An indirect enzymoimmunological assay for hyaluronidase.

The use of a hyaluronic acid-binding proteoglycan (hyaluronectin) as a probe for the detection of hyaluronic acid has facilitated the development of an indirect enzymo-immunological assay for hyaluronidase. Plastic microtest ELISA plates were coated with hyaluronic acid. Incubation with hyaluronidase led to the destruction of insolubilized hyaluronic acid in proportion to the hyaluronidase concentration of samples. Residual hyaluronic acid was assayed by its capacity to bind immune complexes made up of hyaluronectin supplemented with alkaline phosphatase-conjugated anti-hyaluronectin antibodies. The technique was very sensitive and permitted the detection of as little as 10(-10) NFU of bovine testicular hyaluronidase. Hyaluronidase was detected by this technique in human sera, bee venom and culture medium of human hepatoma cell lines.

Phospholipases B from Japanese yellow hornet (Vespa xanthoptera) venom.

Two phospholipases B (Vx I and Vx II) were purified from the venom of the Japanese yellow hornet, Vespa xanthoptera, by sequential chromatography on Sephadex G-100, SP-Sephadex and Mono S columns. They are very similar to each other in molecular and enzymatic properties, though the specific activity of Vx I was one-fifth that of Vx II. They hydrolyze the acyl ester bonds at the 1-position of phosphatidylcholine and lysophosphatidylcholine, therefore, their enzymatic specificities were of the A1 and L1 types.

Interaction of myelin basic protein, melittin and bovine serum albumin with gangliosides, sulphatide and neutral glycosphingolipids in mixed monolayers.

Some parameters that may regulate the miscibility and stability of mixed lipid-protein monolayers at the air-145 mM NaCl interface were studied employing six glycosphingolipids (acidic or neutral), three different types of proteins (soluble, extrinsic or highly amphipathic) and some phospholipids. The results obtained show that the percentage of the total area occupied by the protein at the interface is an important parameter leading to lateral phase separations; the amount and area contribution of the protein accepted in the film before the components become immiscible increase with the complexity of the polar head group of the glycosphingolipids. The interactions occur with progressive reductions of the intermolecular packing as the polar head group of the glycosphingolipid becomes more complex and this is accompanied by more negative values of the excess free energy of mixing. The lipid component seems to be the major responsible for the reduction in mean molecular area.

Continuous measurement of phospholipase A2 activity using the fluorescent probe ADIFAB.

A new method is described for the continuous quantitation of phospholipase A2 (PLA2) activity with greatly improved sensitivity compared to existing techniques. The method utilizes a fluorescent probe to detect the release of fatty acid monomers (free fatty acids) into the aqueous phase. The fluorescent probe ADIFAB, which is the acrylodan derivative of rat intestinal fatty acid binding protein, exhibits a change in the ratio of its fluorescence upon binding medium- to long-chain native fatty acids. ADIFAB was used to measure the hydrolysis of artificial and natural membranes using PLA2s from porcine pancreas, Naja mocambique mocambique, Crotalus durissus terrificus, and bee venom. Total phospholipid hydrolysis was determined from the free fatty acid concentration using membrane/water partition coefficients, also measured using ADIFAB. The results indicate that continuous monitoring of natural substrates can be determined with a sensitivity limit of less than 1 pmol/min, a more than 10(4)-fold increase in sensitivity over the most commonly used pH stat method.