Optical, EPR and M?ssbauer spectroscopic studies on the NO derivatives of cytochrome cd1 from Thiobacillus denitrificans

We have used optical, EPR and M?ssbauer spectroscopies to study the formation of heme-NO complex upon the addition of nitrite to reduced cytochrome cd1 from Thiobacillus denitrificans. The reduced d1 heme binds NO under both alkaline and acidic conditions, but the binding of NO to the reduced c heme was strongly pH-dependent. The M?ssbauer data showed unambiguously that at pH 7.6 the c heme does not complex NO, whereas at pH 5.8 approximately half of the reduced c heme binds NO. This observation was confirmed by EPR studies, which showed that the spin concentration of the heme-NO EPR signal increased from 2 spins/molecule at pH 8.0 to approximately 3 spins/molecule at pH 5.8. Optical absorption study also showed strong pH dependence in the binding of NO to the reduced c heme. We have also analyzed the M?ssbauer spectra of the ferrous d1 heme-NO complex using a spin-Hamiltonian formalism. The magnetic hyperfine coupling tensor was found to be consistent with the unpaired electron residing on a sigma orbital.     

Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding

Chemical modification of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine), with pyridoxal 5'-phosphate suggested that Lys-22 (equivalent to Lys-23 of the Petunia hybrida enzyme) is a potential active site residue (Huynh, Q. K., Kishore, G. M., and Bild, G. S. (1988) J. Biol. Chem. 263, 735-739). To investigate the possible role of this residue in the reaction mechanism, we have used site-directed mutagenesis to replace Lys-23 of the P. hybrida enzyme with 3 other amino acid residues: Ala, Glu, and Arg. Analysis of these mutant enzymes indicates that of these only the Lys-23 to Arg mutant enzyme is active; the other two replacements (Ala and Glu) result in inactivation of the enzyme. Two of the mutant enzymes (Lys-23 to Arg and Ala) were purified to homogeneity and characterized. The purified Lys-23 to Arg mutant enzyme is less sensitive than the wild type enzyme to pyridoxal 5'-phosphate. It showed identical Km values for substrates and a 5-fold higher I50 value for glyphosate in comparison with those from the wild type enzyme. Binding studies using fluorescence measurements revealed that the substrate shikimate 3-phosphate and glyphosate were able to bind the purified Lys-23 to Arg mutant enzyme but not to the purified catalytically inactive Lys-23 to Ala mutant enzyme. The above results suggest that the cationic group at position 23 of the enzyme may play an important role in substrate binding.     

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.     

Evidence for lysosomal processing of amyloid β-protein precursor in cultured cells

Amyloid -protein precursor (ABPP) of Alzheimer's disease (AD) represents a family of proteins which includes the parent protein which generates a small (4 kD) fragment that self-assembles to form amyloid fibrils in AD. Thus, the normal and abnormal proteolysis of ABPP may be directly relevant to AD pathogenesis. We have examined the accumulation of ABPP in cultured rodent and human neuronal cell lines in the presence and absence of a battery of protease inhibitors using immunohistochemistry and Western blot analysis. Here we present evidence for a lysosomal pathway for the turnover of ABPP and discuss the relevance of these results to plaque pathology and abnormal ABPP immunostaining in AD.Special issue dedicated to Dr. Paola S. Timiras     

Characterization of a sulfite reductase from Desulfovibrio vulgaris. Evidence for the presence of a low-spin siroheme and an exchange-coupled siroheme-[4Fe-4S] unit

We have studied a low-molecular-weight (Mr = 27,200) sulfite reductase from Desulfovibrio vulgaris (Hildenborough, NCIB 8303) with M?ssbauer, EPR, and chemical techniques. This sulfite reductase was found to contain one siroheme and one [4Fe-4S] cluster. As purified, the siroheme is low-spin ferric (S = 1/2) which exhibits characteristic EPR resonances at g = 2.44, 2.36, and 1.77. At 150 K, the observed M?ssbauer parameters, delta EQ = 2.49 +/- 0.02 mm/s and delta = 0.31 +/- 0.02 mm/s, for the siroheme are typical for low-spin ferric complexes. The [4Fe-4S] cluster is in the 2+ state. The M?ssbauer parameters, delta EQ = 0.95 +/- 0.02 mm/s and delta = 0.38 +/- 0.02 mm/s, for the cluster are almost identical to those observed for the [4Fe-4S]2+ cluster in the hemoprotein subunit of the sulfite reductase from Escherichia coli. Similar to the hemoprotein subunit of E. coli sulfite reductase, low-temperature M?ssbauer spectra of D. vulgaris sulfite reductase recorded with weak and strong applied fields also show evidence for an exchange-coupled siroheme-[4Fe-4S] unit.     

Photoacoustic imaging of the human placental vasculature

Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies.      

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.