Omega-aminoalkyl agaroses in the resolution of enzymes involved in regulation of glutamine metabolism.

Abstract

Systematic examination of a homologous series of w-aminoalkyl agaroses showed that the pentyl derivative (Seph-Cs-NH2) was best suited for the retention and subsequent separation of several proteins involved in the regulation of glutamine metabolism in Escherichia coli, including: glutamine synthetase [EC 6.3 The term "hydrophobic chromatography" was introduced (1, 2) to describe a new approach to the resolution, purification, and probing of biomolecules using homologous series of hydrocarbon-coated agaroses. The individual columns in each series differ in the length of their hydrocarbon side chains and in their ability to retard or retain different molecules in a mixture. Such columns were shown to be very effective in the purification of several proteins (1-13). Their discrimination power may involve several types of interactions (hydrophobic, electrostatic, hydrogen bonding, dipoledipole, etc). However, hydrophobic interactions seem to play a key role in the resolution, since within the homologous series all the members are identical in all respects except for the length of the hydrocarbon chains, and yet they differ in their retention power. These hydrophobic interactions presumably occur between the hydrocarbon chains on the agarose beads and available hydrophobic "pockets" or regions in the various proteins. One of the major advantages of the above-mentioned approach resides in the fact that it provides a large number of columns from which the most suitable can be chosen in each case. For example, using columns with the general structure Seph-Cn-X (where X = H, NH2, COOH, OH, etc. and n = 0-12), the purification of a given protein can be optimized Abbreviations: Adenylyltransferase, ATP:glutamine synthetase adenylyltransferase; glutamine synthetase(A+D), total glutamine synthetase (i.e., both adenylylated, A, and deadenylylated, D, forms); PIIA, unmodified small protein component which stimulates adenylylation; PIID, uridylylated PIIA which stimulates deadenylylation; Seph-C,,-NH2, Sepharose 4B activated with CNBr and reacted with an a,-diaminoalkane n-carbon-atoms long.by (a) selecting the homologous series to be used (the best X), (b) selecting the most effective member in the series (the best n), and (c) adjusting the loading and eluting conditions (ionic strength, ionic composition, concentration of apolar compounds, pH, temperature, and even the presence of substrates or biospecific effectors).Previous efforts to obtain efficient separation of the several enzymes concerned with synthesis and the regulation of glutamine synthetase activity of Escherichia coli (14,15) were discouraging. This paper illustrates the use of w-aminoalkyl agaroses in the resolution and purification of these proteins. A systematic examination of a homologous series of w-aminoalkyl agaroses (3, 4) showed that Seph-C5-NH2 was best suited for the retention of some of these proteins, and a procedure was designed for detaching them from the column one after the other. MATERIALS AND METHODSw-Aminoalkyl Agaroses. The homologo...