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S L Nance

Publications and source records attributed to S L Nance.

At least 19 recordsLinked to original sources
PubMed1993

Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated proton transport.

Alanine↗
PubMed1992

In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways.

The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.

Binding Sites↗
PubMed1987

Detection of heritable mutations as quantitative changes in protein expression.

A computerized search for the appearance of heritable mutations (as indicated by changes in protein expression) was conducted on three sets of mice, whose sires had been either untreated, exposed to 3 gray units of gamma radiation, or treated with 150 mg/kg ethylnitrosourea. Proteins from the livers of approximately 800 mice were separated by two-dimensional electrophoresis, and abundances were measured by using image analysis techniques. Heritable mutations were detected by the appearance of new proteins or by the quantitative decrease in abundance of normally occurring proteins. Measurements of the variability of the protein abundance indicate that at least 48 proteins are consistent enough to be used in searches when mutations are expected to result in a 50% reduction in the normal amount of protein. New proteins were found in four offspring from ethylnitrosourea-treated mice, and in each case a nearby spot was found to be significantly diminished. These mutations were confirmed in subsequent generations. A computer-assisted search detected three of these mutations on the basis of the abundance decrease alone. These results indicate that two-dimensional electrophoresis can be used to detect mutations reflected as quantitative changes in protein expression, provided that the proteins to be monitored are quantitatively stable when samples from different individuals are compared.

Animals↗
PubMed1987

Effects of toxic agents at the protein level: quantitative measurement of 213 mouse liver proteins following xenobiotic treatment.

By analyzing two-dimensional electrophoretic patterns of mouse liver proteins with a computerized image analysis system, we have observed quantitative changes in the abundance of more than 70 proteins in mice treated with various agents. Aroclor 1254, a mixture of polychlorinated biphenyls known to induce a broad spectrum of microsomal activity, induces the largest group of changes (60 proteins altered at p less than 0.001 significance). Phenobarbital produces a small set of characteristic changes that forms part of the much larger Aroclor 1254 effect. Ibuprofen treatment produces a phenobarbital-like pattern of change, with the addition of at least one protein change not observed with any of the other treatments. Cycloheximide and carbon tetrachloride each induces a different characteristic pattern of protein alteration. We have assigned most of the mouse liver proteins to a specific subcellular fraction, and it appears that the predominant class of proteins altered by each compound is present in the soluble phase, rather than in the microsomal fraction. The ability to survey large numbers of tissue proteins for involvement in pharmacologic and toxic effects may allow a more comprehensive understanding of the mechanisms of action in vivo and provide new markers of tissue damage.

Animals↗
PubMed1984

Two-dimensional electrophoresis used to differentiate the causal agents of American tegumentary leishmaniasis.

American tegumentary leishmaniasis is caused by a diverse group of Leishmania classified within two species complexes: L. mexicana and L. braziliensis. Because distinct disease forms are associated with certain species or subspecies, prognosis requires taxonomic identification of the pathogen. Biological criteria allow identification only to the species level; thus, biochemical and immunological markers are eagerly sought. We have used two-dimensional electrophoresis to examine the relationships among reference strains of New World Leishmania and stocks isolated from Colombian patients. The L. mexicana and L. braziliensis species complexes are shown to be extremely disparate, and the relative affinities of the subspecies reference strains and Colombian Leishmania stocks are documented. The latter displayed a variety of patterns--some clearly identifiable with a particular L. braziliensis subspecies reference strain, others intermediate between those of L.b. panamensis and L.b. guyanensis. Such comparisons are useful both in establishing relatedness and identifying subspecies and variant marker proteins, which may have biological significance.

Animals↗
PubMed1982

A two-dimensional electrophoretic analysis of the heat-shock-induced proteins of human cells.

Using two-dimensional electrophoresis, we have investigated the responses of human cells in culture to heat shock and to various chemical agents producing a similar effect. These treatments result in the induction of increased synthesis of several specific proteins. One (HShock:1, SDS-molecular mass about 65000) is increased by about 350-fold over the amount in untreated cells. Computer analysis of time-course studies indicates, however, that rates of synthesis of various proteins other than the classical "heat shock proteins" are affected, some of these alterations following time courses quite different from the main (HShock) inductions. The heat shock effect is thus much more complicated than previously realized. We purified the HShock:1 protein from heat-shocked human lymphoblastoid cells, and prepared a rabbit antiserum specific for HShock:1 on nitrocellulose two-dimensional gel transfers of total lymphoblastoid cell protein. A survey of mouse tissues shows high concentrations of an HShock:1-like protein in the testis, and human testes also appears to contain substantial (though lower) concentrations. These results are consistent with the hypothesis (derived from the tissue-culture studies) that the heat shock effect is a general response to the need for increased protein catabolism within the cell. Increased concentrations of HShock:1 are also observed in preparations of blood leukocytes collected from patients after surgery, indicating that some types of physiological trauma may induce the heat shock proteins in man. Using the antiHShock:1 antibody in an immunoassay, it will be possible to systematically examine HShock:1 concentrations in plasma and leukocytes, thereby opening up the possibility of a clinical test based for the first time upon an inducible aspect of cellular gene expression.

Animals↗
PubMed1979

Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate proteins.

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

Actins↗
PubMed1969

Ultrastructural and biochemical changes in brown fat in cold-exposed rats.

During the first 3 days of exposure of rats to 5 degrees C, the nitrogen concentration of interscapular brown fat increased by 50% and remained at this elevated level for the duration of the 8-wk observation period, while the mass of tissue increased fourfold. The concentration of both DNA and RNA per unit nitrogen reached a maximum after 3 days, then declined; however, the total quantity of each continued to rise. The concentration of various respiratory enzymes decreased during the first few days and then increased, but at different rates. The morphological changes in mature brown fat cells during cold acclimation were observed to be: a reduction in fat droplet size during the first 3 days, followed by a gradual increase in size through 6 wk in the cold; a continual increase in the amount of intermitochondrial ground substance during the first 3 wk, with increased granularity and glycogen content after 1 wk; initial disappearance of glycogen between mitochondria, followed by the reappearance of a few isolated particles in the intermitochondrial ground substance after 1 wk in the cold; initial increase in the density of intramitochondrial matrix for the first 3-4 days, followed by a gradual return to the control density; loss in integrity of mitochondrial outer membranes during the first 4 days, followed by gradual but incomplete restoration; temporary loss of the dense material in lipid droplets during the first 24 hr, with return after 1 wk in the cold; and a 40% increase in mitochondrial diameter within 1 day, followed by a decrease in diameter within 1 wk to a constant value about 15% larger than the controls.

Adipose Tissue↗