Use of mushroom tyrosinase to introduce michaelis-menten enzyme kinetics to biochemistry students.

An inexpensive enzyme kinetics laboratory exercise for undergraduate biochemistry students is described utilizing tyrosinase from white button mushrooms. The exercise can be completed in one or two three-hour lab sessions. The optimal amounts of enzyme, substrate (catechol), and inhibitor (kojic acid) are first determined, and then kinetic data is collected in the absence and presence of the inhibitor.

Variations in IC(50) values with purity of mushroom tyrosinase.

The effects of various inhibitors on crude, commercial and partially purified commercial mushroom tyrosinase were examined by comparing IC(50) values. Kojic acid, salicylhydroxamic acid, tropolone, methimazole, and ammonium tetrathiomolybdate had relatively similar IC(50) values for the crude, commercial and partially purified enzyme. 4-Hexylresorcinol seemed to have a somewhat higher IC(50) value using crude extracts, compared to commercial or purified tyrosinase.

Tissue printing to visualize polyphenol oxidase and peroxidase in vegetables, fruits, and mushrooms.

A simple tissue-printing procedure to determine the tissue location of the endogenous enzymes polyphenol oxidase and peroxidase in a variety of vegetables, fruits, and mushrooms is described. In tissue printing, cell contents from the surface of a cut section of the tissue are transferred to an adsorptive surface, commonly a nitrocellulose membrane. Because of the considerable expense of nitrocellulose, our procedure utilizes artists' hot-press watercolor paper as a novel and more economical alternative.

Proteolytic processing of polyphenol oxidase from plants and fungi.

Polyphenol oxidase (PPO), a metalloenzyme containing a type-3 copper center, is produced by many species of plants, fungi, and bacteria. There is great variability in the subunit molecular mass reported for PPO, even from a single species. In some cases, experimental evidence (usually protein sequencing by Edman degradation) indicates that the variability in molecular mass for PPO from a given species is the result of proteolytic processing at the N and/or C-termini of the protein.

Enzyme, protein, carbohydrate, and phenolic contaminants in commercial tyrosinase preparations: potential problems affecting tyrosinase activity and inhibition studies.

Commercial mushroom tyrosinase contains other proteins, enzymes, carbohydrates, and phenolic material besides tyrosinase. Carbohydrate and phenolic material comprise a large percentage of the powder resuspensions derived from Agaricus bisporus. Enzyme assays identified the presence of tyrosinase, laccase, beta-glucosidase, beta-galactosidase, beta-xylosidase, cellulase, chitinase, xylanase, and mannanase in the commercial tyrosinase.

Comparative analysis of polyphenol oxidase from plant and fungal species.

Polyphenol oxidase from plants and fungi is a metalloenzyme containing a type-3 copper center and is homologous to oxygen-carrying hemocyanin of molluscs. Molluscan hemocyanin consists of two domains, an N-terminal domain containing the copper center and a smaller C-terminal domain, connected by an alpha-helical linker. It is presumed that the same is true of polyphenol oxidase from plants and fungi although the structure of a polyphenol oxidase containing the C-terminal domain has not been determined.

Use of solid phase extraction in the biochemistry laboratory to separate different lipids.

Solid-phase extraction (SPE) was used to demonstrate how various lipids and lipid classes could be separated in a biochemistry laboratory setting. Three different SPE methods were chosen on their ability to separate a lipid mixture, consisting of a combination of a either a fatty acid, a triacylglycerol, a mono- or diacylglycerol, phospholipid, cholesterol, or cholesteryl ester into distinct lipid fractions. These mini-scale SPE methods used aminopropyl-bonded silica columns or silica Sep-Pak cartridges suitable for completion in a 2- to 3-h time block.

Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms.

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms.