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Inborn errors of monoamine neurotransmitter metabolism are rare diseases characterized by nonspecific neurological symptoms. These symptoms appear in early childhood and correspond to movement disorders, epilepsy, sleep disorders and/or mental disability. Cerebrospinal fluid biomarkers have been identified and validated to allow specific diagnosis of these diseases. Biomarkers of inborn errors of monoamine neurotransmitter metabolites are divided in two groups: monoamine neurotransmitter metabolites and pterins. Biomarkers quantification in cerebrospinal fluid is based on high-performance liquid chromatography separation coupled to electrochemical detection, fluorescence detection, or mass spectrometry. The following article reviews the advances in the proposed routine methods for the measurement of these analytes in cerebrospinal fluid. The purpose of this review is to compare the various proposed methods in terms of sample preparation, chromatographic conditions and detection modes. Despite the broad range of proposed methods, quantification of inborn errors of monoamine neurotransmitter biomarkers remains a great challenge, given the complexity of biological fluids and the low amounts of analytes that are present in cerebrospinal fluid.

Neopterin (NP), biopterin (BP) and monapterin (MP) exist in saliva. The physiological role of salivary NP as well as the pathophysiological role of increased NP in the immune-activated state has been unclear. Saliva is a characteristic specimen different from other body fluids. In this study, we analysed salivary NP and related pterin compounds, BP and MP and revealed some of its feature. High-performance liquid chromatography (HPLC) analysis of saliva and plasma obtained from 26 volunteers revealed that salivary NP existed mostly in its fully oxidized form. The results suggested that salivary NP as well as BP would mostly originate from the oral cavity, perhaps the salivary glands, and that salivary NP levels might not reflect those in the plasma. We also found that a gender difference existed in correlations between concentrations of salivary total concentrations of NP (tNP) and BP (tBP). HPLC analysis of saliva obtained from 5 volunteers revealed that the concentrations of salivary tNP as well as tBP fluctuated in an irregular fashion in various individuals. MP, a diastereomer of NP, might have come from oral cavity NP itself or its precursor. These results indicated that the nature of salivary NP might be different from that of NP in the blood or urine., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Sepiapterin reductase deficiency (SR) is a rare inborn disorder of neurotransmitter metabolism. The early diagnosis of SR disease should be achieved through the determination of the sepiapterin level in body fluids of suspected patients. Here, we report the selection, identification, and characterization of DNA aptamers against sepiapterin. The aptamer selection was achieved via the systematic evolution of ligand by the exponential enrichment technique. After ten rounds of selection, high-affinity aptamers were identified. The binding affinities of the selected aptamers were evaluated using fluorescence binding assays showing dissociation constants ranging from 37.3 to 79.0 nM. The highest affinity aptamer was then integrated into a competitive electrochemical biosensor. The biosensor achieved outstanding sensitivity with a detection limit of 0.8 pg/ml which was much lower than the reported chromatographic method for sepiapterin quantification. The aptasensor has also shown a high degree of selectivity against the closely-related compound. The aptasensor was then challenged by detecting the sepiapterin in spiked serum samples where a good recovery percentage was achieved., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Cerebrospinal fluid (CSF) metabolomic investigations are a powerful tool for studying neurometabolic diseases. We aimed to assess the effect of CSF contamination with blood on the concentrations of selected biomarkers., Methods: CSF samples were spiked in duplicate with increasing volumes of whole blood under two conditions: (A) pooled CSF spiked with fresh whole blood and frozen to cause red blood cell (RBC) lysis; (B) pooled CSF spiked with fresh blood and centrifuged (the supernatant with no RBCs was frozen until the moment of analysis). CSF concentrations of amino acids, biogenic amines, pterins, and vitamins were analysed by HPLC coupled with tandem mass spectrometry, electrochemical and fluorescence detection., Results: Aspartate, glutamate, taurine, ornithine, glycine, citrulline, pyridoxal 5´-phosphate, 5-methyltetrahydrofolate, and thiamine showed higher values when RBCs were lysed when compared with those of CSF with no RBC, while arginine, 5-hydroxyindoleacetic and homovanillic acids showed lower values. When RBCs were removed from CSF, only some amino acids, thiamine and pyridoxal 5´-phosphate showed moderately higher values when compared with the non-spiked CSF sample., Conclusions: CSF-targeted metabolomic analysis is feasible even when substantial RBC contamination of CSF has occurred since CSF centrifugation to remove RBC prior to freezing eliminated most of the interferences observed.

