Enzyme immunoassay of (24R)-brassinosteroids

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.     

Dioxygen activation by putidamonooxin: Substrate-modulated reaction of activated dioxygen

In the presence of the NADH-putidamonooxin oxidoreductase, NADH, and ¹⁸O₂, and with 4-vinylbenzoate as substrate, the monooxygenase putidamonooxin catalyzes a dioxygenase reaction producing mainly 4-glycylbenzoate. Mass spectrometry of the isolated product showed that both atoms of the ¹⁸O₂ molecule were incorporated into the substrate molecule. The rates of NADH oxidation and of O₂ uptake were between those found with benzoate and 4-methoxybenzoate, the physiological substrate of putidamonooxin.     

Visualization of antigenic proteins blotted onto nitrocellulose using the immuno-gold-staining (IGS) method

A new and simple method for the detection of antigenic proteins blotted onto nitrocellulose was developed. After transfer of spinach stromal proteins and purified phosphoribulokinase immunolabeling was performed with phosphoribulokinase antiserum, followed by a) Protein A-labeled colloidal gold particles, and b) by horseradish peroxidase conjugated Protein A and substrate mixture. The Protein A-Gold method is at least twofold more sensitive than the Protein A-peroxidase procedure. Incubation of immunolabeled nitrocellulose replicas with 0.1 M glycine, pH 2.2, removes the antibody-Protein A-Gold complexes quantitatively without influencing the antigenicity of the immobilized proteins. The replicas can be re-used for immunostaining with other antisera. The versatile applicability of the immuno-gold-staining method suggests that it is a true alternative to the peroxidase assay.     

Raman and surface enhanced Raman spectroscopy of 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide labeled proteins: bovine serum albumin and cytochrome c

2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine serum albumin and cytochrome c at different pH values were prepared and investigated using Raman-resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins in the pH 6.7-11 range were compared to those of the corresponding free species. The SERS spectra were interpreted in terms of the structural changes of the tempyo labeled proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be inactive in the Raman-RR and SERS spectra of the proteins. The alpha-helix conformation was concluded to be more favorable as the SERS binding site of bovine serum albumin. In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures

The glucose-6-P dehydrogenase specific activity in rat hepatocytes increases approximately 10-fold when the cells are placed into culture for three days. The induction requires insulin with maximum enzyme levels occurring at 10^?7 M. Pulse-labeling experiments revealed a 10-fold increase in the enzyme's relative rate of synthesis after only 8 hours in culture.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

Particulate and soluble adenylate cyclase activities of mouse male germ cells

Germ cells from the mouse testis possess both a particulate and a soluble form of adenylate cyclase (EC 4.6.1.1). Germ cell adenylate cyclase activity is Mn⁺⁺ dependent and is not stimulable with either NaF or 5′guanylylimidodiphosphate. Both particulate and soluble adenylate cyclase specific activities increase as germ cells progress through their differentiative stages, but epididymal spermatozoa seem to lack a significant amount of soluble activity. Somatic cells of the seminiferous tubule possess only a membrane bound activity, which is Mg⁺⁺ and Mn⁺⁺ dependent, NaF and 5′guanylylimidodiphosphate stimulable. It is suggested that germ cell adenylate cyclases represent incomplete forms of the enzyme, devoid of regulative subunits.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.     

Biochemical characterization of various populations of isolated bovine adrenal chromaffin cells

Various populations of bovine adrenal chromaffin cells were isolated first by successive digestions with collagenase (original cell preparation) followed by sedimentation through a stepwise bovine serum albumin gradient (cell layers I, II and III). At the fine structural level, the ratios between the number of adrenaline-cells and noradrenaline-cells were 1.9 in the original cell preparation and 0.9, 2.0 and 4.6 in cell layers I, II and III, respectively. The catecholamine content of each cell population was also measured by spectrofluorometry. The original cell preparation contained 20.1 and 12.2 nmol per 10⁶ cells of adrenaline and noradrenaline, respectively. Each cell layer had similar total amount of catecholamines (from 38.3 to 40 nmol per 10⁶ cells) but their adrenaline/noradrenaline content ratios varied from 0.6 in cell layer 1 to 1.3 and 3.3 in cell layers II and III, respectively. Incubation of the cells in the presence of acetylcholine (50 μM) induced a release of catecholamines which was proportional to the cell content of each amine. However, the percentage of total cell content released was much higher in cell layer I (20%) than in cell layers II (8%) and III (5%). Finally, each cell population was also analyzed for its ability to respond to a muscarinic stimulation of cyclic GMP level and to bind [³H]etorphine, a highly potent opiate agonist. Acetylcholine induced 3.15-, 2.15- and 4.21-fold increases in the levels of cyclic GMP in the original cell preparation, cell layers II and III, respectively, but not in cell layer I. Conversely, the high affinity opiate binding site for [³H]etorphine was almost exclusively confined to cell layer III (B_max of 28.4 fmol per 10⁶ cells as compared with 2.8–7.5 fmol in the other cell preparations). These results indicate that bovine adrenal chromaffin cells can be separated according to their content in adrenaline and noradrenaline and their response to nicotinic, muscarinic and opiate stimuli.     

