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E A Robinson

Publications and source records attributed to E A Robinson.

At least 37 records · Page 2Linked to original sources
PubMed1989

Murine 86- and 84-kDa heat shock proteins, cDNA sequences, chromosome assignments, and evolutionary origins.

The two forms of the approximately 90-kDa murine heat shock protein, referred to as HSP86 and HSP84, are coded for by separate but related genes. A full-length nucleotide sequence of the cDNA coding for HSP86 from a chemically induced tumor, Meth A, was determined. Sequences from a number of peptides from HSP86 were found to be in complete agreement with the nucleotide sequence. The HSP84 sequence from the same tumor was also completed. HSP86 and HSP84 are acidic polypeptides 733 and 724 amino acids long with calculated molecular weights of 84,796 and 83,290, respectively. The two proteins are 86% homologous. HSP86 was found to contain internal peptide repeats of Glu-Lys-Glu within a region of highly charged amino acid residues. The coding regions of the cDNAs were 76% homologous; however, this homology did not extend to the 5'- and 3'-untranslated regions. The 5'-untranslated region of hsp86 cDNA was considerably longer than that of hsp84 cDNA and, unlike that of hsp84, contained extraneous ATG triplets. Hsp86-related sequences were assigned to chromosomes 12, 11, and 3. An evolutionary tree constructed from HSP90-related protein sequences indicated that HSP86 and HSP84 were likely to have diverged more than 500 million years ago. The findings presented herein suggest that HSP86 and HSP84 may have different functions.

Amino Acid Sequence↗
PubMed1989

Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants.

Two chemoattractants for human monocytes were purified to apparent homogeneity from the culture supernatant of a glioma cell line (U-105MG) by sequential chromatography on Orange A-Sepharose, an HPLC cation exchanger, and a reverse phase HPLC column. On SDS-PAGE gels under reducing or nonreducing conditions, the molecular masses of the two peptides glioma-derived chemotactic factor 1 and 2 were 15 and 13 kD, respectively. Amino acid composition of these molecules was almost identical, and differed from other cytokines that have been reported. The NH2 terminus of each peptide was apparently blocked. When tested for chemotactic efficacy, the peptides attracted approximately 30% of the monocytes added to chemotaxis chambers, at the optimal concentration of 10(-9) M. Potency and efficacy were comparable with that of FMLP, which is often used as a reference attractant. The activity was chemotactic rather than chemokinetic. In contrast to their interaction with human monocytes, the pure peptides did not attract neutrophils. These pure tumor-derived chemoattractants can now be compared with attractants produced by normal cells and evaluated for their biological significance in human neoplastic disease.

Amino Acids↗
PubMed1989

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes.

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.

Amino Acids↗
PubMed1989

Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence.

Human monocyte-derived neutrophil chemotactic factor (MDNCF) was purified from culture supernatant of lipopolysaccharide-stimulated human peripheral blood mononuclear leukocytes on a column of Sepharose-bound murine monoclonal anti-MDNCF. About 65% of the culture fluid chemotactic activity was bound to the column. The unbound 35% probably represents chemotactic activity of other cytokines in the culture fluid. More than 85% of the bound activity was eluted by pH 2.5 glycine buffer. When this material was applied to an HPLC-CM column, gradient elution produced four well-separated A280 peaks, each of which had chemotactic activity. N-terminal amino acid analysis of the four peaks revealed three different sequences. One (MDNCF-c) was identical to the sequence that we reported previously. The other two (MDNCF-a and -b) had seven and five additional amino acids, respectively, at the N-terminus. MDNCF-a, -b and -c accounted for 8, 47 and 45% of the total MDNCF peptide. Alignment with the MDNCF cDNA sequence shows that MDNCF-a results from cleavage of a 20 residue signal peptide. MDNCF-c results from culture fluid proteolytic cleavage of the N-terminal sequences of MDNCF-a and -b at an R-S bond. The three peptides occurred in the four HPLC-CM peaks in different ratios. The bulk of any one peptide was distributed in two adjacent HPLC-CM peaks. This suggests that each peptide exists in a minimum of two states. In contrast to our previous multi-step purification, the immunoaffinity and HPLC-CM column sequence resulted in complete purification of MDNCF in two steps and led to identification of two additional MDNCF peptides, one of which has not heretofore been detected.

