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B W Erickson

Publications and source records attributed to B W Erickson.

At least 55 records · Page 3Linked to original sources
PubMed1984

Active site of C3a anaphylatoxin: contributions of the lipophilic and orienting residues.

Activation of the serum complement cascade generates C3a anaphylatoxin, a primary mediator of inflammation. The active-site pentapeptide from the COOH terminus of C3a, Leu-Gly-Leu-Ala-Arg (residues 73-77), exhibits the inflammatory activities and specificity of the native 77-residue polypeptide. Functionally important features of this active site were studied by testing the ability of 22 synthetic analogues of this pentapeptide to contract isolated muscle strips from guinea pig ileum and to desensitize this tissue to contraction induced by human C3a or C5a. The C3a receptors on mast cells and basophils probably contain lipophilic groups that interact with the lipophilic side chains of Leu-73 and Leu-75 and charged groups that interact with the carboxylate and guanidinium groups of Arg-77. The lipophilic contribution of Leu-73 is modest and sterically nonspecific while that of Leu-75 is substantial and sterically specific. Gly-74 and Ala-76 appear to position and orient the adjacent residues Leu-73, Leu-75, and Arg-77 for optimal receptor binding. The contribution of Gly-74 is neither conformationally nor sterically specific while that of Ala-76 is both conformationally and sterically specific. The cellular C3a receptors evidently interact most efficiently with peptides ending in -Leu-Ala-Arg-OH.

Anaphylatoxins↗
PubMed1984

Contraction of guinea pig lung by synthetic oligopeptides related to human C3a.

Complement-derived human C3a is a 77 residue protein whose biological activities include the contraction of guinea pig ileum and parenchymal lung strips. The C3a molecule is active at submicromolar concentrations and the spasmogenic activities are absolutely dependent on a carboxy-terminal arginyl residue. Studies with synthetic peptide analogues of C3a have localized the active site for all spasmogenic functions at the carboxy-terminal portion of the native molecule. Studies reported here demonstrate that the spasmogenic action of C3a on guinea pig parenchymal lung tissue is mimicked by synthetic peptides based on the carboxy-terminal sequence of C3a. Synthetic peptides with sequences corresponding to the 5, 8, 13 and 21 carboxy-terminal residues of C3a all possess spasmogenic activity on lung tissue. Molar activities of the synthetic peptides relative to that of C3a increase as the length of the peptide increases. The activity of the pentapeptide C3a 73-77 is only 0.5% that of C3a, while those of C3a 70-77 and C3a 65-77 are 3.8 and 7.8%, respectively. A 21 residue peptide, C3a 57-77, exhibits activity equivalent to native C3a. The synthetic peptides, unlike C3a, fail to produce tachyphylaxis. We compared C3a reactivity of guinea pig parenchymal lung strips with those of the synthetic C3a peptides in the presence of various inhibitor combinations. Responses of lung strips to C3a or the C3a peptides were not significantly inhibited by the antihistamine pyrilamine. However, lung responses to synthetic C3a peptides, like those to C3a, were inhibited by indomethacin. Complete inhibition of responses to C3a or the synthetic C3a peptides was produced in the presence of indomethacin, FPL55712 and pyrilamine.

Acetylcholine↗
PubMed1984

Internal duplication in human alpha 1 and beta 1 interferons.

Metric analysis of the nucleotide sequence of the intron-free human interferon beta 1 (IFN-beta 1) gene by using the Sellers TT algorithm revealed that this gene contains two major repeated segments, which span the entire coding region. These repeats are each approximately 300 nucleotides in length and have 45% identical aligned nucleotides (common bases). When these metrically aligned DNA repeats were translated into amino acids, 9 (19%) of the 47 in-phase amino acid residues were identical (common acids). This internal duplication was also apparent on visual inspection of the amino acid sequence of IFN-beta 1. In addition, metric analysis of the nucleotide sequence of the intron-free IFN-alpha 1 gene showed that this gene also contains two repeats, each approximately 300 nucleotides long, having 47% common bases and 19% common acids. Since the IFN-alpha 1 and -beta 1 genes are known to be related (by the present metric analysis they contain 53% common bases and 45% common acids), a consensus DNA sequence was derived from all four of these repeats. Manual alignment of the separate metric alignments corresponding to the two halves of the IFN-alpha 1 and -beta 1 genes provided a composite alignment with 58% of the alignment positions having the same nucleotide in at least three of the four repeats. When this composite nucleotide alignment was translated to define a composite alignment of the four protein segments, 10 (31%) of the 32 in-phase amino acid residues contained the same amino acid in at least three of the four segments. These sequences relationships provide insight into the origin of the IFN-alpha 1 and -beta 1 genes and furnish an additional basis for comparing them with other related genes.

