Addition Of Cofactors Research Articles

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Mechanism of action of the specific nutrient combination Fortasyn® Connect, which is designed to enhance synapse formation and function in Alzheimer's disease

homocysteine levels, which in turn influences the PEMT pathway. This especially is relevant in AD, which is associated with high plasma homocysteine levels and B-vitamin deficiencies. Vitamin C, E, and selenium can serve as antioxidants to protect the membrane precursors DHA and EPA from lipid peroxidation. Dietary phospholipids can also act as a direct source of precursors, whereas selenium may enhance membrane synthesis by increasing the activity of a key enzyme in the Kennedy pathway.Conclusions: The availability of membrane precursors is affected by their nutritional intake as well as by the intake of cofactors that increase precursor availability. Consequently, addition of nutritional cofactors potentially facilitates the synthesis of membrane phospholipids by enhancing the availability of the rate-limiting precursors.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Physical and functional association of lactate dehydrogenase with mitochondria in intact and permeabilized skeletal muscle fibers

To clarify whether mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat red (RG) and white (WG) gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2 in either RG or WG. However, subsequent addition of NAD+ significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α‐hydroxycinnamate. In parallel experiments with subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria isolated from RG, only SS exhibited significant NAD+ ‐ dependent lactate oxidation. To determine whether lactate dehydrogenase (LDH) is physically associated with mitochondria in RG, confocal microscopy was used to detect immunohistochemically stained LDH and mitochondrial proteins. LDH clearly colocalized with mitochondria in intact fibers. In permeabilized fibers, most of the LDH signal was lost in IMF but not SS regions. The LDH signal was absent in SS regions following trypsin treatment; however, colocalization was augmented in both SS and IMF regions with exogenous LDH + washing, suggesting LDH preferentially associates with muscle mitochondria. Collectively, these results suggest that LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Comparison of Two Measures of Missing Cofactor in Cofactor-Dependent Enzymes: Proportion Versus Relative Increase

The degree of missing cofactor in a cofactor-dependent enzyme is widely used as a biomarker of cofactor defi-ciency. The degree of missing cofactor can be expressed either as the proportion of enzyme without cofactor, or as the rel-ative increase in enzyme with cofactor after addition of excess cofactor to the sample. Especially for enzymes with thia-mine pyrophosphate (TPP) as a cofactor, the relative increase (TPP-effect) has been used in a majority of studies, and its use seems to prevail without consideration of the proportion (latency) as a better alternative. In this letter, the statistical properties of the two measures are compared in the context of a thiamine-dependent enzyme. Proportion is a more bal-anced and sensitive measure than relative increase, and simulation shows that proportion is associated with equal or high-er statistical power than relative increase. The power difference can be as high as 0.12.

Catalytic removal of acetaldehyde in saliva by a Gluconobacter strain

Acetaldehyde (AA) accumulates in the oral cavity after alcohol intake and is responsible for an increased risk of alcohol-related upper aerodigestive tract (UDAT) cancer among aldehyde dehydrogenase 2-inactive heterozygotes in particular. Thus, the removal of AA from the saliva to a level below its mutagenic concentration (50 μM) after drinking is a potentially straightforward method for reducing the risk of alcohol-related UDAT cancer. Although microbial cells with AA-decomposing activity could potentially serve as a useful agent for the catalytic removal of AA from the saliva without the supplemental addition of cofactors, these cells generally exhibit strong AA-producing activity from ethanol, which is present in excess (50mM) over AA (100 μM) in the saliva after drinking. In this study, we observed that Gluconobacter kondonii (GK) cells efficiently decomposed salivary AA (100-390 μM) without the supplemental addition of cofactors irrespective of the type of alcoholic beverages consumed, even in the presence of an excess of ethanol (63 mM). Hydrogen peroxide, which is carcinogenic in animal experiments, was not produced because of the AA removal. The GK cells incubated at 45 °C and pH 3.5 for 15 h were killed, but they retained 80% of their original AA-decomposing activity. The treated cells were used as nonviable microcapsules that harbor a membrane-bound AA-decomposing activity.

Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of dehydrocholic acid

Ursodeoxycholic acid is an important pharmaceutical so far chemically synthesized from cholic acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of dehydrocholic acid to 3,12-diketo-ursodeoxycholic acid was observed with the selected recombinant E. coli strain. In the optimal, slightly acidic pH range dehydrocholic acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial dehydrocholic acid concentration, and initial formate concentration. Complete conversion of 72mM dehydrocholic acid was thus made possible at pH6.4 in a whole-cell batch process within a process time of 1h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50mM dehydrocholic acid directly to 12-keto-ursodeoxycholic acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic acid.

