Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate

Background: The enzyme coproporphyrinogen oxidase (copro\u27gen oxidase) converts coproporphyrinogen-Ill (GIII) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro\u27gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (GIII) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purified human copro\u27gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. Material/Methods: Therefore, purified, cloned human copro\u27gen oxidase was incubated with GIII or C-IV at 37 degrees C with various substrate concentrations (from 0.005 mu M to 3.5 mu M). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. Results: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic efficiency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using GIII relative to C-IV. Conclusions: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias

Use of Di- and Tripropionate substrate analogs to probe the active site of human recombinant coproporphyrinogen oxidase

Background: Defects in the enzyme coproporphyrinogen oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human coproporphyrinogen oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrinogen-III. These Substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. Material/Methods: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K-m and V-max, were determined. Results: Relative to the authentic substrate, the K-m values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K-m values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V-max values were an apparent function of the carbon length of the substituent. on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. Conclusions: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme

Investigation of the catalytic and structural roles of conserved histidines of human coproporphyrinogen oxidase using site-directed mutagenesis

Background: The catalytic contribution of four conserved histidines of human coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). Material/Methods: The wild-type and mutant enzyme forms were analyzed for their ability to utilize coproporphyrinogen-III, mesoporphvrinogen-VI, and harderoporphyrinogen as substrates. Results: Wild-type CPO had specific activities of 4.9 +/- 0.9 nmole product/min/mg for coproporphyrinogen-III, 1.7 +/- 0.7 nmole ptoduct/min/mg for mesoporphyrinogen-VI, and 5.1 +/- 1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent With all three substrates, but to varying degrees. The most affected Mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. Conclusions: Thus, His 158 of human CPO may have a role ill the active site, but none of the conserved histidine residues of human coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria

2′-3′-Cyclic Nucleotide 3′-Phosphodiesterase Inhibition by Organometallic Vanadium Complexes: A Potential New Paradigm for Studying CNS Degeneration

The enzyme, 2′-3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) has been known for over fifty years. Nevertheless, the roles this membrane-bound enzyme play have yet to be described completely. Recently, there has been renewed interest in the study of this enzyme due to studies that suggest that CNPase plays a role in the mediation of cellular inflammatory responses in renal and nervous system tissues. Also, this enzyme, found in oligodendrocytes of the nervous system, has been reported to participate in significant regulatory changes associated with age which may be involved in age-related CNS degeneration. Consequently, development of CNPase inhibitors is of interest and should aid in the study of this, as yet, poorly understood enzyme. In this work we utilized a spectrophotometric enzyme assay to determine the effect a panel of organo-vanadium complexes had on isolated hamster myelin CNPase activity. Our group has now identified several potent in vitro CNPase inhibitors that could prove useful in clarifying the important roles of this enzyme

Sensory-based niche partitioning in a multiple predator-multiple prey community

Many predators and parasites eavesdrop on the communication signals of their prey. Eavesdropping is typically studied as dyadic predator-prey species interactions; yet in nature, most predators target multiple prey species and most prey must evade multiple predator species. The impact of predator communities on prey signal evolution is not well understood. Predators could converge in their preferences for conspicuous signal properties, generating competition among predators and natural selection on particular prey signal features. Alternatively, predator species could vary in their preferences for prey signal properties, resulting in sensory-based niche partitioning of prey resources. In the Neotropics, many substrate-gleaning bats use the mate-attraction songs of male katydids to locate them as prey. We studied mechanisms of niche partitioning in four substrate- gleaning bat species and found they are similar in morphology, echolocation signal design and prey-handling ability, but each species preferred different acoustic features of male song in 12 sympatric katydid species. This divergence in predator preference probably contributes to the coexistence of many substrate-gleaning bat species in the Neotropics, and the substantial diversity in the mate-attraction signals of katydids. Our results provide insight into how multiple eavesdropping predator species might influence prey signal evolution through sensory-based niche partitioning