Inborn errors of monoamine neurotransmitter biosynthesis and degradation belong to the rare inborn errors of metabolism. They are caused by monogenic variants in the genes encoding the proteins involved in (1) neurotransmitter biosynthesis (like tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC)), (2) in tetrahydrobiopterin (BH 4 ) cofactor biosynthesis (GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SPR)) and recycling (pterin-4a-carbinolamine dehydratase (PCD), dihydropteridine reductase (DHPR)), or (3) in co-chaperones (DNAJC12). Clinically, they present early during childhood with a lack of monoamine neurotransmitters, especially dopamine and its products norepinephrine and epinephrine. Classical symptoms include autonomous dysregulations, hypotonia, movement disorders, and developmental delay. Therapy is predominantly based on supplementation of missing cofactors or neurotransmitter precursors. However, diagnosis is difficult and is predominantly based on quantitative detection of neurotransmitters, cofactors, and precursors in cerebrospinal fluid (CSF), urine, and blood. This review aims at summarizing the diverse analytical tools routinely used for diagnosis to determine quantitatively the amounts of neurotransmitters and cofactors in the different types of samples used to identify patients suffering from these rare diseases., Competing Interests: The authors declare no conflict of interest.

Folic acid, or pteroyl‑l‑glutamic acid (PteGlu) is a conjugated pterin derivative that is used in dietary supplementation as a source of folates, a group of compounds essential for a variety of physiological functions in humans. Photochemistry of PteGlu is important because folates are not synthesized by mammals, undergo photodegradation and their deficiency is related to many diseases. We have demonstrated that usual commercial PteGlu is unpurified with the unconjugated oxidized pterins 6‑formylpterin (Fop) and 6‑carboxypterin (Cap). These compounds are in such low amounts that a normal chromatographic control would not detect any pterinic contamination. However, the fluorescence of PteGlu solutions is due to the emission of Fop and Cap and the contribution of the PteGlu emission, much lower, is negligible. This is because the fluorescence quantum yield (Φ F ) of PteGlu is extremely weak compared to the Φ F of Fop and Cap. Likewise, the PteGlu photodegradation upon UV-A radiation is an oxidation photosensitized by oxidized unconjugated pterins present in the solution, and not a process initiated by the direct absorption of photons by PteGlu. In brief, the fluorescence and photochemical properties of PteGlu solutions, prepared using commercially available solids, are due to their unconjugated pterins impurities and not to PteGlu itself. This fact calls into question many reported studies on fluorescence and photooxidation of this compound., (Copyright © 2018. Published by Elsevier B.V.)

Folic acid (FA) is the synthetic form of folate (vitamin B9), present in supplements and fortified foods. During ultraviolet (UV) radiation FA is degraded to 6-formylpterin (FPT) and pterin-6-carboxylic acid (PCA) which generate reactive oxygen species (ROS) and may be phototoxic. The aim of the present study was to investigate the production of ROS and phototoxicity of FA, FPT and PCA in skin cells during UVA exposure. The production of ROS and phototoxicity of FA, FPT and PCA were studied in the immortal human keratinocytes (HaCaT) and malignant skin cells (A431 and WM115) during UVA exposure. Increased ROS production and the photoinactivation of cells in vitro were observed during UVA exposure in the presence of FA, FPT and PCA. HPLC analysis revealed that 10 μM FA photodegradation was around 2.1 and 5.8-fold faster than that of 5 μM and 1 μM FA. Photodegradation of FA is concentration dependent, and even non-phototoxic doses of FA and its photoproducts, FPT and PCA, generate high levels of ROS in vitro. FA, FPT and PCA are phototoxic in vitro. The photodegradation of topical or unmetabolized FA during UV exposure via sunlight, sunbeds or phototherapy may lead to ROS production, to the cutaneous folate deficiency, skin photocarcinogenesis and other deleterious skin effects. Further studies are needed to confirm whether UV exposure can decrease cutaneous and serum folate levels in humans taking FA supplements or using cosmetic creams with FA., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A new capillary electrophoretic (CE) method has been developed for analysis of 10 selected derivatives of pterin that can occur in the integument (cuticle) of true bugs (Insecta: Hemiptera: Heteroptera), specifically L-sepiapterin, 7,8-dihydroxanthopterin, 6-biopterin, D-neopterin, pterin, isoxanthopterin, leucopterin, xanthopterin, erythropterin and pterin-6-carboxylic acid. Pterin derivatives are responsible for the characteristic warning coloration of some Heteroptera and other insects, signaling noxiousness or unpalatability and are used to discourage potential predators from attacking. Regression analysis defining the parameters significantly affecting CE separation was used to optimize the system (the background electrolyte (BGE) composition, pH value and applied voltage). The optimized separation conditions were as follows: BGE with composition 2 mmol L(-1) the disodium salt of ethylendiamintetraacetic acid, 100 mmol L(-1) tris(hydroxymethyl)aminomethane and 100 mmol L(-1) boric acid, pH 9.0, applied voltage 20 kV and UV detection at 250 nm. Under these conditions, all the 10 studied derivatives of pterin were baseline separated within 22 min. The optimized method was validated from the viewpoint of linearity (R(2)≥0.9980), accuracy (relative error ≤7.90%), precision (for repeatability RSD≤6.65%), detection limit (LOD in the range 0.04-0.99 μg mL(-1)) and limit of quantitation (LOQ in the range 0.13-3.30 μg mL(-1)). The developed method was used for identification and determination of the contents of pterin derivatives in adults of four species of Heteroptera (Eurydema ornata cream color morph, Scantius aegyptius, Pyrrhocoris apterus and Corizus hyoscyami) and their distribution in the individual species was determined., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Optical techniques toward the realization of sensitive and selective biosensing platforms have received considerable attention in recent times. Techniques based on interferometry, surface plasmon resonance, and waveguides have all proved popular, while spectroscopy in particular offers much potential. Raman spectroscopy is an information-rich technique in which the vibrational frequencies reveal much about the structure of a compound, but it is a weak process and offers poor sensitivity. In response to this problem, surface-enhanced Raman scattering (SERS) has received much attention, due to significant increases in sensitivity instigated by bringing the sample into contact with an enhancing substrate. Here we discuss a facile and rapid technique for the detection of pterins using SERS-active colloidal silver suspensions. Pterins are a family of biological compounds that are employed in nature in color pigmentation and as facilitators in metabolic pathways. In this work, small volumes of xanthopterin, isoxanthopterin, and 7,8-dihydrobiopterin have been examined while adsorbed to silver colloids. Limits of detection have been examined for both xanthopterin and isoxanthopterin using a 10-s exposure to a 12 mW 532 nm laser, which, while showing a trade-off between scan time and signal intensity, still provides the opportunity for the investigation of simultaneous detection of both pterins in solution.

