Interaction of hepatic asialoglycoprotein receptor with dimyristoyl phosphatidylcholine vesicles

The hepatocyte membrane asialoglycoprotein receptor (ASGP-R) was extracted from rabbit liver, purified, and then incubated with preformed vesicles of dimyristoyl phosphatidylcholine. The association of protein with lipid was dependent on vesicle size and the best results were achieved with small vesicles of about 20 nm diameter. The ligand binding capacity of ASGP-R-vesicle complexes was also measured and found to be approximately sevenfold greater than free receptor in aqueous buffer and twofold greater than receptor solubilized in Triton X-100. Most likely, the reconstitution procedure used in these experiments does not result in transmembrane insertion of the receptor. ASGP-R probably resides on the surface of the vesicle, held there primarily by weak hydrophobic forces.     

YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of a 1:1 complex of two homodimeric subunits: alpha and beta. beta contains the diferric-tyrosyl radical (Y*) cofactor essential for the reduction process. In vivo, the mechanism of Y* regeneration from the diferric-beta2 (met-beta2) or apo-beta2 is still unclear. Y* regenerations from met-beta2 and apo-beta2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding alpha and beta) were examined. In 29% of the cases, an open reading frame annotated 2Fe2S ferredoxin (YfaE in Escherichia coli) is located next to nrdB. Thus, YfaE has been cloned, expressed, resolubilized, reconstituted anaerobically with Fe2+, Fe3+, and S2-, and characterized by M?ssbauer, EPR, and visible spectroscopies. Titration of met-beta2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-beta2 and [2Fe2S]2+-YfaE with one Fe reduced/YfaE oxidized. At the end point of the titration, O2 is added to the mixture and the diferrous-beta2 rapidly undergoes reaction to form the diferric-Y* with a stoichiometry of 2Fe/Y* and a specific activity correlated to the amount of Y*. The reducing equivalent required for diferric-Y* cofactor biosynthesis is supplied by beta. Under anaerobic conditions, stopped flow kinetics have been used to monitor the disappearance of the diferric cluster and the formation of [2Fe2S]2+-YfaE. The titrations and kinetic studies provide the first evidence for a protein involved in the maintenance pathway and likely the biosynthetic pathway.