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Rat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one-and twodimensional acid-urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two-dimensional patterns of the nuclear proteins of the Go + GI and Gz + M populations.However, comparison of the two-dimensional autoradiographs of 3H-leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in Gz + M populations, but no noticeable effect in protein labeling was observed in the GO + GI populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the Gz + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.Key Terms: Nuclear protein analysis, sorted nuclei, isoproterenol, nuclear protein electrophoresis Alterations in specific nuclear proteins after the administration of isoproterenol were observed in rat hepatocyte nuclei. These changes were seen in autoradiographs of two dimensional electrophoretic gels using samples obtained by electrostatically sorting nuclei from selected phases of the cell cycle.The development of flow microcytometric systems (FMC) with electrostatic sorting capability allows the separation of cells or nuclei into small homogeneous populations based on detectable cytologic parameters such as DNA content. Methods for collecting and preparing these small homogeneous samples for biochemical analysis have also been developed (37).In the past several years the development and application of new and sensitive gel electrophoretic techniques have resolved the complexity, heterogeneity and metabolic altera-
Rat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one-and twodimensional acid-urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two-dimensional patterns of the nuclear proteins of the Go + GI and Gz + M populations.However, comparison of the two-dimensional autoradiographs of 3H-leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in Gz + M populations, but no noticeable effect in protein labeling was observed in the GO + GI populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the Gz + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.Key Terms: Nuclear protein analysis, sorted nuclei, isoproterenol, nuclear protein electrophoresis Alterations in specific nuclear proteins after the administration of isoproterenol were observed in rat hepatocyte nuclei. These changes were seen in autoradiographs of two dimensional electrophoretic gels using samples obtained by electrostatically sorting nuclei from selected phases of the cell cycle.The development of flow microcytometric systems (FMC) with electrostatic sorting capability allows the separation of cells or nuclei into small homogeneous populations based on detectable cytologic parameters such as DNA content. Methods for collecting and preparing these small homogeneous samples for biochemical analysis have also been developed (37).In the past several years the development and application of new and sensitive gel electrophoretic techniques have resolved the complexity, heterogeneity and metabolic altera-
A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing> 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.
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