Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose. Effect of protein charge on immobilization.

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.

Secretion of very low density lipoproteins enriched in cholesteryl esters by cultured rat hepatocytes during simulation of intracellular cholesterol esterification.

Very low density lipoproteins, newly secreted by cultured rat hepatocytes into a serum-free medium, contain some cholesteryl esters although the percentage of total cholesterol in ester form is less than that in plasma very low density lipoproteins. When acyl CoA:cholesterol acyltransferase activity in hepatocytes was stimulated by the addition of 25-hydroxycholesterol (10 microgram/ml) or mevalonolactone (1 mM), the absolute amount of esterified cholesterol secreted in very low density lipoproteins increased significantly, but the amount of free cholesterol decreased or showed no change. Thus the percentage of very low density lipoprotein cholesterol in ester form increased, in some experiments to as much as 50% of the total. These results provide additional evidence that hepatic acyl CoA:cholesterol acyltransferase plays a role in the generation of some of the cholesteryl ester in newly-secreted lipoproteins. They further suggest that changes in the activity of the enzyme can potentially regulate the fraction of cholesterol secreted in esterified form.