Liver Triton

Receptor-detergent complex (rat liver) Triton X-100 0.777 81S1... 

Gruner R, Seidel A, Winter R. 1981. The initial early distribution of monomeric 239Pu and 241Am in rat liver as determined by triton WR 1339 injection. Radiat Res 85 367-379. 

With bilirubin UDP-glucuronyltransferase from rat liver, Mn-+ was more (HIO), and Ca + less, stimulatory than Mg + (A2, F17, HIO). The behavior was similar when either UDP-glucose or UDP-xylose was used as the glycosyl donor (F3). Enzyme activities were also stimulated by Fe and Co (F3, HIO) Pb + activated glucuronyl transfer but was inhibitory with the other UDP-sugars. The effects of Mg +, Mn +, and Co are in accordance with work of Lucier et al. (L14) on the catalysis of glucuronyl transfer to p-nitrophenol and 1-naphthol by Triton X-100-activated and untreated microsomal material from rat liver. 

Sialyltransferases can be solubilized from their subcellular site by using detergents, and be purified by affinity chromatography on, for example, CDP-6-aminohexanol-agarose,267 as already described. Solubilization of frog- and rat-liver sialyltransferases by means of Triton X-100 has been described.27 Soluble sialyltransferase occurs in colostrum, and is also present in small quantities in normal blood-serum. From the latter source, the enzyme was purified 300-fold by poly (acrylamide) gel-electrophoresis.2 ... 

Gal-(1— 3)-D-GalNAc. By using 0.7 U of STB, as a soluble preparation readily obtained from 300 g of porcine liver, the sialylation of /J-D-Gal-(1 — 3)-d-G1cNAc was performed on a one-mmol scale and sialylated trisaccharide 65 was obtained in 21 % isolated yield. In this respect, the purification of reaction mixtures is still troublesome, especially because of the presence of Triton X-100 from our experience, the use of immobilized enzymes, eliminating the need for detergent, greatly facilitates the purification procedure. 

Figure 18 Effect of nonionic detergent (Triton X-100) on benzydamine N-oxygenation (FMO) and N-demethylation (CYP) by human liver microsomes. Benzydamine (500 pM) was incubated with pooled human liver microsomes (1.0 mg protein/mL) in tricine buffer (50 mM, pH 8.5 at 37°C) with or without Triton X-100 [1% (v/v)]. Reactions were initiated by the addition of an NADPH-generating system and stopped after 10 minute by the addition of an equal volume (500 pL) of methanol. Precipitated protein was removed by centrifugation, and an aliquot (25 pL) of the supernatant fraction was analyzed by HPLC with fluorescence detection. Abbreviations FMO, flavin monooxygenase CYP, cytochrome P450.

For tissues the spectrophotometric method can be used for tissues such as liver and kidney which have a high content of catalase provided all cells and debris can be removed to give a solution that is clear and only slightly coloured. A stock homogenate should be made in buffer containing 1% Triton X-100 (1 g tissue + 9-19 ml buffer), and further dilutions made to reach an appropriate concentration of enzyme which can only be determined by trial. If such dilution is not... 

Rat liver was homogenized in 0.2 M potassium phosphate buffer (pH 7.0) containing 0.8% Triton X-100. The supernate obtained by centrifuging the homogenate at 40 OOOg for 30 minutes at 0°C was used to assay leucine 2,3-aminomutase. 

Figure 9-29. Effect of a previous injection of Triton WR-1339 on the equilibrium densities of particulate enzymes. Density equilibration of mitochondrial fractions from rat liver in an aqueous sucrose gradient. Upper panel control. Lower panel animal injected intravenously with 170 mg of Triton WR-1339 four days prior to sacrifice. [From Wattiaux et al.. Arch. Intern. Physiol. Biochem., 71 140 (1963) Ciba Foundation Symposium, Lysosomes, Little, Brown, Boston, 1963, p. 176.]...

