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Biomedical subjects

P W Morris

Publications and source records attributed to P W Morris.

18 recordsLinked to original sources
PubMed1992

Inactivation of endothelin I by deamidase (lysosomal protective protein).

Deamidase cleaves ester and peptide bonds in various substrates and deamidates protected COOH-terminal amino acids. It preferentially hydrolyzes peptides which contain hydrophobic amino acids in the P1' and/or P1 position. Because the COOH-terminal end of endothelin I contains the hydrophobic sequence -Ile19-Ile20-Trp21-OH, we investigated whether human deamidase, purified from platelets, could inactivate this peptide. We found that deamidase readily cleaved off Trp21 with an acid pH optimum, a Km = 22 microM, a kcat of 1454 min-1, and a kcat/Km of 68 microM-1 min-1. We also found the enzyme to be present in target cells of endothelin, in vascular smooth muscle cells. Extracts of cultured vascular smooth muscle cells cleave both the synthetic fluorescent substrate 5-dimethylaminonaphthalene-1-sulfonyl(Dns)-Phe-Leu-Arg and endothelin I by releasing the COOH-terminal amino acid. The reaction was inhibited by diisopropyl fluorophosphate, benzyloxycarbonyl-Gly-Leu-Phe-CH2Cl, and p-chloromercuribenzenesulfonate, which inhibit the purified deamidase, but not by inhibitors of some other peptidases. The rate of hydrolysis of endothelin I in the soluble, 100,000 x g final supernatant of the homogenized smooth muscle cells was 2.1 mumol/h/mg and 3.1 mumol/h/mg for Dns-Phe-Leu-Arg. Thus, smooth muscles, platelets, and many other tissues which contain the deamidase can inactivate endothelin by cleaving the COOH-terminal tryptophan.

Amidohydrolases↗
PubMed1992

Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.

Amino Acid Sequence↗
PubMed1992

The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence.

Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation. The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter. The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY. The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase. Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations. These results indicate that orfY-tsr encodes aldolase and should be renamed fba1.

Amino Acid Sequence↗
PubMed1992

Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation.

Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [alpha-32P]-GTP. Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.

Amino Acid Sequence↗
PubMed1992

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis.

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.

Adenosine Triphosphate↗
PubMed1990

Production of the rat type 1 insulin-like growth factor-binding protein by well differentiated H4EIIC3 hepatoma cells: identification, purification, and N-terminal amino acid analysis.

We recently identified a 32 K mol wt insulin-like growth factor (IGF)-binding protein (BP) which is markedly increased in the serum of streptozotocin-diabetic rats and recognized by antiserum against the human amniotic fluid IGFBP (hIGFBP-1). In the present study we sought to confirm that this protein represents the rat homolog of IGFBP-1 (rIGFBP-1), and that rIGFBP-1 may, therefore, play an important role in the regulation of IGF bioactivity in experimental diabetes. Since the abundance of related hepatic mRNA is high in diabetic rats, we asked whether well differentiated H4EIIC3 rat hepatoma cells produce rIGFBP-1 and provide sufficient amounts of this protein for purification and further characterization. Specific IGF-binding activity in hepatoma conditioned medium was detected initially by incubation with 125I-labeled recombinant human IGF-II and precipitation with polyethylene glycol. Ligand blotting demonstrated a 32 K BP, identical in size to the major low mol wt IGFBP found in diabetic rat serum. Affinity labeling and immunoprecipitation confirmed that this BP is related to human IGFBP-1 and is distinct from the fetal rat IGFBP, rIGFBP-2. Incorporation of [35S]methionine into 32 K BPs confirmed synthesis by hepatoma cells. For purification of BPs, conditioned medium was collected in roller culture, and BPs were purified by ammonium sulfate precipitation, Sephadex G-75 chromatography, and reverse phase HPLC. Partial amino acid sequencing of purified protein demonstrated 68% identity with the human IGFBP-1 and distinguished this BP from previously characterized rat IGFBPs. Purified protein bound both IGF-I and IGF-II with high affinity. We conclude that the 32 K IGFBP produced by H4EIIC3 hepatoma cells in culture represents the rat form of IGFBP-1 (rIGFBP-1). Regulation of rIGFBP-1 may play an important role in the modulation of IGF bioactivity in experimental animals with metabolic disease. The availability of purified rIGFBP-1 and identification of a cell line that produces this BP will greatly facilitate future studies of IGFBP-1 in the rat model.

Amino Acid Sequence↗
PubMed1985

Separation of nucleosides and nucleotides by reversed-phase high-performance liquid chromatography with volatile buffers allowing sample recovery.

