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T Kleinschmidt

Publications and source records attributed to T Kleinschmidt.

At least 55 records · Page 3Linked to original sources
PubMed1987

The primary structure of the hemoglobin of an Indian flying fox (Cynopterus sphinx, Megachiroptera).

The hemoglobin of the Indian flying fox Cynopterus sphinx contains only one component. In this work, we are presenting its primary structure. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas-phase Edman degradation of the chains and their tryptic peptides, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The alpha-chains show 14 and the beta-chains 19 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1 contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are exchanged. The functional and evolutionary aspects of these findings are discussed.

Amino Acid Sequence↗
PubMed1987

Primary structure and functional properties of the hemoglobin from the free-tailed bat Tadarida brasiliensis (Chiroptera). Small effect of carbon dioxide on oxygen affinity.

The hemoglobin of the Free-Tailed Bat Tadarida brasiliensis (Microchiroptera) comprises two components (Hb I and Hb II) in nearly equal amounts. Both hemoglobins have identical beta-chains, whereas the alpha-chains differ in having glycine (Hb I) or aspartic acid (Hb II) in position 115 (GH3). The components could be isolated by DEAE-Sephacel chromatography and separated into the globin chains by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman degradation with the film technique or the gas phase method (the alpha I-chains with the latter method only), using the native chains and tryptic peptides, as well as the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The comparison with human hemoglobin showed 18 substitutions in the alpha-chains and 24 in the beta-chains. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1-contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. A comparison with other chiropteran hemoglobin sequences shows similar distances to Micro- and Megachiroptera. The oxygenation characteristics of the composite hemolysate and the two components, measured in relation to pH, Cl-, and 2,3-bis-phosphoglycerate, are described. The effect of carbon dioxide on oxygen affinity is considerably smaller than that observed in human hemoglobin, which might be an adaptation to life under hypercapnic conditions.

Amino Acid Sequence↗
PubMed1987

N-terminal amino-acid sequence of a sphingolipid activator protein missing in a new human Gaucher disease variant.

A naturally occurring non-enzymic sphingolipid activator protein (A1a activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide-lipidosis) without glucosylceramidase deficiency was characterized by partial sequence analysis. The N-terminal amino-acid sequence of the A1a activator--a glycoprotein with high carbohydrate content--could be determined up to position 38. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. Position 22 seems to be occupied by a carbohydrate-binding asparagine. The N-terminus of the A1a activator does not show any homology with the activator for the enzymic sulfatide degradation.

Amino Acid Sequence↗
PubMed1987

The primary structure of the pallid bat (Antrozous pallidus, Chiroptera) hemoglobin.

The complete primary structure of the hemoglobin from the Pallid Bat (Antrozous pallidus, Microchiroptera) is presented. This hemoglobin consists of two components with identical amino-acid sequences, differing, however, in the N-terminus which is formylated in 12.5% of the beta-chains. The alpha- and beta-chains were separated by reversed phase high performance liquid chromatography. The sequences of both chains were established by automatic Edman degradation with the film technique or gas phase method using the native chains and the tryptic peptides. The formylation of a part of the N-terminal peptide of the beta-chains was determined by mass spectrometric examination. Compared to the corresponding human chains we found 14 substitutions in the alpha-chains and 21 in the beta-chains. One substitution in the alpha-chains and three in the beta-chains are involved in alpha 1/beta 1-contacts. Among these the exchange beta 123(H1)Thr----Cys is unusual because cysteine was so far not found in this position of mammalian beta-chains. Compared to the hemoglobin of Myotis velifer, another representative of the family Vespertilionidae, 5 residues are replaced in the alpha-chains and 18 in the beta-chains.

Amino Acid Sequence↗
PubMed1987

Reductive alkylation of lysine residues in subtilisin DY.

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.

Alkylation↗
PubMed1987

Preparation and characterization of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative.

Convenient methods for preparative synthesis of N epsilon-(2,3-dihydroxypropyl)-L-lysine and its phenylthiohydantoin derivative are described. The former compound was characterized by elemental analysis, melting point, and ion-exchange chromatography and the latter by elemental analysis, melting point, UV-spectrum, HPLC and thin-layer chromatography. This study was performed for investigations of lysine residues in proteins.

Amino Acids↗
PubMed1987

Complete amino-acid sequence and carbohydrate content of the naturally occurring glucosylceramide activator protein (A1 activator) absent from a new human Gaucher disease variant.

