Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L.

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms.

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.

beta-Mercaptoethanol differentially acts on the rat somatotrophs and lactotrophs in primary culture to suppress the secretion of immunoreactive hormones.

We previously reported that beta-mercaptoethanol (ME) reduced the content of immunoreactive prolactin (iPRL) within the adenohypophyseal cells, primarily through its direct effect on the disulfide bonding of the PRL molecule. Because of the structural similarities between PRL and growth hormone (GH), the effects of ME on the hormonal dynamics of iGH were compared to those of iPRL. ME reduced secretion and intracellular content of both iGH and iPRL in the cultured rat adenohypophyseal cells. However, iGH was more resistant to the suppressive effects of ME than iPRL. This was particularly so with regard to the intracellular hormonal contents. While 0.01% ME caused approximately 90% reduction in the iPRL content of the lactotrophic cells, the highest tested dose of ME, i.e. 0.1%, showed only 20% reduction in the iGH content of the somatotrophic cells. Also, the minimum effective dose of ME to suppress iGH secretion was 10-fold higher than that required for the suppression of iPRL secretion. Significant suppression of iGH secretion was not observed until 120 min after the onset of ME incubation as opposed to 2-9 min for the suppression of iPRL. These findings, together with the lack of any direct effects of ME on the iGH molecule itself, strongly suggested that ME acted via different mechanisms and/or pathways in the somatotrophs and lactotrophs to suppress respective hormonal dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of retinoblastoma cells from clinical specimens: growth-promoting effect of 2-mercaptoethanol.

Establishment of new tumor cell lines is an important first step for biological studies of tumor cells. High success rates in establishing retinoblastoma cell lines have been reported when feeder-layer culture but not liquid-culture techniques were used. Liquid culture is, however, essential for studies in which feeder-layer cells are undesirable. In a previous study, we formulated a medium (RB-- medium), the components of which were almost identical to those of a soft-agar medium developed for colony formation of established retinoblastoma cell lines, in which one cell line from 12 primary retinoblastoma specimens was established. In this study, another medium (RB++ medium), RB-- medium to which 20 microM 2-mercaptoethanol and 375 microM asparagine were added, was tested for its ability to grow retinoblastoma cells from clinical specimens. In the RB++ medium, 6 cell lines from 16 primary sites, 2 from 2 intraocular-recurrent and 3 from 3 metastatic retinoblastomas grew for over a year. The major reason for the apparent improvement of the RB++ medium on the RB-- medium was demonstrated to be the addition of 2-mercaptoethanol.