Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

A histochemical study of the circumtesticular Leydig cells of a teiid lizard, Cnemidophorus tigris

Several oxidative enzymes in the testis of the teiid lizard Cnemidophorus tigris were studied histochemically. The cells of the circumtesticular sheath (Leydig cell tunic) are functionally equivalent to Leydig cells of the interstitium on the basis of similar histochemical reactions for the five enzyme systems studied. Both groups of cells were positive for 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, NADH diaphorase, NADPH diaphorase, and glucose-6-phosphate dehydrogenase. These results support the hypothesis that the circumtesticular sheath has endocrine function as indicated by its vascularity and its ability to catalyze histochemical reactions involving steroid biosynthesis.     

Nitric oxide synthase in invertebrates

Summary The gas nitric oxide is now recognized as an important signalling molecule that is synthesized froml-arginine by the enzyme nitric oxide synthase. This enzyme can be localized by different methods, including immunocytochemistry and the histochemical reaction for NADPH diaphorase. It has been demonstrated in various vertebrate cells and tissues, and recently several studies dealing with the production of nitric oxide in invertebrates have been published. Diploblastic animals, flatworms and nematodes seem to lack NADPH diaphorase activity but it has been found in the rest of the phyla studied. The most frequently reported sites for the production of nitric oxide are the central and peripheral nervous systems and, in primitive molluscs, the muscle cells. In insects, it has also been described in the Malpighian tubules. The roles of nitric oxide in invertebrates are closely related to the physiological actions described in vertebrates, namely, neurotransmission, defence, and salt and water balance. The recent cloning of the first nitric oxide synthase from an invertebrate source could open interesting avenues for further studies.     

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues

 The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes – neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms. Accepted: 2 September 1996     

Comparison of dark- and light-adapted carp retinas with NADPH diaphorase staining

The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

NADPH diaphorase and nitric oxide synthase in the corpus cardiacum-corpus allatum of the cockroach Diploptera punctata

Juvenile hormone synthesis by corpora allata is regulated partly by allatostatin containing nerves from the brain that innervate the corpora cardiaca and the corpora allata. To investigate whether NO also participates in the regulation of juvenile hormone synthesis, antibody against NO synthase and the histochemical test for NADPH diaphorase activity, a marker for NO synthase, were applied to the corpora cardiaca-corpora allata of Diploptera punctata. Strong NADPH diaphorase activity occurred in corpus allatum cells but not in nerve fibers in the corpora allata or corpora cardiaca. In contrast, NO immunoreactivity occurred in nerves in the corpora cardiaca but not within the corpora allata. NO and allatostatin were not colocalized. NO synthase and NADPH diaphorase activity were localized in similar areas of the subesophageal ganglion and cells in the pars intercerebralis of the brain. Positive correlation of the quantity of NADPH diaphorase activity with juvenile hormone synthesis during the gonadotrophic cycle and lack of such correlation in subesophageal ganglia suggest that NADPH diaphorase activity reflects the necessity of NADPH in the pathway of juvenile hormone synthesis. These data suggest that NO is unlikely to play a significant role in the regulation of the corpora allata.     

Dehydrogenation of reduced pyridine nucleotides by Leydig cell tumors of the rat testis.

The activities of the cytochrome c reductases and of the D-T diaphorase in rat Leydig cell tumors have been described. The increase in enzymatic activity of the NADH cytochrome c reductase activity in functional tumors derived from interstitial cells of the rat testis is interpreted as being possibly related to hydroxylation of steroids by the neoplastic cells. Meanwhile, the increase in the activity of the D-T diaphorase in the other tumor is interpreted as being an anaplerotic reaction to substitute for the deficient shuttles for the transfer of reducing equivalents from the cytoplasm to the mitochondria observed in tumors.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Essential requirement of cytosolic phospholipase A(2) for stimulation of NADPH oxidase-associated diaphorase activity in granulocyte-like cells.

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine.

Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with NG-nitro-L-arginine, a NOS inhibitor, or L-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

Trypanosoma cruzi: ultrastructural cytochemistry of mitochondrial enzymes

Mitochondrial enzymes were detected cytochemically in all developmental stages of Trypanosoma cruzi maintained in tissue cultures at the light and electron microscope levels. Cytochrome oxidase (CO) was detected using the diaminobenzidine method. Succinate dehydrogenase (SDH), isocitrate dehydrogenase (ICDH), NADPH diaphorase, α-glycerophosphate dehydrogenase (GPDH), and β-hydroxybutyrate dehydrogenase (HBDH) were detected using the dystyril nitroblue tetrazolium salt. A reaction product indicative of CO, SDH, ICDH, and NADPH diaphorase activities was found either in the inner mitochondrial membrane or in the cristae. β-HBDH and α-GPDH activities, however, were localized only in the inner membrane. No difference in the localization and intensity of the reaction was observed in the various stages of T. cruzi.     

Stimulation of NO synthase activity in the immune-competent lepidopteran Estigmene acraea hemocyte line.

In contrast to the vertebrate immune system, nearly nothing is known about the immunological role of nitric oxide (NO) in invertebrates. This study provides evidence of the presence of a NO synthase (NOS) activity in an immune-competent, macrophage-like insect hemocyte line, previously established from larvae of the lepidopteran insect Estigmene acraea. As proven by photometric determination of nitroblue tetrazolium reduction after cell fixation, the E. acraea cells possess NADPH diaphorase (NADPHd) activity. This NADPH diaphorase activity was NADPH dependent, not inhibitable by superoxide dismutase, influenced by extracellular addition of L-arginine, and inhibited in a dose-dependent manner by the specific NOS inhibitor Nomega-monomethyl-L-arginine. Furthermore, the NADPH diaphorase activity was stimulated within 30 min by the addition of insect pathogenic bacteria (Bacillus thuringiensis var. kurstaki, Photorhabdus luminescens), bacterial lipopolysaccharide, and silica beads. In activated E. acraea cell suspensions strongly increased amounts of L-citrulline and enhanced levels of total nitrite/nitrate (as NO derivates) can be determined. This is the first report on stimulable NOS activity in insect hemocytes.     

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.     

Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenolindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 microM for NADH and 0.2 microM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in the reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 degrees C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.     

Heterogenous staining ofd-amino acid oxidase in peroxisomes of rat liver and kidney

Summary The localization ofd-amino acid oxidase (d-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogencity is obseved in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium thed-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes ford-AAOX suggests that subpopulation of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.