Biomedical subjects

S M Schuster

Publications and source records attributed to S M Schuster.

At least 37 records · Page 2Linked to original sources

PubMed1996

Mutations at histidine 211 of the yeast F1-ATPase beta-subunit affect the stability and assembly of the ATPase and the structure of the active site.

The role of H211 of the yeast F1-ATPase beta-subunit was investigated by site-specific mutagenesis and characterization of the resulting enzymes. Five amino acids (N, D, I, K, and A) were substituted for H211 of the ATP2 gene. The mutated genes were expressed in an atp2::LEU2 host, and only yeast expressing H211N respired aerobically. The respiratory phenotypes of the other four mutants were suppressed by a second site mutation (L203F). The ATPases from the single mutant strains were unstable when removed from the mitochondrial inner membrane, preventing purification. Submitochondrial particles were prepared from each strain and the activities were stable under a variety of conditions, allowing determination of Vmax and Km for ATP hydrolysis. Mutations of H211 caused increases in Km of 3.7- to 7.4-fold, while L203F had little effect. The suppressive effect of the L203F mutation was also expressed in the Km values of the double mutant strains. The ATPases from the H211 mutants had diminished sensitivity to oligomycin, and their pH optima were 1.5-2.0 units less than the wild-type optimum. Values of pKa for the groups involved in catalysis were estimated for the wild-type enzyme and three H211 mutants (N, D, and K). Each mutant enzyme showed a substantial decrease in the pKa of the group(s) which serves as a base in acid-base catalysis. The results of this study demonstrate that H211 is important in maintaining the structure of the wild-type enzyme complex and also contributes to the structure of the active site. L203 is also required for the stability of the enzyme complex and may have a structural or functional interaction with H211. Neither H211 nor L203 is required for catalysis by F1.

Binding Sites↗

PubMed1996

Autoimmune diabetes-prone NOD mice express the Lyt2 alpha (Lyt2.1) and Lyt3 alpha (Lyt3.1) alleles of CD8.

Predisposition to Type I insulin-dependent diabetes (IDD) has a strong underlying genetic basis involving class II major histocompatibility complex (MHC) genes as well as several non-MHC genetic systems. In the non-obese diabetic (NOD) mouse, a model for human IDD, genes associated with the appearance of immune cell infiltrates in the pancreatic islets (insulitis) and/or overt IDD have been mapped to chromosomes 1, 3, 6, 11, and 17. A recent report has suggested that CD8+ lymphocytes of the NOD mouse might be deficient in the expression of the CD8 beta molecule, a protein encoded by a gene on chromosome 6. The CD8 beta molecule is a T-cell surface marker, the lack of which could affect selection in the thymus, possibly permitting auto-reactive T-cell clones to populate the peripheral lymphoid tissues. For this reason, we examined the expression of the CD8 molecule by lymphocytes in the NOD mouse. Results indicate that the NOD mouse is not deficient in its transcription of detectable mRNA encoding either the CD8 alpha or beta subunits. However, the NOD mouse expresses the Lyt2 alpha and Lyt3 alpha alleles, suggesting that a portion of chromosome 6 centromeric to the diabetes-susceptibility genetic region is derived from an ancestry common to AKR and, like AKR, the CD8 alpha and CD8 beta 3.1 (but not CD8 beta 3.2) subunits are detected on the cell surface of T lymphocytes of the NOD mouse. Interestingly, though, the CD8 beta 3.1 molecule may not be expressed in the NOD mouse to the same extent as it is expressed in the AKR/J mouse, suggesting the possibility that the NOD mouse possesses a defect somewhere between transcription and cell surface expression of the CD8 beta molecule.

Alleles↗

PubMed1996

Current status of the Plasmodium falciparum genome project.

The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.

Animals↗

PubMed1994

Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.

