General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes

Abstract

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.

Dual function of calmodulin (δ) in phosphorylase kinase

“…6A). As demonstrated earlier [28], a main contaminant of M , 100000 can be separated; it is detected here as a protein peak which is eluted by about 0.28 M NaCl. The activities A2, Al,hep and are superimposed.…”

“…Phosphorylase kinase was prepared according to [3] as modified by [28]. To the pooled fractions of the DEAEcelluIose chromatography, 0.54 vol.…”

cDNA cloning and complete primary structure of skeletal muscle phosphorylase kinase (alpha subunit).

“…Rabbit skeletal muscle phosphorylase kinase was purified according to Cohen (24) with the modifications introduced by Jennissen and Heilmeyer (25) and Hessova et al (26). The subunits were isolated by preparative reversed-phase HPLC according to Crabb and Heilmeyer (27,28).…”

Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria

“…Cell extract of glucose-cultivated cells was centrifuged at 150,000 ϫ g for 1 h. Solid ammonium sulfate was added to the supernatant fluid to 1.5 M. The mixture was equilibrated for 30 min and then centrifuged at 18,000 ϫ g for 15 min. The supernatant fluid was loaded on a 1-by 10-cm ethylamino-Sepharose column (22,23) and batch eluted with 50 ml of buffer B (100 mM potassium phosphate [pH 7.6] containing 1 M zinc sulfate, 0.1 mM PMSF, 1 mM dithiothreitol, and 1.5 mM ammonium sulfate). The eluate was centrifuged at 12,000 ϫ g for 15 min, and the supernatant fluid was loaded at an injection rate of 0.5 ml …”