Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium

Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.     

Human protoporphyrinogen oxidase: relation between the herbicide binding site and the flavin cofactor

Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants. Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships. A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-[3H]tetrahydrophthalimide ([3H]AzTHP), resulting in high specific activity (2.6 TBq/mmol). Human protox expressed in Escherichia coli and purified by affinity chromatography is used with [3H]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [3H]AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The Bmax establishes one [3H]AzTHP binding site per FAD. Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting [3H]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD. The first step has been taken in photoaffinity labeling the herbicide/substrate site with [3H]AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.     

Inhibition of pig kidney diamine oxidase by nazlinin and nazlinin derivatives

Nazlinin (1-(4-butylamino)-1,2,3,4-tetrahydro-beta-carboline) (1), an alkaloid recently isolated from Nitraria schoberi, and its two derivatives, 1-(4-butylamino)-3,4-dihydro-beta-carboline (2) and 1-(4-butylamino)-beta-carboline (3), were synthesized and their interaction with pig kidney diamine oxidase (PKDO) was studied. Nazlinin appeared to be a very poor substrate while 3 was a good substrate with an apparent Km of 9.3-10(-5) M. The enzyme was inhibited by 1 and 2. With both compounds the mode of inhibition found was non-competitive and inhibition constants calculated from the slopes and intercepts of double-reciprocal plots show that 2 is a much more potent inhibitor than the natural product. The relationship between the structure of these compounds and the results found is discussed.     

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the A rabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G-->A change at 291 in the first putative exon, resulting in a Val-->Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G-->A change at the equivalent position (5751) within exon 10.     

Inhibition of the slow inward current by nifedipine in mammalian ventricular myocardium

An improved micromethod for the determination of inorganic phosphorus in serum and urine is outlined. The procedure requires no deproteinization and yields a stable colour. The new formulation proposed avoids the pitfalls of other techniques. The method is both accurate (recovery 99-100.4 percent) and precise (C.V. 2.19 percent).     

Inhibition by verapamil and diltiazem of agonist-stimulated contractile responses in mammalian ventricular cardiomyocytes

Secretin, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) each exert potent positive contractile responses directly in rat ventricular cardiomyocytes. However, the contractile-coupling mechanisms associated with these responses have not been determined. In the present study, the involvement of L-type calcium channels in the contractile responses elicited by each peptide has been investigated using the selective antagonists at L-type calcium channels, verapamil and diltiazem. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of CaCl2 (2 mM) and adenosine deaminase (5U/ml). Cardiomyocytes were pre-incubated for 3 min prior to stimulation, in the absence of L-type calcium channel antagonist, and in the presence of verapamil (< or = 1 microM) or diltiazem (< or = 1 microM). Verapamil (< or = 1 microM) and diltiazem (< or = 1 microM) inhibited the contractile responses elicited by isoprenaline (100 nM) and forskolin (40 microM), used as positive controls, significantly, and in a concentration-dependent manner, but did not inhibit significantly the contractile response elicited by phenylephrine (2 microM), which was employed as a negative control. Verapamil (< or = 1 microM) and diltiazem (< or = 1 microM) inhibited the contractile responses to secretin (20 nM) and VIP (20 nM) significantly, and in a concentration-dependent manner, but did not inhibit the contractile response to CGRP. These data indicate that the positive contractile responses to secretin and VIP in mammalian ventricular cardiomyocytes involve the influx of calcium ion via L-type calcium channels, while the positive contractile response to CGRP does not.     

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.     

The genetic basis of "Scarsdale Gourmet Diet" variegate porphyria: a missense mutation in the protoporphyrinogen oxidase gene

