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W Konigsberg

Publications and source records attributed to W Konigsberg.

At least 19 recordsLinked to original sources
PubMed2004

The activity of selected RB69 DNA polymerase mutants can be restored by manganese ions: the existence of alternative metal ion ligands used during the polymerization cycle.

Site specific mutants in the pol active center of RB69 DNA polymerase have been produced and studied using rapid chemical-quench techniques. Pre-steady-state kinetic analysis carried out with Mg(2+) and Mn(2+) has enabled us to divide the mutants into two groups. One group had greatly reduced k(pols) values in the presence of Mg(2+) but responded to Mn(2+) which restored the k(pol) values for the nucleotidyl transfer reaction to near wild-type levels. The other group of mutants also had lower k(pol) values, relative to that of the wild-type polymerase, but could not be rescued by Mn(2+). The behavior of these mutants was interpreted in terms of the crystal structures of the available RB69 pol complexes. Our results on the metal ion dependence of the D621A and E686A mutants, together with knowledge of the position of their side chains in two different RB69 pol conformations, suggest that these acidic residues serve as alternative ligands for the metal ions destined to occupy the A and B catalytic sites. We infer that this occurs prior to the conformational change that produces the ternary RB69 pol complex in which the A and B metal ions are ligated by D623 and D411 as the enzyme is poised for phosphoryl transfer.

Binding Sites↗
PubMed2004

Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity.

DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it.

Alanine↗
PubMed1992

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.

Amino Acid Sequence↗
PubMed1991

Molecular cloning, characterization and expression of cDNA for rabbit brain tissue factor.

Tissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factor VII or VIIa. TF has been identified in atherosclerotic plaques and may possibly trigger thrombosis after spontaneous plaque rupture as seen in acute myocardial infarction or angioplasty. We have previously developed an atherosclerotic rabbit model for study of the acute and chronic outcomes following angioplasty. As a first step in developing inhibitors of TF, we have isolated and characterized a rabbit cDNA coding for the mature TF. The sequence comparison of rabbit TF cDNA with those of human and mouse TFs show considerable similarity at both the nucleotide and amino acid levels. The TF cDNA when expressed in E. coli demonstrates a procoagulant activity comparable to that of native rabbit brain TF. The TF activity can be blocked by a polyclonal antibody against rabbit TF.

Animals↗
PubMed1991

A rapid method for purification of synthetic oligoribonucleotides.

A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O-silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide.

Chromatography, High Pressure Liquid↗
PubMed1986

Identification, cloning and sequence determination of the genes specifying hexokinase A and B from yeast.

The hexokinase A (HKA) and hexokinase B (HKB) genes of Saccharomyces cerevisiae have been cloned from a library of yeast genomic DNA. Using an in vitro glucose phosphorylation assay, the HKB gene was located on a plasmid carrying a 13.6 kb fragment of yeast DNA. After subcloning the relevant restriction fragments, the nucleotide sequence of the HKB gene was determined. Using this information, we were able to locate the HKA gene on a plasmid carrying this gene, which we then sequenced. Approximately 43% of the amino acid sequence of HKB was determined directly from 24 tryptic peptides. The results are in complete agreement with those derived from the DNA sequence and are consistent with the results of x-ray crystallography. Comparison of the amino acid sequences of HKA and HKB show that 378 out of 485 residues are identical. The 5' flanking region of the A gene contains nucleotide sequences expected for genes that are expressed at relatively high levels in yeast. The 24 base pair hyphenated palindrome at the 3' end of the HKB gene may be a site for termination of transcription of this gene.

Amino Acid Sequence↗
PubMed1986

Acidic lipids enhance cathepsin D cleavage of the myelin basic protein.