House flies, Musca domestica L., were collected in copula over two summers from six dairies located in three climatically distinct regions in the U.S.A. southern California, Minnesota and Georgia. Ages of males and females from a total of 511 mating pairs were estimated using pterin analysis. Cuticular hydrocarbon profiles and gonotrophic ages of females also were evaluated. Mean age of mating males ranged from 54 to 102 degree-days (DD) (4-10 days based on field air temperatures), depending on the farm. Very young males (< 10-20 DD) and old males (> 200 DD) were rare in mating pairs. Mean female age at mating ranged from 20 to 46 DD (2.5-4 days). All mating females had eggs in the early stages of vitellogenesis and 99.2% were nulliparous. However, some older and parous females were collected, demonstrating that re-mating can occur in the field. Head width measurements of mating pairs suggested that assortative mating by size did not occur. The cuticular hydrocarbon profiles of females were determined, with emphasis on (Z)-9-tricosene (muscalure). Overall, only 55% of mating females had detectable amounts (> 4 etag per fly) of (Z)-9-tricosene. Of the females that had detectable (Z)-9-tricosene, variation in amount per female was high in all fly populations, and thus was not statistically related to the size or age of the mating female. The proportion of mating females with detectable levels of (Z)-9-tricosene varied by geographic region. Seventy-one, 63, and 27% of females from southern California, Minnesota and Georgia had detectable amounts of (Z)-9-tricosene. Principal components analysis of the eight most abundant hydrocarbons from mating females, by state, revealed state-level distinctiveness of hydrocarbons in house fly populations, which may reflect genetic variation associated with environmental stresses in those geographical zones.

Surface enhanced Raman scattering (SERS) has been used to detect specific pterin molecules at sub-nanomolar concentrations. SERS is fast becoming a widely used technique for the sensitive and specific detection of multiple analytes. The information-rich and concentration-dependent spectra obtained from SERS make the technique ideally placed for high speed, low cost analysis of almost any analyte. Further, to show the feasibility of SERS in the detection of biologically relevant targets, a synthetic pterin analogue of the naturally occurring pterin cofactor, tetrahydrobiopterin, has been detected at a series of concentrations and the method used for the successful detection of the synthetic pterin in mouse serum. In this analysis, spectroscopic collection was optimized for water-based pteridine derivatives using two visible wavelengths of excitation (514.5 and 632.8 nm) and differing mesoscopic metal nanoparticles allowing the limits of detection to be calculated.