Aralin,a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: A promising anticancer drug

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.     

Partial purification and characterization of two isoenzymes involved in the sulfurylation of catecholamines

To elucidate the specificity of the enzymatic system involved in the sulfurylation of catecholamines, the purification of the enzyme(s) from canine liver was undertaken. Ion-exchange chromatography led to the resolution of two sulfotransferases A and B with different pH optima for dopamine (6 for A and 9.5 for B). The apparent K_m values for 3′-phosphoadenosine 5′-phosphosulfate and dopamine were 1.7 μM and 17.7 μM for enzyme A and 26 μM and 6.2 μM for enzyme B. Each enzyme has a molecular weight of 60 000 while their isoelectric points differ; 5.7 for A and 4.7 for B. The enzyme B catalyzes the sulfurylation of a wider range of substrates than A which is preferentially active with dopamine. These results suggest the presence of two isoenzyme forms of sulfotransferase in canine liver.     

Antithyroid and antiperoxidase activity of tropolone and 3-hydroxy-4-pyrone.

Tropolone (TR) and 3-hydroxy-4-pyrone were investigated for antithyroid activity following the finding that the 2-hydroxy-oxo pyridine, 3-hydroxy-4(1H)-pyridone (DHP, I), is goitrogenic. Both compounds inhibited the thyroidal uptake of radioiodine in rats and resembled the thioamide drugs in inhibiting the organic binding of iodine by the thyroid gland rather than the trapping of iodide, but were weaker binding inhibitors than 6-methyl-2-thiouracil (MeTU). Both compounds also inhibited the iodination of bovine serum albumin and thyroglobulin, catalyzed by thyroidperoxidase (TPO), lactoperoxidase (LPO), chloroperoxidase (CPO) and horseradish peroxidase (HPO) in vitro. The inhibitory effect of TR but not that of 3-hydroxy-4-pyrone was antagonized by ferrous ions. When fed to mice at levels of intake expected to produce goitre both compounds were toxic and caused severe liver damage. Thyroid enlargement was not observed in any of these feeiding experiments, but the thyroids of mice fed 0.1% TR showed moderate hyperplasia. It was concluded that both compounds are weakly goitrogenic. Hyperactivity was observed in the mice fed TR which may be associated with inhibition of catechol methyl transferase (COMT).     

Mixed chelate copper complex, Casiopeina IIgly, binds and degrades nucleic acids: a mechanism of cytotoxicity

Metal-containing drugs that interact with DNA have been designed and studied for their anticancer activity. In this study, the mixed chelate copper-based anticancer drugs, the casiopeinas, were found to bind to DNA and to degrade DNA and RNA in the presence of reducing agents (e.g. ascorbic acid). Casiopeinas binding to DNA is high affinity, with harsh wash conditions failing to remove the interaction. The reaction requires oxygen, probably involved in the generation of *OH radicals, which would be responsible for the strand breakage. The reaction was diminished by catalase, and was completely abolished by copper chelators (e.g. trientine, EDTA); however, superoxide dismutase (SOD) had no significant effect on casiopeina-mediated DNA degradation. Casiopeina IIgly (casIIgly) in the presence of ascorbate was capable of degrading RNA, plasmid and genomic DNA, and chromatin and intranuclear genetic material. Moreover, catalase and/or SOD partially protected cells, ascorbic acid enhanced and trientine, a copper chelator, abolished the cytotoxicity of casIIgly. The generation of 8-oxodG in cells exposed to casIIgly suggests that the generation of ROS is the major cause of the cytotoxicity observed and underlies the high toxicity and anticancer activity of these compounds.