Elevated serum triglyceride levels may be related to the following clinical features: increased blood coagulation and viscosity, increased serum fibrinogen levels, decreased fibrinolysis, and for serum levels over 1000 mg/dl, a strong increase of acute pancreatitis rate. Pharmacological choice among the numerous drugs to treat hypertriglyceridemias is currently debated. Our study was aimed to assess the therapeutic efficacy of acarbose in the treatment of non-diabetic subjects, affected by familiar hypertriglyceridemia (FH). We studied 18 non-diabetic patients (10 males, 8 females; mean age 57.61+/-6.85 years) without family history of diabetes mellitus affected by familiar hypertriglyceridemia. The study protocol planned a treatment period of 20 weeks, divided into five 4-week courses and made up as follows: diet plus acarbose therapy (4 weeks); diet therapy alone (4 weeks) alternatively. In the second and fourth 4-week courses diet plus acarbose were administered, while diet therapy alone was administered in the first, third, and fifth 4-week courses. Acarbose doses consisted of 50 mg (1/2 pill) twice daily. Mean serum triglyceride levels, after first month of dietary treatment, underwent a significant reduction from 481.5 +/- 67.1 mg/dl to 389.5 +/- 62.7 mg/dl, even if they did not reach the optimal levels to keep on the dietary therapy alone. After the first month of treatment with acarbose associated to diet, we observed a further reduction of serum triglycerides levels (p = 0.02). When diet alone was administered, mean triglyceride serum levels underwent a significant enhancement (p = 0.003). Restarting for the second time the association treatment, we observed a noteworthy reduction of mean serum triglyceride levels (p = 0.0001). Acarbose acts on the pathogenesis of FH, lowering the production of endogenous triglycerides. Our data suggested that acarbose can be considered a valid therapeutic tool in the treatment of familiar hypertriglyceridemias, also in non-diabetic patients.     

Inhibition of brain monoamine oxidase by adducts of 1,2,3,4-tetrahydroisoquinoline with components of cigarette smoke

A series of adducts of 1,2,3,4-tetrahydroisoquinoline (TIQ) and some components of tobacco smoke were investigated for their ability to inhibit rat brain monoamine oxidase. 1-Cyano-TIQ (1CTIQ), N-(1'-cyanoethyl)-TIQ (CETIQ), N-(1'-cyanopropyl)-TIQ (CPTIQ), and N-(1'-cyanobutyl)-TIQ (CBTIQ) were found to act as competitive inhibitors for both MAO-A and MAO-B. Ki values ranged from 16.4 to 37.6 microM. N-(Cyanomethyl)-TIQ (CMTIQ) was not found to be an inhibitor (Ki > 100 microM). These findings may help to explain the in vivo inhibitory effects of tobacco smoke on MAO activity and the suggested protective effect of tobacco smoking against Parkinson's disease. They also appear to reinforce the usefulness of reversible MAO inhibitors in smoking cessation and abstinence. However, different results must be expected between Burley and Bright tobacco.     

Inhibition of mammalian topoisomerase I by 1-nitro-9-aminoacridines. Dependence on thiol activation

It has been suggested that the rate of CD4 cell decline accelerates in parallel with decreasing numbers of cells; however, the statistical literature suggests the opposite. CD4 cells were counted about every 6 months in a cohort of 1264 human immunodeficiency virus-infected subjects (the Italian Seroconversion Study cohort). Kaplan-Meier analysis was used to estimate the time for CD4 cells to decline by 100 cells/mm3, conditional on reaching predefined levels. In addition, CD4 cell counts were modeled as a function of time since seroconversion in individuals with > or = 5 counts. Kaplan-Meier survival times for a 100 cell/mm3 decrease in CD4 cells increased as lower counts were reached (log rank test, P < .001). The shape of the overall fitted curve of the CD4 cell counts does not suggest an increasing rate of decline. Data from the Italian Seroconversion Study cohort do not show a general tendency for accelerating CD4 cell decline in association with lower counts.     

Inhibition of [3H]norepinephrine uptake in peripheral organs of some mammalian species by ouabain

In order to demonstrate the possible involvement of (Na+ + K+)-ATPase in the high affinity uptake of [3H]-norepinephrine in the sympathetic nerve endings, the effect of ouabain on [3H]norepinephrine uptake in spleen and heart slices of five mammalian species was examined. The ouabain sensitivity of [3H]norepinephrine uptake in the heart slices form various species, as determined by the estimation of IC52, was, in increasing order, lamb (2,3 muM) less than calf (2.5 muM) less than guinea pig (4 muM) less than rabbit (10muM) less than rat (greater than 500 muM). The IC50 values in the spleen slices were: lamb (1 muM) less than calf (3.2 muM) less than rabbit (9.5 muM) less than guinea pig (25 muM) less than rat (greater than 500 muM). The IC50 values for the inhibition of specific [3H]ouabain binding in the microsomal fractions of spleen and heart of the five mammalian species by ouabain were similar to the IC50 values for the inhibition of [3H]norepinephrine uptake by the cardiac glycoside. Since ouabain is known to bind exclusively to (Na+ + K+)-ATPase of a microsomal fraction, these results suggest that the inhibition of [3H]norepinephrine uptake in the sympathetic nerve endings by ouabain is mediated by the inhibition of (Na+ + K+)-ATPase.     

