[3H]homoquinolinate binds to a subpopulation of NMDA receptors and to a novel binding site.

NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [3H]homoquinolinate, a potential subtype-selective ligand. Using quantitative receptor autoradiography, NMDA-specific [3H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I-III of cerebral cortex, striatum, hippocampus, and septum). NMDA-specific [3H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA-specific [3H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA-displaceable binding, there was a subpopulation of [3H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or L-glutamate. In contrast, we found that the derivative of homoquinolinate, 2-carboxy-3-carboxymethylquinoline, markedly inhibited the NMDA-insensitive binding of [3H]homoquinolinate without inhibiting the NMDA-sensitive population. [3H]Homoquinolinate may be useful for selectively characterizing NR2B-containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.

A novel kainate receptor ligand [3H]-(2S,4R)-4-methylglutamate: pharmacological characterization in rabbit brain membranes.

Since kainate evokes large non-desensitizing currents at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate is of limited use in discriminating between AMPA and kainate receptors. Following recent reports that (2S,4R)-4-methylglutamate is a kainate receptor-selective agonist, we have radiolabelled and subsequently characterized the binding of [3H]-(2S,4R)-4-methylglutamate to rabbit whole-brain membranes. [3H]-(2S,4R)-4-methylglutamate binding was rapid, reversible and labelled two sites (KD1 = 3.67+/-0.50 nM/Bmax1 = 0.54+/-0.03 pmol/mg protein and KD2 = 281.66+/-12.33 nM/ Bmax2 = 1.77+/-0.09 pmol/mg protein). [3H]-(2S,4R)-4-methylglutamate binding was displaced by several non-NMDA receptor ligands: domoate > kainate >> L-quisqualate > or = L-glutamate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) >> (S)-AMPA = (S)-5-fluorowillardiine > NMDA. Neither the metabotropic glutamate receptor agonists (1S,3R)-ACPD or L-AP4, together with the L-glutamate uptake inhibitor L-trans-2,4-PDC, influenced binding when tested at 100 microM. We conclude that [3H]-(2S,4R)-4-methylglutamate is a useful radioligand for labelling kainate receptors. It possesses high selectivity, and possesses a pharmacology similar to that for rat cloned low-affinity (Glu5 and 6) kainate receptor subunits.

The effects of (RS)-alpha-cyclopropyl-4-phosphonophenylglycine ((RS)-CPPG), a potent and selective metabotropic glutamate receptor antagonist.

1. In this study we describe the potent antagonist activity of a novel metabotropic glutamate (mGlu) receptor antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine ((RS)-CPPG) which exhibits selectivity for mGlu receptors (group II and III) negatively coupled to adenylyl cyclase in the adult rat cortex. 2. Both the L-2-amino-4-phosphonobutyrate (L-AP4) and (2S, 1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1) inhibition of forskolin-stimulated cyclic AMP accumulation were potently reversed by (RS)-CPPG (IC50 values: 2.2 +/- 0.6 nM and 46.2 +/- 18.2 nM, respectively). 3. In contrast, (RS)-CPPG acted as a weak antagonist against group I mGlu receptors. In neonatal rat cortical slices, (RS)-CPPG antagonized (KB = 0.65 +/- 0.07 mM) (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD)-stimulated phosphoinositide hydrolysis. (RS)-CPPG (100 microM) failed to influence L-quisqualate-stimulated phosphoinositide hydrolysis in cultured cerebellar granule cells. 4. In the rat cerebral cortex, (RS)-CPPG is the most potent antagonist of group II/III mGlu receptors yet described (with 20 fold selectivity for group III mGlu receptors), having negligible activity at group I mGlu receptors.

Acemannan, a beta-(1,4)-acetylated mannan, induces nitric oxide production in macrophage cell line RAW 264.7.

Acemannan is a polydispersed beta-(1,4)-linked acetylated mannan with antiviral properties. It is an immunomodulator, and studies in our laboratory have shown that it causes activation of macrophages. Inducible NO synthase is generally expressed after transcriptional induction and is known to mediate some of the cytotoxic action of activated macrophages. Acemannan, in the presence of interferon-gamma, greatly increased the synthesis of NO in RAW 264.7 cells. This increase was preceded by increased expression of mRNA for the inducible form of macrophage NO synthase. Preincubation with pyrrolidine dithiocarbamate inhibited the induction, indicating the involvement of nuclear factor-kappa B. These results suggest that acemannan causes the activation of macrophages by increasing the level of NO synthase at the level of transcription.

Novel potent selective phenylglycine antagonists of metabotropic glutamate receptors.

