Uncoupled Enzyme Research Articles

Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression.

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O2•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O2•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.

The affinity purification and characterization of ATP synthase complexes from mitochondria

The mitochondrial F1-ATPase inhibitor protein, IF1, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

Abstract 2057: Tumor suppressor effects of tetrahydrobiopterin are mediated through beta-catenin in breast cancer cells

Abstract Nitric Oxide (NO) can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2− instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO−), which is produced rapidly by the reaction of O2− with NO. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7 and MDA-231 cells in tissue culture and in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is around 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. To ensure that increasing the BH4:BH2 ratio leads to enhanced NOS coupling, the production of NO was measured by cGMP formation and O2− levels were assessed by HPLC. As hypothesized NO levels went up and O2− levels went down. To directly examine the effects of manipulating the BH4:BH2 ratio cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. To assess the effects of SP on xenografts, we used an ex-vivo clonogenic assay. This assay also showed that colony formation is significantly reduced upon treatment of the animals with SP. Previous studies in colon cancer cells have shown that when PKG, the major pathway activated by NO, is activated β-catenin levels are reduced. In our experiments MCF7 xenografts showed a significant decrease in β-catenin levels after treatment with SP. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2057. doi:1538-7445.AM2012-2057

Abstract 4062: Nitric Oxide synthase dysregulation in solid tumors: The therapeutic potential of tetrahydrobiopterin

Abstract Nitric Oxide (NO), in terms of cancer therapeutics, can be viewed as a double edge sword. Both anti-NO and NO-based anticancer treatments appear effective in several preclinical models. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2- instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO-), which is produced rapidly by the reaction of O2- with NO, and is a highly reactive species that can modify proteins, lipids and DNA. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7, Fadu, and A431 cells in vitro and Fadu and A431 cells in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is less than 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. Increasing the BH4:BH2 ratio leads to enhanced NO production as evidenced by an increase in cGMP formation. To directly examine the effects of manipulating the BH4:BH2 ratio, MCF7 and MDA cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. Current experiments focus on SP's effects in vivo and elucidating the mechanism of cell death and the signaling pathways involved. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2011-4062

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

Aliskiren Improves Nitric Oxide Bioavailability and Limits Atherosclerosis

Inhibition of the renin-angiotensin system (RAS), with angiotensin-converting enzyme inhibitor- (ACEI) and angiotensin receptor blocker (ARB)–based treatment approaches, has emerged as the cornerstone of contemporary cardiovascular risk reduction. These therapies offer robust reductions in cardiovascular mortality in patients postmyocardial infarction, those with systolic heart failure, and in high-risk patients with atherosclerosis. Furthermore, interruption of the RAS system has emerged as a critical modulator of normal and aberrant renal physiology, and interruption of RAS signaling is linked to improved renal structure and function.

The Catalytic Site of Cytochrome P4504A11 (CYP4A11) and Its L131F Mutant

CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.

Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.

Specific killing of human endothelial cells by antibody-conjugated glucose oxidase

Conjugates of antibody with glucose oxidase obtained via the carbohydrate moiety of the enzyme via oxidation with periodate are suggested as a tool for selective killing of the target cell. The conjugate was separated from uncoupled enzyme by repeated precipitation with ammonium sulfate (42% saturation). Purity of the conjugate was estimated by gel filtration on Toya Pearl TP-65. The glucose oxidase conjugated with rabbit antibody against mouse IgG bound specifically to plastic-adsorbed mouse immunoglobulins and to cultured human endothelial cells pretreated with mouse anti-endothelial antiserum. Glucose oxidase targeted to the cells generates hydrogen peroxide in the presence of glucose. This hydrogen peroxide killed the endothelial cells even in the absence of a halide-peroxidase system and in the presence of catalase. Features of the conjugate (specificity, effective cytotoxicity, high stability) make it suitable for prospective in vivo application and for immunoselective segregation of heterogeneous cell populations.

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase.

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.

Activation of ATPase of spinach coupling factor 1. Release of tightly bound ADP from the soluble enzyme.

Several seemingly unrelated procedures used to elicit the latent ATPase activity of soluble spinach coupling factor 1 can be correlated to the release of tightly-bound ADP from the uncoupled enzyme. This ADP release is further enhanced by the presence of medium nucleotides, especially substrate ATP, and may or may not involve release from the catalytic site itself. Similarly, the light/dithiothreitol activation of membrane-bound CF1 ATPase is reported to be accompanied by energy-dependent ADP dissociation. Further indication that ADP release is involved in the ATPase activation mechanism is the observation that a pyruvate kinase phosphoenolpyruvate trap for ADP released during light/dithiothreitol treatment greatly retards the decay of membrane-bound ATPase activity that occurs in the dark, presumably by preventing reassociation of ADP. The time course of CF1 reactivation by light, after light/dithiothreitol activation followed by dark decay, allows a distinction to be made between the apparently rate-limiting dithiol modification and the more rapid dissociation of tightly bound ADP.

Covalent coupling of rat liver phenylalanine hydroxylase

Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (Phe H) to cyanogen bromide-Sepharose 4B. When 8 mg of ligand was reacted with 100 mg of matrix, 20 to 30 percent of the initial enzyme activity was covalently bound along with 90 percent of the protein. The coupled enzyme showed greater thermal stability from 40° to 60°, a broader base of optimal pH activity from 5.8 to 10.0, more resistance to proteolysis and less inhibition by various inhibitors. The uncoupled enzyme exhibited greater storage stability at 25° after 24 hr and at 0° after 18 days. Alteration of the microenvironment by introduction of sulfhydryl groups or of carriers having positive or negative charges had variable effects on the hydroxylase activity.

Covalent coupling of calf brain prolidase.

Calf brain prolidase covalently bound to CNBr-Sepharose 4B, retained about 32% of the activity of the uncoupled enzyme. The free enzyme showed slightly greater stability than the bound preparation when stored at 20 degrees C or at 0 degrees C. However, in either case the free and bound enzymes were more stable at the lower temperature. Greater thermal stability was shown by the free enzyme than by the bound preparation over a temperature range of 25 degrees C-60 degrees C. The free and bound prolidase, with and without Mn+2, had maximal activity at pH 4.0. Although the bound enzyme showed a single maximum, the free preparation exhibited three pH maxima of 4.0, 9.0, and 6.5, in decreasing order of activity. The ions Ag+, Cu+, Hg+2, and Zn+2 were strongly inhibitory on the free enzyme, whereas inhibition of the bound enzyme, with the exception of Zn+2 , was less. Unlike the coupled enzyme, a stimulatory effect was obtained on the free preparation with Co+3, Mg+2, and Mn+2. Various other compounds were studied and their effects were noted.

The use of glutaraldehyde as a coupling agent for ribonuclease and bovine serum albumin

Glutaraldehyde has been used to couple bovine pancreatic ribonuclease to bovine serum albumin. Under the conditions of the reaction 32 per cent of the original enzymatic activity is retained in the coupled product, and less than 1 per cent can be detected as uncoupled enzyme. The molecular weight of the coupled products is estimated to range between 70,000 and over 200,000, with a peak at 80,000. All of the products remain soluble in dilute saline and are stable at 4°C for at least 3 weeks.