A reexamination of the properties of spinach nitrite reductase: protein and siroheme content heterogeneity in purified preparations

Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration.     

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.     

The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study

The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.