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

Objectives: Mitochondrial disorders are clinically heterogeneous. We aimed to describe 5 patients who presented with a clinical picture suggestive of primary neurotransmitter defects but who finally fulfilled diagnostic criteria for mitochondrial disease., Methods: We report detailed clinical features, brain magnetic resonance findings and biochemical studies, including cerebrospinal fluid (CSF) biogenic amine and pterin measurements, respiratory chain enzyme activity, and molecular studies., Results: The 5 patients had a very early onset age (from 1 day to 3 months) and a severe clinical course. They all showed a clinical picture suggestive of infantile hypokinetic-rigid syndrome (hypokinesia, hypomimia, slowness of reactions, tremor), other abnormal movements (myoclonus, dystonia), axial hypotonia, limb hypertonia, feeding difficulties, and psychomotor delay. Abnormal CSF findings among the 4 patients without treatment included low levels of homovanillic acid (HVA) in 3 patients, with associated low 5-hydroxyindoleacetic acid (5-HIAA) concentrations in two of them. Absent or mild and transitory improvement was observed after treatment with L-dopa. A diagnosis of mitochondrial disorder was finally made due to the appearance of hyperlactacidemia, diverse respiratory chain defects, and multisystemic involvement., Conclusions: Secondary neurotransmitter disturbances may occur in mitochondrial diseases. Differential diagnosis of hypokinetic-rigid syndrome presenting in infancy could also include paediatric mitochondrial disorders.

The beads in the wing scales of pierid butterflies play a crucially important role in wing coloration as shown by spectrophotometry and scanning electron microscopy (SEM). The beads contain pterin pigments, which in Pieris rapae absorb predominantly in the ultraviolet (UV). SEM demonstrates that in the European subspecies Pieris rapae rapae, both males and females have dorsal wing scales with a high concentration of beads. In the Japanese subspecies Pieris rapae crucivora, however, only the males have dorsal wing scales studded with beads, and the dorsal scales of females lack beads. Microspectrophotometry of single scales without beads yields reflectance spectra that increase slightly and monotonically with wavelength. With beads, the reflectance is strongly reduced in the UV and enhanced at the longer wavelengths. By stacking several layers of beaded scales, pierid butterflies achieve strong colour contrasts, which are not realized in the dorsal wings of female P. r. crucivora. Consequently, P. r. crucivora exhibits a strong sexual dichroism that is absent in P. r. rapae.

6-Pyruvoyltetrahydropterin Synthase (PTPS) deficiency is the most common cause of hyperphenylalaninemia due to tetrahydrobiopterin deficiency. The presenting symptoms of PTPS deficiency are mental retardation, convulsions, disturbance of tone and posture, drowsiness, irritability, abnormal movements, hypersalivation, and swallowing difficulties(1-3). The authors reported the first two cases of PTPS deficiency in Thailand. Both cases were male infants who showed phenylalanine levels of 25.23 mg/dl and 23.4 mg/dl respectively. The urinary pterins analysis showed low biopterin and high neopterin. The percentage of urinary biopterin was also found to be very low. The mutation analysis of the first case revealed a point mutation of exon 4, a homozygous C to T transition at nucleotide 200 in codon 67 (T67M), and the second case showed a compound heterozygous of exon 4, C to A transition at nucleotide 200, and exon 5, C to T transition at nucleotide 259 of the PTS gene confirming that they had PTPS deficiency. Treatment was started with neurotransmitters and a low phenylalanine diet. Family carriers were detected by means of urinary pterins determination and mutation analysis.

This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28 compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds used as internal standards. The identification of compounds is achieved using the retention times (RT) and the characteristic fragmentations of ionized compounds. The acquisition modes used for the detection of characteristic ions turned out to be complementary: the identification of expected compounds only is feasible by MRM while expected and unexpected compounds are detected by NL. In the first part of this work, the fragmentations characterizing each molecule of interest are described. These fragmentations are used in the second part for the detection by MRM and NL of selected compounds in mixture with and without biological fluids. Any preliminary extraction precedes the analysis of compounds in biological fluids., (Copyright 2006 John Wiley & Sons, Ltd.)

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Background: Previous CSF studies in Rett syndrome suggest reduced turnover of the biogenic monoamines serotonin and dopamine. Because diminished turnover may result from CNS folate depletion, the authors studied transport of folate across the blood-brain barrier., Methods: In four patients with Rett syndrome, the authors measured CSF values of 5-methyltetrahydrofolate (5MTHF), biogenic monoamine end-metabolites, and pterins together with serum and red blood cell folate. In CSF, the overall folate binding capacity by the two soluble folate-binding proteins FBP1 and FBP2 (sFBP) was measured using a radioligand binding method for H3-labeled folate. A specific immunoreactive test (ELISA) detected sFBP1, which normally contributes to 30 to 35% of the total folate binding capacity. Genetic analysis included DNA sequencing of the MECP2, FBP1, and FBP2 genes. Empirical treatment with oral folinic acid was evaluated., Results: Two patients without and two with mutations of the MECP2 gene had normal values for red blood cell folate, serum folate, homocysteine, and methionine. In CSF, all patients had low values for 5MTHF, neopterin, and the serotonin end-metabolite 5-hydroxyindoleacetic acid (5-HIAA). Genetic analysis of FBP1 and FBP2 genes had normal results. Compared to controls, patients with Rett syndrome had normal immunoreactive sFBP1 in CSF, whereas the total folate binding capacity was disproportionately lowered. Empirical treatment with oral folinic acid normalized 5-MHTF and 5-HIAA levels in CSF, and led to partial clinical improvement., Conclusion: Irrespective of the MECP2 genotype, 5MTHF transfer to the CNS is reduced in Rett syndrome. Folinic acid supplementation restores 5MTHF levels and serotoninergic turnover. The lowered folate binding capacity of FBP is not explained by a defect of the FBP1 or FBP2 gene, but most likely occurs as a secondary phenomenon in Rett syndrome.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