Efficient Synthesis of a Chiral Precursor for Angiotensin-Converting Enzyme (ACE) Inhibitors in High Space-Time Yield by a New Reductase without External Cofactors

A new reductase, CgKR2, with the ability to reduce ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), an important chiral precursor for angiotensin-converting enzyme (ACE) inhibitors, was discovered. For the first time, (R)-HPBE with >99% ee was produced via bioreduction of OPBE at 1 M without external addition of cofactors. The space-time yield (700 g·L(-1)·d(-1)) was 27 times higher than the highest record.

Effects of diet and behavioral enrichment on free fatty acids in the aged canine brain

Despite several recent studies suggesting that dysregulation of brain lipid metabolism might contribute to the mechanisms of aging and Alzheimer's disease (AD), lipid metabolism has not been evaluated extensively in the aging brain. Here, we use a lipidomic approach to demonstrate that antioxidants plus mitochondrial cofactors treatment, either alone or in combination with behavioral enrichment, attenuates lipid abnormalities in the frontal cortices of aged canine in a manner correlated with cognitive scores. Our analyses revealed that the levels of free palmitoleic acid and nervonic acid were decreased in frontal cortices of aged dogs (n=5–6/group) treated with antioxidant compared with the control group. The monounsaturated/saturated fatty acid ratio, also known as “desaturation index”—an ex-vivo indicator of stearoyl-CoA desaturase activity, was also reduced in the frontal cortex of dogs treated with antioxidants compared with control groups. Increased palmitoleic acid levels and desaturation index were positively correlated with increased reversal learning errors and decreased cognitive performance. In conclusion, our study indicates that the addition of antioxidants and mitochondrial cofactors to the regular diet alters the composition of free fatty acids in the aged brain. Together with data showing increased palmitoleic acid levels in AD patients, our data suggest that reducing palmitoleic acid levels and desaturation index in the brain may be associated with improved cognitive performance.

Correlation between Intracellular Cofactor Concentrations and Biocatalytic Efficiency: Coexpression of Diketoreductase and Glucose Dehydrogenase for the Preparation of Chiral Diol for Statin Drugs

Dynamic change of intracellular nicotinamide cofactor concentrations, the limiting factor for the bioreductions catalyzed by oxidoreductases, was monitored in Escherichia coli cells coexpressing diketoreductase and glucose dehydrogenase. On the basis of an unexpected observation, a relationship between catalytic efficiency and cofactor concentrations was established to optimize the process for the preparation of a chiral diol for statin drugs. Consequently, compared to previous reactions by E. coli cells expressing diketoreductase alone, exogenous addition of cofactors was completely eliminated to yield an increase of substrate concentration by 15-fold. The present strategy could be employed in the biocatalytic processes catalyzed by nicotinamide-dependent oxidoreductases.

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

The in-vitro metabolism of [14C]pentobarbitone and [14C]phenobarbitone by hamster liver microsomes.

The metabolism of [14C]pentobarbitone and [14C]phenobarbitone has been reinvestigated using an in-vitro hepatic microsomal system (Syrian hamsters, Aroclor 1254 induction). The incubation system was routinely supplemented with EDTA (1 mM) and a substrate concentration study revealed the metabolism of [14C]pentobarbitone to be concentration-dependent, with the greatest overall metabolism (greater than 50%) occurring at 0.054 mumol per 3.5 mL. With [14C]phenobarbitone as substrate, overall metabolism was extremely low (3%) and independent of substrate concentration. Addition of further cofactors to the incubation mixture at 20 min intervals over an extended period resulted in almost complete metabolism of [14C]pentobarbitone (100 min), 3'-hydroxypentobarbitone and 3'-oxopentobarbitone being identified as metabolites together with many minor, unidentified products. With [14C]phenobarbitone as the substrate, cofactor addition up to 120 min resulted in 8% overall metabolism; p-hydroxyphenobarbitone was identified as a product of metabolism; other minor products were unidentified. The metabolism studies failed to produce a metabolite having the properties of the N-hydroxylated product of either [14C]pentobarbitone or [14C]phenobarbitone within the detection limits available (0.02% of 0.5 mumol per incubate).

Induced Conformational Changes in the Activation of the Pseudomonas aeruginosa type III Toxin, ExoU

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca 2+-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

Circular Dichroism Spectroscopy Is a Sensitive Tool for Investigation of Bilirubin−Enzyme Interactions