Although Arg-30, Asn-74, and Asn-79 appear totally conserved throughout the purF glutamine-dependent amidotransferases, their potential roles in catalysis and binding remain unexplored for any member of the enzyme family. Here we report the overexpression, purification, and kinetic characterization of a series of AS-B mutants which allow an examination of the functional roles of these 3 residues in glutamine-dependent nitrogen transfer. While Asn-79 appears to possess no catalytic function in AS-B, site-directed mutagenesis of Asn-74 has implicated this residue as playing a role in catalysis of nitrogen transfer from glutamine. The kinetic properties of the Asn-74 AS-B mutant enzymes appear consistent with both ammonia-mediated nitrogen transfer and two apparently novel mechanistic suggestions for this reaction involving either an oxyanion or imide intermediate (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). We also demonstrate that replacement of Arg-30 by an alanine residue yields an AS-B mutant for which the apparent Km for glutamine is increased in the glutamine-dependent synthesis of asparagine. In addition, ATP-dependent stimulation of the glutaminase activity of AS-B is modified or completely eliminated when Arg-30 is replaced by other amino acids. The latter observation may indicate the existence of a molecular switch involving Arg-30 which coordinates the two half-reactions catalyzed by the glutamine-dependent amidotransferases and synthetase domains of cellular AS-B.

Adenosine Triphosphate↗

PubMed1994

Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.

The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants. In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue. Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis. As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R. D., Richards, N. G. J., and Schuster, S. M. (1993) J. Biol. Chem. 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B. In the case of C1A, the inhibition pattern suggests that an abortive complex is formed. This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Animals↗

PubMed1993

Cloning and characterization of subunit genes of ribonucleotide reductase, a cell-cycle-regulated enzyme, from Plasmodium falciparum.

Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. The sequences of full-length clones of the large and small RNR subunits revealed an open reading frame encoding 806 and 349 amino acids, respectively, and showed significant identity with other RNR sequences in the data base. RNA blot analysis showed that the size of the large and small RNR subunit transcripts are 5.4 kb and 2.2 kb, respectively. Both the RNR subunit transcripts fluctuate in level during the cell cycle, reaching a peak preceding maximal DNA synthesis activity. An oligodeoxynucleotide phosphorothioate that is complementary to sequences around the translational initiation codon of the small RNR subunit showed significant inhibition of growth, as measured by the inhibition in DNA synthesis.

Amino Acid Sequence↗

PubMed1993

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.

Animals↗

PubMed1993

Recombinant bovine heart mitochondrial F1-ATPase inhibitor protein: overproduction in Escherichia coli, purification, and structural studies.

A synthetic gene coding for the inhibitor protein of bovine heart mitochondrial F1 adenosine triphosphatase was designed and cloned in Escherichia coli. Recombinant F1-ATPase inhibitor protein was overproduced in E. coli and secreted to the periplasmic space. Biologically active recombinant F1-ATPase inhibitor protein was recovered from the bacterial cells by osmotic shock and was purified to near homogeneity in a single cation-exchange chromatography step. The recombinant inhibitor protein was shown to inhibit bovine mitochondrial F1-ATPase in a pH-dependent manner, as well as Saccharomyces cerevisiae mitochondrial F1-ATPase. Thorough analysis of the amino acid sequence revealed a potential coiled-coil structure for the C-terminal portion of the protein. Experimental evidence obtained by circular dichroism analyses supports this prediction and suggests F1I to be a highly stable, mainly alpha-helical protein which displays C-terminal alpha-helical coiled-coil intermolecular interaction.

Amino Acid Sequence↗

PubMed1993

Glutamine inhibits the ammonia-dependent activities of two Cys-1 mutants of human asparagine synthetase through the formation of an abortive complex.

Cys-1 mutants of recombinant human asparagine synthetase were constructed and their ability to catalyze the glutamine-dependent nitrogen transfer reaction required for asparagine biosynthesis was determined. In agreement with previous work, altering Cys-1 to either Ala or Ser eliminated the glutamine-dependent activity while only minimally affecting the kinetic properties of the ammonia-dependent reaction. A lack of glutaminase activity in these mutants also allowed examination of glutamine binding in studies of the ability of glutamine to inhibit the ammonia-dependent production of asparagine. In both mutants, analysis of the observed kinetics indicated that glutamine inhibited ammonia-dependent asparagine synthesis through the formation of an abortive complex. This unanticipated observation suggests that the commonly accepted mechanism for nitrogen transfer from the primary amide of glutamine to aspartic acid in asparagine synthetase may have to be re-examined. A novel mechanistic proposal which is consistent with the formation of an abortive complex in the two Cys-1 mutants is presented.