Reversed-phase high-performance liquid chromatography using a C18 column with volatile buffers as the eluant was applied to the separation of a number of nucleosides and nucleotides. Groups of seven nucleosides and five nucleoside monophosphates were separated isocratically employing 0.1 M trimethylammonium acetate and 2% acetonitrile at pH 7.0. Groups of seven nucleoside diphosphates and seven nucleoside triphosphates were separated with 0.1 M triethylammonium bicarbonate and 2% acetonitrile titrated to a pH of 7.1 with acetic acid. The techniques described give resolution and separations comparable to nonvolatile buffers. Moreover, the eluant trimethylammonium acetate or triethylammonium bicarbonate buffer can easily be removed in vacuo from the column effluent, making the technique useful for preparative separations of these compounds. The observed elution pattern of nucleoside phosphates suggests that "paired-ion" chromatography is involved in the separation.

Buffers↗
PubMed1985

Cloning and location of the dgsA gene of Escherichia coli.

The dgsA locus of Escherichia coli was isolated on plasmids obtained from the library of L. Clarke and J. Carbon (Cell 9:91-99, 1976). Restriction fragment analysis and further subcloning demonstrated that the gene is located at kilobase 425 on the Bouché physical map of the terminus region (J. P. Bouché, J. Mol. Biol., 154:1-20, 1982). This corresponds to 35.2 min on the Bachmann genetic map (B. J. Bachmann, Microbiol. Rev. 47:180-230, 1983).

Bacterial Proteins↗
PubMed1979

alpha-Amanitin: inactivation by bovine lactoperoxidase.

The principle amatoxin, alpha-amanitin, is found to be extremely sensitive toward lactoperoxidase catalyzed degradation, rather than iodination, of the indole nucleus. Extensive attenuation of inhibitor potency against eukaryotic DNA-dependent RNA polymerase II accompanies the treatment of alpha-amanitin with lactoperoxidase, iodide and hydrogen peroxide.

Amanitins↗
PubMed1978

Biochemistry of the amatoxins: preparation and characterization of a stably iodinated alpha-amanitin.

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.

Amanitins↗
PubMed1978

DNA polymerase alpha and beta in the California urchin.

DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species.

Animals↗
PubMed1978

Ribonucleotidyl transferase in preparations of partially purified DNA polymerase alpha of the sea urchin.

Three ribonucleotidyl transferase types have been described in the sea urchin: riboadenylate trnasferase, the DNA dependent RNA polymerases, and a DNA polymerase associated ribonucleotidyl transferase (Biochemistry 15:3106-3113, 1976). In the present work this latter ribonucleotidyl transferase was found to purify with DNA polymerase alpha through phosphocellulose, DEAE-Sephadex and DNA cellulose and to cosediment at 6.5 S. This ribonucleotidyl transferase was active with Mn+2, but not Mg+2, on calf thymus DNA and poly(dC). Other synthetic templates elicited DNA polymerase alpha but no ribonucleotidyl transferase activity. From alkaline hydrolysates of the poly(dC) directed GTP polymerization, we found Goh and Gp in a ratio of 1:16 indicating an average chain length of 17 residues after a 20 min reaction. Co-polymerization of GTP (5 micrometer) and dGTP (10 micrometer) yielded a non-random distribution of the ribonucleotide in the deoxyribonucleotide. The properties of this urchin ribonucleotidyl transferase are unlike any previously described eukaryotic transferase and the data is discussed with reference to the known properties of E. coli DNA polymerase I and the primase.

Animals↗
PubMed1976

Nucleic acid polymerizing enzymes in developing Strongylocentrotus franciscanus embryos.

DNA-dependent RNA polymerase, DNA-Dependent DNA polymerase, and terminal riboadenylate transferase (TRT) activities have been measured after DEAE-Sephadex chromatography of whole cell extracts prepared from eggs and staged embryos of the urchin, Stronglyocentrotus franciscanus. Activity of each of these three polymerase classes is present in the egg, and the total activity per embryo is constant throughout embryogenesis to the pluteus stage (approximately 1000 cells). Thus the egg appears to contain sufficient DNA polymerase, RNA polymerase, and TRT TRT for embryogenesis. The increases in the synthesis of DNA, RNA and polyadenylated RNA tracts observed after fertilization must be due to the activation of the preexisting egg enzymes. Separation of the egg into nucleate and anucleate halves demonstrates that the RNA polymerases are not restricted to the egg nucleus. During development, the enzymes become progressively more associated with the cell nucleus. The egg extracts contain low activities (approximately 6% total) of RNA polymerase II as measured by sensitivity to alpha-amanitin. This is confirmed by resolution of the RNA polymerase forms I, II, and III by gradient sievorptive elution on DEAE-Sephadex. Later stage embryos contain more nearly equal activities of RNA polymerase, I, II, and III, although the total RNA polymerase activity per embryo is not changed. Additionally, two chromatographicallly distinct species of RNA polymerase III are detected, one of which is observed only in later stages. Thus interconversion of enzymes via addition of new subunits or coordinate synthesis and loss of enzyme species must occur.

Amanitins↗