Amino Acid Sequence↗
PubMed1984

Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins.

The molecular mechanisms by which calcium regulates cellular processes such as metabolic and mechanochemical events probably involve interactions with a variety of molecules. A large body of evidence suggests that the targets of calcium's regulatory effects inside the cell are calcium-binding proteins. Our work attempts to correlate calcium-binding protein structure with activities. In this chapter we have presented some of our recent studies on these functional domains in calmodulin and calmodulin-binding proteins. Selected chemical modifications of known amino acid sequence positions have demonstrated the presence of multiple functional domains on calmodulin, have allowed the dissociation of calmodulin functions, and have provided the necessary tools for further investigations of the molecular basis of calmodulin action. One of these modifications, iodination of tyrosine-99, has allowed us to develop procedures to reproducibly detect calmodulin-binding proteins by a binding technique. This method is technically simple. It allows us to detect and study calmodulin-binding proteins (e.g., myosin heavy chain and membrane gap junction proteins) that would be difficult to study with immobilized calmodulin. We have developed a library of antibodies to calmodulin and related proteins such as troponin C and S100 beta. Some of these antisera appear to be site-specific gamma globulins. We have demonstrated that reactivity can be contained in an amino acid sequence as short as seven residues. We have demonstrated the feasibility of using an immunochemical mapping approach to study calmodulin and calmodulin-binding proteins. Comparative sequence analyses combined with functional analyses have allowed correlation of function and structure and have suggested logical candidates for functional domains on calmodulin and calmodulin-binding proteins. Although not discussed in detail in this chapter, these calmodulin-binding proteins appear to contain amino acid sequence homologies. This suggests, analogous to the approach used for calmodulin and related proteins, a logical starting point for domain analyses of calmodulin-binding proteins. Interestingly, structural homologies among calmodulin-binding proteins are reminiscent of the different phosphorylation sites found in many of the physiological substrates for protein kinases. Since a number of calmodulin-binding proteins are themselves substrates for protein kinases, these results suggest another possible point of interrelationships between calcium and cyclic nucleotide regulation.

Amino Acid Sequence↗
PubMed1983

Complete nucleotide sequence of an immunoglobulin VH gene homologue from Caiman, a phylogenetically ancient reptile.

Immunoglobulin variable (V) gene regions typify extensive multigenic families in terms of overall size, chromosomal arrangement and presence of large numbers of apparent pseudogenes. A unique mechanism of somatic reorganization involving recombination of VH, D and JH or VL and JL segments accompanies the differentiation of lymphoid cells and together with somatic mutation and other types of recombination accounts for V-region diversity. Although these processes have been well characterized in higher mammals, little is known concerning their origin and diversification during phylogenetic time. Previously, we described the blot-hybridization characteristics of murine VHIII probes with restriction enzyme-digested genomic DNA isolated from several phylogenetically critical species, including Caiman crocodylus, a modern representative of an ancient reptilian subclass. Here we have used a murine probe, S107V, to select homologous clones from a library of Caiman genomic DNA constructed in a lambda bacteriophage. The complete nucleotide sequence of a Caiman gene homologous to the murine VH gene and its adjacent 5' and 3' region is described. Comparison of the sequence with mammalian prototypes shows evidence of considerable organizational and structural homology extending outside the presumed VH-coding region and including elements believed to be involved in somatic recombination. Inferences about the evolution of this multigenic family can now be extended to the level of phylogenetic class.

Alligators and Crocodiles↗
PubMed1983

Radioimmunoassays for the thymic hormone serum thymic factor (FTS).