Amino Acid Sequence↗
PubMed1989

Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions.

In a study of the structural basis for leukocyte specificity of chemoattractants, we determined the complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2), a peptide that attracts human monocytes but not neutrophils. The choice of a tumor cell product for analysis was dictated by its relative abundance and an amino acid composition indistinguishable from that of lymphocyte-derived chemotactic factor (LDCF), the agonist thought to account for monocyte accumulation in cellular immune reactions. By a combination of Edman degradation and mass spectrometry, it was established that GDCF-2 comprises 76 amino acid residues, commencing at the N terminus with pyroglutamic acid. The peptide contains four half-cystines, at positions 11, 12, 36, and 52, which create a pair of loops, clustered at the disulfide bridges. The relative positions of the half-cystines are almost identical to those of monocyte-derived neutrophil chemotactic factor (MDNCF), a peptide of similar mass but with only 24% sequence identity to GDCF. Thus, GDCF and MDNCF have a similar gross secondary structure because of the loops formed by the clustered disulfides, and their different leukocyte specificities are most likely determined by the large differences in primary sequence.

Amino Acid Sequence↗
PubMed1989

Protease inhibitors from Ecballium elaterium seeds.

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of two different insulins from an amphibian, Xenopus laevis.

The South African clawed toad, Xenopus laevis, is a versatile laboratory model of vertebrate development. To study the role of insulin during embryogenesis, we have recently cloned preproinsulin cDNAs from this species. Unexpectedly, we identified two preproinsulin cDNAs corresponding to two different nonallelic genes that code for similar but distinctly different insulins. We now report the isolation, amino acid sequence, and characterization of both of these insulins from pancreatic extracts of adult toads, confirming that both Xenopus preproinsulin genes are expressed. Xenopus insulins represent the first amphibian insulins to be characterized. Xenopus insulin I and Xenopus insulin II are more similar to each other than they are to insulins of other species. In addition, Xenopus insulins are more similar to mammalian and bird insulins, than they are to fish insulins, implying a closer evolutionary link to terrestrial vertebrates than to most aquatic vertebrates. A homogeneous preparation of Xenopus insulin I showed high reactivity in a pork insulin RIA. Xenopus insulin I was approximately 2-fold more potent than pork insulin in binding to insulin receptors on human IM-9 lymphocytes and 1.5-fold more potent than pork insulin in stimulating glucose oxidation in rat adipocytes. We were unable to purify Xenopus insulin II sufficiently for immunological and biological characterization.

Amino Acid Sequence↗
PubMed1988

Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor.

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7-34 and 9-50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7-37, 30-72 and 17-72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.

Alkylation↗
PubMed1988

Structures of amidohydrolases. Amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans and preliminary crystallographic data for an asparaginase from Erwinia chrysanthemi.

The complete amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans, for which a preliminary tertiary structure is available from crystallographic analysis, has been determined by automated Edman degradation of fragments produced by chemical and proteolytic cleavages. The protein consists of 331 amino acid residues and has a molecular weight of 35,500. The pattern of hydrophilic and hydrophobic regions is typical of a globular protein. A new crystal form of an Erwinia chrysanthemi 1125 asparaginase is reported. The space group is monoclinic C2, with unit cell parameters of: a = 107.8, b = 91.7, c = 129.2 A and beta = 91.7 degrees. A Vm of 2.25 A3/dalton was calculated for one tetramer of 35,100-dalton subunits per asymmetric unit. X-ray intensity data have been obtained to 2.2 A resolution. The point group symmetry of the Er. chrysanthemi tetramer is 222 from self-rotation function calculations. The relative orientations of an A. glutaminasificans glutaminase-asparaginase model and the Er. chrysanthemi asparaginase tetramer have been determined with the cross-rotation function, and translation function calculations have revealed a plausible location for the asparaginase tetramer in the crystal.

Acinetobacter↗
PubMed1987

The nucleotide sequence of a human cellular thyroid hormone binding protein present in endoplasmic reticulum.