The metabotropic glutamate (mGlu) receptor antagonist properties of novel phenylglycine analogues were investigated in adult rat cortical slices (mGlu receptors negatively coupled to adenylyl cyclase), neonatal rat cortical slices and in cultured rat cerebellar granule cells (mGlu receptors coupled to phosphoinositide hydrolysis). (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG), (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG), (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-methyl-3-carboxymethyl-4-hydroxyphenylglycine (M3CM4HPG) and (RS)-alpha-methyl-4-hydroxy-3-phosphonomethylphenylglycine (M4H3PMPG) were demonstrated to have potent and selective effects against 10 microM L-2-amino-4-phosphonobutyrate (L-AP4)- and 0.3 microM (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-1)-mediated inhibition of forskolin-stimulated cAMP accumulation in the adult rat cortex. In contrast, these compounds demonstrated either weak or no antagonism at mGlu receptors coupled to phosphoinositide hydrolysis in either neonatal rat cortex or in cultured cerebellar granule cells. These compounds thus appear to be useful discriminatory pharmacological tools for mGlu receptors and form the basis for the further development of novel antagonists.

Agonists of cyclic AMP-coupled metabotropic glutamate receptors in adult rat cortical slices.

A number of potential Group 2 and Group 3 metabotropic glutamate receptor (mGlu receptor) agonists were investigated in adult rat brain cerebrocortical slices. The rank order of their potency in inhibiting forskolin-stimulated adenylyl cyclase was found to be: (S)-2-amino-2-methyl-4-phosphonobutyric acid (MAP4) > (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I) > (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3S-ACPD) > (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R)-ACPD) > (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) > (S) -2-methylglutamate ((S)-MG) > L-glutamate > (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)alanine (MCCG) > L-2-amino-4-phosphonobutyric acid (L-AP4) > L-serine-O-phosphate (SOP). The finding that (S)-2-amino-2-methyl-4-phosphonobutyric acid was the most potent agonist at these metabotropic glutamate receptors is in contrast to its observed potent mGlu receptor antagonist action in the neonatal rat spinal cord.

Structure-activity relationships for a series of phenylglycine derivatives acting at metabotropic glutamate receptors (mGluRs).

1. The actions of a series of twelve phenylglycine derivatives at metabotropic glutamate receptors (mGluRs) linked to both phosphoinositide hydrolysis (PI) and cyclic AMP were investigated. 2. PI hydrolysis was determined by the accumulation of [3H]-inositol-monophosphate ([3H]-IP1) in neonatal ral cortical slices prelabelled with [3H]-myo-inositol. The non-selective mGluR agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid ((1S,3R)-ACPD) produced a concentration-dependent increase in [3H]-IP1 (EC50 approximately 20 microM). This agonist was subsequently used to investigate potential antagonist activity of the phenylglycine derivatives. Of the compounds tested (+)-alpha-methyl-4-carboxyphenylglycine (M4CPG) and (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) were the most active with KP values of 0.184 +/- 0.04 mM and 0.367 +/- 0.2 mM respectively. 3. Activity at adenylyl cylase-coupled mGluRs was investigated by determining the accumulation of [3H]-cyclic AMP in adult rat cortical slices. [3H]-cyclic AMP accumulation, elicited by 30 microM forskolin, was inhibited by (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-1) and L-2-amino-4-phosphonobutanoate (L-AP4) with respective EC50 values of 0.3 microM and 10 microM. Neither agonist was able to inhibit completely forskolin stimulated cyclic AMP accumulation; this is evidence that not all adenylyl cyclase is susceptible to modulation by mGluRs. Phenylglycine derivatives were examined for their ability to antagonize the inhibition of [3H]-cyclic AMP accumulation by L-CCG-1 or L-AP4 at their EC50 concentrations. 4. A rank order of potency of the phenylglycine derivatives as antagonists of L-AP4 and L-CCG-1 was obtained. The most effective compound. (RS)-alpha-methyl-3-carboxymethylphenylglycine (M3CMPG) had IC50 values in the order of 1 microM against L-AP4 and 0.4 microM against L-CCG-1. 5. The results from this study indicate that phenylglycine-derived compounds can discriminate between groups of metabotropic glutamate receptors and may also display some selective activity between subtypes within groups. Future work based on these findings may lead to the development of more selective and potent compounds as important pharmacological tools.

A comparative study of avian reovirus pathogenicity: virus spread and replication and induction of lesions.