Four radioimmunoassays (RIA) are described for the quantitation of serum thymic factor (facteur thymique serique, FTS), a thymic peptide hormone. Each assay employs an antibody specific for FTS, synthetic FTS (Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) as the hormone standard, and a radioiodinated FTS analogue as the tracer. Since FTS lacks a tyrosine residue, 2 FTS analogues were synthesized by the solid-phase method with tyrosyl-alanyl or 3-(2,6-dichlorobenzyl)tyrosyl-alanyl in place of the amino-terminal pyroglutamyl residue (Glp). They showed full FTS immunoreactivity and their radioiodinated derivatives served as FTS tracers. Two assays used the antiserum from a rabbit immunized with an FTS-protein conjugate. Two other assays used a monoclonal antibody against FTS produced by a hybridoma derived from mouse myeloma cells and splenocytes from a BALB/c mouse immunized with an FTS-mouse IgG conjugate (Ohga et al., 1982). All 4 RIAs were specific for FTS. The more sensitive rabbit antiserum can detect as little as 1 pg of FTS in a 50 microliters sample, which may allow quantitation of the FTS circulating in human peripheral blood.

Amino Acid Sequence↗
PubMed1983

Elucidation of a minimal immunoreactive site of vertebrate calmodulin.

The heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2 corresponding to residues 137-143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem. 256, 4205-4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127-144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only seven-residue segment in the 135-145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.

Animals↗
PubMed1983

The active site of human C4a anaphylatoxin.

The human C4 activation peptide C4a has recently been shown to be biologically active and to share common tissue receptors with human C3a anaphylatoxin. Human C3a and C4a each induce contraction and cause cross-desensitization of isolated guinea-pig ileal strips. The essential active site of C3a is comprised in the model peptide containing the five COOH-terminal residues, Leu-Gly-Leu-Ala-Arg. The anaphylatoxic activities of the corresponding C4a pentapeptide, Ala-Gly-Leu-Gln-Arg, and several other synthetic peptides related to the COOH-terminal sequence of human C4a were examined. The C4a pentapeptide induced contraction of guinea-pig ileum at 1 X 10(-3) M and produced a wheal and flare reaction in human or guinea-pig skin when 2-5 mumols were injected intradermally. The corresponding C3a pentapeptide is 500-fold more active, since it induces contraction of guinea-pig ileum at 3-4 X 10(-6) M and only 4-10 nmole induce a visible skin reaction. Although the C4a pentapeptide is relatively inactive compared to the C3a pentapeptide, two analogs of these peptides, Leu-Gly-Leu-Gln-Arg and Ala-Gly-Leu-Ala-Arg, each exhibited significantly greater activity than Ala-Gly-Leu-Gln-Arg and each analog desensitized ileal smooth muscle towards contraction by either C3a or C4a. Thus it is a combination of two amino acid substitutions, the Ala for Leu-73 and Gln for Ala-76, in the COOH-terminal pentapeptide of C3a that accounts for the markedly reduced activity of C4a. The contribution of the COOH-terminal portion of C4a on its activity was further documented by examining the C4a octapeptide, Lys-Gly-Gln-Ala-Gly-Leu-Gln-Arg and a trialanyl analog, Ala-Ala-Ala-Ala-Gly-Leu-Gln-Arg. The C4a octapeptide, C4a (70-77), exhibited 5-fold greater biologic activity than the C4a pentapeptide, while the trialanyl analog was 40-fold more active. Anaphylatoxic activities of the C4a-(73-77) pentapeptide, C4a-(70-77) octapeptide, and the trialanyl octapeptide analog and their ability to specifically block the action of C3a and C4a on smooth muscle tissue support the conclusion that, as in C3a, the essential active site of C4a resides at its COOH terminus. Since C4a functions as an anaphylatoxin and significant quantities of this mediator may be generated in individuals with hereditary angioneurotic edema (HANE), the hypotheses that the kinin-like activity promoting edema in HANE patients is derived solely from component C2 and/or kininogens should be reappraised. The activities previously assigned to C4a and now confirmed by synthetic C4a analog peptides suggest that the kinin-like activity generated in HANE plasma may be derived in part from C4a.

Amino Acid Sequence↗
PubMed1983

Engineering of site-directed antisera against vertebrate calmodulin by using synthetic peptide immunogens containing an immunoreactive site.

