PubMed · 5799142
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M Caplow. 1969. Biotin basicity.. https://doi.org/10.1021/bi00834a061
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The phase diagram of hydrated binary mixtures of dimyristoyl-N-biotinylphosphatidylethanolamine with dimyristoylphosphatidylcholine in 1 M NaCl has been established by differential scanning calorimetry. Identification of the structures of the phases involved has been made by using X-ray diffraction, spin label ESR spectroscopy, and 31P NMR spectroscopy. On the composition axis, the phase diagram is divided into three regions corresponding to formation of compounds in the gel phase with biotinyl lipid to phosphatidylcholine stoichiometries of approximately 1:1 and 1:3 mol/mol. For the first two regions (up to 75 mol % phosphatidylcholine), the lipids in the gel phase have interdigitated chains (L beta i), whereas in the third region the gel phase is not interdigitated (L beta' or L beta). For the first region (up to 50 mol % phosphatidylcholine), the fluid phase is of the novel isotropic type (IMI) composed of aggregated normal micelles that is characteristic of shorter chainlength biotinylated lipids [Swamy, M.J., Würz, U., & Marsh, D. (1993) Biochemistry 32, 9960-9967], whereas for the other two regions a normal fluid lamellar (L alpha) phase obtains. The equimolar mixture, which lies at a stoichiometric phase boundary, melts isothermally and then undergoes a transition from the isotropic IMI structure to the lamellar L alpha structure with increasing temperature in the fluid phase.
Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.
Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) or conventional autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer onto nitrocellulose. Soaking polyacrylamide gels run under nonreducing conditions in beta-mercaptoethanol (2-ME) prior to protein transfer onto nitrocellulose resulted in a 2- to 10-fold augmentation of the resultant signal. This enhancement was observed for both disulfide- and nondisulfide-bonded proteins. Furthermore, 2-ME had no effect on either the activity of the extravidin-horse-radish peroxidase conjugate, used to detect biotin moieties, or the net protein transfer onto nitrocellulose. Thus, we propose that amplification of either ECL or gamma emission following 2-ME treatment is due to its ability to modify protein conformation, which in turn provides greater access of avidin to biotin.