Search PubMed⌕ Search

Biomedical subjects

K G Welinder

Publications and source records attributed to K G Welinder.

71 records · Page 4Linked to original sources
PubMed1986

Amino acid sequence analysis of the glycopeptides from human complement component C3.

Human complement component C3 has been cleaved completely by trypsin in the presence of 2-propanol. The hydrolysate was fully solubilized and fractionated by reversed-phase HPLC. Two peptides only contained glucosamine, Unambiguous sequence analyses identified Asn-63 of the beta-chain and Asn-268 of the alpha-chain as the sites of carbohydrate attachment. A third potential Asn-Xaa-Thr/Ser glycosylation site, Asn-946 of the alpha-chain, is not modified. The different states of glycosylation of the sites cannot be explained by differences in exposure or secondary structure. All three are predicted reverse turn.

1-Propanol↗
PubMed1986

Isolation and characterization of chicken and turkey beta 2-microglobulin.

Chicken and turkey beta 2-m were isolated from citrated plasma in sequential use of three chromatographic steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta 2-m by reaction with a beta 2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta 2-m and 4.7 and 4.8 for turkey beta 2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta 2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. EM280 was 15.9 for chicken beta 2-m and 16.4 for turkey beta 2-m. The amino acid compositions and sequences of the two avian beta 2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N-terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta 2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.

Amino Acid Sequence↗
PubMed1986

Human complement component C2: production and characterization of polyclonal and monoclonal antibodies against C2.

Rabbit antibodies to human complement component C2 were produced by immunization of rabbits with precipitates from line immunoelectrophoresis, and the antibodies were used to monitor a classical chromatographic purification of C2 and for affinity purification of C2. Twelve monoclonal antibodies with specificity for human complement component C2 were produced by fusion of myeloma cells with spleen cells from mice immunized with the affinity purified C2. The specificity of the monoclonal antibodies was confirmed by their reaction with antigen-antibody precipitates where C2 was the antigen, and by their specific reaction with C2 after separation in SDS-PAGE followed by immunoblotting. The affinity of the monoclonal antibodies varied as demonstrated by the titration curves in ELISA. The antibodies will be of importance for immunospecific purification of human C2 and C2 fragments, for specific depletion of C2 from human serum, and for quantification of C2 for clinical purposes.

Animals↗
PubMed1985

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase.

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.

Amino Acid Sequence↗
PubMed1985

Hemolytic plate assay for quantification of active human complement component C3 using methylamine-treated plasma as complement source.

A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbit erythrocytes. Plasma treated with methylamine supplies the essential complement components other than C3. The lytic reaction is complete in 5 h at 37 degrees C and is unchanged by incubation overnight. The dose-response curve, i.e., lysis diameter versus logarithm of C3 concentration, is linear within 0.1-10 times normal plasma concentrations of C3. The standard deviation is below 10%. The hemolytic agarose plates are easy and inexpensive to prepare, and they can be stored at 4 degrees C for 2 weeks before use. This paper describes the optimal conditions of the assay and proves its specificity. Its use in C3 preparation and plasma screening for C3 is discussed.

Animals↗
PubMed1984

Appendix. Structural predictions on alpha 2-macroglobulin from the amino acid sequence.

The composite result from three different methods predicts that alpha 2-macroglobulin has a predominant pattern of alternating beta-strands and turns. Only scattered pieces of alpha-helix are present throughout its sequence. Predicted beta-strands constitute 43.8% and helices 8.6%, in fair agreement with circular dichroism spectra. Circular dichroism spectra and sequence data suggest that the tertiary structure of complement component C3b is similar to that of the alpha 2 macroglobulin monomer. At present, the beta-barrel configuration of prealbumin seems to be a better model than those of soybean trypsin inhibitor, serine proteases, and immunoglobulins. A repetitive pattern of disulfide loops could indicate structural units of about 200 residues in size, but this is not supported by amino acid sequence homology. The hydropathic pattern of the sequence reveals numerous examples of predicted immunogenic peptides and detects an approximately 100-residue hydrophobic core region, which could constitute parts of the tentatively assigned consecutive activation cleavage and thiol ester domains. This core is an obvious candidate of rearrangement transfer between the highly exposed bait region and the partly shielded thiol ester site. The presumed intersubunit half-cystine is moderately buried and has two nearby patches of high charge density which could guide the dimeric assembling.

Amino Acid Sequence↗
PubMed1983

The oxygen binding site of cytochrome oxidase. Structural predictions on subunit I from amino acid sequences.

Analyses of heme-attached amino acid sequences in known hemoprotein superfamilies provide a basis for prediction of such sequences in hemoproteins of unknown three-dimensional structure. Among 11 histidine residues conserved in subunit I of 3 mammalian and 2 fungal cytochrome oxidases the sequence around His-233 (human) is the most conserved and shows remarkable similarity to the sequence of the oxygen binding site in globins. Furthermore, the gene coding for subunit I in Saccharomyces cerevisiae and the gene for leghemoglobin in soybean are both split by introns right after these similar histidine sequences. The predicted distal histidine sequence of subunit I provides for heme a3 and Cua3 binding and has an extraordinarily high content of aromatic residues. These aromatic groups may serve as a molecular electron capacitor. Transmembrane sequences and electron transfer sequences are proposed.

