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A Kornberg

Publications and source records attributed to A Kornberg.

At least 73 records · Page 4Linked to original sources
PubMed1995

Inorganic polyphosphates in the acquisition of competence in Escherichia coli.

A complex of polyhydroxybutyrate (PHB), Ca2+, and inorganic polyphosphate (polyP) was proposed as the membrane component responsible for competence for DNA entry in Escherichia coli (Reusch, R. N., and Sadoff, H. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4176-4180). While chemical and immunological assays and 1H NMR have unequivocally established the identity and content of PHB in the complex, comparable methods were not available for polyP. With specific enzyme assays developed for polyP, we have identified, in chloroform extracts of competent cell membranes, a novel form of polyP of about 60 to 70 residues in a stoichiometric ratio of PHB to polyP of 2:1. In E. coli mutants, incapable of synthesizing the predominant, thousand-long polyP chains, appearance of this short polyP and its inclusion in membranes can account for their capacity to develop competence and indicates an auxiliary pathway for polyP synthesis. A variety of fluorescent lipid probes demonstrate the appearance of extensive rigid domains in membranes of competent cells. We propose that the PHB.Ca2+.polyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA.

Cell Membrane↗
PubMed1995

Inorganic polyphosphate in mammalian cells and tissues.

Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods. Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes). PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally. Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h. Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s). In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours. The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.

3T3 Cells↗
PubMed1995

Wrong move.

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Academies and Institutes↗
PubMed1995

Inorganic polyphosphate: toward making a forgotten polymer unforgettable.

Pursuit of the enzymes that make and degrade poly P has provided analytic reagents which confirm the ubiquity of poly P in microbes and animals and provide reliable means for measuring very low concentrations. Many distinctive functions appear likely for poly P, depending on its abundance, chain length, biologic source, and subcellular location. These include being an energy supply and ATP substitute, a reservoir for Pi, a chelator of metals, a buffer against alkali, a channel for DNA entry, a cell capsule and, of major interest, a regulator of responses to stresses and adjustments for survival in the stationary phase of culture growth and development. Whether microbe or human, we depend on adaptations in the stationary phase, which is really a dynamic phase of life. Much attention has been focused on the early and reproductive phases of organisms, which are rather brief intervals of rapid growth, but more concern needs to be given to the extensive period of maturity. Survival of microbial species depends on being able to manage in the stationary phase. In view of the universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, such as aging and the aberrations of disease. Of theoretical interest regarding poly P is its antiquity in prebiotic evolution, which along with its high energy and phosphate content make it a plausible precursor to RNA, DNA, and proteins. Practical interest in poly P includes many industrial applications, among which is its use in the microbial depollution of P1 in marine environments.

Animals↗
PubMed1995

The gene for a major exopolyphosphatase of Saccharomyces cerevisiae.

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem. 269:10996-11001, 1994) has been isolated from a genomic library. The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.

Acid Anhydride Hydrolases↗
PubMed1994

Induction of tissue factor-like activity in monocytes by anti-cardiolipin antibodies.

Anti-cardiolipin Abs (ACLA) are present in the sera of patients with antiphospholipid syndrome (APLS) and are associated with high incidence of thromboembolic phenomena, fetal loss, thrombocytopenia, and prolongation of the phospholipid-dependent coagulation assays (lupus anticoagulant). Recently, it has been shown that APLS can be induced experimentally by using ACLA. However, the pathophysiology of thrombus formation in this syndrome is unknown. Monocytes generate a potent procoagulant activity (PCA) after stimulation with various substances. Increased PCA has been found in monocytes from patients with diseases that are associated with high incidence of thromboembolic phenomena. In the present study, we report that the monoclonal ACLA that were shown previously by us to induce APLS stimulate mononuclear cells to generate a potent PCA. The PCA resembled tissue factor (TF) in that it accelerated clotting through the extrinsic coagulation pathway, was abolished by phospholipase C, and was inhibited by anti-TF mAbs. The induction of TF-like activity by ACLA in monocytes was dose- and time-dependent. It was induced in monocytes and monocytic cell lines, but not in lymphoid or myeloid cells, and did not require T lymphocytes for expression. The generation of PCA was dependent on protein synthesis inasmuch as it was prevented by adding puromycin to the system and was not affected by cytarabine. The TF-like activity that is induced by ACLA in monocytes may activate coagulation and thereby play a major role in the pathogenesis of thrombus formation in APLS.

