The activation of human type IV collagenase proenzyme. Sequence identification of the major conversion product following organomercurial activation. (41/109)

Type IV collagenase is a metalloproteinase which cleaves type IV collagen in a pepsin-resistant domain. Organomercurial activation of the latent 70-kDa type IV collagenase (type IV procollagenase) results in the autocatalytic removal of an amino-terminal domain resulting in the conversion to a 62-kDa activated form of the enzyme. Synthetic peptides corresponding to domains from the amino terminus (residues 1-17) and an internal domain near the carboxyl terminus (residues 472-490) were used as antigens to generate affinity-purified polyclonal antibodies which recognized their respective domains on the native type IV procollagenase. Western immunoblotting studies of the time course of the organomercurial activation process demonstrate a direct loss of the amino-terminal domain during the conversion to the lower molecular weight form. The amino-terminal sequence of the purified type IV procollagenase before and after activation reveals cleavage at a single locus with removal of residues 1-80, generating a new amino terminus YNFFPRKPKWDKNQ. This results in the removal of three distal cysteine residues located at positions 31, 36, and 73. The type IV collagenase site of autocatalytic cleavage corresponds exactly to the homologous sites of type I collagenase and stromelysin cleavage during their respective organomercurial activation. This site is adjacent to the carboxyl end of a highly conserved region consisting of the sequence PRCGVPDV, which contains an unpaired cysteine residue.  (+info)

Reagents specific for cell surface components. (42/109)

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.  (+info)

Mapping of adenovirus late promoters with nascent mercurated RNA. (43/109)

Nascent RNA molecules were labeled in vivo and elongated in vitro by incubation of the isolated nuclei in the presence of mercurated nucleotides. The RNA molecules initiated and labeled in vivo and elongated in vitro were then selectively purified on a thiopropyl 6-B Sepharose affinity column. The procedure was shown to be free of artifacts since the addition of mercurated nucleotides and the retention on the affinity column is mediated by the endogenous RNA polymerase II (nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), is sensitive to actinomycin D, and is dependent on the presence of all four ribonucleotide triphosphates. This general procedure was applied to the mapping of viral promoters late after adenovirus 2 infection of HeLa cells. RNA purified as described above was hybridized to restriction enzyme fragments attached to nitrocellulose filters. The 5' ends of the nascent RNA chains are located in coordinates 9.5-17 for a rightward transcript, 0-25 for a leftward transcript, and possibly 60-70 for a second rightward transcript. These locations clearly differ from locations of the early promoters and therefore suggest that the transition from early to late functions is controlled at the transcriptional level.  (+info)

Ligands containing heavy atoms: perturbation of phosphorescence of a tryptophan residue in the binding site of wheat germ agglutinin. (44/109)

Information on the structure of binding sites of wheat germ agglutinin was obtained on the basis of fluorescence and phosphorescence changes of tryptophan residues induced by the binding of several thiomercuribenzoate derivatives of glycosides. The thiomercuribenzoate derivatives bind selectively to wheat germ agglutinin in the same way as the corresponding sugars. Using the thiomercuribenzoate of di-N-acetyl-beta-chitobiose, it was found that: (i) the fluorescence of tryptophan residues was drastically quenched at both 298 and 77 K; (ii) the phosphorescence intensity was strongly enhanced at 77 K; (iii) the phosphorescence lifetime was markedly decreased. A similar effect was observed with the thiomercuribenzoate of N-acetyl-beta-D-glucosamine. These changes were completely reversed upon addition of 1-O-methyl-di-N-acetyl-beta-chitobioside. The thiomercuribenzoate of beta-D-glucose had no effect at all, and the thiomercuribenzoate of tri-N-acetyl-beta-chitotriose had a limited effect. These results are interpreted as a specific heavy atom effect due to a close contact between one tryptophan residue of the protein and the heavy atom of the bound ligand. They are consistent with the view that: (i) binding sites of wheat germ agglutinin may be divided in three subsites, A, B, and C; (ii) a tryptophan residue is in the binding site at subsite C; and (iii) this residue and the ligand are in close contact. This new method, using the enhancement of spin-orbit coupling due to the selective perturbation induced in a tryptophan residue by a ligand containing a heavy atom, has proved to be suitable for locating the tryptophan residue in the binding site of wheat germ agglutinin and can probably be extended to other sugar-binding proteins.  (+info)

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate. (45/109)

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.  (+info)

Evidence for changes in the conformational status of rat liver microsomal glucose-6-phosphate:phosphohydrolase during detergent-dependent membrane modification. Effect of p-mercuribenzoate and organomercurial agarose gel on glucose-6-phosphatase of native and detergent-modified microsomes. (46/109)

Comparative studies investigating influences of temperature and time of preincubation on the interactions of an organomercurial agarose gel and p-mercuribenzoate with glucose-6-phosphatase of native and Triton X-114-modified rat liver microsomes were carried out. The effect of p-mercuribenzoate on glucose 6-phosphate hydrolysis is a result of two processes, a moderate membrane perturbation connected with release of some latency and temperature- and time-dependent inhibition of the catalytic activity. Short-term preincubation with both organic mercurials at 37 degrees C is a necessary condition for the entire inhibition of the enzyme activity of native as well as of Triton X-114-modified microsomes. A binding site of the phosphohydrolase itself is accessible to p-mercuribenzoate and the phenyl mercury residue of the affinity gel from the cytoplasmic surface even in native microsomes. Kinetic analyses reveal a formally competitive mechanism of inhibition using native microsomes, but the kinetic picture changes to a noncompetitive pattern of Lineweaver-Burk plots when the inhibitor-loaded microsomes are modified optimally by Triton X-114. This behavior can be evaluated as the first convincing evidence for drastic changes of the conformational status of the phosphohydrolase during the membrane modification process. A combined conformational flexibility-substrate transport model characterizing the microsomal glucose-6-phosphatase as an integral channel-protein embedded within the hydrophobic interior of the membrane is proposed.  (+info)

The activation of human skin fibroblast procollagenase. Sequence identification of the major conversion products. (47/109)

Human skin collagenase is secreted by cultured fibroblasts in a proenzyme form and can be activated to a catalytically competent enzyme by a number of processes. All modes of activation studied lead to conversion of the proenzyme to a stable 42-kDa active enzyme, concomitant with removal of an 81-amino acid peptide from the amino-terminal end of the molecule. The sequence of events leading to the formation of this enzyme form has been determined by analyzing the primary structure of the conversion intermediates. Trypsin-induced activation of procollagenase occurs as a result of the initial cleavage of the peptide bond between Arg-55 and Asn-56, generating a major intermediate of 46 kDa. Treatment of the proenzyme with organomercurials, which have no intrinsic ability to cleave peptide bonds, initially results in activation of the enzyme without loss of molecular weight. This is followed by conversion to two lower molecular weight species of 44 and 42 kDa, the latter corresponding to the stable active enzyme form. The final cleavage producing this form of collagenase is not restricted to a single polypeptide bond but can occur on the amino-terminal side of any one of three contiguous hydrophobic residues, Phe-100, Val-101, Leu-102. The data suggest that both trypsin and organomercurials activate procollagenase by initiating an intramolecular autoproteolytic reaction resulting in the formation of a stable 42-kDa active enzyme species.  (+info)

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358. (48/109)

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA X DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 determinant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg2+-resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C--Hg bond of phenylmercury.  (+info)