W Burkhart

Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, United States

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Publications (43)212.67 Total impact

  • Biochemistry 09/2003; 42(37):11092-11092. · 3.19 Impact Factor
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    ABSTRACT: TNF alpha converting enzyme (TACE) processes precursor TNF alpha between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNF alpha cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNF alpha cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNF alpha release. The specificity constants for TNF alpha cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNF alpha at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNF alpha between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH(2) was not cleaved at all by TACE, while collagenase (MMP 1), gelatinase (MMP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage.
    Biochemistry 08/2002; 41(30):9462-9. · 3.19 Impact Factor
  • Biochemistry 07/2002; 41(30):9462-9469. · 3.19 Impact Factor
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    ABSTRACT: Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.
    Journal of Biological Chemistry 12/2001; 276(47):43958-69. · 4.60 Impact Factor
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    ABSTRACT: Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.
    Journal of Biological Chemistry 07/2001; 276(22):19363-74. · 4.60 Impact Factor
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    ABSTRACT: In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.
    Hybridoma 07/2001; 20(3):149-57. · 0.24 Impact Factor
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    ABSTRACT: Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.
    FEBS Letters 04/2001; 492(1-2):166-70. · 3.34 Impact Factor
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    ABSTRACT: Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.
    Protein Science 02/2001; 10(3):471 - 481. · 2.86 Impact Factor
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    ABSTRACT: Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.
    Journal of Biological Chemistry 11/2000; 275(42):32585-91. · 4.60 Impact Factor
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    ABSTRACT: Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.
    Biochemical and Biophysical Research Communications 01/2000; 266(1):141-6. · 2.28 Impact Factor
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    ABSTRACT: The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.
    Journal of Biological Chemistry 11/1999; 274(43):30563-70. · 4.60 Impact Factor
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    ABSTRACT: Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.
    Nature 03/1997; 385(6618):733-6. · 42.35 Impact Factor
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    ABSTRACT: Tumor necrosis factor-alpha is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-alpha is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76 Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-alpha has previously been identified as a microsomal metalloprotease called TNF-alpha converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.
    Journal of Neuroimmunology 03/1997; 72(2):127-9. · 2.79 Impact Factor
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    ABSTRACT: The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.
    Experimental Parasitology 02/1997; 85(2):158-67. · 1.86 Impact Factor
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    ABSTRACT: Tumor necrosis factor-α is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-α is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76–Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-α has previously been identified as a microsomal metalloprotease called TNF-α converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.
    Journal of Neuroimmunology 02/1997; 72(2):127-129. · 2.79 Impact Factor
  • Akira Suzuki, William Burkhart, Steven Rothstein
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    ABSTRACT: Changes in the levels of ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and its mRNA were investigated in leaves of maize (Zea mays L. cv DEA). In etiolated leaves, detached from nitrogen-starved dark-grown seedlings, Fd-glutamate synthase was present at a low level. The enzyme protein and the activity were induced from 3- to 5-fold during the 35 h after transfer of etiolated leaves to a medium containing either 10 mM KNO3, 10 mM NH4Cl or 10 mM NH4NO3 under the continuous photosynthetic photon flux density of 300 μmol quanta/m2 per s. A slight increase in the activity occurred in the leaves grown in a nitrogen-free medium under continuous light. The increase in the enzyme activity was paralleled by the incorporation of l-[35S]methionine in the medium into the Fd-glutamate synthase polypeptide under the same conditions. The production of the enzyme protein and the uptake of labeled amino acid into the enzyme protein were blocked by adding 71 μM cycloheximide to the medium supplemented with KNO3, NH4Cl or NH4NO3. The addition of the different nitrogenous compounds under continuous darkness did not significantly alter the Fd-glutamate synthase activity and l-[35S]methionine incorporation into the enzyme protein. A partial cDNA of 2479 base pairs long encoding maize Fd-glutamate synthase was cloned and characterized. Using this cDNA as hybridization probe, Fd-glutamate synthase mRNA was observed to be present at a low level in nitrogen-starved etiolated leaves. The mRNA increased about 5-fold when etiolated leaves were incubated under continuous light in a medium containing either 10 mM KNO3, 10 mM NH4Cl or 10 mM NH4NO3. The level of mRNA was also slightly enhanced in the leaves incubated in a nitrogen-free medium under the light. The exogenous nitrogen compounds did not increase the level of the mRNA under continuous darkness. The presence of 71 μM cycloheximide in either of the media did not significantly change the level of the transcript during the initial 6 h. The induction of mRNA in the presence of exogenous nitrogen under the light was consistent with a protein synthesis-independent process in maize leaves.