9-[(E)-2-phenylethenyl]anthracene and 9-[(E)-2-(naphthalen-2-yl)ethenyl]anthracene as traps for singlet oxygen: photosensitized oxidation and photodynamic effect on Leishmania tarentolae parasites

Introducción: El oxígeno singulete es una especie reactiva que se obtiene mediante t r a n s f e r e n c i a e n e r g é t i c a u s a n d o u n fotosensibilizador. Su cuantificación directa requiere de instrumentación costosa, por lo cual es necesario recurrir a métodos indirectos que tengan suficiente selectividad y bajo costo. Estos procedimientos se basan en la interceptación química del oxígeno singulete produciendo una especie que se pueda detectar por métodos analíticos convencionales. En este artículo se describe la utilización del 9-[(E)-2-feniletenil] antraceno 1 (PEA) y del 9-[(E)-2-(naftalen-2-il) etenil]antraceno 2 (NEA), como alternativas viables y económicas para la cuantificación indirecta del oxígeno singulete, en medios acuosos. Su ventaja radica en la fácil detección de la desactivación de su fluorescencia una vez son oxidados por el oxígeno singulete. Materiales y Métodos: Los compuestos se sintetizaron y caracterizaron siguiendo procedimientos previamente reportados. Su capacidad para atrapar oxígeno singulete se determinó siguiendo su oxidación fotosensibilizada en solución de H2O/THF y en parásitos de Leishmania tarentolae, empleando azul de metileno o rosa bengala como fotosensibilizadores. Las muestras experimentales se iluminaron con una lámpara de emisión de luz visible, y se utilizaron métodos espectroscópicos (absorción UV-Vis, fluorescencia, RMN-1H) y espectrometría de masas para monitorear el atrapamiento y fotooxidación. Resultados y Discusión: Las pruebas espectroscópicas demostraron la capacidad que tienen los compuestos PEA 1 y NEA 2 para atrapar oxígeno singulete en solución acuosa y dentro de parásitos de L. tarentolae. Estudios de viabilidad parasitaria demuestran que PEA 1 es citotóxico en la oscuridad y cuando los cultivos son expuestos a la luz, mientras que NEA 2 no es citotóxico en la oscuridad, pero sí lo es cuando el cultivo es expuesto a la luz. En conclusión, los compuestos estudiados pueden servir como sondas para detectar y medir la producción de oxígeno singulete en medio acuoso y potencialmente en cultivos celulares, aunque es recomendable evaluar su actividad citotóxica en la oscuridad y bajo iluminación en estos casos.Introduction: Singlet oxygen is a reactive species obtained via energy transfer using a photosensitizer. Its direct quantification requires expensive instrumentation, so it is necessary to use indirect methods having sufficient selectivity and low cost. These procedures are based on the chemical interception of singlet oxygen producing a species that can be detected using conventional analytical methods. This article describes the utilization of 9-[(E)-2- phenylethenyl]anthracene 1 (PEA) and 9-[(E)-2- (naphtalen-2-yl)ethenyl]anthracene 2 (NEA) as suitable and economic alternatives for the indirect quantification of singlet oxygen in aqueous media. Their advantage is the easy detection of their fluorescence once they are oxidized by singlet oxygen. Materials and Methods: Compounds were synthesized and characterized following procedures previously reported. Their capacity to trap singlet oxygen was determined by monitoring their photosensitized oxidation in either a H2 O/THF solution or within Leishmania tarentolae parasites, utilizing methylene blue or rose bengal as photosensitizers. Experimental samples were illuminated with a lamp emitting visible light, while spectroscopical techniques (absorption, fluorescence, 1 H-NMR) and mass spectrometry were used to monitor trapping and photooxidation. Results and Discussion: Spectroscopical evidence demonstrates that both PEA 1 and NEA 2 are capable of trapping singlet oxygen in both aqueous media and within L. tarentolae parasites. Viability studies demonstrate that PEA 1 is cytotoxic in the dark and when parasite cultures were exposed to light, while NEA 2 does not show dark cytotoxicity, but is toxic when cultures were exposed to light. It can be concluded that both compounds under study may be utilized as probes to detect and quantify the production of singlet oxygen in aqueous media and potentially in cell cultures, although it is recommended to evaluate their cytotoxic activity both in the dark and upon light exposure in these cases

