Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14-16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
Carrageenans are sulfated galactans found in the cell walls of numerous red seaweeds (Rhodophyta). They are classified according to the number and the position of sulfate ester groups and the occurrence of 3,6-anhydro-galactose. Although the carrageenan biosynthesis pathway is not fully understood, it is usually accepted that the last step consists of the formation of a 3,6-anhydro ring found in k-and i-carrageenans through the enzymatic conversion of D-galactose-6-sulfate or D-galactose-2,6-disulfate occurring in m-and n-carrageenan, respectively. We purified two enzymes, sulfurylase I (65 kD) and sulfurylase II (32 kD), that are able to catalyze the conversion of n-into i-carrageenan. We compared their sulfate release rates (i.e. arising from the formation of the anhydro ring) with the viscosity of the solution and demonstrated two distinct modes of action. In addition, we found that some mixtures of sulfurylase I and II lead to the formation of carrageenan solutions with unexpectedly low viscosities. We discuss the implication of these findings for the assembly of a densely aggregated matrix in red algal cell walls.Agars and carrageenans are the most abundant components of the cell walls in numerous red algae (Rhodophyta) and can represent up to 50% of algal dry weight. These sulfated galactans are densely packed in the cell wall in a three-dimensional solid network of pseudocrystalline fibers, which assemble during the deposition of cell wall macromolecules (Craigie, 1990).
Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.
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