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B W Erickson

Publications and source records attributed to B W Erickson.

72 records · Page 4Linked to original sources
PubMed1981

Solid-phase peptide synthesis under continuous-flow conditions.

A system is described for solid-phase synthesis of peptides under continuous-flow conditions with liquid chromatographic equipment, conventional polystyrene supports, and well-defined chemistry. The model tetrapeptide Leu-Ala-Gly-Val was assembled in 99.3% purity in about 4 hr on microporous copoly(styrene-1% divinylbenzene). During coupling, the preformed symmetric anhydrides were conserved by being recycled. Relative yields of the peptide products were determined quantitatively in 20 min by reverse-phase high-pressure liquid chromatography. This rapid assay system was used to examine the influence on product yields of (i) the time and number of couplings per cycle, (ii) microporous versus macroporous polystyrene, and (iii) tert-butoxycarbonyl (Boc) group versus 9-fluorenylmethoxycarbonyl for amine protection. Use of microporous polystyrene and two 30-min couplings of Boc-amino acids per cycle gave the best results. This continuous-flow system provides a rapid and efficient approach to solid-phase peptide synthesis. A 17-residue peptide from chicken ovalbumin was obtained in similar purity and yield from a discontinuous synthesis and from a continuous-flow synthesis.

Amino Acid Sequence↗
PubMed1981

Molecular basis for the temperature-dependent insolubility of cryoglobulins. X. The amino acid sequence of the heavy chain variable region of McE.

The amino acid sequence of the VH region of McE, a monoclonal IgM cryoimmunoglobulin, has been determined employing automated sequencing methodology. Digestion of the intact Fab mu component, derived by trypsin cleavage of the parent protein at elevated temperature with CNBr, followed by complete reduction and alkylation, yielded the intact light chain as well as the 2 CNBr fragments that constituted the VH. N-terminal sequencing of the larger unblocked CNBr fragment, along with a component fragment derived by cleavage by BNPS-Skatole, established the structure of the VH from position 88 through the V leads to C switch region. Citraconylation of the smaller, blocked fragment effected sufficient solubilization for enzymatic deblocking and direct sequencing of the N-terminal 20 residues of the VH. Complete trypsin digestion of the N-terminal CNBr fragment yielded 9 peptides that could be isolated by preparative cation exchange chromatography and gel filtration. The complete sequence of these peptides along with 4 chymotryptic peptides completed the primary structure of the VH region. The primary structure of McE appears to resemble that of He, previously identified as belonging to the VH II subgroup. The presence of characteristic CDR and FR regions as well as the identification of a probable site of glycosylation suggest that the cryoimmunoglobulin closely resembles noncryoglobulins in terms of overall structural composition. The cryoglobulin property may arise through alterations in individual residues or unfavorable arrangements of CDR and FR segments.

Amino Acid Sequence↗
PubMed1981

Inhibition of C1-mediated immune hemolysis by monomeric and dimeric peptides from the second constant domain of human immunoglobulin G.

Activation of the classical complement cascade by immunoglobulin G involves binding of the first complement component (C1) to a multivalent antigen-IgG complex. Binding occurs by interaction of a site on the surface of the C gamma 2 domain of IgG with a globular head of the C1q subcomponent of C1. Previously we found that the synthetic decapeptide consisting of residues 281-290 from the second constant domain of the gamma-chain of the human IgG1 protein Eu inhibited the binding of human C1 to sensitized erythrocytes. The present study describes inhibition of monomeric and dimeric peptides containing residues 289-292 or 282-292: (formula: see text). On a molar basis, monomeric peptide 282-292 is just as active an inhibitor of C1 binding as peptide 281-290, whereas monomeric peptide 289-292 (tuftsin) is 4 times less active. Peptides 282-292 and 289-292 were each cross-linked at the amino terminus through terephthaloyl-bis(iminodiacetic acid) (Tid). Each dimeric peptide is twice as active on a molar basis as the corresponding monomeric peptide. Dimeric peptide Tid(282-292)2 is just as active on a molar basis as the monomeric 7S form of human IgG1 and 60% as active as the Fc fragment of IgG in inhibiting the binding of human C1 to sensitized erythrocytes. These results suggest that the positively charged residues His-285, Lys-288, Lys-290, and Arg-292, which are located on the outer surface of the C gamma 2 domain, may be involved in the C1q-binding site of human IgG.

Animals↗
PubMed1980

The active site of C3a anaphylatoxin.

