream take about one day. 

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Pathogenesis and complications (A). Type I diabetes mellitus typically manifests in childhood or adolescence (juvenile onset diabetes mellitus) it is caused by the destruction of insulin-producing B cells in the pancreas. A genetic predisposition together with a precipitating factor (viral infection) could start an autoimmune reaction against B-cells. Replacement of insulin (daily dose-40U, equivalent to -1.6 mg) becomes necessary. 

Therapeutic principles. In healthy subjects, the amount of insulin is automatically matched to carbohydrate intake, hence to blood glucose concentration. The critical secretory stimulus is the rise in plasma glucose level. Food intake and physical activity (increased glucose uptake into musculature, decreased insulin demand) are accompanied by corresponding changes in insulin secretion. 

The DNP method (p. 5) may also be used for the identification of the A-terminal residues in peptides. It is rather more laborious than the above deamination procedure, but the results are usually more clear-cut. Two methods are available. Either the terminal DNP amino acid is identified by chromatography or the remaining unsubstituted amino acids are identified by paper chromatography. The former method has the advantage that it may be carried out quantitatively but requires rather more material than is usually available from the elution of one paper chromatogram. 

Bowman (1950) has shown that amino acids and peptides may be quantitatively converted to their dimethyl derivatives by reductive alkylation in the presence of formaldehyde, and has used the method to detect the iV-terminal residues of peptides. A micro-modification was described by Ingram (1950) who identified the amino acid which is absent from a hydrolyzate of the dimethyl peptide. 

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Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Circular dicliroism has been a useful servant to tire biophysical chemist since it allows tire non-invasive detennination of secondary stmcture (a-helices and P-sheets) in dissolved biopolymers. Due to tire dissymmetry of tliese stmctures (containing chiral centres) tliey are biaxial and show circular birefringence. Circular dicliroism is tlie Kramers-Kronig transfonnation of tlie resulting optical rotatory dispersion. The spectral window useful for distinguishing between a-helices and so on lies in tlie region 200-250 nm and hence is masked by certain salts. The metliod as usually applied is only semi-quantitative, since tlie measured optical rotations also depend on tlie exact amino acid sequence. 

Biological infonnation is also concerned witli tire analysis of biological messages and tlieir import. The fundamental premise of tire protein-folding problem section C2.14.2.2 is tliat tire full tliree-dimensional arrangement of tire protein molecule can be predicted, given only tire amino acid sequence, together witli tire solvent composition, temperature and pressure. One test of tire validity of tliis premise is to compare tire infonnation content of tire sequence witli tire infonnation contained in tire stmcture [169]. The fonner can be obtained from Shannon s fonnula ... 

The protein folding problem is the task of understanding and predicting how the information coded in the amino acid sequence of proteins at the time of their formation translates into the 3-dimensional structure of the biologically active protein. A thorough recent survey of the problems involved from a mathematical point of view is given by Neumaier [22]. 

The forces in a protein molecule are modeled by the gradient of the potential energy V(s, x) in dependence on a vector s encoding the amino acid sequence of the molecule and a vector x containing the Cartesian coordinates of all essential atoms of a molecule. In an equilibrium state x, the forces (s, x) vanish, so x is stationary and for stability reasons we must have a local minimizer. The most stable equilibrium state of a molecule is usually the... 

Example Molecular dynamics simulations of selected portions of proteins can demonstrate the motion of an amino acid sequence while fixing the terminal residues. These simulations can probe the motion of an alpha helix, keeping the ends restrained, as occurs n atiirally m transmembrane proteins. You can also investigate the conformations of loops with fixed endpoints. 

Chou P Y and G D Fasman 1978. Prediction of the Secondary Structure of Proteins from Tlieir Amino Acid Sequence. Advances in Enzymology 47 45-148. 

Needleman S B and C D Wunsch 1970. A General Method Applicable to the Search for Similarities in the Amino Acid Sequences of Two Proteins. Journal of Molecular Biology 48 443-453. 

An amino acid sequence is ambiguous unless we know the direction m which to read It—left to right or right to left We need to know which end is the N terminus and which IS the C terminus As we saw m the preceding section carboxypeptidase catalyzed hydrolysis cleaves the C terminal ammo acid and so can be used to identify it What about the N terminus ... 

As described in the preceding sections protein synthesis involves transcription of the DNA to rtiRNA followed by translation of the mRNA as an amino acid sequence In addition to outlining the mechanics of transcription we have described the relationship among mRNA codons tRNA anticodons and ammo acids... 

Biosynthetic Human Insulin from E. coli. Insulin [9004-10-8] a polypeptide hormone, stimulates anaboHc reactions for carbohydrates, proteins, and fats thereby producing a lowered blood glucose level. Porcine insulin [12584-58-6] and bovine insulin [11070-73-8] were used to treat diabetes prior to the availabiHty of human insulin [11061 -68-0]. AH three insulins are similar in amino acid sequence. EH LiHy s human insulin was approved for testing in humans in 1980 by the U.S. EDA and was placed on the market by 1982 (11,12). 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

The amino acid sequences of hCS-A, hCS-B, and hCS-V are shown in relation to GH in Figure 1. The sequence of hCS-V is predicted from the DNA coding sequence and apparentiy does not possess amino acids 8—55 relative to GH and the other hCS molecules. It is not certain whether hCS-V is expressed or what function it may have. Human CS-A and hCS-B share approximately 85% identity with GH and also possess the disulfide bonds between Cys 53—165 and Cys 182—189 which produce the long and short S—loops characteristic of the PRL/GH family. 

Fig. 1. Stmcture of oxytocin and arginine vasopressin. Numbers indicate approximate location of amino acids sequences found in Table 1. A, oxytocin B,...

Fig. 1. Primary stmcture of hGH showing the amino acid sequence and the disulfide bonds.

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