Nicotinamide Dinucleotide Phosphate Diaphorase Research Articles

NO-dependent mechanisms of amygdalocortical influence

The brain amygdala complex (AC) participates in awide spectrum of motivational and emotional behav-ioral responses, which are accompanied by changes inthe visceral and endocrine systems [1]. AC projectionsto the anterior limbic cortex are an important pathwayof AC effects on the integrative brain functionsinvolved in visceral regulation [2–4].Numerous neuroanatomical data indicate that ACprojections run from the parvicellular region of the ACbasal nucleus to deeper layers of the prelimbic andinfralimbic cortices [5]. However, the neurophysiolog-ical mechanisms of the amygdalocortical effects medi-ated by this connections remain unclear., To date, theinhibitory and excitatory AC effects on cells of the ante-rior limbic cortex have been described in a few reports[3, 6–8], though nothing is known about the neurotrans-mitters of these processes.At the same time, nitric oxide (NO) is generallyknown to be one of the most important neurotransmit-ters and/or neuromodulators in the central nervous sys-tem [9, 10]. NO is synthesized by some cortical cells[11, 12] and plays an important role in the regulation ofcortical processes; in particular, it modulates the releaseof γ-aminobutyric acid and acetylcholine [10, 13].The nitroergic activity of cortical neurons may bealso involved in the mechanisms underlying the ACeffects on the anterior limbic cortex. Nitroergic activitymodulation may change the level of neuronal excitabil-ity in the prelimbic and infralimbic regions and, hence,the sensitivity of these regions to the regulatory influ-ences of other brain structures, including the ACnucleus.We verified this hypothesis in a series of histochem-ical and neurophysiological experiments performed onWistar male rats weighing 250–360 g; the animals wereanesthetized with urethane (1.5 g/kg body weight;intraperitoneal administration). In response to electricalstimulation of the parvicellular part of the AC basalnucleus, the NO-synthase activity in various regions ofthe anterior limbic cortex was studied histochemically(detection of intracellular nicotinamide dinucleotidephosphate diaphorase (NADP-d). In neurophysiologi-cal experiments, the effects of the same AC nucleusstimulation on the neuronal activity of the anterior lim-bic cortex were studied before and after the administra-tion of 7-nitroindasol, a blocker of the neuronalNO-synthase, into the left lateral brain ventricle. Two groups of animals, each containing four rats(the control one without stimulation and the group ofelectrically stimulated animals) were used in his-tochemical studies. The surgical preparation was thesame in both groups. The parvicellular part of the basalAC nucleus was subjected to a monopolar electricalstimulation in a region with the following coordinates:3.3 mm caudal of bregma, 5.0 mm lateral of the medianline, and about 7.0 mm away from the dorsal brain sur-face; tungsten electrodes in varnish insulation wereused (tip diameter, 50–60 µm; resistance, 80–100 kΩ).Three series of 0.5-ms rectangular pulses were used(current, 30–50 µA). The pulse frequency in a serieswas 50 pulses per second, the series length was 10 s,and the interval between series was 20 s.On completion of the stimulation, the animals weretranscardially perfused with 4% paraformaldehydesolution; 24 h later, 30-µm frontal sections across theanterior limbic cortex were made using a freezingmicrotome. The sections were treated histochemicallyto detect NADP-d according to a modified method ofScherer-Singler et al. [14]. NADP-d-positive neuronswere located, and their number and optical densitywere determined on the cortical sections using animage-analyzing device, which was connected with apersonal computer; the program packages Video TestMaster 4.0 and Image Tool were used. The neuronaloptical density was expressed in arbitrary units(arb. un.) within a range from 0 (a white object) to 1.55(a black object).Neurophysiological experiments were performed on34 rats. In this experimental series, the parvicellularpart of the AC basal nucleus was stimulated by solitaryrectangular pulses with the same parameters. The neu-ronal responses from the anterior limbic cortex were

Differential distribution of NADPH-diaphorase histochemistry in human cerebral cortex

