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PubMed2000

Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia.

BACKGROUND: The role of protein phosphorylation in the Pasteur effect--the phenomenon whereby anaerobic conditions stimulate glycolysis--has not been addressed. The AMP-activated protein kinase (AMPK) is activated when the oxygen supply is restricted. AMPK acts as an energy-state sensor and inhibits key biosynthetic pathways, thus conserving ATP. Here, we studied whether AMPK is involved in the Pasteur effect in the heart by phosphorylating and activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. RESULTS: Heart PFK-2 was phosphorylated on Ser466 and activated by AMPK in vitro. In perfused rat hearts, anaerobic conditions or inhibitors of oxidative phosphorylation (oligomycin and antimycin) induced AMPK activation, which correlated with PFK-2 activation and with an increase in fructose 2,6-bisphosphate concentration. Moreover, in cultured cells transfected with heart PFK-2, oligomycin treatment resulted in a parallel activation of endogenous AMPK and PFK-2. In these cells, the activation of PFK-2 was due to the phosphorylation of Ser466. A dominant-negative construct of AMPK abolished the activation of endogenous and cotransfected AMPK, and prevented both the activation and phosphorylation of transfected PFK-2 by oligomycin. CONCLUSIONS: AMPK phosphorylates and activates heart PFK-2 in vitro and in intact cells. AMPK-mediated PFK-2 activation is likely to be involved in the stimulation of heart glycolysis during ischaemia.

AMP-Activated Protein Kinases↗
PubMed2001

The Brix domain protein family -- a key to the ribosomal biogenesis pathway?

Six (one archaean and five eukaryotic) protein families have similar domain architecture that includes a central globular Brix domain, and optional N- and obligatory C-terminal segments, both with charged low-complexity regions. Biological data for some proteins in this superfamily suggest a role in ribosome biogenesis and rRNA binding.

Amino Acid Sequence↗
PubMed2003

Activation of epidermal growth factor receptor signals induction of nitric oxide synthase-2 in human optic nerve head astrocytes in glaucomatous optic neuropathy.

Glaucoma is an optic neuropathy that is associated with elevated intraocular pressure in most patients. We have previously demonstrated that the mechanism by which pressure damages optic nerve axons involves excessive nitric oxide generated by inducible nitric oxide synthase (NOS-2). We have now found that activation of the epidermal growth factor receptor (EGFR) induces NOS-2 in astrocytes of the human optic nerve head (ONH) in vitro and EGFR is significantly upregulated and tyrosine phosphorylated in reactive astrocytes in human glaucomatous ONHs in vivo. Furthermore, in response to elevated hydrostatic pressure, EGFR rapidly becomes phosphorylated in the nucleus. This pressure-dependent activation of EGFR is necessary for NOS-2 induction. Our results suggest that activation and nuclear localization of EGFR may be needed for induction of NOS-2 in response to elevated intraocular pressure in glaucomatous optic neuropathy. Identification of this key signaling pathway provides new therapeutic approaches to pharmacological neuroprotection for glaucoma.

Adult↗
PubMed2000

S-Adenosylmethionine.

S-Adenosyl-Lmethionine (SAM) is an important molecule in normal cell function and survival. SAM is utilized by three key metabolic pathways: transmethylation; transsulfuration; and polyamine synthesis. In transmethylation reactions, the methyl group of SAM is donated to a large variety of acceptor substrates including DNA, phospholipids and proteins. Thus, interference of these reactions can affect a wide spectrum of processes ranging from gene expression to membrane fluidity. In transsulfuration, the sulfur atom of the SAM is converted via a series of enzymatic steps to cysteine, a precursor of taurine and glutathione, a major cellular anti-oxidant. Polyamines are required for normal cell growth. Given the importance of SAM in tissue function, it is not surprising that this molecule is being investigated as a possible therapeutic agent for the treatment of various clinical disorders.

Animals↗
PubMed2000

Sterols and gene expression: control of affluence.

Intracellular and extracellular cholesterol levels are tightly maintained within a narrow concentration range by an intricate transcriptional control mechanism. Excess cholesterol can be converted into oxysterols, signaling molecules, which modulate the activity of a number of transcription factors, as to limit accumulation of excess of cholesterol. Two key regulatory pathways are affected by oxysterols. The first pathway involves the uptake and de novo synthesis of cholesterol and is controlled by the family of sterol response element binding proteins, whose activity is regulated by a sterol-dependent feedback mechanism. The second pathway, which only recently has become a topic of interest, involves the activation by a feedforward mechanism of cholesterol utilization for either bile acid or steroid hormone synthesis by oxysterol-activated nuclear receptors, such as liver X receptor and steroidogenic factor-1. Furthermore, biosynthesis and enterohepatic reabsorption of bile acids are regulated by the farnesol X receptor, a receptor activated by bile acids. Both the feedback inhibition of cholesterol uptake and production and the stimulation of cholesterol utilization will ultimately result in a lowering of the intracellular cholesterol concentration and allow for a fine-tuned regulation of the cholesterol concentration.

