Exfoliated ECTO-NOX3 (ENOX3) proteins, are members of the human TM9 superfamily of transmembrane proteins that generate superoxide, are present in blood and other body fluids, and increase activity with age beginning ...Exfoliated ECTO-NOX3 (ENOX3) proteins, are members of the human TM9 superfamily of transmembrane proteins that generate superoxide, are present in blood and other body fluids, and increase activity with age beginning about age 30, hence age-related NOX (arNOX or ENOX3). 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 26 min and capable of generating superoxide. The cDNA was cloned from a yeast over expression library using NADH as an impermeant substrate with analysis by Fast Fourier Transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 26 min rather than a 24 or 25 min period length. The finding identified YER113C as the yeast ENOX3 protein with a 26 min period and capable of generating superoxide. The encoded protein was expressed in bacteria and characterized. Gel slices of expressed proteins revealed a protein of ca. 81,545 kDa with properties paralleling those of human ar-NOX (periodic NADH oxidation, protein disulfide thiol interchange, inhibited by mammalian arNOX inhibitors and superoxide production inhibited by superoxide dismutase). The YER113C sequence exhibited a 44% similarity and a 26% identity with the mammalian ENOX3 SF4 (arNOX SF4) of the TM9 superfamily of transmembrane proteins1. The YER113C deletion mutant lacked arNOX activity.展开更多
The yeast, Saccharomyces cerevisiae, has an ENOX1 activity with a period length of 24 min similar to that of other eukaryotes. In contrast to other eukaryotes, however, Saccharomyces cerevisiae has a second ENOX1-like...The yeast, Saccharomyces cerevisiae, has an ENOX1 activity with a period length of 24 min similar to that of other eukaryotes. In contrast to other eukaryotes, however, Saccharomyces cerevisiae has a second ENOX1-like activity with a period length of 25 min. The latter is distinguishable from the traditional ENOX1 on the basis of the longer period length along with resistance to an ENOX1 inhibitor, simalikalactone D, and failure to be phased by melatonin. In addition, two periods are apparent in measurements of oxygen consumption indicating that the consumption of oxygen to water occurs independently by homodimers of both of the two forms of ENOX. Based on the measurements of glyceraldehyde-3- phosphate dehydrogenase, S. cerevisiae exhibits circadian activity maxima at 24 and 25 h together with a 40 h period possibly representing the 40 min metabolic rhythm of yeast not observed in our measurement of oxygen consumption and normally observed only with continuous cultures. The findings are indicative of at least three independent time-keeping systems being operative in a single cell.展开更多
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 p...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 D2O 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.展开更多
A yeast (Saccharomyces cerevisiae) deletion library was screened based on NADH fluorescence using a 384 well plate assay and robotics to identify a yeast isolate lacking the 24 min periodic cell surface oxidase. The o...A yeast (Saccharomyces cerevisiae) deletion library was screened based on NADH fluorescence using a 384 well plate assay and robotics to identify a yeast isolate lacking the 24 min periodic cell surface oxidase. The oxidase was shown previously to be a candidate ultradian oscillator of the yeast’s biological clock. The cDNA was cloned from a yeast overexpression library and the encoded protein was expressed in bacteria and characterized. Glyceraldehyde-3-phosphate dehydrogenase activity was used as the cellular circadian indicator. The identified gene was YML117W which encodes a ca 126 kDa putative RNA-binding protein. The candidate ENOX1 activity from yeast had functional characteristics similar to those of other constitutive ENOX1 proteins of eukaryotes exhibiting oscillating activities with a temperature independent period length of 24 min phased by melatonin and low frequency electromagnetic fields and susceptible to inhibition by the ENOX1 inhibitor, simalikalactone D. The YML117W deletion mutant cells lacked the ENOX1 clock output present in wild type yeast. The findings identify YML117W as the ENOX1 of Saccharomyces cerevisiae and support its proposed function as an ultradian oscillator of the yeast biological clock.展开更多
ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze oxidation of both NADH and hydroquinones as well as carry out protein disulfidethiol interchange. They exhibit both pr...ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze oxidation of both NADH and hydroquinones as well as carry out protein disulfidethiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a plant candidate constitutive ENOX (CNOX or ENOX1) protein from Arabidopsis lyrata. The gene encoding the 335 (165) amino acid protein is found in accession XP-002882467. Functional motifs characteristics of ENOX proteins previously identified by site-directed mutagenesis and present in the candidate ENOX1 protein from plants include adenine nucleotide and copper binding motifs along with essential cysteines. However, the drug binding motif (EEMTE) sequence of human ENOX2 is absent. The activities of the recombinant protein expressed in E. coli were unaffected by capsaicin, EGCg, and other ENOX2-inhibiting substances. Periodic oxidative activity was exhibited both with NAD(P)H and reduced coenzyme Q as substrate. Bound copper was necessary for activity and activity was inhibited by the ENOX1-specific inhibitor simalikalactone D. Addition of melatonin phased the 24-min period such that the next complete period began 24 min after the melatonin addition as appeared to be characteristic of ENOX1 activities in general. Periodic protein disulfide-thiol interchange activity also was demonstrated along with the 2 oxidative plus 3 interchange activity pattern characteristics of the 24-min ENOX1 protein period. Concentrated solutions of the purified plant ENOX1 protein formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid. Activity was restored to aggregate preparations by isoelectric focusing.展开更多
ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prion-like and time-keeping (clock) ...ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression and characterization of a constitutive plant ENOX protein activated by both natural (Indole-3-acetic acid, IAA) and synthetic (2,4-dichlorophenoxyacetic acid, 2,4-D) auxin plant growth regulators with an optimum of about 1 μM, higher concentrations being less effective. The gene encoding the 213 amino acid protein (ABP20) is found in EMBL accession number U81162. Functional motifs characteristic of ENOX1 proteins, previously identified by site-directed mutagenesis, are present in the candidate auxin-activated ENOX (dNOX, ENOX5), including adenine nucleotide and copper binding motifs along with essential cysteines and a motif having homology with a previously identified auxin-binding motif. Periodicity was exhibited by both the oxidative and protein disulfide-thiol inter-change activities as is characteristic for other ENOX proteins. Activity was blocked by the ENOX2-specific quassinoid inhibitor glaucarubolone and other ENOX2 inhibitors but not by the ENOX1-specific quassinoid inhibitor simalikalactone D. Activity required both auxin and bound copper. The inactive auxin 2,3-D was without effects.展开更多
文摘Exfoliated ECTO-NOX3 (ENOX3) proteins, are members of the human TM9 superfamily of transmembrane proteins that generate superoxide, are present in blood and other body fluids, and increase activity with age beginning about age 30, hence age-related NOX (arNOX or ENOX3). 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 26 min and capable of generating superoxide. The cDNA was cloned from a yeast over expression library using NADH as an impermeant substrate with analysis by Fast Fourier Transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 26 min rather than a 24 or 25 min period length. The finding identified YER113C as the yeast ENOX3 protein with a 26 min period and capable of generating superoxide. The encoded protein was expressed in bacteria and characterized. Gel slices of expressed proteins revealed a protein of ca. 81,545 kDa with properties paralleling those of human ar-NOX (periodic NADH oxidation, protein disulfide thiol interchange, inhibited by mammalian arNOX inhibitors and superoxide production inhibited by superoxide dismutase). The YER113C sequence exhibited a 44% similarity and a 26% identity with the mammalian ENOX3 SF4 (arNOX SF4) of the TM9 superfamily of transmembrane proteins1. The YER113C deletion mutant lacked arNOX activity.