β−Nicotinamidedinucleotide phosphate diaphorase (NADPH-d) colocalizes with NOS in the central nervous system. Two types of NADPH-d-positive neurons are present in the primate cerebral cortex: type 1, intensely and Golgi-like labeled neurons, a subset of GABAergic interneurons; type 2, lightly labeled neurons (divided into two subclasses, a first one having a lightly stained cell body bearing only one short process, and a second one showing intense NADPH-d staining with short processes extending radially). We have analyzed the distribution of NADPH-d activity in human frontal, temporal, and occipital cortical areas, finding remarkable laminar and interareal differences in cell size and distribution of the different cell types. There was a clear bias for type 1 neurons in infragranular layers in all areas considered; both in supra- and infragranular layers, their density was highest in frontal, and lowest in temporal cortex. The density of type 2 neurons was lower supragranularly in temporal cortex and infragranularly in occipital cortex. The overall density of type 2 cells was remarkably higher in occipital cortex than in the temporal and frontal ones. Type 1 neurons were significantly larger than type 2, and were smaller in the supragranular than in the infragranular subzone in occipital and temporal cortex. Type 1 cells were significantly larger in frontal cortex than in occipital and temporal cortex, and type 2 cells were significantly smaller in occipital than in temporal and frontal cortex. These area-related differences might reflect differences between heterotypic and homotypic cortex in the regulation of cortical blood flow.

Anatomical relationships between aromatase-immunoreactive neurons and nitric oxide synthase as evidenced by NOS immunohistochemistry or NADPH diaphorase histochemistry in the quail forebrain

In Japanese quail ( Coturnix japonica), previous studies indicated that the distribution of reduced nicotinamide dinucleotide phosphate (NADPH) diaphorase overlaps with steroid-sensitive areas that contain dense populations of aromatase-immunoreactive (ARO-ir) cells. We investigated here the anatomical relationships between aromatase (ARO) and nitric oxide synthase (NOS)-containing cells that were visualized both by NOS-immunohistochemistry and NADPH-histochemistry. The distribution of ARO-ir and of NADPH-positive cells in the forebrain observed here matched exactly the distribution previously reported. The distribution of NOS-immunoreactive material in the vicinity of ARO-ir cell groups appeared similar to the distribution of NADPH-positive structures previously identified by histochemistry. The number of NOS-immunoreactive cells was similar to the number of NADPH-positive cells and they were found in the same brain regions. In contrast, few NOS-immunoreactive fibers were observed whereas numerous NADPH-positive fibers and punctuate structures were present in many areas. Major groups of NOS-immunoreactive/NADPH-positive neurons were adjacent to the main ARO-ir cell groups, such as the medial preoptic nucleus, the bed nucleus of the stria terminalis and the nucleus ventromedialis hypothalamic. However, examination of adjacent sections indicated that there is very little overlap between the NOS-immunoreactive and ARO-ir cell populations. This notion got further support by double-labeled sections where no double-labeled cells could be identified. In sections stained simultaneously by histochemistry for NADPH and immunohistochemistry for ARO, many NADPH-positive fibers and punctate structures were closely associated with ARO-ir perikarya. Taken together, the present data indicate that NOS is not or very rarely colocalized with ARO but that NOS inputs are closely associated with ARO-ir cells. Based on previous work in a variety of model systems, it can be hypothesized that these inputs modulate the expression or activity of ARO in the quail brain.

Nitric oxide-containing neurons in the bovine gut, with special reference to their relationship with VIP and galanin.