A common method of aging adult flies, fluorescence spectrometry, was used to monitor the increase of overall pterine titer in head extracts of Anastrepha ludens (Loew). Accumulation of fluorescent compounds was measured as a function of chronological age of flies maintained at 17 and 27 degrees C. Although relative fluorescence increased with age, field studies revealed that this phenomenon could not be used for accurate age estimation, as relative fluorescence did not increase predictably with age over the entire life span. Accumulation of individual pterins, deoxysepiapterin and sepiapterin, were studied in a similar manner. These two specific compounds were separated by high-pressure liquid chromatography and their accumulation was followed at 15 and 30 degrees C in the laboratory and under caged field conditions. While titer of deoxysepiapterin increased steadily in a curvilinear fashion, sepiapterin quickly reached a maximum and then maintained a constant level for the rest of the life of the flies. Based on the physiological response of deoxysepiapterin to chronological time and ambient thermal conditions, this compound was determined to be an age specific biological parameter for the Mexican fruit fly and should allow age estimation in field-collected flies.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

Simple binary mixtures composed of very similar pteridines (neopterin and pterin) have been resolved by derivative spectrophotometry. Detection limits of 0.30 microgram ml-1 and 0.12 microgram ml-1 for pterin and neopterin, respectively, have been calculated. Also, different mixtures of pteridines considered as disease markers, such as pterin, neopterin, xanthopterin and isoxanthopterin, have been determined by using a partial least-squares (PLS-2) model. Calibration set containing 0-7 micrograms ml-1 for each component was used. The resolution of several mixtures and single determination was tested in artificial samples.

The tetrahydrobiopterin (BH4) cofactor is essential for the aromatic amino acid hydroxylases that are involved in phenylalanine degradation and catecholamine and serotonin biosynthesis. Furthermore, BH4 is an essential and limiting cofactor for all types of nitric oxide synthases. BH4 deficiency results in hyperphenylalaninemia and monoamine neurotransmitter depletion associated with progressive mental retardation and is most commonly due to autosomal recessive mutations in 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme for cofactor biosynthesis. Due to the relatively poor blood-brain barrier penetration of the cofactor, conventional therapy requires, besides oral doses of synthetic BH4, administration of neurotransmitter precursors and an aromatic amino acid decarboxylase inhibitor. The outcome of this therapy is not always beneficial. In this study we transduced into primary patient fibroblasts the human cDNAs for the BH4 biosynthetic enzymes GTP cyclohydrolase I and PTPS, expressed from different retroviral vectors. This allowed BH4 biosynthesis in originally PTPS-deficient cells. Moreover, the double-transduced fibroblasts released between 200 and 800 pmol of BH4/10(6) cells/day. Such engineered fibroblasts may be grafted into the central nervous system and used as depository cells for constitutive delivery of BH4.

The basic scheme for the biosynthesis of methanopterin (MPT) in Methanobacterium thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg, has been shown to be the same as that recently determined for Methanosarcina thermophila strain TM-1. This scheme has, as one of its unique steps, the condensation of 4-aminobenzoic acid with 5-phospho-alpha-d-ribosyl diphosphate (PRPP) to form 4-(beta-d-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P). Labeling experiments with each of these organisms have established that the sites in the overall sequence of reactions from beta-RFA-P to MPT, where the S-adenosylmethionine-dependent C-9 and C-7 methylations of the pterin-containing intermediates occur, are organism related. In this work, cell extracts of M. thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg were found to contain significant amounts of methanopterin lacking the phosphate and 2-hydroxyglutaric acid groups., (Copyright 1998 Elsevier Science B.V.)

A sensitive and simple liquid chromatographic method to determine glyoxal, methylglyoxal, and diacetyl is reported. The method is based on the conversion to the corresponding pteridin derivatives (pterin, 6-methylpterin, and 6,7-dimethylpterin). The proposed method using fluorometric detection has been applied to the determination of the three alpha-dicarbonyl compounds in human urine. Linearity (peak area vs concentration of alpha-dicarbonyl) was observed at least up to 43 microM. Detection limits of 32 pmol for glyoxal, 11 pmol for methylglyoxal, and 99 pmol for diacetyl were calculated (20 microliters was injected). Levels of 132 microM for glyoxal and 15 microM for methylglyoxal were determined in normal urine samples, while diacetyl was not detected.

