Enzymatic Determination of Bile Acids. The NADP-specific 7α-Hydroxysteroid Dehydrogenase from P. testosteroni (ATCC 11996)

Abstract

By use of the specific substrates, 7α-hydroxy-5β-cholanic acid and 3α, 7α, 12α-trihydroxy-3,12-diacetyl-5β-cholanic acid methyl ester, crude extracts of P. testosteroni grown on a steroid-containing medium have been shown to exhibit 7α-hydroxysteroid: NADP-oxidoreductase activity. The enzyme is highly specific for NADP. Both free and conjugated 7α-hydroxy bile acids can act as substrates, but those of low polarity (few hydroxyl groups) seem to be preferred, judging from initial reaction velocity studies. Optimal conditions appears to be at pH 8.5-9.5 and at 25 °C. Free SH-groups are essential for maximum catalytic activity, since the enzyme is inhibited by SH-reacting substances such as p-chloromercuribenzoate and monoiodoacetic acid. Also the ketone-trapping agents hydrazine hydrate and semicarbazide act as inhibitors. Upon dilution, the storage stability is severely reduced, but this effect may be counteracted by the addition of glycerol at concentration of 20% or more. By gel filtration experiments on Sephadex G-100, the molecular weight was estimated to about 80,000.

Key-words:

• Bile acids
• enzyme assay
• 7α-hydroxysteroid dehydrogenase
• 3α-hydroxysteroid dehydrogenase