Ammonia↗

PubMed1993

Peptide analogs of the beef heart mitochondrial F1-ATPase inhibitor protein.

Peptide analogs which correspond to the conserved region of the natural ATPase inhibitor protein from beef heart, Candida utilis, and Saccharomyces cerevisiae mitochondria were synthesized by solid-phase methodologies and tested for ATPase inhibitory activity. These peptides were found to be potent inhibitors of F1-ATPase-catalyzed ATP hydrolysis in acidic reaction media, having I50 values of 1.1 +/- 0.4 microM, 10 +/- 5 microM, and 48 +/- 19 microM, respectively. These results closely match those obtained for the naturally occurring inhibitor proteins. Additional peptides that correspond to the beef heart beta-subunit near the binding site of the beef heart inhibitor protein and that possess a substantial homology with the conserved region of the inhibitor protein were synthesized. Several of these peptides were found to be inhibitors of the ATPase activity. The best inhibitor, with an I50 value of 20 +/- 3 microM, was the peptide resembling the beef heart beta-subunit comprising amino acids 394-413. This peptide most closely resembles the peptides derived from the conserved region of the inhibitor protein. The insertion of five glycine residues between the charge clusters in the beta-394-413 peptide resulted in a peptide which was able to stimulate the hydrolysis of ATP.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of Saccharomyces cerevisiae DNA damage-responsive protein 48 (DDRP 48).

A yeast protein was purified from wild type Saccharomyces cerevisiae (S. cerevisiae) to near homogeneity using an ethanolamine affinity chromatography procedure. The N-terminal amino acid sequencing and the amino acid composition analyses identified this protein as the product of the second open reading frame of S. cerevisiae DNA Damage-responsive gene 48 (DDR48) (Treger, J.M., and McEntee, K. (1990) Mol. Cell. Biol. 10, 3174-3184) The first methionine residue encoded by the translation starting codon was not present in the mature protein which is designated as DDRP 48. DDRP 48 was found to be a negatively charged and highly hydrophilic glycoprotein. The glycosidase cleavage analyses suggested that DDRP 48 was mainly N-link-glycosylated. The apparent molecular mass of DDRP 48 was estimated to be approximately 65 kilodaltons. DDRP 48 was found able to hydrolyze ATP and GTP yielding PPi. The Km values for ATP and GTP are 0.29 mM and 0.58 mM, respectively. The Western blot analysis demonstrated that DDRP 48 was expressed to various concentrations in different S. cerevisiae strains. Increased DDRP 48 abundance was observed after yeast cells carrying the wild type RAD 52 gene were exposed to either ethylmethane sulfonate or heat shock treatments. After similar DNA-damaging treatments, however, no significant inductions of DDRP 48 were found in a rad 52 mutant strain. These observations are consistent with the predictions resulting from previous studies on transcriptional regulation of the DDR 48 gene (Maga, J.A., McClanahan, T.A., and McEntee, K. (1986) Mol. & Gen. Genet. 205, 276-284; McClanahan, T., and McEntee, K. (1986) Mol. Cell. Biol. 6, 90-96).

Amino Acid Sequence↗

PubMed1993

Interaction of the eucaryotic peptide chain initiation factor eIF-4A with the specific elements at the 5'-untranslated sequence of human asparagine synthetase mRNA.