Two naturally occurring non-enzymic glucosylceramide activator proteins (A1a and A1b activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide lipidosis) without glucosylceramidase deficiency, were characterized by amino-acid sequence and carbohydrate content. The complete amino-acid sequence of the A1a activator was determined. The protein consists of 80 amino-acid residues including three disulfide bridges lacking arginine and tryptophan. The molecular mass is 8.95 kDa. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. The A1b activator was characterized by the amino-acid compositions of all tryptic peptides and of the entire protein; sequencing was performed of the regions 1-34 and 42-56. Identical results were obtained for the polypeptide chains of both A1 activators. This suggests that they do not differ in their primary structures which is in agreement with the immunochemical results. The difference between A1a and A1b activator is due to the carbohydrate part. The total amount of 49% carbohydrate in A1a and 76.7% in A1b consists mainly of hexoses. Both chains contain two moles of N-acetylglucosamine per mole protein bound to asparagine in position 22. A comparison of the primary structure of the A1 activator with the sulfatide activator sequence revealed an interesting similarity, especially of the cysteine residues and the carbohydrate-binding asparagine. Sequence homology was also found between a part of the A1 activator sequence and the hemagglutinin neuraminidase of influenza virus as well as to a hypothetical glycoprotein of the Epstein-Barr virus. The comparison with human lysosomal glucosylcerebrosidase showed no sequence similarity.

Amino Acid Sequence↗
PubMed1986

Paenungulata: a comparison of the hemoglobin sequences from elephant, hyrax, and manatee.

Inspection of the amino acid differences among hemoglobin sequences of a wide range of mammalian species suggested that at alpha 19, alpha 110, alpha 111, beta 23, beta 44, and beta 56, synapomorphies group manatee (Trichechus inungius, Sirenia), Indian and African elephant (Elephas maximus and Loxodonta africana, Proboscidea), and rock hyrax (Procavia habessinica, Hyracoidea) into a monophyletic clade. Results obtained by parsimony analysis provide evidence for this grouping--and thus support for the genealogical validity of Simpson's superorder Paenungulata, which contains as the extant orders Proboscidea, Sirenia, and Hyracoidea. All of the 39 most, or nearly most, parsimonious of 10,395 trees constructed from a tandemly combined alpha- and beta-hemoglobin sequence for 103 vertebrate species (of which 79 were mammals from 16 extant orders), depicted Paenungulata as one of the most anciently separated branches of Eutheria. It was found on examining thousands of alternative trees that to not group Proboscidea, Hyracoidea, and Sirenia in a monophyletic clade required at least four additional substitutions.

Amino Acid Sequence↗
PubMed1986

Interaction of allosteric effectors with alpha-globin chains and high altitude respiration of mammals. The primary structure of two tylopoda hemoglobins with high oxygen affinity: vicuna (Lama vicugna) and alpaca (Lama pacos).

Polyacrylamide gel electrophoresis and ion-exchange chromatography revealed one hemoglobin component for vicuna (Lama vicugna) and alpaca (Lama pacos). Following chain separation by chromatography on carboxymethyl-cellulose, the amino-acid sequences were elucidated for the alpha- and beta-chains of both hemoglobins using automatic Edman degradation of the chains and the tryptic peptides. Vicuna and alpaca have identical beta-chains showing no substitutions to llama (Lama glama) either. In the alpha-chains alpaca differs from llama by the exchange of one amino-acid residue: alpha 122(H5)Asp----His. The same substitution is present in vicuna too, but in addition we found two more exchanges: alpha 10(A8)Ile----Val and alpha 130(H13)Ala----Thr. The close relationship between llama and alpaca suggests that they both originate from the wild guanaco, and there is no domesticated form of vicuna. The sequence data show that the higher oxygen affinity in vicuna compared to llama and alpaca must be due to the alpha-chains as the beta-chains are identical. The significance of the substitutions in alpha 122(H5), an alpha 1/beta 1-contact, and alpha 130(H13) is discussed.

Altitude↗
PubMed1986

The primary structure of the hemoglobin of the European marmot (Marmota marmota marmota, Rodentia).