The essential active site responsible for the inflammatory activities of C3a, an anaphylatoxin derived from the serum complement system, has been elucidated using C3a peptides synthesized by the solid phase method and assayed for their ability to contract guinea pig ileal tissue and to produce a wheal and flare response in human skin. The COOH-terminal C3a pentapeptide (Leu-Gly-Leu-Ala-Arg) common to rat, pig, and man shows vascular and smooth muscle activity as well as specificity similar to natural human C3a. The porcine C3a octapeptide is 3 times more active than the common pentapeptide, but the human octapeptide (Ala(70)-Ser-His-Leu(73)-Gly-Leu(75)-Ala-Arg(77) is 12 times more active than the pentapeptide. Replacement of the serine and histidine by alanine or acetylation of the NH2 terminus provides analogues with the same activity as the octapeptide. Thus, the increased activity of the human C3a octapeptide over the pentapeptide appears to be related to the backbone of residues 70 to 72 and is not due to the presence of the hydroxyl group of serine-71, the imidazole ring of histidine-72, or a positive charge at or near the NH2 terminus. Since the COOH-terminal tetrapeptide is 40 times less active than the pentapeptide, an adequate model of the essential active site of C3a anaphylatoxin is the common COOH-terminal pentapeptide region. A C3a active site analogue containing a COOH-terminal lysyl residue is devoid of ileal activity. In addition, the [alanine-73]pentapeptide is 9 times less active and the [alanine-75]pentapeptide is at least 70 times less active than the active site pentapeptide in the ileal assay. Thus, the hydrophobic side chains of leucine-73 and leucine-75 and the guanidinium group of arginine-77 are important for the contractile activity of the active site COOH-terminal pentapeptide of human C3a anaphylatoxin.

Amino Acid Sequence↗
PubMed1979

Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.

Anaphylatoxins↗
PubMed1979

The T-lymphocyte as a diploid reference standard for flow cytometry.

T-lymphocytes prepared from the peripheral blood of normal individuals exhibit uniform postmitotic DNA contents that are constant from individual to individual when fixed in ethanol and stained with mithramycin, or when fixed in formalin and stained with acriflavine-Feulgen and analyzed by flow microfluorometry. T-lymphocytes fixed by either method are stable on storage for several months. These cells are suitable for use as an external diploid reference standard for flow cytometric detection of cells with abnormalities in postmitotic content exceeding 5% of diploid reference. Cells with abnormalities in postmitotic DNA content of less than 5% can be detected in mixtures containing both normal and malignant cells by multiparameter analysis.

DNA↗
PubMed1977

Synthetic peptides with the biological activities and specificity of human C3a anaphylatoxin.

Two peptides identical to the COOH-terminal sequence of human C3a anaphylatoxin and two analogs were synthesized by the solid-phase method and tested for biological activity. The synthetic COOH-terminal octapeptide, C3a-(70-77) or Ala-Ser-His-Leu-Gly-Leu-Ala-Arg, caused contraction of guinea pig ileum and uterus, release of vasoactive amines from rat mast cells, and increased vascular permeability in guinea pig and human skin. On a molar basis, the synthetic octapeptide possessed 1-2% of the biological activities of C3a and specifically desensitized smooth muscle to stimulation by C3a. Like natural C3a, the synthetic C3a=(70-77) was inactivated by digestion with carboxypeptidase B [peptidyl-L-lysine(-L-arginine) hydrolase, EC 3.4.12.3], which removed the essential COOH-terminal arginine. A synthetic nonapeptide [C3a-(70-77)-Gly], containing a glycyl instead of an arginyl COOH terminus, was approximately 1% as active as the octapeptide when assayed with smooth muscle. The COOH-terminal 13-residue peptide of C3a, C3a-(65-77), was equal in activity to C3a=(70-77); similarly, C3a-(65-77)-Gly expressed the same activity as C3a-(70-77)Gly. It is concluded that both the biological specificity and the activity of human C3a anaphylatoxin are determined by eight or fewer residues located at the COOH terminus of the natural protein. However, expression of full activity requires additional groups and the secondary conformational integrity of the C3a molecule.

Amino Acid Sequence↗
PubMed1976

Affinity purification of synthetic peptides.

A general strategy and a specific tactic for affinity purification of polypeptides synthesized on solid supports are desbribed and demonstrated. The desired peptide chains were distinguished from terminated peptide chains before removal from the support by attachment of an affinity reagnet (cysteinyl-methionine) bearing an affinity group (thiol) and a binding group (carboxylic acid). After cleavage from the synthetic support, the affinity-labeled peptides (Cys-Met-peptides) were bound to an affinity receptor (organomercurial-agarose) and thus separated from terminated peptides and all other peptides lacking the affinity group. The desired synthetic peptide was obtained by separation of the affinity reagent (loss of Cys-Met by cyanogen bromide cleavage). This general affinity purification strategy is independent of the length or amino acid sequence of the desired peptide. After assembly of ribonuclease-(111-124)-tetradecapeptide, using radiolabeled acetic anhudride for termination of uncoupled in termediates, essentially all (greater than 98.5%) of the acetylated delection peptides were removed by employing the organomercurial Cys-Met tactic. Similarly, the purity of crude synthetic histone H4-(1-37)-heptatriacontapeptide was increased six-fold by using this tactic to remove terminated peptides. A related dimeric Cys-Met tactic is outlined for affinity purification of peptides containing internal cysteine and methionine residues.

Amino Acids↗