The resistance of certain tumor cells to the chemotherapeutic agent L-asparaginase has often been found to be associated with the presence of asparagine synthetase activity. In an attempt to study the translational regulation of the asparagine synthetase gene, the 5'-untranslated region of human asparagine synthetase cDNA was mapped by antisense oligonucleotide-mediated hybrid arrest translation in reticulocyte lysate. Three consecutive cis-acting regulatory elements, spanning from -60 to -120 bases from the initiation codon, in the 5'-untranslated region of the asparagine synthetase gene, were identified. T1 RNase footprinting analysis showed that those regulatory elements can be protected from T1 digestion when incubated with reticulocyte lysate. A 46-kDa trans-acting protein factor that interacts with the cis-acting regulatory element of asparagine synthetase mRNA was detected. This 46-kDa protein factor is most likely to be the eucaryotic peptide chain initiation factor eIF-4A as determined by immunoprecipitation experiments using a monoclonal antibody raised against reticulocyte eIF-4A.

Aspartate-Ammonia Ligase↗

PubMed1993

A specific quantitative colorimetric assay for L-asparagine.

When an aqueous L-asparagine solution was mixed with a dilute ethanolic ninhydrin solution and incubated at temperatures lower than 37 degrees C, the resulting mixture exhibited an ultraviolet (uv)-visible absorption spectrum with the maximum absorption at 340-350 nm. In contrast, the mixtures of several other amino acids with ninhydrin yielded Ruhemann purple (S. Ruhemann, 1910, J. Chem. Soc. 97, 1438-1449, 1910; S. Ruhemann, 1910, J. Chem. Soc. 97, 2025-2031) and the corresponding uv-visible spectra had absorption maxima at 405 and 570 nm. The effects of several factors including the reaction temperature, the ninhydrin concentration, and pH on the asparagine-ninhydrin reaction were investigated to optimize the specificity and sensitivity. As a result, a simple and specific colorimetric asparagine assay was developed. Using the assay protocol, the absorption of the asparagine-ninhydrin mixtures at 340 nm had a linear relationship with the asparagine concentration in the range of 50 microM to 50 mM, even in the presence of a high background of other amino acids. The application of this assay could be easily extended to more complex enzymatic reaction systems. When the enzyme activities of L-asparagine synthetases from different species and commercial L-asparaginase were measured with both the ninhydrin colorimetric procedure and the HPLC amino acid analysis, comparable results were obtained. While the chemistry of this novel asparagine-ninhydrin reaction is not fully understood, the colorimetric asparagine assay reported herein is of great practical value because it is specific, sensitive, simple, and extremely inexpensive.

Asparaginase↗

PubMed1993

Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.

A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

Amino Acid Sequence↗

PubMed1992

An alternative mechanism for the nitrogen transfer reaction in asparagine synthetase.

In the absence of crystallographic data, the mechanism of nitrogen transfer from glutamine in asparagine synthetase (AS) remains under active investigation. Surprisingly, the glutamine-dependent AS from Escherichia coli (AsnB) appears to lack a conserved histidine residue, necessary for nitrogen transfer if the reaction proceeds by the accepted pathway in other glutamine amidotransferases, but retains the ability to synthesize asparagine. We propose an alternative mechanism for nitrogen transfer in AsnB which obviates the requirement for participation of histidine in this step. Our hypothesis may also be more generally applicable to other glutamine-dependent amidotransferases.

Amino Acid Sequence↗

PubMed1992

The topology of the glutamine and ATP binding sites of human asparagine synthetase.

Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.

Adenosine Triphosphate↗

PubMed1992

Monoclonal antibodies specific for mercuric ions.

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by injection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCl, but not with BSA-glutathione, were isolated from the spleen of a mouse given multiple injections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb 1F10. The binding of both antibodies to immobilized BSA-glutathione-HgCl was inhibited by soluble HgCl2, and dissociation constants for mercuric chloride binding were 2.3 and 3.7 nM for mAbs 4A10 and 1F10, respectively. Both antibodies bound mercuric acetate with similar affinities, demonstrating that the antibodies were capable of binding to mercuric ions in the presence of a different counterion than the one used in the immunogen. Reactions were not observed with other metal cations by either antibody. These data demonstrate the successful induction of antibodies that react very specifically with mercuric ions in solution regardless of the presence of a carrier.

Amino Acid Sequence↗