Noncovalent complex formation of unconjugated bilirubin with various enzymes has been demonstrated by measuring induced circular dichroism (ICD) peaks associated with the pigment VIS absorption band. Preferential binding of the P- or M-helical conformer of bilirubin to dehydrogenases, catalase, alkaline phosphatase, and α-chymotrypsin is responsible for the characteristic exciton CD couplet that undergoes remarkable changes upon the addition of enzymatic cofactors (NADH, AMP) and an inhibitor (acridine). Alterations of the ICD spectra refer to a direct binding competition between bilirubin and NADH for a common binding site on alcohol dehydrogenase and catalase, suggesting a potential mechanism for the inhibitory effect of BR reported on NAD(P)H dependent enzymes. NADH and bilirubin form a ternary complex with glutamate dehydrogenase indicated by peculiar CD spectral changes that are proposed to be generated by allosteric mechanism. α-chymotrypsin binds bilirubin in its catalytic site, as indicated by CD displacement experiments performed with the competitive inhibitor acridine. Surprisingly, the closely related trypsin does not induce any CD signal with bilirubin. Taking into consideration the clinically relevant but controversial and poorly understood areas of bilirubin biochemistry, the fast and simple CD spectroscopic approach presented here may help to unfold diverse physiological and pathophysiological roles of BR on a molecular level.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Mutagenic activation and detoxification of benzo[ a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals

The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[ a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using α-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione- S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals.

Improved Synthesis of (S)-1-Phenyl-2-Propanol in High Concentration with Coupled Whole Cells of Rhodococcus erythropolis and Bacillus subtilis on Preparative Scale

Bioreduction of 1-phenyl-2-propanone to prepare (S)-1-phenyl-2-propanol, a useful pharmaceutical intermediate, was performed with growing cells of Rhodococcus erythropolis JX-021, giving 14 mM (1.9 g/L) product in 99% e.e. at 5 h in the catalysis of 15 mM substrate. The reduction stopped afterwards due to strong inhibition of substrate and formed product, a problem that is often encountered in biotransformation. While the substrate inhibition was solved by stepwise feeding, product inhibition was tackled by different methods: repeated removal of the product by centrifugation, by absorption with Amberlite XAD-7 resin, and by the use of dodecanol as the second phase gave the final product in 58, 68, and 61 mM in the catalysis of 80 mM substrate, respectively. The inhibition was caused by the partial permeabilization of cell membrane of R. erythropolis JX-021, and addition of NADPH or glucose 6-phosphate to such cell culture retained the reduction activity. Therefore, higher productivity in the reduction of 1 with resting cells of R. erythropolis JX-021 was achieved through cofactor regeneration and recycling by the addition of glucose and permeabilized cells of Bacillus subtilis BGSC 1A1 containing a glucose dehydrogenase, giving the product in 62 mM without addition of cofactor and 78 mM with the addition of 0.01 mM NADP(+) in the catalysis of 120 mM substrate. The product e.e. retained 99% during the process which showed industrial possibility.

Characterization of a whole‐cell catalyst co‐expressing glycerol dehydrogenase and glucose dehydrogenase and its application in the synthesis of L‐glyceraldehyde

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 mM NADP(+). Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde.

Cell-free Production of the Extracellular Domain of the Nicotinic Acetylcholine Receptor

he nicotinic acetylcholine receptor (nAchr) is a li-gand-gated ion channel which is incorporated into the postsynaptic membrane of neurons [1]. nAchr is composed of five homologous subunits, whose transmem-brane domains form an ion pore and the n-terminal do-mains contain the binding sites for the ligand (Fig.1). One of the most common subtypes of nAchr receptor in mam-malian nervous systems is a homopentameric, α7 nAchr (α7nAchr), and several neurodegenerative disorders are associated with its dysfunction [2]. An effective sys-tem for the production of the individual subunits and oth-er domains of nAchr is a prerequisite for studies focused on the receptor itself, and for the design of biomedical drugs to be used in the treatment of the disease. the ten-dency of these proteins to form insoluble aggregates in so-lution makes the development of such systems difficult [3] .recently, a research group from the uSA published the x-ray structure of acetylcholine binding protein (AchBP) from Lymnaea stagnalis [4]. AchBP is a water-soluble protein com-posed of five identical subunits. this protein shares 25% se-quence homology with the extracellular domain of α7nAchrr (α7eD), and it is capable of interacting with some of the nAchr ligands (for example, acetylcholine, α-conotixins, and α-neurotoxins) [4]. It has been shown that replacing the frag-ment of α7eD which is located between cys128 and cys142 (the so-called cys-loop) with a homologous loop from AchBP increases the solubility of the domain [5].During the past 10 years, cell-free systems, especially continuous-z exchange cell-free systems, have been success-fully used in the production of recombinant proteins [7, 8]. these systems have some advantages over host-based gene expression systems: (i) the direct addition of special agents or co-factors into the reaction mixture can prevent the ag-gregation of the target protein, and (ii) the method permits the synthesis of selectively labeled proteins which can then be used in structural studies. the main objective of the present study was the develop-ment of an effective cell-free system for the production of

Nitric Oxide Synthase: “Enzyme Zero” in Air Pollution–Induced Vascular Toxicity

Nitric Oxide Synthase: “Enzyme Zero” in Air Pollution–Induced Vascular Toxicity