The presence and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d)-containing neurons have been studied by means of NADPH-d histochemistry in different regions of the adult cow gut, from the esophagus to the rectum. NADPH-d and nitric oxide synthase (NOS) were constantly recognized to be colocalized in the same neuron. The colocalization of vasoactive intestinal polypeptide (VIP) and galanin in such nitrergic neurons was also studied by means of combined histochemical and immunofluorescence techniques. NADPH-d-positive neurons were present along the myenteric plexus of the entire gut, and in the submucous plexus from the abomasum to the rectum. Notably, they formed two types of nerve networks in the submucous connective tissue of the jejunum-ileum. NADPH-d-positive innervation of the muscle layers occurred throughout the tract, and sometimes a clear correspondence was noted between the number of reactive fibres and the thickness of the muscle. Nitrergic fibres also occurred in the mucosa and often were in relation to glands and blood vessels. The nitrergic neurons varied in size, shape, and intensity of staining, and often their terminals were seen to surround unstained perikarya. Various types of neurons were recognized on the basis of their chemical content; one of them contained galanin, VIP and NOS simultaneously. The present results suggest that the nitrergic neurons of the bovine gastrointestinal tract play roles presumably for controlling the motility of the gut and the conduction of interneuronal impulses.

Nitric Oxide–Mediated Effects on Myometrial Contractility at Term During Prelabor and Labor

In Brief Objective To assess the existence of a nitric oxide (NO) system in the human myometrium and the effects of mediators of this system on contractile activity in vitro. Methods Myometrial tissue was obtained before the onset of labor and during labor at term. Production of NO was assessed by the use of nicotinamide dinucleotide phosphate diaphorase staining and by immunoblots for NO. Effects of NO were examined by adding L-arginine (the substrate for NO synthesis); NG-nitro-L-arginine methyl ester (an inhibitor of NO synthase); two NO donors, sodium nitroprusside and spermine NONOate; as well as 8-bromo cyclic guanosine monophosphate (8-bromo cGMP) (a second messenger analogue) to organ baths. Results Myometrial NO production was indicated by positive nicotinamide adenine dinucleotide phosphate diaphorase staining. Immunoblots detected endothelial NO synthase, whereas only a weak signal for inducible NO synthase was seen. The addition of L-arginine (10−4–10−3 mol/L) did not result in any change of contractility. NG-nitro-L-arginine methyl ester (10−3 mol/L) caused a minor increase of contractility in half of the specimens. Sodium nitroprusside, spermine NONOate, and 8-bromo cGMP resulted in a concentration-dependent inhibition of contractility (10−7–10−6 mol/L for sodium nitroprusside, 10−6–10−5 mol/L for spermine NONOate, and 10−5–10−3 mol/L for 8-bromo cGMP). However, at 10−5–10−4 mol/L, sodium nitroprusside exhibited a dose-dependent increase in the frequency of contractions. Women in prelabor did not differ from those in active labor. Conclusion The myometrium produces NO at term. Nitric oxide inhibits myometrial contractile activity. The responsiveness to NO is similar in nonlaboring and laboring women. Nitric oxide synthase is present in human myometrial tissue, and nitric oxide–donating substances inhibit spontaneous contractility of myometrial strips.

Nitric oxide–mediated effects on myometrial contractility at term during prelabor and labor

Objective: To assess the existence of a nitric oxide (NO) system in the human myometrium and the effects of mediators of this system on contractile activity in vitro. Methods: Myometrial tissue was obtained before the onset of labor and during labor at term. Production of NO was assessed by the use of nicotinamide dinucleotide phosphate diaphorase staining and by immunoblots for NO. Effects of NO were examined by adding l-arginine (the substrate for NO synthesis); N G-nitro- l-arginine methyl ester (an inhibitor of NO synthase); two NO donors, sodium nitroprusside and spermine NONOate; as well as 8-bromo cyclic guanosine monophosphate (8-bromo cGMP) (a second messenger analogue) to organ baths. Results: Myometrial NO production was indicated by positive nicotinamide adenine dinucleotide phosphate diaphorase staining. Immunoblots detected endothelial NO synthase, whereas only a weak signal for inducible NO synthase was seen. The addition of l-arginine (10 −4–10 −3 mol/L) did not result in any change of contractility. N G-nitro- l-arginine methyl ester (10 −3 mol/L) caused a minor increase of contractility in half of the specimens. Sodium nitroprusside, spermine NONOate, and 8-bromo cGMP resulted in a concentration-dependent inhibition of contractility (10 −7–10 −6 mol/L for sodium nitroprusside, 10 −6–10 −5 mol/L for spermine NONOate, and 10 −5–10 −3 mol/L for 8-bromo cGMP). However, at 10 −5–10 −4 mol/L, sodium nitroprusside exhibited a dose-dependent increase in the frequency of contractions. Women in prelabor did not differ from those in active labor. Conclusion: The myometrium produces NO at term. Nitric oxide inhibits myometrial contractile activity. The responsiveness to NO is similar in nonlaboring and laboring women.