A novel pterin compound, designated as tepidopterin, was detected from a thermophilic photosynthetic green sulphur bacterium, Chlorobium tepidum. The amount of tepidopterin inside the cell was estimated to be 2-5 micromol g(-1) dry weight of cell. This compound was purified through a high performance liquid chromatography using preparative DeltaPak C18 column. This compound was characterized by chromatographic behavior and by absorption and fluorescence properties. Its structure was determined to be 1-O-(L-threo-biopterin-2'-yl)-beta-N-acetylglucosamine by 1D- and 2D-NMR spectroscopy, mass spectrometry and CD. The relative amount of tetrahydrotepidopterin was estimated to be 96.7% inside the cell, that of dihydrotepidopterin 2.9%, and that of fully oxidized tepidopterin 0.4%. The amount of tepidopterin within the cell increased continuously until the beginning of the stationary phase of the cell growth.

A high performance liquid chromatographic procedure was developed to determine folic acid and its photodegradation products, p-aminobenzoic acid, pterine-6-carboxylic acid, p-aminobenzoyl-L-glutamic acid, and pteroic acid in the presence of riboflavin. The method involves reversed phase, paired-ion chromatography on mu-BondaPak C18 column using a UV detector (254 nm), and isocratic solvent system (at ambient temperature) comprising 0.017 M monobasic potassium phosphate, tetrabutyl ammonium hydroxide solution (20%, aqueous) and methanol (870:15:250, v/v). The range of quantitation for the individual compounds was found to be: p-aminobenzoic acid, 0.01 - 1.25 x 10(-5) M; pterine-6-carboxylic acid, 0.01-2.0 x 10(-5) M; p-aminobenzoyl-L-glutamic acid, 0.02-2.0 x 10(-5) M; pteroic acid, 0.02-2.5 x 10(-5) M; folic acid, 1.0-5.0 x 10(-5) M; riboflavin, 1.0-5.0 x 10(-5) M. Linear regression analysis of the data demonstrates adequate performance of the method in terms of accuracy and precision (R. S. D. 3%). The method is specific, rapid and convenient and has been applied to photodegradation studies of folic acid in the presence and absence of riboflavin.

Methanol-grown Amycolatopsis methanolica NCIB 11946 contains a molybdoprotein dehydrogenase, active with aldehydes and formate esters as substrates and with Wurster's blue as electron acceptor, the so-called formate ester dehydrogenase (FEDH) (van Ophem et al., 1992, Eur. J. Biochem. 206, 519-525). It appears now that another molybdoprotein dehydrogenase is present in this organism. This enzyme, indicated here as dye-linked aldehyde dehydrogenase (DL-AlDH), has the same set of cofactors and converts the same type of substrates but with different specificity, and uses 2,6-dichlorophenol-indophenol as sole artificial electron acceptor for those conversions. The enzymes also differ in their quaternary structure, FEDH having an alpha, beta, gamma and DL-AlDH having an alpha, beta, gamma 2 composition. Furthermore, differences exist with respect to the sizes and the N-terminal amino acid sequences of their subunits, indicating that the enzymes derive from different genes. However, neither their substrate specificity nor their induction pattern give a clear indication for distinct physiological roles. Just like other bacterial molybdoprotein dehydrogenases, DL-AlDH consists of three different subunits (87, 35, and 17 kDa) and contains FAD, molybdopterin-cytosine-dinucleotide cofactor, Fe, and acid-labile sulfide in a molar ratio of 1:1:4:4. Although eukaryotic xanthine oxidase and dehydrogenase differ from these prokaryotic dehydrogenases in size and number of their subunits, certain stretches of amino acid sequences show similarity and the magnetic coupling between the Mo and the [2Fe-2S]-1 cluster in DL-AlDH and bovine milk xanthine oxidase is of the same magnitude. In view of this similarity, the topology of the cofactors in the active site of this type of molybdoproteins might be conserved among enzymes from prokaryotic as well as eukaryotic organisms.

Chemical analysis of dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans has shown that its molybdenum center contains two molybdopterin guanine dinucleotide molecules and a single atom of molybdenum. The enzyme, which exists as a monomer of 86 kDa, was shown to contain 1 mol of molybdenum, 4 mol of organic phosphate, and 2 mol of guanine per mole of protein. In addition, the relative yield of Form A, a fluorescent derivative of molybdopterin, was twice that obtained from sulfite oxidase, a protein which contains a single molybdopterin per molybdenum. These findings correlate with the recent report of the presence of two molybdopterin ligands in the tungsten cofactor of aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus, providing the first example of a bis(pterin)molybdenum cofactor and extending this structural motif to the molybdopterin dinucleotide enzymes.