The hemoglobin of the European marmot Marmota marmota marmota has been found to consist of only one component. In this work, we are presenting its primary structure. The globin chains have been separated by high performance liquid chromatography and the sequences have been determined by automated Edman degradation of the chains and their tryptic peptides, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. In the alpha-chains we have found 13 and in the beta-chains 34 exchanges compared with the human alpha- and beta-chains, respectively. The amino acids which are substituted in the alpha-chains are not involved in any contacts, whereas in the beta-chains, one exchange involves a heme contact, two alpha 1/beta 1- and one alpha 1/beta 2-contacts. The functional and evolutionary aspects of these findings are discussed.

Amino Acid Sequence↗
PubMed1986

Preparation and characterization of 5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantoin, the phenylthiohydantoin derivative of 3-nitrotyrosine.

The phenylthiocarbamoyl derivative of 3-nitrotyrosine was synthesized according to the known Edman method and then converted to its phenylthiohydantoin derivative [5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantion] by incubation in 0.5M HCl for 24 h at room temperature. After drying over P2O5 the chromatographically pure substance could be obtained by double recrystallization from hot acetic acid. It could be established that a shorter incubation time leads to an incomplete conversion and higher temperatures cause polymerization of the product. The compounds could be characterized by thin-layer and high-performance liquid chromatography, melting point, elemental analysis as well as NMR- and absorption spectroscopy.

Chromatography, High Pressure Liquid↗
PubMed1986

The primary structure of a mouse-eared bat (Myotis velifer, Chiroptera) hemoglobin.

The hemoglobin of the Mouse-Eared Bat Myotis velifer consists of one component. We present the primary structures of the alpha- and beta-globin chains which have been separated by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman-degradation with the film technic or the gas phase method, using the native chains and the tryptic peptides, as well as the C-terminal prolyl-peptides obtained by acid hydrolysis of the Asp-Pro-bonds. Compared to the corresponding human chains we found only 13 substitutions in the alpha-chains, but 27 in the beta-chains. The amino-acid residues substituted in the alpha-chains are not involved in any contacts, whereas in the beta-chains, one exchange involves a heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts, the latter [beta 43(CD2)-Glu----Thr] brings for the first time threonine in this position of the beta-chains. Comparison with the Egyptian Fruit Bat (Rousettus aegyptiacus) shows 12 and 25 substitutions in the alpha- and beta-chains, respectively, suggesting a large phylogenetic distance between Micro- and Megachiroptera. We consider this primary structure as a contribution towards solving the problem of the origin of bats and their relation to primates.

Amino Acid Sequence↗
PubMed1986

Topography of all tyrosine residues in subtilisin DY.

The extracellular alkaline proteinase subtilisin DY was nitrated with increasing amounts of tetranitromethane. At 2-fold molar excess of the reagent with respect to the tyrosine residues in the enzyme, when 1.3 residues were modified, a peak of the caseinolytic activity (13% increase) was observed. Evidence is provided that the diminishing of the pK of the phenolic hydroxyl group in Tyr(3NO2)104 causes this phenomenon. The products obtained after nitration of the enzyme with 5-fold and 200-fold molar excess of tetranitromethane were cleaved by trypsin and cyanogen bromide and the peptides obtained were studied by analysis with respect to the tyrosine and 3-nitrotyrosine residues. Their degree of substitution was established. Tyrosine-104 was the first modified residue, then follow the residues with numbers 57, 143, 206, 262 and somewhat later 21, 209, 263, all fully modified by 200-fold molar excess of the reagent. Partial modification was observed at numbers 91, 167, 214, 238 and no modification at numbers 6 and 171. It has been established that the nonmodified residues are buried inside the molecule and the partially modified residues are screened by the side chains of lysine, valine, leucine, and tryptophan as seen on a working video three-dimensional model of subtilisin Carlsberg. The approach for characterization of tyrosyl groups in proteins based on peptide sequencing and HPLC quantitation of the phenylthiohydantoin derivatives of tyrosine and 3-nitrotyrosine was further developed with respect to the quantitation of the HPLC-separated peptides using fragments of the protein studied.

Amino Acid Sequence↗
PubMed1985

Functional multiplicity and structural correlations in the hemoglobin system of larvae of Chironomus thummi thummi (Insecta, Diptera): Hb components CTT I, CTT II beta, CTT III, CTT IV, CTT VI, CTT VIIB, CTT IX and CTT X.