文摘The yeast, Saccharomyces cerevisiae, has an ENOX1 activity with a period length of 24 min similar to that of other eukaryotes. In contrast to other eukaryotes, however, Saccharomyces cerevisiae has a second ENOX1-like activity with a period length of 25 min. The latter is distinguishable from the traditional ENOX1 on the basis of the longer period length along with resistance to an ENOX1 inhibitor, simalikalactone D, and failure to be phased by melatonin. In addition, two periods are apparent in measurements of oxygen consumption indicating that the consumption of oxygen to water occurs independently by homodimers of both of the two forms of ENOX. Based on the measurements of glyceraldehyde-3- phosphate dehydrogenase, S. cerevisiae exhibits circadian activity maxima at 24 and 25 h together with a 40 h period possibly representing the 40 min metabolic rhythm of yeast not observed in our measurement of oxygen consumption and normally observed only with continuous cultures. The findings are indicative of at least three independent time-keeping systems being operative in a single cell.
文摘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 D2O 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.
文摘A yeast (Saccharomyces cerevisiae) deletion library was screened based on NADH fluorescence using a 384 well plate assay and robotics to identify a yeast isolate lacking the 24 min periodic cell surface oxidase. The oxidase was shown previously to be a candidate ultradian oscillator of the yeast’s biological clock. The cDNA was cloned from a yeast overexpression library and the encoded protein was expressed in bacteria and characterized. Glyceraldehyde-3-phosphate dehydrogenase activity was used as the cellular circadian indicator. The identified gene was YML117W which encodes a ca 126 kDa putative RNA-binding protein. The candidate ENOX1 activity from yeast had functional characteristics similar to those of other constitutive ENOX1 proteins of eukaryotes exhibiting oscillating activities with a temperature independent period length of 24 min phased by melatonin and low frequency electromagnetic fields and susceptible to inhibition by the ENOX1 inhibitor, simalikalactone D. The YML117W deletion mutant cells lacked the ENOX1 clock output present in wild type yeast. The findings identify YML117W as the ENOX1 of Saccharomyces cerevisiae and support its proposed function as an ultradian oscillator of the yeast biological clock.
文摘ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze oxidation of both NADH and hydroquinones as well as carry out protein disulfidethiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a plant candidate constitutive ENOX (CNOX or ENOX1) protein from Arabidopsis lyrata. The gene encoding the 335 (165) amino acid protein is found in accession XP-002882467. Functional motifs characteristics of ENOX proteins previously identified by site-directed mutagenesis and present in the candidate ENOX1 protein from plants include adenine nucleotide and copper binding motifs along with essential cysteines. However, the drug binding motif (EEMTE) sequence of human ENOX2 is absent. The activities of the recombinant protein expressed in E. coli were unaffected by capsaicin, EGCg, and other ENOX2-inhibiting substances. Periodic oxidative activity was exhibited both with NAD(P)H and reduced coenzyme Q as substrate. Bound copper was necessary for activity and activity was inhibited by the ENOX1-specific inhibitor simalikalactone D. Addition of melatonin phased the 24-min period such that the next complete period began 24 min after the melatonin addition as appeared to be characteristic of ENOX1 activities in general. Periodic protein disulfide-thiol interchange activity also was demonstrated along with the 2 oxidative plus 3 interchange activity pattern characteristics of the 24-min ENOX1 protein period. Concentrated solutions of the purified plant ENOX1 protein formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid. Activity was restored to aggregate preparations by isoelectric focusing.
文摘ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression and characterization of a constitutive plant ENOX protein activated by both natural (Indole-3-acetic acid, IAA) and synthetic (2,4-dichlorophenoxyacetic acid, 2,4-D) auxin plant growth regulators with an optimum of about 1 μM, higher concentrations being less effective. The gene encoding the 213 amino acid protein (ABP20) is found in EMBL accession number U81162. Functional motifs characteristic of ENOX1 proteins, previously identified by site-directed mutagenesis, are present in the candidate auxin-activated ENOX (dNOX, ENOX5), including adenine nucleotide and copper binding motifs along with essential cysteines and a motif having homology with a previously identified auxin-binding motif. Periodicity was exhibited by both the oxidative and protein disulfide-thiol inter-change activities as is characteristic for other ENOX proteins. Activity was blocked by the ENOX2-specific quassinoid inhibitor glaucarubolone and other ENOX2 inhibitors but not by the ENOX1-specific quassinoid inhibitor simalikalactone D. Activity required both auxin and bound copper. The inactive auxin 2,3-D was without effects.