NADPH-diaphorase activity in normally developing and intracranially transplanted retinas.

The activity and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d), an enzyme that is widely distributed in the central nervous system and involved in the production of the free radical nitric oxide, were investigated histochemically in the normal developing and intracranially transplanted retinas. In the normal rat retina, NADPH-d activity was first detected in cells in the ganglion cells layer (GCL) and blood vessels on the first postnatal day (P0). A small but distinct population of NADPH-d positive cells were observed along the inner border of the inner nuclear layer at P7. NADPH-d positive sublaminae began to appear in the inner plexiform layer during the second postnatal week, and several strongly reactive sublaminae resembling those observed in the adult were observed by the fourth postnatal week. The overall spatio- temporal sequence of development of NADPH-d positive cells in the transplanted retina was similar to that of the normal retina, except a lack of reactive in the inner plexiform layer in more mature transplants as compared with normal retinas of corresponding ages. These results indicate that the time course of development and distribution of NADPH-d cells in early postnatal retina requires signals mainly of intraretinal origin and is independent of influence from the surroundings. While this finding is supportive to the notion that neurons that are rich in NADPH-d are resistant to injury or perturbation, the observation of a lack of well organized NADPH-d reactive sublaminae in the inner plexiform layer in older transplants suggests a possible alteration in the synaptic circuitry in the inner retina with increasing postgrafting survival time.

Cytochrome oxidase and NADPH‐diaphorase on the afferent relay branch of the optokinetic reflex in the opossum

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO)<0B> <0R>was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system. J. Comp. Neurol. 398:206–224, 1998. © 1998 Wiley-Liss, Inc.

Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in the opossum

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO) was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system.

Nitric oxide synthase and vasopressin in rat circumventricular organs. An immunohistochemical study.

The distribution of immunoreactivity to neuronal nitric oxide synthase (nNOS) and vasopressin (AVP) was studied in the circumventricular organs of the female rat. The occurrence of NOS immunoreactivity showed correspondence to nicotinamide dinucleotide phosphate diaphorase reactivity, a previously used but less specific marker for neuronal NOS. nNOS immunolabeling was detected in the two most rostrally located circumventricular organs - the organum vasculosum of the lamina terminalis and the subfornical organ. In the latter, AVP immunoreactivity was observed in some cell bodies, which also were nNOS-immunoreactive. In the median eminence and the neurohypophysis there were large amounts of nNOS- and AVP-immunoreactive nerve fibers, which often displayed similarities in distribution and morphology. Within the pineal gland, only very few nNOS-immunoreactive varicose terminals were observed, which ran along blood vessels. nNOS immunoreactivity was also seen in the epithelium of the choroid plexus, whereas no nNOS immunoreactivity could be found in the subcommissural organ or in the area postrema. The present demonstration of nNOS and AVP immunoreactivity in the subfornical organ, median eminence, and neurohypophysis, and the occurrence of nNOS immunoreactivity also in the choroid plexus and organum vasculosum of the lamina terminalis, provides a morphological background for a functional role for nitric oxide in water homeostatic mechanisms, both as executed through the hypothalamohypophyseal system and via the production of cerebrospinal fluid.

Activation of nitric oxide-synthesizing neurones during precipitated morphine withdrawal.

