digitonin and zwittergent-3-12

Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 μM) and the neutral digitonin (IC50 37 μM). A 2 h incubation of rat brain membranes at 24°C with 100 μM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 μM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 μM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 μM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation.

Topics: Animals; Cell Membrane; Cerebral Cortex; Cetrimonium; Cetrimonium Compounds; Cholic Acids; Deoxycholic Acid; Detergents; Digitonin; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Male; Octoxynol; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

The molecular masses of the UDP-glucuronyltransferases were determined by means of radiation-inactivation analysis of sodium dodecyl sulfate-treated lyophilized rat liver microsomal preparations using a calibrated 60Co source. Bilirubin glucuronidation with formation of bilirubin monoglucuronide is catalyzed by a 41,500-Da enzyme; conversion of bilirubin mono- to diglucuronide is catalyzed by a 175,000-Da enzyme. The glucuronidation of estrone and testosterone is mediated by enzymes with molecular masses of 73,500 and 142,000 Da, respectively, and the glucuronidation of p-nitrophenol and phenolphthalein is mediated by enzymes with molecular masses of 109,000 and 159,000 Da, respectively. Our results show that UDP-glucuronyltransferase consists of a heterogenous group of enzymes with strikingly different molecular masses. Our data furthermore suggest that these enzymes may be oligomers composed of one to four subunits with similar molecular masses. Based on these findings, a molecular model of bilirubin UDP-glucuronyltransferase is proposed, consisting of four subunits. For bilirubin diglucuronide formation, the complete tetrameric enzyme is required, whereas formation of monoglucuronide can be mediated by a single subunit. The monomeric monoglucuronide-forming enzyme is resistant to sodium dodecyl sulfate, treatment whereas the tetrameric diglucuronide-forming enzyme is labile, but once inactivated, the diglucuronide-forming enzyme can be reconstituted by decreasing the sodium dodecyl sulfate concentration by means of dialysis.

Topics: Animals; Bilirubin; Detergents; Digitonin; Glucuronosyltransferase; Hexosyltransferases; Male; Microsomes, Liver; Models, Molecular; Molecular Weight; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Sodium Dodecyl Sulfate