Search PubMed⌕ Search

Biomedical subjects

G T Mullenbach

Publications and source records attributed to G T Mullenbach.

At least 19 recordsLinked to original sources
PubMed1998

Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality.

Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo.

Amino Acid Sequence↗
PubMed1995

Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity.

A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln. This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site. This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme. The analog described here has afforded for the first time a monomeric superoxide dismutase with substantial activity. This point mutation does not significantly influence the protein structure but interactions with anions, including superoxide, are altered with respect to the monomeric form. The present monomeric Glu133Gln mutant has partially restored enzymic activity. The diminished activity of the monomeric analogs is discussed in the light of possible minor structural changes and some of their characteristics are compared with those of naturally occurring mutants associated with various neurological diseases.

Binding Sites↗
PubMed1995

Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat.

Transfection of murine NIH3T3 fibroblasts and human MCF7 breast carcinoma cells with a pSV2-derived eukaryotic expression vector for human cytosolic glutathione peroxidase resulted in clones with increased glutathione peroxidase activity. This heterologous expression indicates that murine cells recognize the human "selenocysteine insertion sequence" in the 3' untranslated region of the mRNA which facilitates insertion of selenocysteine directed by the opal codon. Though most clones from both cell lines eventually lost their enhanced glutathione peroxidase activity despite continuous selection on G418, some NIH3T3 clones retained enhanced enzyme activity without continuous G418 exposure. Transfection of MCF7 cells with an Epstein-Barr virus (EBV)-derived episomally replicating expression vector carrying the glutathione peroxidase gene also revealed increased glutathione peroxidase activity. These MCF7 cells, however, all required exposure to G418 to maintain enhanced glutathione peroxidase activity. Detailed biochemical analysis of a stably expressing NIH3T3 clone and MCF7 expressing cells revealed no alterations in activities of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, phospholipid-glutathione peroxidase, glutathione reductase, glutathione transferase, or NADPH-P450 reductase. Both pSV2- and EBV-derived glutathione peroxidase-expressing clones exhibited enhanced resistance to paraquat as well as to peroxides.

3T3 Cells↗
PubMed1995

Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain.

The human urokinase plasminogen activator (uPA) epidermal growth factor-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast. Purified human uPA 1-48 and uPA 1-48glu complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.

Amino Acid Sequence↗
PubMed1994

Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis.

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[Ser]Sec did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA[Ser]Sec.

Acylation↗
PubMed1994

A spectroscopic characterization of a monomeric analog of copper, zinc superoxide dismutase.

A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and 1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog. Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.

Circular Dichroism↗
PubMed1993

Glutathione peroxidase protects cultured mammalian cells from the toxicity of adriamycin and paraquat.

Dihydrofolate reductase-minus mutants of Chinese hamster ovary cells were depleted of glutathione peroxidase by transcription of the transfected bovine cDNA in inverted orientation upstream from the cDNA for dihydrofolate reductase to engender a bicistronic mRNA. In a clone of cells selected for expression of dihydrofolate reductase by the ability to grow in nucleoside-free medium the activity of glutathione peroxidase was reduced to 20% of the activity in the untransfected parental line of cells (DG44). The cells depleted of glutathione peroxidase were more sensitive to the toxicities of paraquat and adriamycin than the untransfected parental cells from which they derived but not more sensitive to bleomycin, menadione, or phenazine methosulfate. That the mildly increased sensitivity to paraquat and adriamycin was the consequence of the diminished cellular content of glutathione peroxidase was confirmed by the increase in sensitivity of untransfected cells after treatment with buthionine sulfoximine, an agent which depletes cells of glutathione. These and other data strongly suggest that the enzymic action of glutathione peroxidase protects cells from the toxicity of paraquat and adriamycin. The toxin which these agents engender is likely to be hydrogen peroxide or another hydroperoxide upon which glutathione peroxidase acts.

Animals↗
PubMed1992

Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1).

Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium.

Amino Acid Sequence↗
PubMed1991

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines.

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.

Amino Acid Sequence↗
PubMed1989

Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives.

CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons. Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits. The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene. The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence. Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli. The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface. The polymers are soluble, thermostable, and of near normal specific activity. Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits. The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active. Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats. The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min.

