Age-related NADH oxidase (arNOX) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited arNOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids.
A yeast deletion library was screened based on NADH fluorescence using a 384-well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 25 min and resistant to the ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX = ENOX4). The cDNA was cloned from a yeast over expression library using NADH fluorescence analyzed by Fast Fourier transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min period length (YNOX). The finding identified YDR005C as the yeast ENOX protein with a temperature-independent 25 min period length and insensitive to inhibition by simalikalactone D. The encoded protein was expressed in bacteria and characterized. Gel slices corresponding to 55 kDa and 39 kDa His-tagged proteins exhibited 25 min oscillatory patterns not inhibited by 1 µM simalikalactone D for both NADH oxidation and reduced coenzyme Q10 oxidation as well as a protein disulfide-thiol interchange activity which alternated with the oxidative activities. Activities were phased by low-frequency electromagnetic fields but, in contrast, to yeast ENOX1, not by addition of melatonin. The assay in the presence of D 2 O shifted the length of the oscillatory period from 25 min to 32 min. The YDR005C deletion mutant cells lacked the ENOX4 clock output present in the wild type yeast.
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