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The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M(r) 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin. The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH(2) and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M(r) 181,000. These results demonstrate that SsuD is a broad range FMNH(2)-dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH(2).

Acute and chronic cocaine administration has been reported to change endocrine and neurochemical functions in animals and human drug abusers. This study examined the effects of acute cocaine administration on anterior pituitary and gonadal hormones in male human volunteers without a history of drug abuse. Using a double-blind, randomized study design, luteinizing hormone, follicle-stimulating hormone (FSH), prolactin and testosterone levels were measured in 12 healthy men before and after intranasal administration of 2 mg/kg cocaine or placebo. Each subject was studied twice, serving as his own control. Compared to placebo, both luteinizing hormone, and, to a lesser degree, follicle-stimulating hormone levels increased significantly after cocaine, reaching a peak value 60 min after the administration of the study drug. This pattern is consistent with a possible cocaine induced rise in gonadotropin-releasing hormone and subsequent rise in luteinizing hormone and follicle-stimulating hormone due to stimulation of gonadotroph cells in the pituitary gland. Neither cocaine nor placebo induced a change in testosterone levels. Prolactin levels showed a decrease from base line after both placebo and cocaine administration, with a significantly more pronounced decrease after cocaine. This likely reflects the combination of the physiologic diurnal variation in prolactin secretion and an added inhibitory effect on prolactin due to cocaine. These findings show that the acute administration of cocaine significantly alters anterior pituitary hormonal release patterns.

The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation. The sequence relatedness of the TauD protein to the alpha-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an alpha-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438-5446). TauD was overexpressed in E. coli to approximately 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent Mr of 81,000 of the native protein and the subunit Mr of 37,400 were consistent with a homodimeric structure. The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine. The reaction also consumed equimolar amounts of oxygen and alpha-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction. The properties and amino acid sequence of this enzyme thus define it as a new member of the alpha-ketoglutarate-dependent dioxygenase family. The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at -20 degrees C for several weeks. Taurine (Km = 55 microM) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates. Among the cosubstrates tested, only alpha-ketoglutarate (Km = 11 microM) supported significant dioxygenase activity.