Activation of nitric oxide (NO)-synthesizing neurones in the hypothalamus and brain stem was studied during naloxone-precipitated morphine withdrawal in rats. In animals that underwent behavioural changes consistent with withdrawal, Fos and nicotinamide dinucleotide phosphate-diaphorase (NADPH-D)-positive neurones were identified within the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei as well as the brain stem nucleus tractus solitarius (NTS). A larger proportion of NADPH-D-positive neurones were activated in the PVN than in the NTS. While NO has been implicated in the genesis of opioid withdrawal, the present data provide evidence for the activation of select populations of NO-synthesizing neurones during the opiate withdrawal syndrome.

Expression of nitric oxide synthase by motor neurones in the spinal cord of the mutant mouse wobbler

The expression of nitric oxide synthase (NOS) has been studied in the spinal cord of the mutant mouse wobbler, a recessive mutation in which there is motor neurone degeneration, using nicotinamide dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Abnormal NOS positive large neuronal profiles could be found in the ventral horn of affected mutant animals but not their unaffected littermate controls. The number of abnormal profiles observed was dependant upon the age of the animal. A small number of these NOS positive large neuronal profiles were seen at the onset of the disease at 3–4 weeks of age, larger numbers were found in animals aged 5–8 weeks coincident with the main period of motor neurone death, whilst in the spinal cords of older animals aged 9–12 months, when motor neurone death is minimal, none were found. These NOS positive profiles seen in younger wobbler mouse ventral horn had a morphology and size similar to that of degenerating motor neurones seen in Nissl stained preparations. It was concluded that these NOS positive profiles were degenerating motor neurones. These observations provide further evidence that induction of nitric oxide synthase expression may play a role in motor neurone death. Though no NOS positive motor neurones were found in the spinal cords of older wobblers increased numbers of NOS positive varicose axons were observed in the ventral horn often forming tangled accumulations on the border of the grey and white matter.

Expression of nitric oxide synthase by motor neurones in the spinal cord of the mutant mouse wobbler

The expression of nitric oxide synthase (NOS) has been studied in the spinal cord of the mutant mouse wobbler, a recessive mutation in which there is motor neurone degeneration, using nicotinamide dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Abnormal NOS positive large neuronal profiles could be found in the ventral horn of affected mutant animals but not their unaffected littermate controls. The number of abnormal profiles observed was dependant upon the age of the animal. A small number of these NOS positive large neuronal profiles were seen at the onset of the disease at 3–4 weeks of age, larger numbers were found in animals aged 5–8 weeks coincident with the main period of motor neurone death, whilst in the spinal cords of older animals aged 9–12 months, when motor neurone death is minimal, none were found. These NOS positive profiles seen in younger wobbler mouse ventral horn had a morphology and size similar to that of degenerating motor neurones seen in Nissl stained preparations. It was concluded that these NOS positive profiles were degenerating motor neurones. These observations provide further evidence that induction of nitric oxide synthase expression may play a role in motor neurone death. Though no NOS positive motor neurones were found in the spinal cords of older wobblers increased numbers of NOS positive varicose axons were observed in the ventral horn often forming tangled accumulations on the border of the grey and white matter.

Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine

The presence and topographical distribution of nitrergic neurons in the enteric nervous system (ENS) of the pig small intestine have been investigated by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide dinucleotide phosphate diaphorase (NADPHd) histochemistry. Both techniques yielded similar results, thus confirming that within the pig ENS the neuronal isoform of NOS corresponds to NADPHd. Intrinsic nitrergic neurons were not confined to the myenteric plexus; considerable numbers were also present in the outer submucous plexus. In the inner submucous plexus, NOS immunoreactivity or NADPHd staining was restricted to a few nerve fibres and nerve cell bodies. The nitrergic neurons displayed a wide variety in size and shape, but could all be characterized as being multidendritic uniaxonal. Nerve lesion experiments showed that the majority of the myenteric nitrergic neurons project in an anal direction. Evidence is at hand to show that a substantial proportion of these neurons contribute to the dense nitrergic innervation of the tertiary plexus and the circular smooth muscle layer. Some of the nitrergic neurons of the outer submucous plexus were equally found to send their axons towards the circular muscle layer. In some of the nitrergic enteric neurons, VIP, neuropeptide Y, galanin or protein 10 occurred colocalized, but not calbindin or serotonin. The present findings provide morphological evidence for the presence of NOS in a proportion of the enteric neurons in the small intestine of a large omnivorous mammal, i.e. the pig. The topographical features of the staining patterns of NOS and NADPHd are in accord with the results of neuropharmacological studies and argue for the existence of distinct nitrergic subpopulations acting either as interneurons or as motor neurons.