Larvae of the dipteran insect Chironomus thummi thummi that burrow in fresh-water muds, contain at least 12 hemoglobin (Hb) components of which the functional properties have not been systematically documented, although their amino acid sequences have been elucidated, showing mutually distinct primary structures. We isolated eight components (the monomeric Hbs CTT I, CTT III and CTT IV and the dimeric Hbs CTT II beta, CTT VI, CTT VIIB, CTT IX and CTT X) and measured in each O2 affinity and cooperativity and their pH dependence, and the effects of temperature, NaCl and ATP. The O2 affinities, Bohr- and temperature effects of the isohemoglobins are discussed in relation to mode of life and the microenvironmental conditions to which the larvae are subjected in nature, and with regard to the molecular mechanisms underlying the Hb-oxygenation reactions.

Animals↗
PubMed1985

Isolation of two forms of an activator protein for the enzymic sphingomyelin degradation from human Gaucher spleen.

Two activator proteins for sphingomyelin degradation were isolated from heat-treated extracts of human Gaucher spleen. The separation was based on the degree of affinity of the activators for ConA-Sepharose. Activator A1, which had affinity for ConA-Sepharose, was purified 1 430-fold, and activator A2, which had no affinity for ConA-Sepharose, 2 140-fold as compared with the original heat-treated extracts. The molecular masses of activator A1 and activator A2 were 6 000 and 3 500 Da, respectively, as determined by dodecyl sulfate electrophoresis, and approximately 5 000 Da as measured in the presence of 8M urea. The two activators had similar properties and a similar but not identical amino-acid composition. Both were shown to form a complex with sphingomyelin and stimulate the degradation of sphingomyelin by normal fibroblast homogenates and by an approximately 1 430-fold purified sphingomyelin phosphodiesterase ("acid sphingomyelinase") from normal human urine. This stimulation was greatly reduced after incubation with pronase E. The enzymic degradation of glucosylceramide and galactosylceramide was not affected by these activators.

Amino Acids↗
PubMed1985

Mole rat hemoglobin: primary structure and evolutionary aspects in a second karyotype of Spalax ehrenbergi, Rodentia, (2n = 52).

The hemoglobins of the four karyotypes of Spalax ehrenbergi (2n = 52, 54, 58, 60) did not show any differences in their electrophoretic pattern and in high performance liquid chromatography. The complete amino-acid sequence of mole rat hemoglobin (Spalax ehrenbergi), chromosome species 2n = 52, is presented. It was elucidated by automatic Edman degradation of the chains, the tryptic peptides, and the C-terminal peptide obtained by acid hydrolysis of the Asp-Pro bond in beta-chains. The alpha- and beta-chains are identical with those of the chromosome species 2n = 60. A comparison of the hemoglobins of mole rat, mouse, and other rodents shows homology but no indication of adaptation to subterranean life. In all probability alpha 11(A9)Arg and alpha 120(H3)Gly, unique in mole rat among all mammalian hemoglobins, are not involved in high oxygen affinity. The construction of a phylogenetic tree by the maximum parsimony method, based on hemoglobin sequences, made it possible to show that Rodentia originated as a monophyletic clade, and to find the phylogenetic relationship of Spalacidae to other Rodentia (Mus, Rattus, Ondatra, Mesocricetus, Citellus, and Cavia). Among all rodents the slowest rate of nucleotide replacements occurred in the lineage to Spalax (20%) and the fastest in the lineage to Cavia (59%).

Amino Acid Sequence↗
PubMed1985

The primary structure and functional properties of the hemoglobins of a ground squirrel (Spermophilus townsendii, Rodentia).

The hemoglobin of the ground squirrel Spermophilus townsendii consists of two components which are present in a ratio of ca. 2:1. The two hemoglobins have identical alpha-chains, but differ in their beta-chains. We present the primary structures of the alpha- and the two beta-globin chains. Following chain separation by chromatography on carboxymethyl-cellulose CM-52, the amino-acid sequences were established by automatic Edman degradation of the globin chains and the tryptic peptides, as well as of a peptide obtained by acid hydrolysis of the Asp-Pro bond of the beta-chains. The two beta-chains differ by only one amino-acid residue, Ala being present in the main and Asp in the minor component in position 58 (E2). The comparison with human hemoglobin showed only 14 exchanges in the alpha-chains but 33 in the beta-chains. Whereas no contact positions are affected in the alpha-chains, we found four such substitutions in the beta-chains, including one heme contact, two alpha 1/beta 1-contacts, and one alpha 1/beta 2-contact. It seems however, that the substitution found in the beta-chains has no effect on the oxygen affinity.

Amino Acid Sequence↗