Method for Determining Antineuraminidase Activity

A test was developed to screen drugs for antineuraminidase (influenza sialidase) activity in vitro. Neuraminidase prepared from Vibrio cholerae was added to a substrate containing ganglioside, prepared from calf brain. Sialic acid is a split product in the reaction. The presence of sialic acid was detected colorimetrically by use of Warren's Thiobarbituric Acid Assay after drugs had been added to inhibit the action of neuraminidase on the calf brain substrate.     

Interactions of native and modified human low density lipoproteins with human skin fibroblasts.

125I-labeled low density lipoprotein (LDL) covalently bonded to Sepharose beads was not degraded by normal human fibroblasts nor did it trigger inhibition of sterol synthesis. The Sepharose beads loaded with LDL bound very tightly to the surface both of normal fibroblasts and fibroblasts from a subject with homozygous familial hypercholesterolemia; control Sepharose beads (activated sites covered with glycine) did not adhere to either cell type. LDL was extracted by a modification of the method of Gustafson (Gustafson, A. (1965) J. Lipid Res. 6, 512-517), so as to remove essentially all cholesterol, cholesterol ester and triglyceride. This modified LDL was bound, internalized and degraded as well as or better than native LDL. However, it failed to suppress sterol synthesis. These results provide additional evidence that the sterol moiety of the LDL is the key component affecting sterol synthesis. They also imply that the neutral lipids of LDL play a minor role in the binding of LDL to cell membranes and that the apoprotein rather than molecular size and shape is the critical factor.     

Cholesterol esterase--a polymeric enzyme

A direct interaction of cholic acid and pancreatic cholesterol esterase has been shown using labeled bile salt of high specific activity and Sephadex G-50 chromatography. This interaction results in the formation of a protein of M.W. 400,000 which appears to be a hexamer of the protein in pancreatic juice (M.W. 65,000 – 69,000). Rechromatography of the polymer on Sephadex G-200 results in dissociation to the monomeric form. Evidence is provided, suggesting that the polymeric form of cholesterol esterase represents the active enzyme responsible for sterol ester synthesis and hydrolysis.