n-octyl-beta-d-thioglucopyranoside and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

We have determined the major ingredients of the commercially available reagents M-PER, Y-PER, and B-PER from Pierce Chemical Co. using electrospray mass spectrometry. These three proprietary reagents have been widely used in the biochemical community as cell membrane dissolving tools during the initial step of protein purification. However, the identity and mechanism of these reagents remained unknown. In this paper, we identified these reagents as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-octyl-beta-d-thioglucopyranoside, respectively. In addition, we wish to stress here the increasing importance of the role of electrospray mass spectrometry in the analysis of such proprietary biological preparations which are increasingly finding their way into the biochemical literature.

Topics: Cholic Acids; Detergents; Quaternary Ammonium Compounds; Spectrometry, Mass, Electrospray Ionization; Thioglucosides

Insulin receptor activities, i.e., insulin binding and tyrosine kinase activation depend on the lipid environment of the receptor. As detergent may disrupt or interfere with this environment, we investigated the effect of various common detergents on insulin receptor properties. Experiments were carried out (i) on solubilized and partially purified insulin receptor and (ii) on the receptor reconstituted into phosphatidylcholine vesicles. The detergents tested, Triton X-100, octyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps), and Na deoxycholate affected the insulin receptor properties differently when compared with the control receptor in the absence of detergent. On the partially purified insulin receptor, Na deoxycholate inhibited both insulin receptor activities; octyl-beta-D-glucopyranoside and octyl-beta-D-thioglucopyranoside decreased insulin binding and kinase activation as their concentration increased, particularly above their respective critical micellar concentration (CMC). Triton X-100 was the only detergent which allowed an increase of insulin binding and kinase activation throughout the whole range of concentrations assayed. Reconstitution of the receptor into phosphatidylcholine vesicles protected the receptor from the direct effects of the detergents, for both the stimulation observed with Triton X-100 and the inhibition produced by the other detergents. In order to determine the effect of detergents on the oligomeric forms of the soluble insulin receptor, we investigated a new rapid sucrose gradient centrifugation technique. Insulin receptors were detected on the gradient by 125I insulin binding. For low concentrations of detergent, i.e., near the CMC, octylglucoside, Chaps, and Triton X-100 favored the (alpha 2 beta 2)2 oligomeric form of the receptor. Higher concentrations of Triton X-100 did not modify the polymeric state of the receptor. In contrast, octylglucoside and Chaps induced an increase in the sedimentation coefficient of the receptor which appeared as (alpha 2 beta 2)3 and (alpha 2 beta 2)4 forms. These alterations in the oligomerization status of the insulin receptor may explain the deleterious effects observed with both Chaps and octylglucoside at higher concentrations.

Topics: Centrifugation, Density Gradient; Cholic Acids; Deoxycholic Acid; Detergents; Female; Glucosides; Humans; Insulin; Liposomes; Macromolecular Substances; Placenta; Protein-Tyrosine Kinases; Receptor, Insulin; Thioglucosides

Triton X-100* was replaced in the purification of the H(+)-ATPase from chloroplasts by either octyl glucoside, octyl thio glucoside or the zwitterionic detergent CHAPS. The results indicate that the enzyme can be purified without Triton X-100 and retain full activity.

Topics: Chloroplasts; Cholic Acids; Detergents; Glucosides; Octoxynol; Plants, Edible; Polyethylene Glycols; Proton-Translocating ATPases; Solvents; Thioglucosides