Amino Acid Sequence↗
PubMed1989

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini.

Mutations were directed to specific regions of the human tissue-type plasminogen activator (t-PA) gene in an effort to better define structure-function relationships of the enzyme. Three types of modifications were effected by in vitro mutagenesis: elimination of glycosylation sites; substitutions of amino acids at the cleavage site for conversion of single-chain t-PA to two-chain t-PA; and truncations of the N- and C-termini. Thirteen variants were purified from permanent CHO cell lines and analyzed for specific activity, fibrin stimulation, fibrin binding, inhibition by plasminogen activator inhibitor-2 (PAI-2) and half-life. The results of these analyses are: (i) variants with carbohydrate-depleted kringle domains possessed higher specific activities than wild-type t-PA; (ii) a cleavage site variant substituted at Arg275 with Gly had greatly reduced specific activity; (iii) two variants substituted at Lys277 exhibited altered interactions with PAI-2; (iv) the variant with a truncated C-terminus had reduced activity in the absence of fibrin; and (v) no variants had significantly altered half-lives. In order to test the effects of combining mutations, four additional variants were produced. Each combination variant retained at least one of the altered properties observed in the original variants, and in three of the variants the diverse properties were additive.

Amino Acid Sequence↗
PubMed1988

Superoxide mediates the toxicity of paraquat for cultured mammalian cells.

To establish some of the necessary steps in the toxic action of paraquat for cultured mammalian cells, we isolated paraquat-resistant HeLa cells after lethal increments in concentration of paraquat in the medium. The paraquat-resistant cells had increased the cellular content of both the Mn-containing and the CuZn-containing superoxide dismutases. The effect of paraquat on the consumption of oxygen by the wild-type and the resistance cells was similar; in both cases exposure to paraquat for 24 or more hours produced similar proportions of cyanide-resistant consumption of oxygen, suggesting that paraquat entered both cells, underwent reduction, and donated electrons to molecular oxygen. When cultivated for 5 months in the absence of paraquat the paraquat-resistant cells maintained the increased cellular content of superoxide dismutases and remained resistant to paraquat. This observation suggested that resistance to paraquat might be caused by enrichment for the two superoxide dismutases and, further, that the increased cellular content of the two enzymes was not a response to growth under stressful conditions. NIH/3T3 cells whose content of superoxide dismutase was increased by transcription of the transfected cDNA for the human CuZn superoxide dismutase were also resistant to paraquat, suggesting strongly that paraquat promotes the formation of O2- as a necessary part of its cytotoxic effects in two types of cultured mammalian cells.

Base Sequence↗
PubMed1988

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases.

The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.

Animals↗
PubMed1987

Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis.

The active site arginine-143 of human Cu,Zn superoxide dismutase has been replaced by lysine or by isoleucine. The mutant proteins were expressed at high levels in yeast, purified, and the amino acid substitution explored through the use of group specific reagents. The specific activities of these enzymes, measured by the xanthine oxidase/cytochrome c method and by using dry weight determination to establish protein concentration, were: native enzyme, 6570 units/mg; Lys-substituted enzyme, 2840 units/mg, Ile-substituted enzyme, 708 units/mg. The active site arginine thus plays an important, but not an essential, role in the catalytic process.

Amino Acid Sequence↗
PubMed1986

Chemical synthesis and expression in yeast of a gene encoding connective tissue activating peptide-III. A novel approach for the facile assembly of a gene encoding a human platelet-derived mitogen.

A synthetic gene encoding the platelet-derived factor, connective tissue activating peptide-III (CTAP-III) (Castor, C.W., Miller, J.W., and Walz, D.A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 765-769), has been expressed and secreted from Saccharomyces cerevisiae by using a yeast expression vector and an alpha-factor leader segment. Mitogenic activity reported for naturally derived CTAP-III has been demonstrated here for recombinant CTAP-III. Active CTAP-III has been purified to apparent homogeneity. Structural studies have confirmed its identity. A general approach for the facile synthesis of genes is presented which has permitted the assembly of the entire structural gene and flanking regions (280 base pairs) from 20 oligomers in a single annealing and ligation reaction pool.

Amino Acid Sequence↗