    Plant Science. 01/1996;
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    ABSTRACT: The bovine liver mitochondrial protein synthesis elongation factor Tu.Ts complex (EF-TU.Tsmt) has been purified and partial peptide sequence information has been obtained for EF-Tumt. A complete cDNA has been obtained encoding bovine EF-Tumt and a nearly complete cDNA has been obtained for human EF-Tumt. The bovine cDNA has a 5' untranslated leader, an open reading frame of 1356 nucleotides and a 3' untranslated region of 189 base pairs. NH2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length. The human and bovine factors are 95% identical. The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences. At least two genes for EF-Tumt are present in the bovine system. Northern analysis indicates that EF-Tumt is synthesized in all tissues but that the level of expression varies over a wide range. EF-TUmt has been expressed in E. coli as a His-tagged protein and purified to near homogeneity. The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu.Tsmt complex.
    Biochimica et Biophysica Acta 01/1996; 1264(3):347-56. · 4.66 Impact Factor
  • H Xin, V Woriax, W Burkhart, L L Spremulli
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    ABSTRACT: The sequences of the cDNAs for the mitochondrial translational elongation factor Ts (EF-Tsmt) from bovine and human liver have been obtained. The deduced amino acid sequence of bovine liver EF-Tsmt is 338 residues in length and includes a 55-amino acid signal peptide and a mature protein of 283 residues. The sequence of the mature form of bovine EF-Tsmt is 91% identical to that of human EF-Tsmt and 29% identical to Escherichia coli EF-Ts. Southern analysis indicates that there are two genes for EF-Tsmt in bovine liver chromosomal DNA. A 224-base pair intron is located near the 5'-end of at least one of these genes. Northern analysis using a human multiple tissue blot indicates that EF-Tsmt is expressed in all tissues, with the highest levels of expression in skeletal muscle, liver, and kidney. Both the mature and precursor forms of bovine liver EF-Tsmt have been expressed in E. coli as histidine-tagged proteins. The mature form of EF-Tsmt forms a complex with E. coli elongation factor Tu. This complex is active in poly(U)-directed polymerization of phenylalanine. The precursor form is expressed as a 42-kDa protein, which is rapidly degraded in the cell.
    Journal of Biological Chemistry 08/1995; 270(29):17243-9. · 4.60 Impact Factor
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    ABSTRACT: A 33 kDa peroxidase was purified from intercellular wash fluids of cucumber leaves expressing systemic acquired resistance. Partial amino acid sequence data of the peroxidase were used to obtain a full length 1218 base pair (bp) cDNA clone (pCS1) which encoded the peroxidase. Deduced amino acid sequences from pCS1 matched observed amino acid sequences of the purified protein and also indicated that pCS1 was typical of known plant peroxidases. Three other cDNAs (pCS2, pCS3 and pCS4) were also isolated and found to be closely related to pCS1. RNA gel blots, using a 153 bp fragment of pCS1 as a probe, demonstrated accumulation of peroxidase mRNA in the second leaf of cucumber seedlings at 18 h after inoculation of the first leaf with the hypersensitive response-inducing bacterium Pseudomonas syringae pv. syringae. Detaching the inoculated leaf at intervals after inoculation demonstrated that this leaf only needed to be on the plant for 4 h to result in systemic induction of the peroxidase mRNA. This indicated that the systemic signal that leads to the expression of peroxidase was mobilized out of the inoculated leaf by 4 h. Exogenous application of salicylic acid, a putative endogenous signal for resistance, also induced accumulation of peroxidase mRNA.
    Physiological and Molecular Plant Pathology 05/1995; 46(5):389-400. · 1.99 Impact Factor
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    ABSTRACT: A stable DNA/protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.
    Archives of Microbiology 04/1995; 163(5):337-344. · 1.86 Impact Factor

Publication Stats

3k Citations
212.67 Total Impact Points

Institutions

  • 2002
    • Hospital of the University of Pennsylvania
      Philadelphia, Pennsylvania, United States
  • 1994–2001
    • University of North Carolina at Chapel Hill
      • Department of Chemistry
      Chapel Hill, NC, United States
  • 1996
    • Research Triangle Park Laboratories, Inc.
      Raleigh, North Carolina, United States
  • 1991–1993
    • Bowling Green State University
      • Department of Biological Sciences
      Bowling Green, OH, United States