Some of these enzymes were found in the blister fluid produced arti-fically with cantharidin and the activities were most likely released from the lysosomes of epidermal cells (S14). There are, however, considerable differences between the reactions of liver lysosomes and epidermal lysosomes. Dicken and Decker (Dl) found that while Triton X-100 caused release of latent acid phosphatase activity from epidermal lysosomes acid pH, hypotonic solutions, and freezing and thawing which are effective disrupters of liver lysosomes did not disrupt epidermal lysosomes. Smith et al. (S16) found that capsaicin and cantharidin also would not lyse epidermal lysosomes and they substantiated Dicken s finding that Triton X-100 did cause lysis. 

Fig. 3. Quantification of Bcl-2 family activities with the long-format cytochrome c release assay. Results are the average of duplicate measurements in (A) and are averages of triplicate measurements +/-SEM in (B-D) (many error bars in panels [B-D] are obscured by the symbols). In panels (A-D) open circles indicate cytochrome c detected when Triton X-100 was added to mitochondria and open diamonds indicate cytochrome c detected when no Bcl-2 family proteins were added to mitochondria. All incubations of mitochondria except those in (B) were for 30 min. (A) Recombinant human Bid (solid circles) and caspase-8-cleaved human Bid (squares) induce release of cytochrome c from isolated mouse liver mitochondria in a dose- dependent manner. (B) Kinetics of 52 nM (solid squares) and 5.2 nM (solid circles) human cleaved Bid-induced cytochrome c release from isolated mouse liver mitochondria. (C) Bcl-xL inhibition of caspase-8-cleaved human Bid and cleaved mouse Bid induced cytochrome c release. Mitochondria were incubated with 52 nM cleaved human Bid without (solid diamond) or with ( solid squares) the indicated concentrations of mouse Bcl-xL. Mitochondria were also incubated with 17 nM cleaved mouse Bid without (open square) or with (solid circles) the indicated concentrations of mouse Bcl-xL. (D) A synthetic peptide (GQVGRQLAIIGDDINR) corresponding to the amino acid 72-87 BH3 region of Bak prevents Bcl-xL from inhibiting caspase-8-cleaved mouse Bid induction of cytochrome c release. Mitochondria were incubated with 17 nM caspase-8-cleaved mouse Bid without (triangle) or with (open square) 155 nM mouse Bcl-xL and the indicated concentrations of Bak-BH3 (solid circle) or the corresponding Bak peptide (GQVGRQAAIIGDDINR) with a L to A substitution (solid squares).

De Ferreyra, E. C., De Fenos, O. M., and Castro, J. A., Modulation of galac-tosamine-induced liver injury by some amino acids or Triton WR1339, Toxicol. 

Arylsulfohydrolase C has been purified to homogeneity from human placental and rat liver microsomes (Moriyasu et al., 1982 Burns, 1983 Noel et al., 1983) by using multiple-column chromatographic procedures. The purified human enzyme is a glycoprotein with Stake s radius and sedimentation coefficient values of 56 A and 4.85 S, respectively. In the purified state this enzyme still has traces of Triton X-100 and has a molecular weight of... 

Other binding agents seem to have no effect on channel behavior. Concanavalin A binds to rat liver VDAC in the triton XIOO-solubilized form (4, 43). Antibodies against N. crassa VDAC inhibit VDAC insertion into planar membranes and bind to VDAC crystals in membranes, but do not affect channel behavior (44). These agents may interact with a surface domain on VDAC that does not respond to membrane potentials. 

Microsomal Incubation Conditions Incubations in animal or human liver microsomes are the most common way to determine activity in the presence of added substrate, UDPGA, Mg, and a buffer. As there is no method available to directly determine enzyme concentration, the incubations are standardized by addition of the same amount of protein (typically 0.25-1.0 mg protein/ImL) after determination of linearity of product formation with respect to protein concentration and time. In general, the enzyme is stable up to 45 min to 1 h. Because of the location of the enzyme, a portion of the microsomal vesicle will be obtained in the normal configuration with the enzyme active site entrapped within the vesicle. Since UDPGA must have access to the active site, and the UDPGA influx transporter is not operative without ATP, it may be necessary to activate or remove latency of the enzyme. In the past this has been achieved by a variety of methods, but most commonly by addition of detergents such as Brij 58, Lubrol, or Triton X... 

Smigel, M. and Fleischer, S. (1977). Characterization of Triton X-lOO-solubilized prostaglandin E binding protein of rat liver plasma membranes. ]. Biol. Chem., 252, 3689-3696... 

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