S100B is selectively expressed by gray matter protoplasmic astrocytes and myelinating oligodendrocytes in the developing CNS.

Studies on the development of central nervous system (CNS) primarily rely on the use of specific molecular markers for different types of neural cells. S100B is widely being used as a specific marker for astrocytes in the CNS. However, the specificity of its expression in astrocyte lineage has not been systematically investigated and thus has remained a lingering issue. In this study, we provide several lines of molecular and genetic evidences that S100B is expressed in both protoplasmic astrocytes and myelinating oligodendrocytes. In the developing spinal cord, S100B is first expressed in the ventral neuroepithelial cells, and later in ALDH1L1+/GS+ astrocytes in the gray matter. Meanwhile, nearly all the S100B+ cells in the white matter are SOX10+/MYRF+ oligodendrocytes. Consistent with this observation, S100B expression is selectively lost in the white matter in Olig2-null mutants in which oligodendrocyte progenitor cells (OPCs) are not produced, and dramatically reduced in Myrf-conditional knockout mutants in which OPCs fail to differentiate. Similar expression patterns of S100B are observed in the developing forebrain. Based on these molecular and genetic studies, we conclude that S100B is not a specific marker for astrocyte lineage; instead, it marks protoplasmic astrocytes in the gray matter and differentiating oligodendrocytes.

Expression and significance of ETFDH in hepatocellular carcinoma.

The ETFDH (electron transfer flavoprotein dehydrogenase) gene mutations are reported to be a major cause of riboflavin-responsive multiple acyl-coenzyme A dehydrogenation deficiency (MADD). However, the role of ETFDH in the prognosis of hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the expression of ETFDH in HCC. Immunohistochemical staining of the 207 HCC tissue microarray showed that expression of ETFDH was significantly decreased in HCC compared with the matching noncancerous hepatic tissues (P < 0.001). Moreover, ETFDH expression levels were found to be correlated with AFP levels (P = 0.011). Intriguingly, ETFDH expression levels were significantly lower in poorly differentiated or undifferentiated HCCs as compared to the well or moderately differentiated cases (P = 0.001). Kaplan-Meier analysis revealed that low tumor expression of ETFDH was associated with a poorer overall survival in patients with HCC (P = 0.024). Furthermore, multivariate analysis showed that ETFDH (P = 0.047) was an independent predictor of overall survival. Our findings may shed new light on the identification of new prognostic marker for HCC.

Visualizing the brain's astrocytes.

Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contribution to neural processing remains unknown. This is, in part, due to the lack of methods available for astrocyte identification and analysis. Existing strategies employ genetic tools like the astrocyte specific promoters glial fibrillary acidic protein (GFAP) or Aldh1L1 to create transgenic animals with fluorescently labeled astrocytes. Recently, small molecule targeting moieties have enabled the delivery of bright fluorescent dyes to astrocytes. Here, we review methods for targeting astrocytes, with a focus on a recently developed methylpyridinium targeting moiety's development, chemical synthesis, and elaboration to provide new features like light-based spatiotemporal control of cell labeling.

Spermine oxidase promotes bile canalicular lumen formation through acrolein production.

Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H2O2) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

[Preparation and identification of monoclonal antibody against human spermine oxidase].

AIM: To prepare mouse anti-human spermine oxidase (anti-hSMO) monoclonal antibody (mAb) and testify its application in the biological techniques including Western blotting and immunohistochemistry. METHODS: Plasmid pET-15b/SMO was first transferred into BL21 (DE3), and then SMO recombinant protein with 6×His tag was induced to express by IPTG and purified by Ni-NTA resin. The purified recombinant SMO was used to immunize BALB/c mouse. The spleen cells from the immunized mouse were harvested and hybridized with Sp2/0 myeloma cells to obtain a hybridoma cell line that could efficiently synthesize and secret anti-SMO mAb. The titer and antigen specificity of this antibody were identified by ELISA, Western blotting and immunohistochemistry. RESULTS: We successfully obtained the hybridoma cell line which could stably secret anti-SMO mAb. The mAb was of a high titer and antigen specificity and could be used in ELISA, Western blotting, and immunohistochemistry for SMO. CONCLUSION: The mouse anti-hSMO mAB with a high antigen specificity has been prepared successfully and used for a variety of bio-analytical techniques.

Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT.

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS-PAGE, or 13 920 Da as determined by MALDI-TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.

Polyamine catabolism is enhanced after traumatic brain injury.

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

[Physiological functions of polyamines and regulation of polyamine content in cells].