This study examined the relationship of avian reovirus spread and replication to induction of lesions and the relevant role of the S1 segment encoding a virus-neutralizing antigen. One-day-old broiler chickens were infected via footpad or orally with two virus strains (883 and 176) that differ greatly in virulence and a reassortant (R44) that has the S1 segment from 176 and the remaining genome segments from 883. Virus replication and histological lesions in various tissues (heart, liver, spleen, kidney, bursa, hock joint, and bone marrow) were measured at 2-day intervals until day 8 postinoculation. The virulent strain 176 spread to and replicated efficiently in all tissues examined and caused extensive and severe lesions, whereas the mild strain 883 was detected only in tissues near inoculation sites and caused only minimal lesions. The appearance of lesions correlated with the presence of viral replication in each tissue tested. Together, these results indicate that induction of lesions, or pathogenicity, is directly related to virus spread and replication. Reassortant R44 behaved like strain 176 in chicken embryo fibroblasts (CEFs), i.e., both replicated much faster and produced larger plaques than strain 883. In broiler chickens, however, R44 behaved like strain 883, replicating and inducing lesions to an extent that was fat lower than that of strain 176. These results suggest that the S1 segment alone is capable of determining viral replication and plaque formation in cultured CEFs but is not sufficient to determine the virus spread and replication and the pathological change in broiler chickens.

Subgenomic S1 segments are packaged by avian reovirus defective interfering particles having an S1 segment deletion.

Two subgenomic segments derived from the S1 genome segment (SGS1-1 and SGS1-2) were identified in avian reovirus defective interfering (DI) particle preparations having an S1 segment deletion. Both SGS1-1 and SGS1-2 were composed of double-stranded RNA (dsRNA) with an estimated size of 400 and 380 bp, respectively. Their segment of origin was identified as the S1 by hybridization analysis. The subgenomic segments were associated with the virus fraction following CsCl density gradient centrifugation, indicating that they are packaged. The subgenomic segments were also shown to be replicated. Therefore, sequence(s) required for replication and packaging are retained. The relative amounts of subgenomic segments were shown to be inversely proportional to that of the S1 segment. The presence of subgenomic segments and concurrent reduction in the relative amount of the S1 segment were found to be directly associated with the decrease in infectious titers. These results suggest that subgenomic segments are responsible for induction of interference by specifically competing with the S1 segment during replication and/or packaging. The competitive relationship between the subgenomic segments and the S1 segment implies that segment-specific sequence(s) or factor(s) are involved in the replication and/or packaging of each individual genome segment.

Identification of proteins encoded by avian reoviruses and evidence for post-translational modification.

Twelve avian reovirus strain 176 proteins, 10 structural (lambda 1, lambda 2, lambda 3, mu 1, mu 2/mu 2C, sigma 1, sigma 2, sigma 3, and sigma 4), and 2 nonstructural (mu NS and sigma NS) were identified and characterized by electrophoretic analysis of purified virions and infected cell lysates. Three of the identified proteins (mu 1, mu NS, and sigma 4) have not been previously described. In pulse-chase experiments, 10 of the proteins (lambda 1, lambda 2, lambda 3, mu 1, mu 2, mu NS, sigma 1, sigma 2, sigma NS, sigma 3) were shown to be primary translation products, whereas mu 2C was shown to be a post-translational cleavage product of mu 2.

Strain-specific selection of genome segments in avian reovirus coinfections.

To determine whether selection of genome segments in coinfections is strain-specific, chicken embryo fibroblasts were coinfected with avian reovirus strain 883 and one of three other avian reovirus strains (176, S1133 and 81-5). Viral progeny from each coinfection (883 x 176, 883 x S1133 or 883 x 81-5) was serially passaged at a low m.o.i. The electropherotypes of the coinfection progeny and those of the plaque-derived clones obtained from passages 1 and 20 were analysed. Two 883 segments (M2 and S2) were found to be selected in the 883 x 176 coinfection, three 883 segments (M2, M3 and S2) in the 883 x S1133 coinfection, and only one 883 segment (M3) in the 883 x 81-5 coinfection, i.e. different 883 genome segments were selected in the three coinfections. It was, therefore, concluded that selection of genome segments in a coinfection of a given cell line is virus strain-specific. The selection of genome segments in coinfections was shown to be due to enhanced infectivity of the reassortants that were formed in the coinfections. In addition, defective interfering particles that lack the S1 segment were identified in the 883 x 81-5 coinfection progeny following serial passage. Selection of genome segment(s) in coinfections as described herein may have potential importance on the effect and production of divalent or multivalent vaccines.

Antigen dependent adjuvant activity of a polydispersed beta-(1,4)-linked acetylated mannan (acemannan).

