lithium-chloride and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The effects of a variety of detergents and neutral salts on the structure of the eukaryotic high molecular mass aminoacyl-tRNA synthetase complex have been directly determined by observing alterations in the composition, sedimentation behavior, and electron microscopic appearance of the rabbit reticulocyte complex. The intact complex is shown to exhibit the enzymatic activities, polypeptide composition, relative stoichiometry, and morphological features that are characteristic of this eukaryotic multienzyme particle. The structure of the high molecular mass aminoacyl-tRNA synthetase complex is seen to be resistant to both ionic and nonionic detergents. However, both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and deoxycholate induce formation of large protein aggregates. In contrast, the chaotropic salts LiCl and NaSCN both selectively remove individual polypeptides from the high molecular mass aminoacyl-tRNA synthetase complex and promote formation of specific particulate subcomplexes which have distinct sizes, polypeptide compositions, and structural features. These data support the view that many of the protein interactions within the high molecular mass amino-acyl-tRNA synthetase complex are hydrophobic in nature. This study also provides direct evidence that the complex contains a core of tightly interacting synthetases onto which the remaining polypeptides are arrayed. The structural alterations observed here may account for the ability of these reagents to markedly inhibit several enzymatic activities within the complex.

Topics: Amino Acyl-tRNA Synthetases; Animals; Chlorides; Cholic Acids; Cyanides; Detergents; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Lithium; Lithium Chloride; Molecular Weight; Protein Conformation; Rabbits; Reticulocytes

Nonspecific adsorption of proteoglycans to chromatography media and surfaces is demonstrated. This adsorption is highly dependent on the nature of the chromatography media and the precise buffer conditions. For a given buffer the amount of adsorption decreases as the pH of the buffer is increased. It is also highly dependent on buffer concentration and increases as the buffer concentration is increased. The effect of salts such as LiCl, NaCl, KCl, and MgCl2 was generally small and complex so that the presence of the salt both increased and decreased the amount of adsorption depending on the buffer conditions. In contrast, the effect due to the presence of guanidine hydrochloride (Gdn-HCl) was relatively large and complex. At low Gdn-HCl concentrations there generally was a large increase in the amount of adsorption, reaching a maximum at approximately 0.5 M Gdn-HCl and decreasing with further increases in Gdn-HCl concentration. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and sodium dodecylsulfate generally reduced the amount of nonspecific adsorption, although in the presence of both the detergent and Gdn-HCl, the effect due to Gdn-HCl predominated. In commonly used buffers such as 0.5 M sodium acetate (NaOAc), pH 7.0 (buffer F), and 4 M Gdn-HCl in 0.05 M NaOAc, pH 5.8 (buffer D), adsorption to surfaces and chromatography media such as Sepharose CL-2B, cellulose, and controlled pore glass (CPG) is highly significant and it is particularly large for cellulose and CPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adsorption; Buffers; Chlorides; Cholic Acids; Chromatography, Gel; Detergents; Guanidine; Guanidines; Hydrogen-Ion Concentration; Lithium; Lithium Chloride; Magnesium Chloride; Potassium Chloride; Proteoglycans; Sodium Chloride