Search PubMed⌕ Search

Biomedical subjects

J Byrd

Publications and source records attributed to J Byrd.

9 recordsLinked to original sources
PubMed1988

X-ray absorption studies of yeast copper metallothionein.

The local structures of the metal sites in copper metallothionein from Saccharomyces cerevisiae have been investigated by x-ray absorption spectroscopy at the copper and sulfur K edges. Analysis of the EXAFS (extended x-ray absorption fine structure) data indicates that each copper is trigonally coordinated to sulfur at a distance of 2.23 A. Cu-Cu interactions at 2.7 and 3.9 A have also been tentatively identified. Sulfur K edge data are compatible with cysteinyl thiolates bridging each of the eight Cu(I) ions. The data support a model for the copper cluster in yeast metallothionein consisting of a Cu8S12 core. EXAFS data on two specifically engineered carboxyl-terminal truncated mutants reveal that the copper coordination in the mutants is similar to that observed in the wild-type protein.

Algorithms↗
PubMed1988

Effect of mutation of cysteinyl residues in yeast Cu-metallothionein.

Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1 mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant metallothioneins, pairs of cysteinyl residues have been converted to seryl residues. The mutant proteins differ only in the positions of the double substitutions; each mutant molecule contains 10 cysteinyl residues. Each mutant protein lacks the first 8 residues at the amino terminus from the decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53 residues analogous to the wild-type metallothionein and are designated 9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of the Cys----Ser conversions). The properties of the mutant metallothioneins are vastly different, and host cells harboring the different plasmid-encoded mutant molecules show marked differences in sensitivity to CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells containing protein 49/50. A CuSO4 concentration of 5 mM was required to inhibit the growth of yeast containing either 49/50 or the wild-type metallothionein. In the purified proteins the copper binding stoichiometry of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26 bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine disulfonate reacted with over 50% of the copper ions in proteins 9/11, 24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50 and wild-type metallothionein were reactive. The thiolates in 9/11, 24/26, and 36/38 were also more reactive in a disulfide exchange reaction with dithiodipyridine compared with the sulfhydryls in 49/50 and the wild-type molecules. The four mutant copper proteins are luminescent and exhibit a similar quantum yield. The cluster structures contributing to the particular electronic transitions are markedly more sensitive to oxygen in proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type molecules. The air-sensitive proteins exhibit a tertiary fold not recognized by polyclonal antibodies directed to a conformational epitope on yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a concentration-dependent fashion similar to the wild-type protein. Mutation of 2 cysteinyl residues in the carboxyl portion of metallothionein does not significantly alter properties of the molecule, whereas mutation of several cysteines in the amino-terminal portion of the molecule yields a different conformation.

Carrier Proteins↗
PubMed1988

Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants.

Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.

Amino Acids↗
PubMed1988

The effect of internal visualization on digit span performance.

Fifty-two subjects were randomly assigned to one of four conditions according to a 2 x 2 factorial design in order to determine the effect of internal visualization on short-term memory for digits as measured by the Wechsler Digit Span subtest. Surprisingly, internal visualization (imaging with eyes closed) was found not to affect Digit Span performance significantly.

Adult↗
PubMed1987

Structural and functional studies of the amino terminus of yeast metallothionein.

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.

Amino Acid Sequence↗
PubMed1986

Cooperative cluster formation in metallothionein.

An ion-exchange chromatography procedure was used to resolve apometallothionein from the metallo- form in a study of metal-thiolate cluster formation. Chromatography of metallothionein reconstituted with Cd(II), Zn(II), or Cu(I) at neutral pH on carboxymethyl-cellulose led to removal of apoprotein from a solution without effect on recovery of the metalloprotein. Analysis of the effluent revealed apparent cooperative binding of these metal ions to the protein. Addition of 1-4 mol eq Cd(II) ions led to the recovery of metallothionein with around 4 mol eq Cd bound. The yield of this form increased with increasing starting metal ion equivalency. These results were obtained with two different ion-exchange resins. The cooperativity of binding was not total, but was initially confined to the carboxyl-terminal alpha domain. The results of metal and protein yields are inconsistent with random, noninteractive binding. Similar data were obtained with Zn(II) and Cu(I) ions although Cu(I) exhibited initial cooperative binding within the amino-terminal beta domain with over 5 mol eq Cu(I) bound.

Allosteric Site↗
PubMed1986

Cancer-associated colonic mucin in cultured human tumor cells and athymic (nude) mouse xenografts.

Mucins derived from colonic cancers differ immunologically and chemically from those in normal colonic epithelium. It has been demonstrated that the lectin from the peanut will bind to mucins present in colonic cancers and other neoplastic lesions but not to those from the normal colon. It was hypothesized, therefore, that in transformed colonic epithelium the glycosylation of mucins occurs differently than in normal epithelium. To rule out the possibility that the differences in oligosaccharide structure were due to postsecretory degradation, studies were designed to evaluate cancer-associated colonic mucins produced under more controlled conditions. We studied nine different cancer cell lines first in monolayer culture and then as xenografts in athymic or nude mice. Eight of the nine cell lines in monolayer culture synthesized glycoconjugates that were labeled by fluorescein-conjugated lectins. After injection into nude mice, eight of the nine cell lines produced tumors typical of human colonic cancer, and six of nine secreted mucin. The mucins produced by the xenografts were labeled at fluorescence microscopy by peanut lectin and other lectins, characteristic of what had been seen in other primary human colonic cancers. One cell line, LS174T, produced large amounts of mucin in the xenograft model. Mucin was purified from these tumors and characterized biochemically. It was demonstrated that mucin purified from the xenografts bound peanut lectin. Therefore, we have concluded that cancer-associated mucins are present in cultured colorectal tumor cells. The cancer-associated mucins are also found in nude mouse xenografts, indicating that they are not the result of postsecretory degradation by colonic flora or by tumor cell necrosis. The cell culture and xenograft can therefore be useful for studying the biosynthesis of cancer-associated mucins.

Animals↗