The adjuvant properties of a polydispersed beta-(1,4)-linked acetylated mannan, acemannan (ACE-M), were evaluated. Day-old broiler chicks were randomly selected and allocated to four flocks (Vac 1-4). The Vac 1 flock was sham vaccinated with saline. The Vac 2 flock was vaccinated with an oil-based vaccine (Breedervac III; Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious bronchitis virus). The Vac 3 flock was vaccinated with a vaccine-ACE-M mixture, and the Vac 4 flock was vaccinated with vaccine and ACE-M at separate anatomical sites. ELISA titres to NDV and IBDV were determined. The immune response to NDV at 21 days postvaccination (PV) was significantly enhanced (P less than or equal to 0.05) by the addition of ACE-M to the vaccine, compared with vaccination without ACE-M. Subsequently, the vaccine-ACE-M mixture appeared to suppress the immune response to NDV. However, at day 35 PV, 95% of the Vac 3 chicks compared with 90% of the Vac 2 and 89% of the Vac 4 chicks exhibited protective titres. The response to IBDV differed from that to NDV. At day 21 PV the immune response to IBDV was essentially the same for all flocks that received vaccine, i.e. addition of ACE-M to the vaccine did not significantly enhance the immune response; however, it did significantly (P less than or equal to 0.05) sustain the immune response at days 28 and 35. In addition to the observed effect on titres to NDV and IBDV, ACE-M also had an effect on flock immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro evaluation of the synergistic antiviral effects of acemannan in combination with azidothymidine and acyclovir.

The antiviral effects of selected combinations between acemannan (ACE-M), a long-chained, polydispersed, beta-(1,4)-acetylated mannan, were tested in combination with azidothymidine (AZT) and acyclovir (ACY) in vitro. The rationale for such combinations was based on the antiviral and immunomodulatory properties exhibited by ACE-M. In addition, the observed antiviral effects of ACE-M against human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses appear to be related to modification of the glycosylation of viral glycoproteins. Therefore, the inhibitory effect of ACE-M does not overlap with that of AZT or ACY. The studies presented herein show that ACE-M combined with suboptimal noncytotoxic concentrations of AZT or ACY act synergistically to inhibit the replication of HIV-1 and herpes simplex virus type 1 (HSV-1), respectively. The median effect method was not applicable for analysis because the test compounds show mutually nonexclusive drug effects. For a meaningful evaluation and interpretation of the effects of drug combinations, the biological significance of combinations must be considered, that is, the protective effect of the combination, the noncytotoxicity of the combination, the mechanism(s) of action of the individual compounds comprising the combination, and so forth. With respect to effects on U1 cells latently infected with HIV-1, treatment with combinations of AZT and ACE-M does not potentiate virus replication.

Inhibition of AIDS virus replication by acemannan in vitro.

Acemannan (ACE-M), a beta-(1,4)-linked acetylated mannan, was evaluated for in vitro activity against human immunodeficiency virus type 1 (HIV-1). Castanospermine (CAS), deoxymannojirimycin (DMN), swainsonine (SWS), azidothymidine (AZT), and dideoxythymidine (DDC) were tested in parallel as control compounds. In vitro antiviral efficacy of ACE-M was evaluated in a variety of cell lines including human peripheral mononuclear, CEM-SS1 and MT-2(2) cells. The virus strain, number of infectious units per cell, and target cell line were important factors in determining the degree of inhibition of viral cytopathic effect in the presence of ACE-M and other control compounds tested. Maximum inhibitory effect was observed in CEM-SS cells infected with the RFII strain of HIV-1. This inhibitory effect was determined to be concentration-dependent. Assay design included primary screening to measure cell viabilities of infected target cells in the presence and absence of test compounds. When tested on HIV-1/RFII-infected CEM-SS cells, the 50% inhibitory effect of CAS (IC50 = 28), an inhibitor of alpha-glucosidase I, was determined to be similar to that observed for ACE-M (IC50 = 45). However, DMN and SWS, inhibitors of mannosidase I and II, tested in parallel to CAS and ACE-M, exhibited no IC50 values. Antiviral potential of ACE-M as an inhibitor of syncytia formation was also explored using CEM-SS cells. Suppression of syncytia formation was observed at an ACE-M concentration of 31.25 micrograms/ml, and complete inhibition was observed at 62.5 micrograms/ml. In addition, HIV-1 RNA levels were studied to establish the antiviral potential of ACE-M in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of genome segments following coinfection of chicken fibroblasts with avian reoviruses.

