The binding of copper ions to copper-free bovine superoxide dismutase. Copper distribution in protein samples recombined with less than stoicheiometric copper ion/protein ratios.

Samples of superoxide dismutase containing less than stoicheiometric amounts of Cu2+ were obtained by either partial re-addition of Cu2+ to the Cu2+-free protein or partial removal of Cu2+ by controlled CN-treatment. In these samples the distribution of the metal between the two identical sites on the two subunits was studied by quantitative gel electrophoresis and found to be statistical only in the process of copper removal by CN-. In the other case the distribution fits a model of co-operative interaction between the two sites, where the sites are equivalent for the binding of the first Cu2+ ion, but the occupation of the first site lowers the activation energy of the binding of the second Cu2+ ion. This indicates that binding of Cu2+ ion at its site on one subunit brings about conformational changes that facilitate Cu2+ binding on the other subunit. These results may relate to possible intersubunit interactions during the catalytic activity.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Dosage effect of SOD-A gene in 21-trisomic cells

Summary SOD-A activity was measured by the polarographic method of catalytic currents in 30 subjects 10–24 years of age with a karyotype 47, +21, and 30 age matched normal subjects. The ratio of the average amount of SOD-A in 21-trisomic cells to the average amount in normal cells is 1.4495. Statistical analysis confirms that this ratio does not differ significantly from the ratio 1.5 expected in the case of a simple gene dosage effect.This work was supported by a grant of the Ministry of Health, No. 500.6/1143.     

A rotating disk electrode for kinetic studies of superoxide dismutases: applicability in a wide pH range and for continuous monitoring of enzyme inactivation

A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.     

Single cell studies of the cell cycle and some models

Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models.     

An approach to large scale identification of non-obvious structural similarities between proteins

Background

A new sequence independent bioinformatics approach allowing genome-wide search for proteins with similar three dimensional structures has been developed. By utilizing the numerical output of the sequence threading it establishes putative non-obvious structural similarities between proteins. When applied to the testing set of proteins with known three dimensional structures the developed approach was able to recognize structurally similar proteins with high accuracy.

Results

The method has been developed to identify pathogenic proteins with low sequence identity and high structural similarity to host analogues. Such protein structure relationships would be hypothesized to arise through convergent evolution or through ancient horizontal gene transfer events, now undetectable using current sequence alignment techniques. The pathogen proteins, which could mimic or interfere with host activities, would represent candidate virulence factors.The developed approach utilizes the numerical outputs from the sequence-structure threading. It identifies the potential structural similarity between a pair of proteins by correlating the threading scores of the corresponding two primary sequences against the library of the standard folds. This approach allowed up to 64% sensitivity and 99.9% specificity in distinguishing protein pairs with high structural similarity.

Conclusion

Preliminary results obtained by comparison of the genomes of Homo sapiens and several strains of Chlamydia trachomatis have demonstrated the potential usefulness of the method in the identification of bacterial proteins with known or potential roles in virulence.