Polyamines (putrescine, spermidine, and spermine) are essential for normal cell growth. The polyamine level in cells is regulated by biosynthesis, degradation, and transport. The role of antizyme on polyamine biosynthesis and transport in mammalian cells and characteristics of polyamine transport in Escherichia coli and yeast are described briefly in this review. In addition, the effects of polyamines on protein synthesis and the NMDA receptor are outlined. Finally, the correlation between acrolein produced from polyamines by polyamine oxidase and chronic renal failure and brain stroke is summarized. Increased levels of polyamine oxidase and acrolein are good markers of chronic renal failure and brain stroke.

The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.

Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.

Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum.

Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.

[The relations between polyamine types and forms and polyamine oxidase activities in barley seedlings under salt stress].

The three polyamines (Put, Spd and Spm) and their different forms (free, perchioric acid soluble and insoluble conjugated) together with polyamine oxidase (PAO) activities were measured in barley (Hordeum vulgare L. KP7) seedlings under salt stress. The results showed that the free polyamines were dominant forms among three polyamines, account about 2/3 total polyamines. All three polyamines decreased in roots and leaves after 8 d under NaCl 200 mmol/L stress. The free polyamine content decreased dramatically as well as soluble conjugated polyamine content, while the insoluble ones changed little. Different concentration salt stress increased markedly the ratios of (Spd+Spm)/Put and decreased significantly the total polyamine content in roots and leaves. PAO activities in roots was increased at first and then decreased under NaCl stress, while in leaves the activities changed in opposite direction. It has been noted that the sum of free Spd and Spm (Spd+Spm) content is reversely correlated with PAO activity changes, which suggests that PAO might regulate the free polyamine content, therefore, the contents of the soluble and insoluble conjugated polyamines were affected.

Heterologous expression and characterization of mouse spermine oxidase.

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Prevalence of factor V G1691A (Leiden), prothrombin G20210A, and methylene tetrahydrofolate reductase C677T thrombophilic mutations in children with inflammatory bowel disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have an increased incidence of thromboembolic events. This risk may be caused by an increased frequency of thrombophilic mutations such as factor V Leiden G1691A (FVL), prothrombin G20210A (PT), or methylene tetrahydrofolate reductase C667T (MTHFR). Prevalence rates of heterozygous mutations in FVL, PT, and MTHFR are reported for whites (1.8%, 1.3%, 26.6%, respectively), blacks (0.8%, 0.3%, and 12.4%, respectively), and Hispanics (1.2%, 2.4%, and 41.5%, respectively). We sought to determine the prevalence of these thrombophilic mutations in a large cohort of children with IBD. METHODS: Children aged 21 years or younger with IBD were genotyped for FVL, PT, and MTHFR mutations by polymerase chain reaction amplification and restriction enzyme digestion. Prevalence rates were compared with established rates in the respective populations. RESULTS: Of 92 patients enrolled, 89 (62 with Crohn disease, 24 with ulcerative colitis, and 3 with indeterminate colitis) had genotype results available. The mean age was 13.3 +/- 4.2 years (range, 2-21 years). Statistical analysis was performed on 89 FVL, PT, and MTHFR allele pairs. Polymerase chain reaction genotyping identified 5 patients with heterozygous FVL mutations, 3 patients heterozygous for the PT mutation, and 36 patients heterozygous and 4 patients homozygous for the MTHFR mutation. The thrombophilic allele mutation frequencies in our sample were not significantly different from predicted weighted average values: FVL, 2.8% versus 1.5%; PT, 1.7% versus 1.1%; and MTHFR, 24.7% versus 24.4%. In 24 patients with a family history of thrombosis, 1 was heterozygous for FVL and for MTHFR, 1 was heterozygous for FVL and homozygous for MTHFR, 2 were heterozygous for PT, and 9 were heterozygous MTHFR. There was no significant correlation between family history of thrombosis and presence of a thrombophilic mutation. The four patients with homozygous mutations for MTHFR, two of whom also were heterozygous for FVL, did not have either a personal history of thrombosis or a family history of thrombotic events. Two patients had thrombotic events without mutations in these genotypes: one had protein S deficiency and the other had no identifiable cause. CONCLUSIONS: The presence of genetic mutations that predispose to hypercoagulable states does not appear to correlate with the prevalence of IBD or to thromboembolic events in patients with IBD. There was no statistical difference between the proportions of the mutated allele frequency in our study patients and the general population.

Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580.

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.

Assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring 5-methyltetrahydrofolate and tetrahydrofolate using high-performance liquid chromatography with fluorescence detection.

We developed a method for assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-methyltetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate) directly, using high-performance liquid chromatography with fluorescence detection. Folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both H(4)folate and 5-CH(3)-H(4)folate in our assay system. The detection limit of this method was less than 20 nM H(4)folate or 5-CH(3)-H(4)folate in the enzyme assay system. This analytical method, therefore, has a sensitivity high enough to obtain accurate parameters of Michaelis-Menten kinetics and for assays of crude extracts from various biological samples. In addition, the analytical procedure is very simple and economical; it may be a useful tool for studying methylenetetrahydrofolate reductase and methionine synthase activities.

