Protein, acetylated precipitants

The first experimental support for this scheme was the identification of the intermediate formation of a / -keto-acyl-enzyme, namely acetoacetyl-enzyme. When l- C-labeled acetyl-CoA and malonyl-CoA were incubated with stoichiometric amounts of the purified yeast enzyme and the protein was precipitated by the addition of trichloroacetic acid, it contained bound radioactive acetoacetate, which could be released by mild alkaline hydrolysis and which yielded radioactive acetone upon decarboxylation. This result proved unequivocally that the condensation of acetyl-CoA and malonyl-CoA had occurred in the manner depicted in equations (8) and (9). 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

The prompt removal of acetyl glutamate from the preincubation mixtures, by dialysis at room temperature or by precipitation of the protein with ammonium sulfate, prevented the cold inactivation. The presence of ammonium sulfate, in concentrations as high as 0.17M, did not protect the enzyme against cold inactivation (at higher molarities of the salt, precipitation began to occur). Once the cold inactivation had taken place, the enzymatic activity could not be restored either by precipitation with ammonium sulfate, or by reincubation with acetyl glutamate or with ATP and Mg+2. 

This conception works out as shown in Scheme 10 CTP 23 formed by the above described sequence is directly consumed by -acetyl neuraminic acid 26 under the catalytic influence of cytidine-5 -monophosphosialate synthase (E.C. 2.7.7.43). This enzyme is isolated from calf brain by ammonium sulfate precipitation (2 5) and subsequent affinity chromatography. The stationary phase consists of CNBr-activated Sepharose 4B reacted with p-[3-(2-amino ethylthio)propyl]-iV-acetyl neuraminic acid 27, which is synthesized by radiating a mixture of the allyl glycoside and cysteamine to achieve radical C-S bond formation (24), The behavior of methyl p-N-acetyl-neuraminic acid as an inhibitor is in accordance with Zbiral s findings (25), where the methyl a-glycoside has been shown to compete with the native substrate for the enzyme, and thus 27 is recommended to be an ideally suited ligand (Scheme 9). A typical analytical run is shown in Scheme 9. Due to elution of the protein fraction by a salt gradient, the transfer to a preparative scale is rather difEcult denaturation occurs and thus a drop in activity down to 6% is observed. 

With loose structures of linear molecules the exponent for instance to cellulose nitrate in acetone, precipitated by water, to Polyvinyl acetate in toluene, precipitated by a methanol-water mixture-, and to the methyles-ters of polymethacrylic acid in benzene, precipitated by cyclohexane With compact spherical particles we must expect n-values in the neighbourhood of 2/3, since in this case only the outer surface of the particles is subject to the action of the medium. Examples can be found in some proteins. Finally, if the long-chain molecule shows a pronounced ramification or if the randomly kinked structure is comparatively close-packed, n may assume values between 0.7 and 1. This is shown for instance, by branched polystryrene h by acetyl-starch and by glycogene An accurate check on the value of n, however, is usually impossible. Husemann s experiments with glycogene, for instance, can be equally well described by n = 2/3 as by n — J (Fig. 5). This is due to the fact that the value of P in this method is not unlimited. In practice the upper limit of the molecular weight lies in the neighbourhood of 5.10, the lower limit lies in the further to be noted that the constant b in equation... 

With a purified enzyme preparation from Clostridium perfringens, Roovers et al. (36) cleave conjugated bile acids directly in plasma. Proteins are then precipitated with Ba(OH)2-saturated ethanol (123) and the supernatant is taken to near dryness. The residue is dissolved in a toluene-isopropanol-methanol-30 % aqueous NaOH (10 20 20 6, v/v) mixture, water is added, and neutral lipids removed by light petroleum extraction. Bile acids are then obtained by acidification and diethyl ether extraction. The bile acids are then methylated and analyzed as above except that the bile acid methyl esters are acetylated before chromatography on 1 % XE-60 columns at 250°C. With this column the following retention times relative to that of the diacetoxy derivative of methyl deoxycholate were found for the following acetate methyl ester derivatives lithocholic, 0.60 23-nor-deoxycholic acid (internal standard), 0.77 chenodeoxycholic acid, 1.24 and cholic acid, 1.88. The deoxycholic acid derivative was eluted after 9.0 min and methyl 5 3-cholanoate after 0.32 min. 

PCCase is a biotinylated protein that catalyzes a reaction required in the catabolism of amino acids and fatty acids of odd-numbered chain length, and in the catabolism and anabolism of branched-chain fatty acids. In order to characterize the structure of this enzyme from plants we undertook its purification. PCCase activity was purified from extracts of maize leaves by a four step scheme that included PEG precipitation, hydrophobic interaction chromatography, anion exchange chromatography and affinity chromatography. This purification scheme achieved a nearly 250-fold purification of PCCase activity. However, throughout this purification of PCCase, ACCase copurified. Indeed, SDS-PAGE analysis of the final purified PCCase preparation identified two biotinylated polypeptides of about 240 and 230 kDa. These polypeptides have previously been described as subunits of ACCase (7). Furthermore, mixed substrate kinetic studies (8) with the purified PCCase/ACCase preparation indicated that the carboxylation of propionyl-CoA and acetyl-CoA were carried out by the same enzyme. Furthermore, both PCCase and ACCase activities were similarly affected by a variety of inhibitors. 

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