Laminar distribution and morphology of NADPH-diaphorase containing neurons in the superior colliculus and underlying periaqueductal gray of the rat

We have studied the laminar distribution of reduced nicotinamide dinucleotide phosphate diaphorase (NADPH-d) activity and the morphology of positive neurons in the superior colliculus (SC) and the underlying periaqueductal gray (PAG) of the rat. The morphology of NADPH-d-positive neurons has been compared to that of Golgi-impregnated cells. The highest activity occurs in the stratum zonale and stratum griseum superficiale, contrasting with the pale neuropil in the stratum opticum, where only a few positive neurons are found. In the stratum griseum intermedium positive neurons are grouped in patches separated by narrow, NADPH-d-negative bands. In the deeper layers, the neuropil is NADPH-d-negative, and few neurons show enzymatic activity. In contrast, numerous neurons in the dorsolateral part of the PAG are intensely positive. They are continuous with the positive neurons in the stratum album profundum, with no clear border between the two centers. In both SC and PAG, only small and medium sized neurons are NADPH-d-positive. In comparison with Golgi material, all types of small neurons in the superficial layers show NADPH-d activity; NADPH-d histochemistry, however, does not visualize the characteristic dendritic appendages of these neurons. The large neurons of the SC and PAG, probably representing the long-projecting neurons of these centers, do not contain the enzyme.

Reduced Nicotinamide Dinucleotide Phosphate Diaphorase from Bacillus subtilis

NADPH‐diaphorase was extensively purified from extracts of Bacillus subtilis N. Y.. FMN is most probably the prosthetic group of this enzyme. NADPH is oxidized by oxygen as the terminal acceptor only in the presence of catalytic amounts of free flavins (Km for FMN was 1.6 × 10−6 M at pH 8.0). NADPH was directly oxidized in the presence of various electron acceptors such as quinones, ferricyanide and tetrazolium salts. Cytochrome c was a very poor acceptor in this system. Km for NADPH was 2.0 × 10−4 M and Ki for NADP+ as a competitive inhibitor was 1.1 × 10−3 M at pH 7.2. Mn++ or Mg++ at mM concentration showed some stimulation of enzyme activity. The enzyme was partially inhibited by various chelating agents and p‐hydroxymercuribenzoate but not by several typical inhibitors of the respiratory chain. It is suggested that this flavoprotein can serve as a useful tool for NADP+ regeneration in coupled enzyme reactions.

Reduced nicotinamide dinucleotide phosphate diaphorase from Bacillus subtilis.

NADPH-diaphorase was extensively purified from extracts of Bacillus subtilis NY. FMN is most probably the prosthetic group of this enzyme. NADPH is oxidized by oxygen as the terminal acceptor only in the presence of catalytic amounts of free flavins (K m for FMN was 1.6 × 10−6 M at pH 8.0). NADPH was directly oxidized in the presence of various electron acceptors such as quinones, ferricyanide and tetrazolium salts. Cytochrome c was a very poor acceptor in this system. K m for NADPH was 2.0 × 10−4M and K i for NADP+ as a competitive inhibitor was 1.1 × 10−3 M at pH 7.2. Mn++ or Mg++ at mM concentration showed some stimulation of enzyme activity. The enzyme was partially inhibited by various chelating agents and p-hydroxymercuribenzoate but not by several typical inhibitors of the respiratory chain. It is suggested that this flavoprotein can serve as a useful tool for NADP+ regeneration in coupled enzyme reactions.