Phytochrome regulation of peroxidase activity in maize

Phytochrome (Pfr) regulates peroxidase activity in maize in a manner characteristic of photomodulation. The increase in peroxidase activity under continuous far-red (FR) light is sensitive to puromycin but not to chloramphenicol and actinomycin D. Cycloheximide (CHI) enhanced the peroxidase activity, both in dark-grown and FR-irradiated seedlings. However, the kinetics of enhancement of peroxidase activity by continuous FR light and CHI is different.     

Phytochrome regulation of peroxidase activity in maize IV. Photosynthetic independence of peroxidase enhancement

The continuous far-red light mediation of the enhancement ofperoxidase activity was repressed only partially by inhibitorsof cyclic and non-cyclic photophosphorylation. Under far-redlight the chlorophyll development was minimal and plastids weredifferentiated only partially. Isolated plastids from seedlingsgrown under far-red light developed photosystem I and II activityafter a lag of 4 hr, cyclic photophosphorylation after 8 hr,and non-cyclic photophosphorylation at 24 hr. These seedlings,however, failed to show CO²-dependent oxygen evolution upto24 hr of irradiation. The magnitudes of the various photochemicalactivities developed under far-red light were considerably lowerthan those developed under white light. Photosynthetic participationin the far-red mediated ‘high irradiance reaction’was excluded as it had a longer lag than the onset of the enhancementof peroxidase activity, and its magnitude was minimal. ¹Present address: School of Life Sciences, University of Hyderabad,Hyderabad-500001, India. (Received February 26, 1979; )     

Heligmosomoides polygyrus: peroxidase activity

Peroxidase activity in Heligmosomoides polygyrus was located primarily in the mitochondrion. The enzyme was active with a range of organic and inorganic electron donors and, in addition to hydrogen peroxide, it could utilize cumene peroxide, but the highest activity was obtained with linoleic acid peroxide. The effects of electron chain substrates and inhibitors on H. polygyrus mitochondrial peroxidase activity was consistent with the enzyme being linked functionally to cytochrome c, although in vivo, this may not be the only electron donor. The interaction of the peroxidase with electron transport is discussed.     

Human placental deoxyadenosine and deoxyguanosine phosphorylating activity

We studied deoxyadenosine and deoxyguanosine phosphorylating activities in human placental cytosol. The specific activities of nucleoside kinase enzymes in nanomoles per h per mg +/- SD were as follows: adenosine kinase, 30 +/- 14; deoxyadenosine kinase, 12 +/- 2; deoxycytidine kinase, 0.30 +/- 0.04; and deoxyguanosine kinase, 27 +/- 16. Three major activities were resolved by ion exchange and affinity chromatography: deoxyguanosine-deoxycytidine kinase, deoxycytidine-deoxyadenosine kinase, and adenosine-deoxyadenosine kinase. Two other activities contained significant quantities of deoxyadenosine kinase. Deoxyguanosine-phosphorylating activity eluted as a single peak in association with deoxycytidine kinase. This deoxyguanosine-deoxycytidine kinase had an apparent molecular weight of 54,000, a Stokes radius of 31 A, and apparent Km values of 10, 130, and 14 microM for deoxyguanosine, deoxycytidine, and ATP, respectively. Four peaks of deoxyadenosine phosphorylating activity were resolved by affinity chromatography with AMP-Sepharose 4B. Adenosine-deoxyadenosine kinase had an apparent molecular weight of 38,000, a Stokes radius of 27.4 A, and apparent Km values of 0.4, 510, and 75 microM for adenosine, deoxyadenosine, and ATP, respectively. Attempts to distinguish whether adenosine-deoxyadenosine kinase was one enzyme with these two activities or two separate enzymes suggested that the former was the case. Deoxycytidine-deoxyadenosine kinase had apparent Km values of 0.7, 670, and 12 microM for deoxycytidine, deoxyadenosine, and ATP, respectively. Its apparent molecular weight was estimated to be 49,000 and its Stokes radius 30 A. Two other minor peaks of deoxyadenosine-phosphorylating activity had characteristics different from either deoxycytidine kinase or adenosine kinase-associated deoxyadenosine kinase. Our studies indicate that human placental cytosol contains a complex mixture of nucleoside kinase enzymes.     

Conformational aspects of antiviral activity of deoxyguanosine acyclic analogues.

Conformational possibilities of a series of deoxyguanosine analogues possessing or lacking antiviral activity were evaluated using methods of the molecular mechanics. Comparison of the spatial structures of acyclic analogues with one another and with the spatial structures of deoxyguanosine demonstrates restricted conformational mobility for compounds devoid of activity. The level of sterically allowed superposition of functional groups from the acyclic moieties of analogues and the corresponding atomic centres of deoxyribose could serve as a criterion of activity. The superposition could be performed in two different ways through either of the nonhydrogen substituents at the C1' atom in the five-membered ring.     

Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.     

