ABSTRACT
Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes. Although PCs have been detected, PC- metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes. Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5). This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples. These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants. Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance.
Subject(s)
Cadmium/metabolism , Metalloproteins/chemistry , Metalloproteins/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Chromatography, Liquid/methods , Glutathione , Mass Spectrometry/methods , Phytochelatins , Plants/metabolism , Spectrometry, Mass, Secondary Ion/methodsABSTRACT
Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) is an important enzyme that catalyzes the final step of de novo pyrimidine nucleotide biosynthesis. The mechanism of this unique enzyme and whether metal ions play any role in catalysis have been topics of intense research interest. In this report, the role of Zn in ODCase was reexamined. Atomic absorption (AA) and X-ray absorption (XAS) spectroscopic studies did not detect zinc in active enzyme samples at high concentration. The XAS results also indicated the absence of other transition metal ions in ODCase.
Subject(s)
Orotidine-5'-Phosphate Decarboxylase/chemistry , Zinc/analysis , Catalysis , Molecular Structure , Saccharomyces cerevisiae , Spectrophotometry, Atomic , Spectrum AnalysisABSTRACT
M NET, the University of Michigan Medical Center's statewide referring physician computer network, was implemented as a pilot project in 1990. After three years of design, development and implementation, M NET has progressed from its pilot project status to a production system within the institution's strategic plan for hospital networking. This paper describes the evolution of M NET from a small pilot project to its current production status encompassing all clinical departments and hundreds of physicians across the state. The lessons learned from the pilot, the requirements of both the referring physicians and the Medical Center, and the development of a flexible and robust network architecture to allow network expansion are addressed.