Site-directed antisera against vertebrate calmodulin were elicited in rabbits by injection of a synthetic immunogen containing the pentadecapeptide Gly-Gln-Val-Asn-Tyr-Glu-Glu-Phe-Val-Gln-Met-Met-Thr-Ala-Lys-OH, which corresponds to residues 134-148 of vertebrate calmodulin. A major immunoreactive region (residues 127-144) of calmodulin is found in the COOH-terminal structural domain and an immunoreactive site for one antiserum is contained in the heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2, which corresponds to residues 137-143 of vertebrate calmodulin. This immunoreactive heptapeptide was conjugated to a carrier protein by adding a cysteine residue to the NH2 terminus of the peptide and coupling the Cys-heptapeptide to the carrier through the thiol group of the cysteine residue. Injection of this Cys-heptapeptide-protein conjugate into rabbits yielded antisera that react with the heptapeptide but not with native calmodulin. Thus, the immunoreactive heptapeptide that is exposed on the surface of calmodulin is immunogenic, but it is not sufficient to elicit antibodies that react with native calmodulin. However, when the Cys-pentadecapeptide corresponding to residues 134-148 and containing the immunoreactive heptapeptide sequence was conjugated to a carrier protein and injected into rabbits, antisera were elicited that react with the intact calmodulin molecule. The affinities and specificities of these antisera for calmodulin are similar to those of antisera elicited by injection of the intact protein and are sufficient for their use in radioimmunoassays. These results indicate that the successful engineering of site-directed antisera against proteins by using synthetic peptide immunogens may require an appropriate intramolecular environment that allows the peptide region to closely approximate the spatial orientation it adopts in the intact protein.

Amino Acid Sequence↗
PubMed1983

Synthesis of 2-methyl and 2-carboxymethyl derivatives of ornithine and arginine.

Synthesis and characterization of a dozen derivatives of ornithine and arginine bearing 2-methyl or 2-carboxymethyl substituents are described. These substituents were introduced by dilithiation of 3-(4-methoxybenzylidineamino)-2-piperidinone with lithium diisopropylamide followed by regiospecific alkylation at C-3 with iodomethane or ethyl bromoacetate. 2-Methyl-D,L-ornithine was obtained in three steps from 3-amino-2-piperidinone in 68% overall yield, and 2-carboxymethyl-D,L-ornithine was isolated in 56% overall yield. 2-Methyl- and 2-carboxymethyl-D,L-arginine were obtained by mild acid hydrolysis to remove the 4-methoxybenzylidine group, N-acylation with 4-toluenesulfonyl chloride, mild alkaline hydrolysis of the lactam ring and the ester group, guanidination of the 5-amino group with O-methylisourea, and strong acid hydrolysis to remove the 4-tolunenesulfonyl group. Several of these compounds are inhibitors of carboxypeptidase B.

Arginine↗
PubMed1983

Suppression of humoral immune responses by synthetic C3a peptides.

Synthetic oligopeptides based on the COOH-terminal sequence of native human C3a were examined for the ability to suppress antigen-specific and polyclonal antibody responses. The synthetic peptides were found to qualitatively mimic the suppressive actions of human C3a, but proved to be less active on a molar basis. Comparison of the activity of the peptides with native C3a indicates that the most potent peptide is C3a 57-77 with C3a 65-77, C3a 70-77, C3a[Ala 71,72]70-77, and C3a 73-77 being less active, respectively. These results indicate that the region of the C3a molecule responsible for immunosuppression is located in the COOH region.

Anaphylatoxins↗
PubMed1982

An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4.