Pterins in face flies, Musca autumnalis DeGeer, were measured spectrofluorimetrically to estimate the chronological ages of adults in natural summer populations. The age distributions showed a systematic undersampling of young males. Exponential, Gompertz, and Weibull models were fitted to the age distributions. A 1-parameter exponential model suggested mean daily survival rates of 0.83 for female face flies, and a mean life expectation of 5.5 d. But the Weibull model best fit the age distributions, providing mean life expectations of 10.1 and 11 d for males and females, respectively. Ovarian dynamics were examined with respect to chronological age and delays were detected in vitellogenesis and oviposition. Face fly populations seemed to reproduce at less than the maximum rate allowed by prevailing temperatures, at which it was estimated that an average female would undergo 2.2 ovarian cycles and have a lifetime fecundity of 28.5 female progeny.

Spectrofluorimetric methods were used to measure pterin quantities in the head capsules of adult face flies, Musca autumnalis De Geer. Quantities at eclosion were greater in males than in females, and were proportional to the head capsule width in each sex. Pterin accumulated as a linear function of time at constant temperature, at rates of 3.8 relative fluorescence units per degree-day in males and 1.68 units in females, both above a common base of 9.8 degrees C. A mark-release-recapture experiment revealed that pterin accumulated at a rate of 6.40 units per degree-day among males, twice as fast as in the laboratory. In contrast, accumulation among females was 1.35 units per degree-day, approximating their laboratory rate. Gonotrophic age grading of recaptured females indicated median development rates corresponded with expectations based on earlier laboratory studies, but substantial variation among females led to imprecise estimates of chronological age. Calibration curves for each sex are presented to estimate degree-day age from head capsule width and pterin content. The pterin age-grading method allows the field study of longevity of male face flies, and complements gonotrophic methods for the study of longevity and reproductive success of females.

The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with gav 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with gav values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via O or N, for a cysteine co-ordinating via S. Since the two signals with gav 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2-oxocarboxylates at the expense of reduced artificial redox mediators to (2R)-hydroxycarboxylates has been purified to a specific activity of up to 1800 mumol.min-1.mg-1 for the reduction of phenylpyruvate. The membrane-bound non-pyridine nucleotide-dependent enzyme appears in the form of various oligomers of the 80-kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo-labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin-6-carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron-sulphur clusters.

The content of specific methanogenic cofactors was assessed for a range of hydrogenotrophic and methylotrophic methanogenic bacteria grown on different substrates using high performance liquid chromatography. In general, all methanogens were found to contain coenzyme F420 analogues, methanopterin (MPT) analogues and 5-hydroxybenzimidazolylcobamide (vitamin B12-HBI). In hydrogenotrophic methanogens of the genera Methano-bacterium and Methanobrevibacter, as a rule, coenzymes F420-2 and F420-3 as well as MPT were present. Members of the closely related genera Methanospirillum, Methanogenium, Methanoculleus and Methanoplanus contained the same coenzyme F420 analogues but tatiopterin and/or thermopterin were present instead of MPT. In contrast, methylotrophic methanogens predominantly contained coenzymes F420-5 and F420-4, and sarcinapterin (SPT). In Methanolobus tindarius, both MPT and SPT were found, whereas no MPT analogue could be detected in Methanosphaera stadtmanae. In the hydrogenotroph Methanococcus voltae, SPT occurred as the sole MPT analogue. The levels of the various cofactors varied markedly among different methanogens and also for individual methanogens as a function of growth substrate or batch number. A correlation of cofactor levels and substrate utilized was not established. However, with methylotrophic methanogens it was noticed that the ratio of the contents of vitamin B12-HBI and SPT was independent of growth substrate.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

All known molybdoenzymes other than nitrogenase contain the metal in association with molybdopterin or one of its dinucleotide variants. All eukaryotic molybdoproteins have been found to contain only molybdopterin, whereas the majority of bacterial enzymes contain one or another of the dinucleotides of molybdopterin. In contrast, xanthine dehydrogenase from Pseudomonas aeruginosa contains molybdopterin rather than a dinucleotide. To examine whether P. aeruginosa contains any dinucleotide of molybdopterin, cells were subjected to an analytical procedure which converts molybdopterin variants to the highly fluorescent Form A derivatives. The results showed that P. aeruginosa cells do contain molybdopterin guanine dinucleotide. The same procedure showed that rat liver does not contain any of the dinucleotides of molybdopterin.

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, < 0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.