A cDNA clone encoding a human cellular thyroid hormone binding protein (p55) has been isolated. The cDNA sequence contains a single open reading frame of 1524 nucleotides which encodes a polypeptide of 491 amino acids and a signal sequence of 17 amino acids. Two partial protein sequences determined from the purified protein correspond to the deduced amino acid sequence. In vitro translation of mRNA prepared by transcription of the cDNA clone yielded a protein of the size of an unprocessed precursor (Mr 57,000) as well as smaller fragments which were immunoprecipitated by antibodies against p55. The isolation of the cDNA clone should allow elucidation of the cellular function of this thyroid hormone binding protein which is present in the endoplasmic reticulum and nuclear envelope.

Amino Acid Sequence↗
PubMed1987

Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain.

We have determined the sequence of the sites phosphorylated by protein kinase C in the turkey gizzard smooth muscle myosin light chain. In contrast to previous work (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072), two-dimensional tryptic peptide maps of both heavy meromyosin and the isolated myosin light chain showed two major phosphopeptides, one containing phosphoserine and the other phosphothreonine. We have purified the succinylated tryptic phosphopeptides using reverse phase and DEAE high pressure liquid chromatography. The serine-containing peptide, residues 1-4 (Ac-SSKR), is the NH2-terminal peptide. The phosphorylated serine residue may be either serine 1 or serine 2. The threonine-containing peptide, residues 5-16, yielded the sequence AKAKTTKKRPQR. Analysis of the yields and radioactivity of the products from automated Edman degradation showed that threonine 9 is the phosphorylation site.

Amino Acid Sequence↗
PubMed1987

The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases.

Previous studies have shown that the region of human urokinase-type plasminogen activator (uPA) responsible for receptor binding resides in the amino-terminal fragment (ATF, residues 1-135) (Stoppelli, M.P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R.K. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 4939-4943). The area within ATF responsible for specific receptor binding has now been identified by the ability of different synthetic peptides corresponding to different regions of the amino terminus of uPA to inhibit receptor binding of 125I-labeled ATF. A peptide corresponding to human [Ala19]uPA-(12-32) resulted in 50% inhibition of ATF binding at 100 nM. Peptides uPA-(18-32) and [Ala13]uPA-(9-20) inhibit at 100 and 2000 microM, respectively. The human peptide uPA-(1-14) and the mouse peptide [Ala20]uPA-(13-33) have no effect on ATF receptor binding. This region of uPA is referred to as the growth factor module since it shares partial amino acid sequence homology (residues 14-33) to epidermal growth factor (EGF). Furthermore, this region of EGF is responsible for binding of EGF to its receptor (Komoriya, A. Hortsch, M., Meyers, C., Smith, M., Kanety, H., and Schlessinger, J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1351-1355). However, EGF does not inhibit ATF receptor binding. Comparison of the sequences responsible for receptor binding of uPA and EGF indicate that the region of highest homology is between residues 13-19 and 14-20 of human uPA and EGF, respectively. In addition, there is a conservation of the spacings of four cysteines in this module whereas there is no homology between residues 20-30 and 21-33 of uPA and EGF. Thus, residues 20-30 of uPA apparently confer receptor binding specificity, and residues 13-19 provide the proper conformation to the adjacent binding region.

Amino Acid Sequence↗
PubMed1987

DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy.

We have determined the sequence of the E. coli gyrB gene, using a new sequencing approach in which transposition from a mini-Mu plasmid into the DNA provides random start points for dideoxynucleotide sequence analysis. The gyrB sequence corresponds to a protein 804 amino acids long; a previously isolated protein fragment with partial enzymatic activity has been identified as the C-terminal half-molecule. A plausible terminator of gyrB transcription is located just beyond the structural gene.

Amino Acid Sequence↗
PubMed1987

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences.

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.

Amino Acid Sequence↗
PubMed1987

Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.

Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.

Amino Acid Sequence↗
PubMed1986

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA.

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.

Actins↗
PubMed1986

Amino acid sequence of the active site of Acanthamoeba myosin II.

We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.

Affinity Labels↗