Adult↗
PubMed1994

Hyperactive initiation of chromosomal replication in vivo and in vitro by a mutant initiator protein, DnaAcos, of Escherichia coli.

DnaA protein initiates genomic replication in Escherichia coli. A cold-sensitive dnaAcos mutant caused excessive initiation at a restrictive temperature without an increase in the level of DnaA protein. The chromosomal origin (oriC) was essential for the lethality caused by the dnaAcos product. Increased initiation activity was neutralized by multiple copies of oriC on a plasmid (pBR322). OriC plasmids were replicated efficiently in vitro in a crude extract prepared from a dnaAcos mutant, with a specific activity for the DnaAcos protein 8-fold greater than that for the DnaA+ protein in a wild-type extract. OriC-dependent replication in the dnaAcos extract was inhibited by rifampicin and by gyrase inhibitors as was replication in the dnaA+ extract. As a control, replication of single-stranded phage phi X174 DNA, which did not require DnaA protein, was similar in extracts prepared from dnaA+ and dnaAcos cells. Thus, initiation at oriC by DnaAcos protein appears to be highly activated both in vivo and in vitro.

Bacterial Proteins↗
PubMed1994

A soluble exopolyphosphatase of Saccharomyces cerevisiae. Purification and characterization.

A soluble polyphosphatase of Saccharomyces cerevisiae, purified to apparent homogeneity, is monomeric with a molecular mass of 40 kDa. It acts as an exoenzyme in a processive mode releasing orthophosphate residues from long polyphosphate chains until pyrophosphate is reached. Polyphosphates of all the lengths examined are used as substrates with a preference for those of about 250 residues. These are degraded with a kcat/Km near the limit for diffusion-controlled reactions. At 37 degrees C, the enzyme releases about 500 phosphate residues/s. It does not act on pyrophosphate, ATP, or the cyclic form of tripolyphosphate. For optimal activity the enzyme requires magnesium, manganese, or cobalt.

Acid Anhydride Hydrolases↗
PubMed1994

Genetically altered levels of inorganic polyphosphate in Escherichia coli.

The ppk gene encoding polyphosphate kinase (PPK), the enzyme in Escherichia coli that makes long chains of polyphosphate (polyP) reversibly from ATP, was disrupted by insertion of a kanamycin resistance gene. Expression of the exopolyphosphatase gene (ppx) immediately downstream of ppk in the operon was likewise disrupted. Cells were also transformed with a high-copy-number plasmid bearing ppk. Genetically altered polyP levels were estimated in cell extracts by the PPK conversion of ADP to ATP. PolyP levels (microgram/10(11) cells) near 2.0 were reduced in the ppk(-)-ppx- mutants to 0.16 and increased more than 100-fold (e.g. 220) in cells transformed with multiple copies of ppk. Mutant cells, lacking the long polyP chains, showed a growth lag following dilution of a stationary-phase culture. PolyP-deficient cells exhibit a striking phenotype in their failure to survive in stationary phase and loss of resistance to heat (55 degrees C) and to oxidants (42 mM H2O2). High polyP levels are also associated with reduced survival.

Adenosine Triphosphate↗
PubMed1994

Unusual bleeding manifestations of amyloidosis in patients with multiple myeloma.

We describe two patients who developed severe haemorrhagic complications. One manifested massive vaginal bleeding and the second haemopericardium with cardiac tamponade. In both cases histopathological examination showed subendothelial deposits of amyloid. Since other local and systemic causes of the haemorrhagic complications were excluded the bleeding was most probably due to amyloid angiopathy. Therefore, amyloid angiopathy should be considered in the differential diagnosis of bleeding in patients with amyloidosis associated with multiple myeloma.

Aged↗
PubMed1993

Fluid membranes with acidic domains activate DnaA, the initiator protein of replication in Escherichia coli.