Animals↗
PubMed2002

Pancreatic beta-cell growth and survival--a role in obesity-linked type 2 diabetes?

Obesity-linked type 2 diabetes is a disease of insulin resistance combined with pancreatic beta-cell dysfunction. Although a role for beta-cell mass in the pathogenesis of obesity-linked type 2 diabetes has recently gained prominence, the idea is still being developed. It is proposed that in early obesity an increase in beta-cell mass and function might compensate for peripheral insulin resistance. However, as time and/or the severity of the obesity continue, there is decay in such adaptation and the beta-cell mass becomes inadequate. This, together with beta-cell dysfunction, leads to the onset of type 2 diabetes. It is becoming evident that elements in insulin and insulin growth factor (IGF)-1 signal-transduction pathways are key to regulating beta-cell growth. Current evidence indicates that interference of insulin signaling in obesity contributes to peripheral insulin resistance. This article examines whether a similar interference of IGF-1 signaling in the beta-cell could hinder upregulation of beta-cell mass and/or function, resulting in a failure to compensate for insulin resistance.

Animals↗
PubMed2002

Role of Ca2+ signaling in the regulation of endothelial permeability.

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.

Animals↗
PubMed2004

Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.

Amino Acid Sequence↗
PubMed2003

Molecular basis of hepatic fibrosis.

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent can lead to cirrhosis and liver failure. Activation of hepatic stellate cells (HSCs), leading to accumulation of extracellular matrix, is the central event of fibrogenesis. Exciting progress has been made in understanding the molecular basis of this process. Major advances include: (1) elucidation of the effects (and signalling pathways) of key cytokines on HSCs; (2) understanding the transcriptional regulation of HSC activation; (3) characterisation of matrix proteases and their inhibitors; (4) demonstration of apoptosis as an important event in the resolution of hepatic fibrosis, and identification of its mediators;(5) elucidation of the complex and dynamic interaction between HSCs and matrix; and (6) understanding the role of other cellular elements in hepatic fibrosis and their interaction with HSCs. Ongoing research with gene analysis using cDNA or oligonucleotide microarrays, or transcriptional profiling, will further increase our knowledge of the regulation of the process. Ultimately,advances in the understanding of the molecular biology of hepatic fibrosis are critical to the development of effective, targeted antifibrotic therapy that might benefit millions of patients with chronic liver disease worldwide.

Apoptosis↗
PubMed2002

Micropatterned substrates: approach to probing intercellular communication pathways.

Intercellular signaling is critical for the normal development and physiology of the central nervous system (CNS). To study such signaling, it is vital to control where and when the cells make contact with one another. It is also important to determine whether the process used for cell localization has an impact on signaling. This paper describes a technique that controls the location for cell growth in vitro and demonstrates that the technique has minimal (if any) impact on intercellular signaling. By using photolithographic methods, poly(dimethylsiloxane) molds were fabricated to function as templates for micrometer-level patterning of a nonadhesive agar (agarose) onto glass coverslips coated with a cell adhesive film (poly(L-lysine)). This process yields a surface composed of well-defined adhesive and nonadhesive microdomains. When endothelia or astrocytes are plated onto these substrates, confluent domains of endothelia or astrocytes grow on the poly(L-lysine) domains. Cocultures of astrocytes and neurons can also successfully be used to form interwoven networks on the adhesive domains. Moreover, studies of calcium signaling revealed that astrocytes grown on such patterns retain their native physiological activity. This conclusion is based on the observed propagation rate for calcium waves within individual astrocyte domains and across neighboring, but spatially disconnected, astrocyte domains. The potential to apply these micropatterned substrates as platforms for interrogating communication pathways in key components of the CNS is discussed.

Astrocytes↗
PubMed2006

Structural basis for stereoselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases.