Two avian reoviruses (883 and 176) shown to have distinct growth kinetics were used to coinfect chicken embryonic fibroblasts asynchronously to generate reassortants. More than 300 plaque-derived clones were obtained from passage 3 of two separate coinfections made at different m.o.i. and time intervals between infection and superinfection. The genome electropherotype of each plaque-derived clone was determined, and a diverse group of reassortants were detected. Genome segments 883 M2 and 176 S1 were shown to be preferentially selected. The preferential selection of the 176 S1 segment was shown to be a virus growth-determined nonrandom event conferred by the function of 176 S1 segment, whereas the data suggest that a factor(s) other than viral growth properties was involved in the preferential selection of 883 M2 segment.

Characterization of avian reovirus strain-specific polymorphisms.

Avian reoviruses have been associated with several pathologic conditions, but correlative relationships between genotypes and specific diseases have not been demonstrated. Six avian reoviruses (883, 176, 81-5, S1133, FC, and TX) were selected for this study, and a comparative study of the pathogenic properties of the viruses in chickens, following peroral and footpad inoculation, was carried out, along with a comparison of the electrophoretic mobility of viral genomic segments and viral proteins encoded by the gene segments. The pathogenic properties of the viruses were shown to be diverse, with three distinct pathotypes being defined: Pathotype I (883) caused only a syndrome that we have termed "transient digestive system disorder" (TDSD); Pathotype II (FC, TX, and S1133) caused only "viral arthritis syndrome" (VAS), whereas Pathotype III (176 and 81-5) caused both TDSD and VAS. Likewise, the genomes of the viruses were shown to be extremely polymorphic, with a maximum of five segments co-migrating between any two strains. Considerable variation in the electrophoretic mobility of the encoded proteins also was demonstrated with pronounced variation in the molecular size of the sigma 4 protein, the purported viral attachment protein, being evident. These results show that the genomes of avian reoviruses were extremely polymorphic, preventing correlation between genotypes and pathotypes. But these studies have provided us with the genetic elements needed to characterize the gene functions involved in viral pathogenesis.

Genotypic transitions among bluetongue viral isolates from domestic ruminants in Colorado during 1981-1984.

Two predominant electropherotypes of bluetongue virus (BTV) serotype 11 isolates from cattle during a 1981-1984 field study in eastern Colorado were characterized. The genomes of strains isolated from the first 2 years of the study had 1 predominant electropherotype (CO81), with the exception of 1 isolate that differed only in the migration of segment 3. A second electropherotype (CO83), with differences in the migration of 4 segments, coexisted in the same region during 1983 and 1984 with strains having the CO81 RNA profile. The genomes of CO81 and CO83 were also distinguishable from those of the US prototype of BTV 11. Analysis of the polypeptides of representative strains of each electropherotype by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the proteins were very similar. The occurrence of the CO81 electropherotype was apparently the result of multiple viral infections since the positions of 7 segments had faint second bands and single-banded variants were isolated after serial plaque purifications. In addition, protein 7 of 1 of the CO81 isolates and protein 7 of the single-banded variant differed as shown by reverse phase-high performance liquid chromatography of 35S-methionine-labeled tryptic peptides.

The biological activities of mannans and related complex carbohydrates.

Complex polymers containing mannose (mannans) possess significant biological activity when administered to mammals. When given orally, they inhibit cholesterol absorption and induce hypocholesterolemia. If administered by other routes, they bind to mannose-binding proteins and induce macrophage activation and interleukin-1 release, inhibit viral replication, stimulate bone marrow activity, promote wound healing and inhibit tumor growth. This range of activities makes the mannans, potentially important biological-response modifiers and therapeutic agents.

Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase.

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.

Demonstration and biochemical characterisation of rat brain NADPH-dependent diaphorase.

An enzyme responsible for the NADPH-dependent reduction of nitroblue tetrazolium HCl (NBT) has been isolated from rat brain. Although other tetrazolium salts could be utilised, NBT was the preferred substrate, and the enzyme had an absolute requirement for NADPH. An in vitro assay was developed and used to determine the kinetic constants: Km NBT = 17.3 microM; Km NADPH = 1.9 microM, Vmax = 30.8 mumol product produced/min/mg protein. Substrate inhibition by NADPH was observed in some instances. Brain subcellular fractionation indicated highest enzyme activities in the microsomal fraction. Activity was present in all brain regions and in a variety of peripheral tissues. Relative molecular mass determinations of the native enzyme yielded an Mr = 170-180,000. It seems likely that the enzyme activity described in this study relates directly to the histochemical demonstration of brain NADPH-diaphorase-positive neurons. As yet, the natural substrate for the enzyme is unknown. However, the isolation and purification of NADPH-dependent diaphorase may be anticipated to assist in the elucidation of its function in the brain, and in the special characteristics of those neurons that contain the enzyme in abundance.