Acidic phospholipids in a fluid phase dissociate ADP or ATP tightly bound to DnaA protein and, in the presence of ATP and DNA, can restore an inactive ADP form to full activity (Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135). Further studies of the interactions between DnaA protein and lipids have used two functional assays: 1) release of ADP or ATP from DnaA and 2) DNA replication upon rejuvenation of an inactive ADP-DnaA protein complex. Among a variety of phospholipids tested were pure synthetic compounds and the mixtures from Escherichia coli auxotrophs (fabA), which are unable to synthesize unsaturated fatty acids and can be supplemented with different acyl derivatives. Fatty acid composition was determined by gas-liquid chromatography and membrane fluidity by fluorescence spectroscopy using 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid requirements of DnaA protein were shown to be: 1) phospholipids in a fluid phase (i.e. above the transition temperature), 2) a charged polar head group, 3) a lamellar phase (i.e. hexagonal II structures were inactive), and 4) a certain degree of fluidity imparted by the fatty acids esterified to the glycerol backbone. This conclusion was based on the incorporation of: 1) cholesterol, known to increase the packing of lipids, or 2) a branched fatty acyl derivative, which exhibits a fluidizing effect similar to that of a cis double bond. Both agents demonstrated that membrane fluidity is required for DnaA protein function in vitro, consistent with early studies of chromosome initiation in growing cells.

Adenosine Diphosphate↗
PubMed1993

Assembly of the primosome of DNA replication in Escherichia coli.

Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, acting at a primosome assembly site (pas) on an SSB-coated single-stranded (ss) DNA. Assembly is initiated by interactions of PriA and PriB with ssDNA and the pas. PriC, DnaB, DnaC, and DnaT then act on the PriA-PriB-DNA complex to yield the primosome. In the primosome, the dATPase (ATPase) of PriA becomes hyper-activated. In addition, the assembled primosome appears to block the pas, preventing it from activating additional PriA molecules. Either ATP alone or dATP in combination with GTP is sufficient for primosome assembly, while ATP or GTP provides for its maintenance during isolation. These nucleotide requirements can be reconciled with the need for ATP or dATP for DnaB-DnaC complex formation and hydrolysis of ATP or GTP by DnaB when it binds ssDNA. Such isolated primosomes contain a dATPase, the hallmark of PriA, and a GTPase indicative of DnaB. Further studies indicate that the isolated primosome contains the PriB replication activity in addition to PriA and DnaB.

Bacterial Proteins↗
PubMed1993

Replicatively active complexes of DnaA protein and the Escherichia coli chromosomal origin observed in the electron microscope.

DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.

Adenosine Diphosphate↗
PubMed1993

Strand switching of a replicative DNA helicase promoted by the E. coli primosome.

The E. coli primosome assembles at an origin on a single-stranded DNA, like that of phi X174, to promote replication of that template. Upon conversion to the duplex form, the primosome can generate a rolling circle product from this template. Rolling circle synthesis implies the transfer of the DnaB helicase from its initial loading site on the viral strand to a displaced complementary strand. Isolated primosomes promote only unit-length synthesis; supplementation with PriC, DnaC, and DnaT is necessary to reconstitute rolling circle synthesis. Rolling circle replication is sensitive to salts, whereas primosome assembly and unit-length synthesis are not. Thus, the primosome promotes two distinct reactions: assembly for first-round synthesis and strand switching for rolling circle synthesis.

Ammonium Sulfate↗
PubMed1993

Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.

An exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase. The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure. The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate [poly(P)] chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot"). The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1. These and other findings direct attention to possible functions of poly(P) in the response of E. coli to stresses and deprivations.

Amino Acid Sequence↗
PubMed1993

A novel binding protein of the origin of the Escherichia coli chromosome.

The replication origin of Escherichia coli (oriC) was probed for specific binding proteins, using the gel shift assay. A 33-kDa protein that binds specifically to the right border of oriC, next to the rightmost of the binding sites of the initiator protein, DnaA, was identified and purified. The stoichiometry of protein to DNA is about 6 to 1. Around 5000 monomers of the protein, named Rob (right oriC binding), are present per cell. The rob gene has been located near 99.8 min on the E. coli map, cloned, and overexpressed. The total protein sequence reveals strong homologies to several regulatory proteins with which it shares the helix-turn-helix motif.

Amino Acid Sequence↗