Epoxide metabolism in Xanthobacter autotrophicus Py2 results in the conversion of epoxypropane to acetoacetate. Epoxide metabolism is initiated by the nucleophilic addition of coenzyme M to the (R)- and (S)-enantiomers of epoxypropane which forms the respective enantiomers of 2-hydroxypropyl-coenyme M. The (R)- and (S)-enantiomers of 2-hydroxypropyl coenzyme are oxidized to the achiral product 2-ketopropyl-CoM by two stereoselective dehydrogenases. The dehydrogenases catalyzing these reactions, termed (R)-hydroxypropyl-coenzyme M dehydrogenase (R-HPCDH) and (S)-hydroxypropyl-coenzyme M dehydrogenase (S-HPCDH), are NAD(+)-dependent enzymes belonging to the short chain dehydrogenase/reductase (SDR) family of enzymes. In this study, the crystal structure of R-HPCDH cocrystallized in the presence of (S)-hydroxypropyl-coenzyme M has been determined using X-ray diffraction methods and refined to 1.8 A resolution. The structure of R-HPCDH is tetrameric and stabilized by the interaction of the terminal carboxylates of each subunit with divalent metal ions. The structure of the presumed product-bound state reveals that binding interactions between the negatively charged oxygen atoms of the sulfonate moiety have striking similarities to sulfonate interactions observed in the previously determined structure of 2-ketopropyl-CoM oxidoreductase/carboxylase, highlighting the utility of coenzyme M as a carrier molecule in the pathway. The key elements of the aforementioned interactions are electrostatic interactions between the sulfonate oxygen atoms and two arginine residues (R152 and R196) of R-HPCDH. The comparison of the structure of R-HPCDH with a homology model of S-HPCDH provides a structural basis for a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for hydride abstraction.

Alcohol Oxidoreductases↗
PubMed2001

Polychlorinated naphthalenes in U.K. soils: time trends, markers of source, and equilibrium status.

Polychlorinated naphthalenes (PCNs) have been analyzed in archived soil from the U.K. to investigate historical trends. Samples were obtained from the Broadbalk experiment plots (1944-1986) and Luddington experiment station (1968-1990). Luddington samples also include a set of soils that received a one-time sludge treatment in 1968, and duplicate samples of this archived sludge were also analyzed. Peak residues of sumPCN (sum of all PCN congeners quantified) were approximately 9000 pg g(-1) dry weight in 1956, declining to approximately 300 pg g(-1) in contemporary soils. The one-time sludge application resulted in elevated soil residues that were 1.5-6 times higher than the control plot over the time series. This increase is consistent with the known application rate and the amount of sumPCN in the applied sludge (approximately 250,000 pg g(-1) dry wt). Half-lives forthe Luddington control soil and the sludge-amended soil for the period 1972-1990 were 5.3 and 9.9 years, respectively. Investigation of time trends revealed differences between homologue groups with the higher molecular weight congeners peaking earlier in the time series (pre-1950) and the lower molecular weight congeners peaking later, ca. 1970. Time trends of individual congeners were investigated in terms of their relative mass percent contribution to the sum of their homologue group. Significant (p < 0.05) increasing trends were observed for several congeners associated with combustion sources (CN-29, -51, -52/60, -54, and -66/67) suggesting that combustion related sources are more important now than they were in the past. However, no decreasing trend was observed for congeners that were thought to be susceptible to degradation by photolysis suggesting that this may not be a key elimination pathway of PCNs in the environment. A simple calculation of the fugacity status of PCNs in air and soil showed that the tri-CNs are exhibiting net outgassing, while the penta-CNs are still being deposited to soil. Interestingly, the penta-CNs associated with combustion show the largest gradient for air-to-soil transfer, supporting the notion that combustion sources are important contributors to contemporary air burdens of these congeners.

Air Pollutants↗
PubMed2002

Direct evidence of atmospheric secondary organic aerosol formation in forest atmosphere through heteromolecular nucleation.

Atmospheric aerosols play a central role in climate and atmospheric chemistry. Organic matter frequently composes aerosol major fraction over continental areas. Reactions of natural volatile organic compounds, with atmospheric oxidants, are a key formation pathway of fine particles. The gas and particle atmospheric concentration of organic compounds directly emitted from conifer leaf epicuticular wax and of those formed through the photooxidation of alpha- and beta-pinene were simultaneously collected and measured in a conifer forest by using elaborated sampling and GC/ MS techniques. The saturation concentrations of acidic and carbonyl photooxidation products were estimated, by taking into consideration primary gas- and particle-phase organic species. Primary organic aerosol components represented an important fraction of the atmospheric gas-phase organic content Consequently, saturation concentrations of photooxidation products have been lowered facilitating new particle formation between molecules of photooxidation products and semi-volatile organic compounds. From the measured concentrations of the above-mentioned compounds, saturation concentrations (Csat,i) of alpha- and beta-pinene photooxidation products were calculated for nonideal conditions using a previously developed absorptive model. The results of these calculations indicated that primarily emitted organic species and ambient temperature play a crucial role in secondary organic aerosol formation.

Aerosols↗
PubMed2005

Molecular structure in soil humic substances: the new view.