Inhibition of [3H]quinpirole binding by a monoamine oxidase inhibitor in subcellular fractions of rat striatum

[3H]Quinpirole is a dopamine agonist with high affinity for D2-like dopamine receptors. A number of non-dopaminergic compounds, most notably monoamine oxidase inhibitors (MAOIs), inhibit the binding of [3H]quinpirole, but not other D2-like agonists and antagonists, in rat striatal membranes by a mechanism that does not involve the enzymatic activity of MAO. To further characterize this novel interaction, the subcellular distribution of spiperone-displaceable, "D2-like" [3H]quinpirole-labeled sites in rat striatum was assessed and compared with the distribution of MAOI-displaceable [3H]quinpirole binding (MQB). "D2-like" [3H]quinpirole binding exhibited similar nanomolar affinity in the crude synaptosomal (P2), crude microsomal (P3), and ribosomal, post-microsomal (P4) fractions. Total binding activity (fmol bound/fraction) of "D2-like" [3H]quinpirole binding was concentrated in the synaptosomal fraction (P2B). The subcellular distribution of MQB paralleled that of "D2-like" [3H]quinpirole binding. This suggests that "D2-like" [3H]quinpirole binding and MQB occur at a common membrane-bound binding site.     

Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential sample mammalian assay for mutagenesis

In an infant with galactosemia high levels of galactose-1-phosphate in red blood cells and of blood galactose were observed under a "galactose-free' diet. The child did not thrive and developed a liver cirrhosis. At the age of 5 months he died unexpectedly. Post mortem examination revealed in the pancreas and the small intestine changes suggestive of a cystic fibrosis. Since the exogenous administration of galactose by diet could be excluded the endogenous production of significant amounts of galactose-1-phosphate has to be considered.     

Inhibition of the Mr 70,000 S6 kinase pathway by rapamycin results in chromosome malsegregation in yeast and mammalian cells

The antifungal and immunosuppressive drug rapamycin arrests the cell cycle in G1-phase in both yeast and mammalian cells. In mammalian cells, rapamycin selectively inhibits phosphorylation and activation of p70 S6 kinase (p70(S6K)), a protein involved in the translation of a subset of mRNAs, without affecting other known kinases. We now report that rapamycin causes chromosome malsegregation in mammalian and yeast cells. Chromosome malsegregation was determined by metaphase chromosome analysis of human lymphocytes and lymphoblasts, detection of CREST-positive micronuclei in human lymphoblasts and Chinese hamster embryonic fibroblast (CHEF) cells, and selection of doubly prototrophic cells in a specially constructed yeast strain. The number of ana-telophases with displaced chromosomes and interphase and mitotic cells with an irregular number of centrosomes was also determined in CHEF cells. In quiescent mammalian cells (human lymphocytes and CHEF cells) induced with growth factor to re-enter the cell cycle, rapamycin was effective when cells were exposed at the time of p70(S6K) activation. In yeast, rapamycin was more effective when treatment was started in G1- than in G2-synchronized cells. Cells from ataxia telangiectasia (A-T) patients are characterized by chromosome instability and have recently been found to be resistant to the growth-inhibiting effect of rapamycin. We found that an A-T lymphoblastoid cell line was also resistant to the induction of chromosome malsegregation by rapamycin, but the level of spontaneous aneuploidy was higher than in normal cells. In yeast, the induction of chromosome malsegregation was dependent on the presence of a wild-type TUB2 gene, encoding the beta-subunit of tubulin. The finding that rapamycin acts in different cell types and organisms suggests that the drug affects a conserved step important for proper segregation of chromosomes. One or more proteins required for chromosome segregation could be under the control of the rapamycin-sensitive pathway.