Analytical performance characteristics of thin and thick film amperometric microcells.

The analytical performance of amperometric microcells with different electrode geometries is compared for enzyme activity measurements. The microcells were fabricated with thin film photolithography or thick film screen-printing in four different designs. The cells made with the thin film process used flexible substrate with microelectrode array or a circular, disk-shaped working electrode. The screen-printed working electrodes had semicircle or disk shape on ceramic chips. Putrescine oxidase (PUO) activity measurement was used as a model. The determination of PUO activity is important in the clinical diagnosis of premature rupture of the amniotic membrane. An electropolymerized m-phenylenediamine size-exclusion layer was used to eliminate common interferences. The size exclusion layer revealed also to be advantageous in protecting the electrodes from fouling by putrescine (enzyme substrate). The electrode fouling of bare electrodes was insignificant for screen-printed electrodes, but very severe for electroplated platinum working electrodes. The microelectrode array electrodes demonstrated smaller RSD and higher normalized sensitivities for hydrogen peroxide and PUO activity. All the other electrodes were demonstrating comparable analytical performances.

Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.

Apolipoprotein E, methylenetetrahydrofolate reductase (MTHFR) mutation and the risk of senile dementia--an epidemiological study using the polymerase chain reaction (PCR) method.

We examined apolipoprotein E (Apo E) polymorphism and methylenetetrahydrofolate reductase (MTHFR) 677 C to T mutation by using the polymerase chain reaction (PCR) method in 100 elderly Japanese aged 60 or more, and assessed whether these genetic factors are associated with an increased risk for the clinical phenotypes of senile dementia, Alzheimer's disease (AD) and vascular dementia (VD) by cross-sectional survey. It was found that the Apo E epsilon 4 allele were associated with an increased prevalence of AD as previously reported. Although, it was not strongly related to the severity of senile dementia, a weak association between the ApoE genotype and the severity of dementia was suggested. The proportion of patients with senile dementia was higher in the group of carriers of MTHFR mutation than in the group of noncarriers. Furthermore, the proportion of male patients with senile dementia was higher in the group of homozygous for the mutation (+/+) than the group without the mutation (-/-). Notably in VD patients, 5 of 7 males had the +/+ genotype. The results suggest that the ApoE epsilon 4 genotype and the MTHFR mutation are associated with the clinical phenotype and the clinical onset of senile dementia.

Comparative study of enzymes related to proline metabolism in tepary bean (Phaseolus acutifolius) and common bean (Phaseolus vulgaris) under drought and irrigated conditions, and various urea concentrations.

There are several mechanisms used by plants for survival in adverse environments such as drought, high temperature and salinity. The objective of this study was to evaluate the drought tolerance of tepary bean as a function of biochemical processes linked to isozyme synthesis and changes in enzymatic activity related to proline metabolism. Mature seeds of common beans var. flor de mayo, Phoseolus vulgaris and tepary beans Phaseolus acutifolius were grown under two water conditions (irrigation and drought), and four levels of urea. Vertical electrophoresis and spectrophotometric techniques were used to evaluate protein patterns, glutamate dehydrogenase (GDH), proline oxidase (PO) and pyrroline-5-carboxylate reductase (P5C reductase) enzyme activities. These enzymes were studied because they are directly related to protein synthesis. Electrophoretic patterns showed more proteins in tepary beans than in common beans with limited irrigation. GDH showed only one isozyme, with a molecular weight between 240) to 270 kDa. A decrease in PO activity was observed in common beans under drought stress with a value of 237 micromol/min, in comparison to irrigation conditions of 580 micromol/min. GDH and P5C reductase enzymes have had higher activity in common beans than in tepary beans under water stress. There was a significant difference only in glutamate dehydrogenase enzyme with respect to urea level. The results suggest that drought tolerance of tepary beans is due to biochemical processes related to proline metabolic enzymes.

Redox potential of the haem c group in the quinocytochrome, lupanine hydroxylase, an enzyme located in the periplasm of a Pseudomonas sp.

The quinocytochrome c, lupanine hydroxylase, was shown to be located in the periplasm of a Pseudomonas sp. The midpoint redox potential of the haem in the purified enzyme was measured by potentiometric titration and shown to be +193 mV. PQQ was removed from the enzyme by isoelectric focusing to give inactive apoenzyme. This resulted in a shift in the midpoint redox potential of the haem to +98 mV. Full activity was recovered by the addition of PQQ to apoenzyme that also restored the original potential.