A critical examination of published data obtained primarily from recent nuclear magnetic resonance spectroscopy, X-ray absorption near-edge structure spectroscopy, electrospray ionization-mass spectrometry, and pyrolysis studies reveals an evolving new view of the molecular structure of soil humic substances. According to the new view, humic substances are collections of diverse, relatively low molecular mass components forming dynamic associations stabilized by hydrophobic interactions and hydrogen bonds. These associations are capable of organizing into micellar structures in suitable aqueous environments. Humic components display contrasting molecular motional behavior and may be spatially segregated on a scale of nanometers. Within this new structural context, these components comprise any molecules intimately associated with a humic substance, such that they cannot be separated effectively by chemical or physical methods. Thus biomolecules strongly bound within humic fractions are by definition humic components, a conclusion that necessarily calls into question key biogeochemical pathways traditionally thought to be required for the formation of humic substances. Further research is needed to elucidate the intermolecular interactions that link humic components into supramolecular associations and to establish the pathways by which these associations emerge from the degradation of organic litter.

Humic Substances↗
PubMed2004

Antioxidant capacity manipulation in transgenic potato tuber by changes in phenolic compounds content.

The main goal of this study was to generate potato tubers with increased levels of flavonoids and thus modified antioxidant capacities. To accomplish this, the vector carrying multigene construct was prepared and several transgenic plants were generated, all overexpressing key biosynthesis pathway enzymes. The single-gene overexpression or simultaneous expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) resulted in a significant increase of measured phenolic acids and anthocyanins. The increase in phenolic compounds synthesis is accompanied by decreases in starch and glucose levels in transgenic plants. The flavonoids-enriched plants showed improved antioxidant capacity; however, there is a complex relationship between antioxidant capacity and flavonoids content, suggesting the great participation of other compounds in the antioxidant potential of the plants. These other compounds are not yet recognized.

Acyltransferases↗
PubMed2001

seco-Cyclothialidines: new concise synthesis, inhibitory activity toward bacterial and human DNA topoisomerases, and antibacterial properties.

seco-Cyclothialidines are a promising class of bacterial DNA gyrase B subunit inhibitors. A new seco-cyclothialidine derivative containing a dioxazine moiety, BAY 50-7952, was synthesized through a new concise pathway. One key step of the synthesis is the straightforward formation of the 2-aminothiazole derivative of S-tritylcysteine. In biological tests, BAY 50-7952 and other known seco-cyclothialidines exhibited high and selective activity toward bacterial DNA gyrase and toward Gram-positive bacteria. The dioxazine moiety and other similar groups were found to be important for the ability of the seco-cyclothialidines to penetrate bacterial membranes. The opposite enantiomer ((S)-form) of BAY 50-7952 was also synthesized, and neither significant target activity nor in vitro antibacterial activity were found, suggesting a highly selective fit of the (R)-form. Despite promising in vitro activity, only poor activity was found in the murine infection model.

Animals↗
PubMed2005

Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N'-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine.

N'-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. alpha-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine alpha-hydroxylation. Therefore, a structure-activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for alpha-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, alpha-hydroxylation can occur at the 2'- or 5'-carbon. P450 2As catalyzed 5'-hydroxylation of (R)- or (S)-NNN with Km values of 0.74-69 microM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2'-hydroxylation with Km values of 0.73-66 microM. (S)-NNN 2'-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5'-hydroxylation, respectively. The catalytic efficiencies (kcat/Km) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5'-hydroxylation with significantly lower Km and higher kcat/Km values than NPIP or NPYR alpha-hydroxylation (p <0.05). Furthermore, P450 2As were better catalysts of NPIP alpha-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower Km and higher kcat/Km values for NPIP than NPYR alpha-hydroxylation (p <0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR alpha-hydroxylation.

Animals↗
PubMed2005

Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-(3-pyridyl)-1-butanone by cytochrome P450 2A13 in human fetal nasal microsomes.

Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-hydroxylation. This reaction is a key bioactivation pathway in NNK-induced carcinogenesis. P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo. Therefore, we studied NNK alpha-hydroxylation in human fetal nasal mucosal microsomes, which have been shown to contain high levels of P450 2A protein, presumed to be a mixture of P450 2A6 and 2A13. The microsomes efficiently catalyzed NNK alpha-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited alpha-hydroxylation by these microsomes greater than 90%. K(m) and V(max) values for alpha-methylene hydroxylation were 6.5 +/- 1.1 muM and 3.0 +/- 0.1 pmol/min/mg; for alpha-methyl hydroxylation, they were 6.7 +/- 0.8 microM and 0.85 +/- 0.03 pmol/min/mg. The K(m) values agree closely with those for NNK metabolism by P450 2A13. Using a new technique, we separated P450 2A13 from P450 2A6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Quantitative immunoblot analysis indicated that the level of P450 2A13 in the pooled fetal nasal microsome sample used for kinetic analysis was approximately 1.6 pmol/mg protein. In the same sample, P450 2A6 was not detected (detection limit, 67 fmol/mg protein). These kinetic, immunoinhibition, and immunoblot data confirm that P450 2A13 is a functional enzyme and the catalyst of NNK alpha-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK alpha-hydroxylation in a human tissue.

Adult↗