Two cyclic structures, the 15-membered thiolactone A and the 5-membered lactam P, have been proposed for the metastable binding sites of the serum proteins C3, C4, and alpha 2-macroglobulin. Neither structure alone adequately explains two unusual reactions of these sites, namely, covalent attachment to nucleophiles with liberation of a thiol group and spontaneous hydrolysis (autolysis) of an internal peptide bond. The metastable binding sites of these proteins were modeled with the 15-membered thiolactone 1 (Khan, S. A., and Erickson, B. W. (1981) J. Am. Chem. Soc. 103, 7374-7376) and the isomeric 5-membered lactam 2, which contains an internal pyroglutamyl (Glp) residue. Under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C), thiolactone 1 and lactam 2 exist in dynamic equilibrium. Since the molar ratio of 2/1 is 11:1 at equilibrium, lactam 2 is 15 kcal/mol more stable than thiolactone 1. The activation energy for isomerization of 1 into 2 is 18 kcal/mol, which is about 5 kcal/mol lower than that for hydrolysis of the acyclic thiolester N,S-diacetyl-L-cysteine methylamide. Part of the chemistry of the metastable binding sites can be explained by an analogous equilibrium between protein structures A and B. Lactam B may be a key intermediate in the biosynthesis of thiolactone A. Under denaturating conditions, thiolactone A could either bind covalently to a receptive surface or isomerize into lactam B, which could undergo spontaneous hydrolysis of the Glu-Glp peptide bond.

Binding Sites↗
PubMed1982

Antibody response in Heterodontus.

Appropriately selected phylogenetic models are capable of providing insight into genetic mechanisms which may have become obscured during the passage of evolutionary time. In higher vertebrates a complex multigenic family encodes immunoglobulin-variable regions. The mechanisms involved in the expansion of the gene family and the stable maintenance of large numbers of individual genes presently are not understood. By defining the nature of antibody diversity in lower vertebrate species, it may be possible to approach such issues at a more fundamental level. Analyses of the immunoglobulins in Heterodontus francisci (horned shark), a representative phylogenetically primitive elasmobranch, indicate that this species may represent a useful developmental model.

Animals↗
PubMed1982

Molecular basis for the temperature-dependent insolubility of cryoglobulins--XI. Sequence comparison of the heavy-chain variable regions of the human cryoimmunoglobulins McE and Hil by metric analysis.

The amino acid sequences of the VH-domains from two human cryoimmunoglobulins have been compared with one another and with the VH-domains of noncryoglobulins by the mathematical method of metric analysis. The VHII sequence of McE [Gerber-Jenson et al. (1981), J. Immun. 126, 1212-1216] and the VHIII sequence of Hil [Chiu et al (1979), Biochemistry, 18, 553-560] resemble much more closely the VH-sequences of noncryoglobulins from their own subgroups than they resemble one another. Neither cryoglobulin sequence contains an unusual insertion or deletion of residues. Based on the crystallographic structure of the VHII domain in the Fab fragment of the human noncryoglobulin Newm [Saul et al. (1978), J biol. Chem., 253, 585-597], McE and Hil each contain two unprecedented residues in the outer beta-sheet structure of the VH-domain. The inwardly directed sidechains of Gly-71 and Ile-84 in McE may perturb the internal hydrophobic interactions and normal folding of adjacent segments of the outer beta-sheet from the third framework region. In contrast, the outwardly directed sidechains of Ile-23 and Arg-77 in Hil may perturb the external hydrophilic properties and folding of adjacent segments of the outer beta-sheet from the first and third framework regions. Thus, the monoclonal immunoglobulins McE and Hil may display cold-induced insolubility by different perturbations of the outer surface of the VH-domain. In each case, the unprecedented framework residues that mnay be responsible could have arisen by two point mutations involving single base changes.

Amino Acid Sequence↗
PubMed1982

Complete nucleotide sequence of the neuraminidase gene of influenza B virus.

The complete nucleotide sequence of the neuraminidase gene of influenza virus B/Lee/40 was derived from a cloned cDNA copy of virion RNA segment 6 and its corresponding mRNA. The RNA segment contains 1,557 virus-specific nucleotides, and the protein encoded by the longest open reading frame has a total of 466 amino acids with a molecular weight of 51,721. As is the case with the influenza A virus neuraminidases, the deduced amino acid sequence of the influenza B protein includes a single hydrophobic region near the amino terminus which would be capable of spanning the lipid bilayer of the viral or cell membrane. There are four potential glycosylation sites in the protein, two of which are near the amino-terminal hydrophobic region. Comparisons of the nucleotide and amino acid sequences with those of influenza A virus neuraminidases revealed seven regions of extensive homology within the central portion of the molecules, including 12 conserved cysteine residues. Five other cysteine residues in the terminal portions were also conserved.

Base Sequence↗