A rapid and sensitive reversed-phase ion-pair liquid chromatographic system with fluorimetric detection by post-column sodium nitrite oxidation was established for measuring six pterin compounds (6R-5,6,7,8-tetrahydrobiopterin, 6S,5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, biopterin, pterin and D-neopterin). The coefficients of variation for these pterins were 0.705-3.714%, and the minimum detectable amount was ca. 10-20 pg at a signal-to-noise ratio of 3. A linear detector response was also verified. The concentrations of the pterin compounds in rat tissues were measured by the described method. Furthermore, by means of brain microdialysis, the output of pterin compounds from rat striatum was detected. Therefore, these results demonstrate that this system can be applied to analyses not only of various rat tissues but also of dialysates collected in vivo.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. Its growth is stimulated by tungsten, and a tungsten-containing iron-sulfur protein that has formaldehyde ferredoxin oxidoreductase (FOR) activity has been purified. FOR is a homotetramer with a subunit M(r) of 70,000. It contains approximately four irons, four acid-labile sulfides, and one tungsten atom per subunit. The tungsten appears to be present as a pterin cofactor, and the Fe/S seems to comprise an unusual [4Fe-4S] cluster that in the reduced state exists in a pH-independent S = 3/2 form and a pH-dependent S = 1/2 form. FOR catalyzed the oxidation of C1-C3 aldehydes with a temperature optimum > or = 90 degrees C and used T. litoralis ferredoxin as an electron acceptor. It did not oxidize aldehyde phosphates, utilize CoASH, or reduce NAD(P). The N-terminal sequence of FOR shows homology with the tungsto-iron-sulfur aldehyde ferredoxin oxidoreductase previously purified from the saccharolytic, hyperthermophilic archaeon Pyrococcus furiosus, in which it is proposed to function in a novel pyroglycolytic pathway (Mukund, S., and Adams, M. W. W. (1991) J. Biol. Chem. 266, 14208-14216). We show here that P. furiosus, which will also grow on peptides, albeit poorly, contains a second aldehyde-oxidizing enzyme analogous to FOR. Similarly, T. litoralis, which utilizes saccharides if limited for peptides, contains low concentrations of an enzyme analogous to AOR. It is proposed that formaldehyde (apparent Km, 62 mM) is not the true substrate for FOR; rather, the enzyme has an as yet unknown role in peptide fermentation in hyperthermophilic archaea.

The pterins neopterin and xanthopterin have been measured together with the cytokines IFN-gamma, TNF-alpha and TNF-beta in the supernatants of 20 allogeneic mixed lymphocyte reactions. We have calculated rank correlation coefficients between these analytes and the immune response. Our findings confirm that neopterin is a sensitive early marker of immune activation and show that another pterin, xanthopterin could be used likewise. Cytokine production was also proportional to immune activation but none could be used consistently as early markers of immune activation.

Urinary excretion of neopterins and biopterins was measured in 23 patients with severe depression before and after receiving electroconvulsive therapy (ECT) and 26 healthy control subjects. Patients with psychotic depression and those responding to ECT had neopterin:biopterin (N:B) ratio significantly higher than controls before commencing ECT and positive therapeutic response was associated with reduction of N:B ratio towards control values. As a raised N:B ratio implies failure to convert neopterin to biopterin it is possible that reduced availability of tetrahydrobiopterin, the essential cofactor for the formation of noradrenaline, serotonin and dopamine, may exert rate limiting control over the synthesis of monoamines implicated in the pathogenesis of depressive disorders. The N:B ratio may be a marker for certain depressive subtypes and response to ECT.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.

Pterins accumulate in the head capsules of horn flies, Haematobia irritans irritans (L.), as a linear function of time and temperature. Pterin concentrations were used to estimate chronological ages and to establish correlations between chronological age and ovarian development and reproductive success in 12 horn fly populations in 1988 and 1989. Male ages were estimated spectrofluorometrically. There were statistically significant differences between years in population age structure measured by pterins. Survival rates estimated from pterin concentration distributions were consistent with a one-parameter exponential model with constant survival rate. Mean daily survival rates were 0.81 for females and 0.84 for males in 1988 and 0.66 and 0.75 in 1989. Mean lifetime egg production was approximately 26 eggs per female in 1988 and 8 in 1989. Female reproductive success was close to the maximum possible, i.e., there were no net delays in oviposition. Analysis of gonotrophic age distributions provided survival estimates that suggested an increasing risk of mortality with age or age-related biases in sampling.

Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.

Judged by properties observed during the purification and based on the sequence of the first 25 amino acids, the enzyme from Clostridium formicoaceticum catalysing the reversible reduction of non-activated carboxylic acids to aldehydes at the expense of reduced viologens, is astonishingly different from that found by us in C. thermoaceticum. According to native and SDS gel electrophoresis the reductase is nearly homogeneous after only 26-fold purification. The specificity for various substrates and artificial electron carriers is also broad, but V of the purified aldehyde dehydrogenase activity (54 U/mg enzyme for butanal) is about 1 order of magnitude lower than that of the enzyme from C. thermoaceticum. The reductase is a dimer of two identical subunits with an Mr of 67,000 each. Increased enzyme concentrations seem to lead to higher oligomers. Per dimer 11 +/- 1 iron, 16 +/- 1 acid labile sulphur, 1.4 tungsten and after permanganate oxidation 1.6 mol pterin-6-carboxylic acid have been found.