Amino Acid Sequence↗
PubMed1983

A simple alternative pathway for hemolytic assay of human complement component C3 using methylamine-treated plasma.

A quantitative, alternative pathway (AP) hemolytic assay for human complement component C3 has been developed. This AP-C3 assay is inexpensive, rapid, simple, reproducible and insensitive to C3 degradation products. The AP-C3 assay uses rabbit erythrocytes as complement activator and methylamine-treated plasma, depleted of C3 and C4, as complement source. Rabbit erythrocyte sensitivity varies little from batch to batch, and remains unaltered for at least one month in Alsever's solution. Methylamine plasma may be stored at -20 degrees C for 3 months. AP-C3 lysis of 5-25% of erythrocytes is complete in 20 min and does not change subsequently. The AP-C3 assay is optimally stable at 2 mM Mg2+, 5 mM EGTA and at 5 X 10(7) erythrocytes/ml, yet insensitive to at least 20% deviation in these concentrations. The AP-C3 assay is specific to functional C3 and well suited for determination in plasma samples and during C3 preparation. Adding excessive amounts of C3c or plasma components other than C3 does not change the hemolytic response. The level of native C3 in plasma from 14 donors relative to a reference plasma pool ranged from 0.78 to 1.23. The standard deviation of relative C3 determinations did not exceed 2%.

Animals↗
PubMed1980

Covalent structure of turnip peroxidase 7. Tryptic peptides.

Turnip isoperoxidase TP 7 had its hemin group removed and was cleaved by trypsin. The digest was fractionated by gel filtration and high-voltage paper electrophoresis and yielded 24 peptides, which counted for all 296 amino acid residues of the enzyme. Sequence analyses on the tryptic peptides and, when necessary, on their thermolytic derivatives were carried out by manual Edman degradation followed by dansylation or by quantitative amino acid analysis of thiazolinones converted by HI. The four disulfide bridges and the only site of carbohydrate attachment of turnip peroxidase 7 are located. The present analysis of tryptic peptides has been complemented by cyanogen bromide fragments cleaved by chymotrypsin as described in the accompanying paper.

Amino Acid Sequence↗
PubMed1980

Covalent structure of turnip peroxidase 7. Cyanogen bromide fragments, complete structure and comparison to horseradish peroxidase C.

The complete amino acid sequence of turnip peroxidase TP 7, the principal isoperoxidase during winter in turnip, Brassica napus L., variety Blanc dur d'hiver, has been determined by sequence analysis of cyanogen bromide fragments and of tryptic peptides. The turnip peroxidase TP 7 enzyme is composed of 296 amino acids, one hemin group and one neutral carbohydrate side chain attached through asparagine. The molecular weight of the polypeptide part is 31,060, and including hemin and carbohydrate the molecular weight of the native enzyme is close to 33,400. The isoelectric point of turnip peroxidase TP 7 is 11.6. Comparison of turnip peroxidase TP 7 and horseradish peroxidase HRP C shows that they contain four similarly located disulfide bridges and have pyrrolidone carboxylyl N termini. Their common evolutionary origin is distant as their amino acid sequences are only 49% identical. Furthermore, turnip peroxidase TP 7 differs significantly from three other isoperoxidases of turnip root, turnip peroxidases TP 1, TP 2 and TP 3, and from horseradish peroxidase HRP C in its physico-chemical and enzymatic properties, and its pronounced season-dependent appearance. All these differences of turnip peroxidase TP 7 and of the others suggest they serve separate biological functions.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C.

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44 000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.

Amino Acid Sequence↗
PubMed1977

Amino-acid sequences of heme-linked, histidine-containing peptides of five peroxidases from horseradish and turnip.

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences nearthe peroxidase heme prostetic group. In the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heme, and 34 residues around the probably distally located histidine, and compare them with the histidine-containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3, and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physio-chemical and enzymic properties. Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site.

Amino Acid Sequence↗
PubMed1975

Similarities and differences of five peroxidases from turnip and horseradish. Peptide mapping studies on glycoproteins.

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to alysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.

Amino Acids↗
PubMedDate not supplied

Amino acid sequences and structures of chicken and turkey beta 2-microglobulin.

The complete amino acid sequences of chicken and turkey beta 2-microglobulins have been determined by analyses of tryptic, V8-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta 2-microglobulin supports the sequence-derived Mr of 11,048. The higher apparent Mr obtained for the avian beta 2-microglobulins as compared to human beta 2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta 2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta 2-microglobulin at 46 and 47 positions, respectively, and to bovine beta 2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta 2-microglobulins. The known X-ray crystallographic structures of bovine beta 2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of beta 2-microglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta 2-microglobulin can substitute the chicken beta 2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.

Amino Acid Sequence↗