Phytochrome regulation of peroxidase activity in maize V. Role of RNA and protein synthesis

The phytochrome mediated enhancement of peroxidase activityin maize leaves was repressed by inhibitors of cytoplasmic proteinsynthesis, whereas inhibitors of RNA and organelle protein synthesiswere ineffective. Continuous far-red light had no effect onthe DNA level in the leaves, but it increased the RNA levelafter a lag of 2 hr. Under continuous far-red light the totalcontent of polyribosomes also increased after a lag of 2 hr.Isolated polyribosomes from far-red grown plants showed an enhancedrate of the in vitro incorporation of amino acids into proteinsas compared to dark grown plants. These results indicate thatthe phytochrome regulation of peroxidase activity occurs atthe translational level. ¹Present address: School of Life Sciences, University of Hyderabad,Hyderabad-500001, India (Received July 25, 1979; )     

Engineering ascorbate peroxidase activity into cytochrome c peroxidase

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.     

Light-activated chimeric GPCRs: limitations and opportunities

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NADH peroxidase activity of rubrerythrin

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.     

Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5'-nucleotidase I activity in MOLT-4 cells

Activation of nucleoside analogues is dependent on kinases and 5'-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5'-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5'-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.     

Glutathione peroxidase activity in insects: A reassessment

In vertebrate species, cytotoxic H₂O₂ and other lipid or organic hydroperoxides (ROOH) formed in aerobic metabolism are removed by a selenoprotein, glutathione peroxidase (GPOX). The GPOX activity in most rat tissues ranges from 100 to 1,000 units (1 unit = 1 nmol NADPH oxidized·mg protein^?1·min^?1), except for muscles (20–30 units). In contrast, GPOX activities of two strains of the housefly (Musca domestica), cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail butterfly (Papilio polyxenes), were found to be in the range 2–12 units. Trivial GPOX activity was detected in the confused flour beetle (Tribolium confusum). In the earthworm (Lumbricus terrestris), banana slug (Ariolimax columbianus), and market squid (Loligo opalescens), the GPOX activity ranged from 1 to 5 units. Tissue selenium concentrations were about 500–1,000 ppb for adult M. domestica, 600 ppb in T. confusum, 32 ppb in T. ni, 17 ppb in S. eridania, and 31 ppb in P. polyxenes larvae. The form of selenium incorporated at such high levels in tissues of invertebrates such as M. domestica remains an unresolved issue. Peroxidase activity of non-selenium glutathione-S-transferase (GT) against ROOH may compensate for the low GPOX activity. Catalase (CAT) has high activity and wide subcellular distribution in insects. This may be an evolutionary adaptation to GT's inability to catalyze the reduction of H₂O₂. The GT's peroxidase and CAT activities were not assessed for other invertebrate species, and warrants an investigation due to their reported low GPOX levels.     

Stabilization and photochemical regulation of antisense agents through PEGylation

Oligonucleotides are effective tools for the regulation of gene expression in cell culture and model organisms, most importantly through antisense mechanisms. Due to the inherent instability of DNA antisense agents, various modifications have been introduced to increase the efficacy of oligonucleotides, including phosphorothioate DNA, locked nucleic acids, peptide nucleic acids, and others. Here, we present antisense agent stabilization through conjugation of a poly(ethylene glycol) (PEG) group to a DNA oligonucleotide. By employing a photocleavable linker between the PEG group and the antisense agent, we were able to achieve light-induced deactivation of antisense activity. The bioconjugated PEG group provides stability to the DNA antisense agent without affecting its native function of silencing gene expression via RNase H-catalyzed mRNA degradation. Once irradiated with UV light of 365 nm, the PEG group is cleaved from the antisense agent leaving the DNA unprotected and open for degradation by endogenous nucleases, thereby restoring gene expression. By using a photocleavable PEG group (PhotoPEG), antisense activity can be regulated with high spatial and temporal resolution, paving the way for precise regulation of gene expression in biological systems.     

Nitrogen regulation of lignin peroxidase and manganese-dependent peroxidase production is independent of carbon and manganese regulation in Phanerochaete chrysosporium

In this study, a N-deregulated mutant (der8-5) of Phanerochaete chrysosporium was used as a tool to investigate the interrelationships between N, C, and Mn(II) regulation of LIP and MNP production in this organism. The results showed that LIP and MNP production by der8-5 was blocked in excess C medium but not in excess N medium. Furthermore, LIP and MNP production in this organism was subject to Mn(II) regulation regardless of the fact whether it is grown in low N medium or in high N medium. These and other results indicate that N regulation of LIP and MNP production in P. chrysosporium is independent of C and Mn(II) regulation.Abbreviations LIP lignin peroxidase - MNP manganese-dependent peroxidase - WT wild-type - der8-5 nitrogen-deregulated mutant     

Induction of uterine peroxidase: Correlation with estrogenic activity

A good correlation was obtained between the uterotrophic activity of a number of steroids and related compounds and their ability to induce peroxidase in the immature rat uterus in vivo. It is proposed that this biochemical test gives a better indication of an intact receptor-acceptor system in target cells than assays based on the binding to high affinity estrogen receptors.     

Light-activated translation of chloroplast mRNAs

The integrated regulation of mRNA stability, processing and translation facilitates the expression of several chloroplast genes, particularly in response to changes in illumination. Nuclear and chloroplast-encoded factors that mediate the expression of specific chloroplast messages have been characterized from green algae and plants. Recent studies suggest that the chloroplast might have recruited eukaryotic proteins, which are usually found in the cytoplasm or the endoplasmic reticulum, to couple the level of photosynthetic activity to gene expression via translational activation. Consequently, elements required for translational initiation of chloroplast messages differ from their prokaryotic ancestors. These results suggest that chloroplast translational regulation is a hybrid between prokaryotic and eukaryotic systems.