Some acidic lipids including sulfatide and phosphatidylinositol were found to increase greatly the rate of cathepsin D cleavage of the myelin basic protein. Since a similar effect was seen when the substrate was changed to cytochrome C, but not when the enzyme was changed to pepsin, these acidic lipids seem to be acting on cathepsin D rather than on myelin basic protein itself. Even so, chemical modification studies suggest that this phenomenon is only seen when the myelin basic protein is in its native conformation. Succinylation of MBP increases its rate of cleavage by cathepsin D by at least tenfold and, in addition, with this modified and presumably denatured MBP as substrate, activation of cathepsin D is no longer seen with acidic lipids. These findings suggest that the native conformation of MBP is both an important determinant of its rate of cleavage by cathepsin D and is also essential for observing activation of this reaction by acidic lipids. The acidic lipids seem to alter the "extended active site" of cathepsin D in such a way as to enable this enzyme better to utilize the native myelin basic protein as a substrate. Cathepsin D has previously been implicated as the protease responsible for the release into cerebrospinal fluid in multiple sclerosis patients of an encephalitogenic fragment derived from myelin basic protein. It is possible that the elevated levels of cathepsin D and sulfatide that have previously been found associated with multiple sclerosis plaques in vivo act in concert to bring about the rapid cleavage and subsequent loss of the myelin basic protein from these localized regions in the myelin sheath.

Animals↗
PubMed1986

Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles.

Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.

Biological Assay↗
PubMed1985

Isolation of altered recA polypeptides and interaction with ATP and DNA.

In this paper we describe the partial proteolytic digestion of recA proteins from Escherichia coli and Proteus mirabilis and the production and isolation of truncated recA polypeptides. A proteolytic fragment of the P. mirabilis recA protein bound single-strand DNA and ATP normally but has altered duplex DNA binding properties. This protein was shown to initiate but not complete DNA strand transfer from a DNA duplex to a complementary single strand. The product of the E. coli recA1 allele bound but could not hydrolyze ATP and the protein bound single-strand but not double-strand DNA. This protein did not appear to initiate the transfer of a strand from a linear duplex to a single-strand circle and inhibited the wild-type recA protein from performing strand transfer. We report that recA protein binds linear duplex DNA in a manner that enhances the rate of ligation by T4 DNA ligase. When heterologous single-strand DNA was added in addition to the duplex DNA large stable aggregates of protein and DNA were formed that could easily be sedimented from solution.

Adenosine Triphosphate↗
PubMed1984

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Amino Acid Sequence↗
PubMed1984

1H NMR (500 MHz) of gene 32 protein--oligonucleotide complexes.

In concentrated solutions, gene 32 single-stranded DNA binding protein from bacteriophage T4 (gene 32P) forms oligomers with long rotational correlation times, rendering 1H NMR signals from most of the protons too broad to be detected. Small flexible N- and C-terminal domains are present, however, the protons of which give rise to sharp resonances. If the C-terminal A domain (48 residues) and the N-terminal B domain (21 residues) are removed, the resultant core protein of 232 residues (gene 32P) retains high affinity for ssDNA and remains a monomer in concentrated solution, and most of the proton resonances of the core protein can now be observed. Proton NMR spectra (500 MHz) of gene 32P and its complexes with ApA, d(pA)n (n = 2, 4, 6, 8, and 10), and d(pT)8 show that the resonances of a group of aromatic protons shift upfield upon oligonucleotide binding. Proton difference spectra show that the 1H resonances of at least one Phe, one Trp, and five Tyr residues are involved in the chemical shift changes observed with nucleotide binding. The number of aromatic protons involved and the magnitude of the shifts change with the length of the oligonucleotide until the shifts are only slightly different between the complexes with d(pA)8 and d(pA)10, suggesting that the binding groove accommodates approximately eight nucleotide bases. Many of the aromatic proton NMR shifts observed on oligonucleotide complex formation are similar to those observed for oligonucleotide complex formation with gene 5P of bacteriophage fd, although more aromatic residues are involved in the case of gene 32P.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-Binding Proteins↗
PubMed1983

Primary structure analysis of the mutant recA 441 and recA 430 proteins.

The mutant recA 441 and recA 430 proteins from Escherichia coli have single amino acid substitutions which distinguish each of them from the wild type recA protein. recA 441 mutants express SOS functions at 42 degrees C even without DNA damage whereas recA 430 mutants are totally unable to express SOS functions at any temperature. Both mutant recA proteins have altered protease functions. However, the amino acid substitutions in each protein are located at a considerable linear distance from one another in their respective polypeptide chains, the recA 441 protein having a lysine in place of a glutamic acid residue at position 68, while the recA 430 protein has a serine in place of a glycine at position 204.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.

Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.

Amino Acid Sequence↗
PubMed1983

Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli.

Amino acid sequence and composition data of Escherichia coli dnaG primase protein and its tryptic peptides have confirmed that the dnaG gene contains an unusually high number of codons that are not frequently used in most E. coli genes. In 25 E. coli proteins analyzed the codons AUA, UCG, CCU, CCC, ACG, CAA, AAT, and AGG are infrequently used, occurring as 4% of the total codons in the reading frame and 11% and 10% in the nonreading frames. In dnaG they occur as 11% in the reading frame and 12% in the nonreading frames. The rpsU and rpoD genes, which flank the dnaG gene [Smiley, B. L., Lupski, J. R., Svec, P. S., McMacken, R. & Godson, G. N. (1982) Proc. Natl. Acad. Sci. USA 79, 4550-4554], however, have normal codon usage. Translational modulation using isoaccepting tRNA availability may therefore be part of the mechanism of keeping the dnaG gene expression low, while expression of the adjacent rpsU and rpoD genes on the same mRNA transcript is high.

Amino Acid Sequence↗
PubMed1982

Photochemical cross-linking of the gene 5 protein.fd DNA complex from fd-infected cells.

The gene 5 protein.fd DNA complex, which is an intermediate in the life cycle of fd bacteriophage, was isolated from fd-infected cells. After ultraviolet irradiation, approximately 10% of the gene 5 protein was covalently linked to fd DNA via the sulfhydryl group of the single cysteine residue at position 33 in the gene 5 protein. When intact, infected cells were first irradiated with ultraviolet light and the gene 5 protein.DNA complex subsequently isolated, less than 1% of the bound protein in the complex was found to be cross-linked. While these results agree with our previous findings based on irradiation of the gene 5 protein.fd DNA complex formed from purified gene 5 protein and fd DNA, they differ from the results of Lica and Ray ((1977) J. Mol. Biol. 115, 45-59) who found high yields of cross-linked gene 5 protein.fd DNA complex after ultraviolet irradiation of intact cells.

Amino Acids↗
PubMed1981

Purification and characterization of bovine tissue factor.

Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain. The protein is an integral membrane glycoprotein with an apparent molecular weight of 43,000 as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The apoprotein was first purified by extraction with Triton X-100 and repeated preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Antiserum was produced against a few micrograms of purified apoprotein and was used to construct an immunoadsorbent column. The column was then used for affinity purification of the apoprotein directly from the Triton X-100 extract, thereby significantly increasing the amount of purified protein produced. The purification scheme may be generally useful for the rapid and large scale purification of membrane proteins. Tryptic digestion of the apoprotein in Triton X-100 cleaved a peptide of approximately 3000 daltons without affecting the activity. The activity was recovered directly from stained SDS polyacrylamide gels, and the profile of recovered activity corresponded directly with the stained bands. The activity shifted along with the protein band following tryptic digestion, thus demonstrating that the protein observed on the gels is tissue factor. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipid. Optimal activity was observed at phospholipid to protein ratios (w/w) greater than 450:1.

Amino Acids↗
PubMed1981

Structure of the filamentous bacteriophage fl. Location of the A, C, and D minor coat proteins.

The location within the virion of the A, C, and D minor coat proteins of the filamentous bacteriophage fl has been analyzed. The A protein is present in approximately 5 copies/particle and is located at the tip of normal length phage, miniphage, and fl/pBR322 chimeric phage, a longer than normal length phage. The mole ratios of the A, C, and D proteins are the same for each type of particle, consistent with a model of phage organization in which the minor coat proteins are clustered near or at the ends of the phage. Normal length phage were fragmented by passing them through a French press, and those fragments that contained the A protein were separated from those that did not by treating the mixture with anti-A protein antibody. Analysis of the protein compositions of the two populations of fragments showed that the A and D proteins were found together in one population of fragments and that most, it not all, of the C protein was found in the other. These results show that the D protein is located near or at the A protein end of the phage and that the C protein is located in a region near or at the opposite end. Treatment of the virion with proteases which lowered the infectivity of the phage resulted in particles in which only the A protein was cleaved to any detectable extent. These particles remained resistant to the action of micrococcal nuclease.

Capsid↗