Vanadium Complexes Are in vitro Inhibitors of Leishmania Secreted Acid Phosphatases

Leishmaniasis is a parasitic disease caused by the protozoa Leishmania. These organisms secrete acid phosphatases during their growth cycle as an important part of cell targeting to host macrophage cells thus allowing for a successful infection. Secreted acid phosphatases (SAP) are reported to play a significant role in the survival of Leishmania cells, thus evaluation of these enzymes is of interest. The inhibition of SAP can be the focus of a new drug therapy. We tested for SAP activity from Leishmania tarentolae following the addition of a series of vanadium complexes including decavanadate. Cell cultures at different stages in their growth curve were harvested by centrifugation and supernatant was collected. The SAP activity in the supernatant was assayed with the artificial substrate p-nitrophenylphosphate (pNPP). Incubation with orthovanadate resulted in a decrease in activity of 18% ± 1 relative to the control, in comparison to decavanadate, which resulted in a 35% ± 4 decrease in activity. Other vanadium complexes showed smaller inhibitory effects than orthovanadate. Some vanadium complexes appeared to have an effect on reducing cell clumping when compared to control cells. The SAP was partially isolated through anion exchange chromatography and results indicate that SAP isozyme forms are present in the supernatant from cells. Future work is focused on obtaining recombinant enzyme which can be more completely characterized for inhibition by vanadium complexes

Studies of the Effectiveness of Bisphosphonate and Vanadium-Bisphosphonate Compounds In Vitro

Leishmaniasis is a disease that is a significant problem for people, especially in tropical regions of the world. Current drug therapies to treat the disease are expensive, not very effective, and/or of significant side effects. A series of alkyl bisphosphonate compounds and one amino bisphosphonate compound, as well as alendronate and zoledronate, were tested as potential agents against Leishmania tarentolae. Also, two polyoxometalates (POMs) with nitrogen-containing bisphosphonate ligands, vanadium/alendronate (V5(Ale)2) and vanadium/zoledronate (V3(Zol)3), were tested against L. tarentolae and compared to the results of the alendronate and zoledronate ligands alone. Of the compounds evaluated in this study, the V5(Ale)2 and V3(Zol)3 complexes were most effective in inhibiting the growth of L. tarentolae. The V5(Ale)2 complex had a larger impact on cell growth than either alendronate or orthovanadate alone, whereas zoledronate itself has a significant effect on cell growth, which may contribute to the activity of the V3(Zol)3 complex

The NANOGrav 11-Year Data Set: Arecibo Observatory Polarimetry And Pulse Microcomponents

We present the polarization pulse profiles for 28 pulsars observed with the Arecibo Observatory by the North American Nanohertz Observatory for Gravitational Waves (NANOGrav) timing project at 2.1 GHz, 1.4 GHz, and 430 MHz. These profiles represent some of the most sensitive polarimetric millisecond pulsar profiles to date, revealing the existence of microcomponents (that is, pulse components with peak intensities much lower than the total pulse peak intensity). Although microcomponents have been detected in some pulsars previously, we present microcomponents for PSRs B1937+21, J1713+0747, and J2234+0944 for the first time. These microcomponents can have an impact on pulsar timing, geometry, and flux density determination. We present rotation measures for all 28 pulsars, determined independently at different observation frequencies and epochs, and find the Galactic magnetic fields derived from these rotation measures to be consistent with current models. These polarization profiles were made using measurement equation template matching, which allows us to generate the polarimetric response of the Arecibo Observatory on an epoch-by-epoch basis. We use this method to describe its time variability, and find that the polarimetric responses of the Arecibo Observatory's 1.4 and 2.1 GHz receivers vary significantly with time.Comment: 41 pages, 20 figure