Threonine purification

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

DL-threonine and L-threonine crystals were supplied from Ajinomoto Co. Inc. and were used without further purification. Excess amounts of DL-threonine crystalline particles were dissolved in water kept at 55, 57, 58 or 60 C. After decantation and filtration each saturated solution was placed in the crystallizer maintained at 50 C. The difference between the saturation temperature and the crystallization temperature was defined as the initial supersaturation in terms of supercooling of the solution and was the driving force for the crystallization. 

Azevedo, R.A. Smith, R.J. Lea, P.J. Aspartate kinase regulation in maize Evidence for co-purification of threonine-sensitive aspartate kinase and homoserine dehydrogenase. Phytochemistry, 31, 3731-3734 (1992)... 

Paris, S. Wessel, P.M. Dumas, R. Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana. Protein Expr. Purif., 24, 105-110 (2002)... 

The methyl ester 84 was dissolved in a Na2C03 soln (dioxane/H20 2 1) at 0°C and treated with Boc20 according to standard literature protocol 135 to give the threonine derivative 90 as an oil (yield 86%), which was used without further purification in the next step. 

Preparation of Human Insulin. Porcine insulin can be converted to the human insulin sequence by an enzyme-catalyzed transpeptidation reaction (10,11). Under appropriate conditions trypsin acts preferentially at LysB29 rather than ArgB22 to yield a covalent des[B30]insulin/trypsin complex (acyl—enzyme intermediate). In the presence of high concentrations of organic co-solvents and the /-butyl ester of threonine, transpeptidation predominates over hydrolysis to yield the /-butyl ester of human insulin. Following appropriate purification steps and acidolytic removal of the ester, human insulin suitable for treating patients is obtained. 

Many serine/threonine kinases require phosphorylation by an upstream kinase for activation. This requires cloning the upstream kinase as well as additional purification steps after phosphorylation [38, 39, 58]. In some cases, the active... 

Af-(D-Biotinyl)-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl)-L-threonine te/t-Butyl Ester 23 [34], A mixture of D-biotin (150 mg, 0.6 mmol), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC 580 mg, 3 mmol), and 1-hydroxy-benzotriazole (HOBt 540 mg, 4 mmol) in dimethylformamide (DMF 2 mL) is stirred under exclusion of moisture at 22 C. After 45 min, the biotin is dissolved, and a solution of freshly prepared glycosyl threonine ester 22 (0.3 mmol, preceding procedure) in dichloro-methane (2 mL) is added at 0°C. After stirring for 16 h at room temperature, the solvent is evaporated in vacuo, the remainder dissolved in dichloromethane (50 mL), extracted with ice-cold 0.2 N HCl (3 x 25 mL), water (25 mL), and saturated NaHCOj solution (2 x 25 mL), dried with MgSO, and concentrated in vacuo. Purification by flash chromatography on silica gel (20 g) in dichloromethane-ethanol (25 1) yields 23 200 mg (93%) [a] 96.5° (c 1, CHClj) Rf 0.29 (toluene-acetone 4 1). 

The most accurate method of analysis for serine, threonine and tyrosine in proteins is to hydrolyze the proteins for several different times (e.g. 24, 48 and 72 hr) and extrapolate the values to 0 time. In general, the more times that are utilized, the more accurate will be the extrapolation. Extrapolation of the NHj content of these hydrolysates to 0 time will also give an estimate of the number of amides present in the protein, if care has been used to exclude NHj during purification of the protein. The inclusion of phenol in the hydrolysates has generally decreased the rates of destruction, particularly for tyrosine. If serine phosphate ( 2.12.4) or similar derivatives are present, the extrapolated curve will be more complex due to different rates of destruction. 

The type II topoisomerase from Drosophila has been found to copurify with a protein kinase activity (Sander et al., 1984). That this kinase activity resides in the same polypeptide as the topoisomerase activity was demonstrated by the appearance of a single band on a denaturing polyacrylamide gel after extensive purification, and by the parallel inactivation of the kinase and topoisomerase activities by heat and V-ethyl-maleimide treatment. The protein kinase activity will phosphorylate several proteins, including histones and the topoisomerase itself. The phosphorylated residues were found to be threonine and serine serine was the predominant residue phosphorylated in the topoisomerase (Sander et al., 1984). 

The final reaction in the biosynthesis of threonine involves a /8-y rearrangement and the loss of phosphate from O-phosphohomoserine (Fig. 2). Threonine synthases have been isolated from Lemna (Schnyder et al., 1975) radish, sugarbeet (Madison and Thompson, 1975), peas (Schnyder et al., 1975 Thoen et al., 1978b), and barley (Aames, 1978). None of these enzymes has been extensively characterized but a requirement for pyridoxyl-5 -phosphate was demonstrated after partial purification of the barley and pea enzymes. Unlike several other enzymes associated with threonine synthesis, the activity of threonine synthase was not stimulated by monovalent cations. However, all of the plant enzymes are strongly activated by 5-adeno-sylmethionine (Section III,B,5). 

This enzyme, catalyzing Eq. (1), has been demonstrated in a number of plants (Smith and Thompson, 1%9 Ngo and Shargool, 1974 Ascano and Nicholas, 1977). The 54-fold purified enzyme from kidney bean seedlings (Smith and Thompson, 1971) had values of 6 x lO IW for serine and 2 x Qr M for acetyl-CoA. The enzyme does not catalyze the acetylation of homoserine or threonine. Activities with other acyl-CoA derivatives were not tested. During purification, this enzyme was separated fix>m cysteine synthase, in contrast to the serine acetyltransferase from SalmoneUa typhimurium which has been isolated predominantly complexed with cysteine synthase (Kredich et al., 1969). 

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