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B S Cooperman

Publications and source records attributed to B S Cooperman.

At least 109 records · Page 6Linked to original sources
PubMed1986

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding.

In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.

Affinity Labels↗
PubMed1986

Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase.

Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].

Amino Acids↗
PubMed1985

Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center.

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

Acyltransferases↗
PubMed1985

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin.

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.

Affinity Labels↗
PubMed1985

On the structural specificity of puromycin binding to Escherichia coli ribosomes.

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.

Binding Sites↗
PubMed1985

Photoaffinity labeling of the tetracycline binding site of the Escherichia coli ribosome. The uses of a high intensity light source and of radioactive sancycline derivatives.

[3H]Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome. However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity. In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of [3H]TC with a common, rather low intensity, lamp. On the basis of the results of this study we next explored the usefulness for photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself. Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site.

Affinity Labels↗
PubMed1984

Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits.

We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and function of Escherichia coli ribosomes. The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins. Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles. The reconstituted subunits contain a full complement of proteins and are active in ribosomal function assays, whereas the slower sedimenting particles lack several proteins and have little or no activity.

Affinity Labels↗
PubMed1984

Yeast inorganic pyrophosphatase. A model for active-site structure based on 113Cd2+ and 31P NMR studies.

Equilibrium dialysis and 113Cd2+ NMR studies in the presence of inorganic phosphate (Pi) provide clear evidence for the existence of four well-defined Cd2+ sites per yeast inorganic pyrophosphatase subunit. Parallel 31P NMR studies demonstrate the existence of two binding sites per subunit for Pi and provide strong confirmatory evidence for a small amount of enzyme-bound inorganic pyrophosphate in equilibrium with enzyme-bound Pi. Such inorganic pyrophosphate formation was demonstrated by chemical analysis earlier [Welsh, K.M., Armitage, I.M., & Cooperman, B.S. (1983) Biochemistry 22, 1046-1054]. In this same earlier paper, we provided evidence for inner-sphere contact between enzyme-bound Cd2+ and Pi bound in the higher affinity of the two Pi sites. We now present heteronuclear decoupling evidence that one to three different Cd2+ ions make such contact. The divalent metal ion cofactor conferring the highest activity on inorganic pyrophosphatase is Mg2+, and we present evidence from competition experiments that such inner-sphere contact is also likely for the Mg2+-enzyme. On the other hand, these experiments also show that some metal ion binding sites on the enzyme bind Mg2+ and not Cd2+, and some bind Cd2+ and not Mg2+. These results are considered along with others obtained recently in proposing an active-site structure for inorganic pyrophosphatase.

Binding Sites↗
PubMed1984

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate.

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.

Adenosine Triphosphate↗
PubMed1984

Mechanism of tetracycline phototoxicity.

Studies were made to determine factors important in the phototoxicity mechanism of 7 clinically used tetracyclines (TC). The clinical phototoxicity, the rates of photochemical degradation, and the in vitro phototoxicity of the TCs were qualitatively but not quantitatively correlated. Phototoxicity in vitro was partially oxygen-dependent and possibly singlet oxygen is involved. The contribution of photoproducts to the phototoxic process may be the basis for the reported differences between the in vivo action spectrum and the absorption spectrum of demethylchlorotetracycline. A mechanistic model for in vivo phototoxicity is proposed where the absorption of UVA radiation by TC leads to at least two main processes: (i) photosensitization by the drug of biologic molecules to cause phototoxicity; (ii) production of one or more photoproducts which photosensitize by absorption of visible radiation.

Humans↗
PubMed1983

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components.

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins. Characteristics of the separation of a complex protein mixture.

We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A. R. Kerlavage, L. Kahan and B. S. Cooperman, Anal. Biochem., 123 (1982) 342-348; A. R. Kerlavage, T. Hasan and B. S. Cooperman, J. Biol. Chem., in press). In the present studies RP-HPLC is shown to yield much greater resolution of these proteins than does size-exclusion HPLC. In addition, we report on various aspects of RP-HPLC of ribosomal proteins including column capacity, resolution, reproducibility, recovery, separation of irreversibly denatured protein, and analysis of affinity-labeled ribosomal protein. The capacity of analytical columns was found to range from several micrograms to several milligrams with minimal loss in resolution and highly reproducible retention values. Recovery varied from protein to protein and ranged from 27% to 91%, with an average total protein recovery of 70%. The partitioning of several proteins between two peaks was shown to be due to irreversible denaturation of a small fraction. Finally, the utility of RP-HPLC in the study of the ribosome was demonstrated by analyses of [3H]puromycin-labeled ribosomal proteins, and the demonstration that labeling slightly alters protein elution.

Bacterial Proteins↗
PubMed1983

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms.

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.

Chromatography, High Pressure Liquid↗