An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro.
After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series. (+info)
The interaction of rhodium(II) carboxylates with enzymes.
The effect of rhodium(II) acetate, propionate, and methoxyacetate on the activity of 17 enzymes was evaluated. The enzymes were preincubated with the rhodium(II) complexes in order to detect irreversible inhibition. All enzymes that have essential sulfhydryl groups in or near their active site were found to be irreversibly inhibited. Those enzymes without essential sulfhydryl groups were not affected. In each case, the rate of inactivation closely paralleled the observed toxicity and antitumor activity of rhodium(II) carboxylates; that is, rhodium(II) propionate greater than rhodium(II) acetate greater than rhodium(II) methoxyacetate. In addition, those enzymes that have been demonstrated to be most sensitive to established sulfhydryl inhibitors, such as glyceraldehyde-3-phosphate dehydrogenase, were also most sensitive to rhodium(II) carboxylate inactivation. Proton nuclear magnetic resonance measurements made during the titration of rhodium(II) acetate with cysteine showed that breakdown of the carboxylate cage occurred as a result of reaction with this sulfhydryl-containing amino acid. (+info)
Glycopeptides from the surgace of human neuroblastoma cells.
Glycopeptides suggesting a complex oligosaccharide composition are present on the surface of cells from human neuroblastoma tumors and several cell lines derived from the tumors. The glycopeptides, labeled with radioactive L-fucose, were removed from the cell surface with trypsin, digested with Pronase, and examined by chromatography on Sephadex G-50. Human skin fibroblasts, brain cells, and a fibroblast line derived from neuroblastoma tumor tissue show less complex glycopeptides. Although some differences exist between the cell lines and the primary tumor cells, the similarities between these human tumors and animal tumors examined previously are striking. (+info)
The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout.
1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history. (+info)
Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.
1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined. (+info)
Carbon 13 NMR study of nonenzymatic reactions of pyridoxal 5'-phosphate with selected amino acids and of related reactions.
Carbon 13 nuclear magnetic resonance spectroscopy has been used to monitor the nonenzymatic reactions of pyridoxal 5'-phosphate with glycine, alanine, valine, serine, and with several other model compounds. Isotopically enriched amino acids were employed so that low concentrations could be utilized while still allowing relatively rapid acquisition of spectral data. The results for alanine and serine are particularly noteworthy in that alanine is deaminated to pyruvate and pyruvate is aminated to alanine, but contrary to the enzymatic reactions of various serine dehydratases wherein serine is converted to pyruvate, the nonenzymatic reaction utilizing serine results in hydroxypruvate rather than pyruvate formation. In the reverse reaction, hydroxypyruvate is aminated to serine but very inefficiently relative to the amination of pyruvate to alanine. The experimental results have been formulated into a proposed reaction mechanism for deamination of amino acids by pyridoxal-P. (+info)
Herpetic keratitis. Proctor Lecture.
Although much needs to be learned about the serious clinical problem of herpes infection of the cornea, we have come a long way. We now have effective topical antiviral drugs. We have animal models which, with a high degree of reliability, clearly predict the effect to be expected clinically in man, as well as the toxicity. We have systemically active drugs and the potential of getting highly active, potent, completely selective drugs, with the possibility that perhaps the source of viral reinfection can be eradicated. The biology of recurrent herpes and stromal disease is gradually being understood, and this understanding may result in new and better therapy of this devastating clinical disease. (+info)
Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.
The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c. (+info)