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T M Stepanik

Publications and source records attributed to T M Stepanik.

12 recordsLinked to original sources
PubMed1990

Intercomparison of small biomolecules by gel filtration and small angle scattering.

This paper describes how small angle neutron scattering (SANS) can be used to confirm that gel filtration results are free of dimerization effects. After characterization by analytical gel filtration, concentrated solutions (in heavy water, D2O) of a cobra neurotoxin, a cytotoxin, and a cytotoxin analog are studied by SANS. Small differences in shape are shown to be discernible by means of least-square fits to ellipsoidal models. The parallel axis theorem is then invoked to assess dimerization levels statistically. The results are briefly discussed on the basis of function in relation to structure.

Cobra Neurotoxin Proteins↗
PubMed1990

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes.

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.

Catalase↗
PubMed1988

Lipid-induced changes in the secondary structure of snake venom cardiotoxins.

The secondary structures of three snake venom cardiotoxins (from Hemachatus hemachatus, Naja naja atra, and Naja naja naja), in aqueous solution and in a lipid-bound form, were investigated by Fourier-transform infrared spectroscopy. The conformation-sensitive protein infrared bands in the amide I region were analyzed using deconvolution and band-fitting procedures. The spectra of the three cardiotoxins in aqueous buffer are very similar; they indicate a high content of both antiparallel beta-sheet structure and unordered conformation. Moreover, component bands characteristic of turns can also be identified. The binding of cardiotoxins to bilayers of dimyristoylphosphatidyl-glycerol results in an increased content of a beta-structure at the expense of the nonordered conformation. It is suggested that lipid-induced conformational transitions to a beta-structure, similar to that observed with cardiotoxins, may be operative also in membrane interaction of other proteins and peptides, particularly with those which have a small tendency to form alpha-helices.

Animals↗
PubMed1985

Common evolutionary origin of alpha 2-macroglobulin and complement components C3 and C4.

A comparison of the sequence of the subunit of human alpha 2-macroglobulin (alpha 2M; 1451 amino acid residues) with that of murine complement component pro-C3 (1639 amino acid residues) reveals eight extended regions of sequence similarity. These regions contain between 19% and 31% identically placed residues and account for 75% and 67%, respectively, of the polypeptide chains of alpha 2M and pro-C3. Published sequence data for complement component C4 show that segments of this protein match well with corresponding stretches in alpha 2M and pro-C3. It is proposed that alpha 2M, C3 and C4, which all contain a unique activatable beta-cysteinyl-gamma-glutamyl thiol ester, have a common evolutionary origin and are homologous proteins. Several larger regions of low sequence similarity indicate the presence of structural domains in each of these proteins that specifically modify an underlying common gross structure. The quartets of basic residues in pro-C3 and pro-C4, at which cleavage takes place to produce the mature subunits of these proteins, and most of the residues forming the anaphylatoxin peptides of C3 and C4 (C3a and C4a) are absent in alpha 2M. In addition, C3 and C4 contain large portions, which extend beyond the COOH terminus of alpha 2M.

Amino Acid Sequence↗
PubMed1984

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments.

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.

Amino Acid Sequence↗
PubMed1984

Primary structure of human alpha 2-macroglobulin. II. Primary structure of eight CNBr fragments located in the NH2-terminal half of alpha 2-macroglobulin, accounting for 603 amino acid residues.

The amino acid sequence has been determined for eight medium-sized CNBr fragments from the NH2-terminal part of human alpha 2-macroglobulin. The fragments are: CB2 (10-78), CB5 (129-223), CB6 (224-295), CB7 (296-392), CB8 (393-441), CB11 (498-584), CB12 (585-643), and CB17 (691-775). These fragments account for 603 of the 1451 residues of the subunits of alpha 2-macroglobulin. CB2 contains two glucosamine-based carbohydrate groups attached to Asn-23 and Asn-38, and one internal disulfide bridge connecting Cys-16 with Cys-54. CB6 contains one glucosamine-based carbohydrate group attached to Asn-1 and two internal disulfide bridges (Cys-5 bound to Cys-53 and Cys-23 bound to Cys-41, respectively); Cys-32 is bound to Cys-16 in CB8. CB7 contains two glucosamine-based carbohydrate groups attached to Asn-78 and Asn-92, CB8 contains 1 Cys residue (Cys-16), bridged to Cys-32 of CB6. CB11 contains 1 Cys residue (Cys-75), bridged to Cys-58 of CB17. CB12 contains 1 Cys residue (Cys-35), bridged to Cys-1 of CB15. CB17 contains 1 Cys residue (Cys-58), bound to Cys-75 of CB11.

Amino Acid Sequence↗
PubMed1984

Primary structure of human alpha 2-macroglobulin. III. Primary structure of three large disulfide-bridged CNBr fragments, located in the COOH-terminal part of alpha 2-macroglobulin and accounting for 301 residues.

The amino acid sequences have been determined for three CNBr fragments of human alpha 2-macroglobulin which form a disulfide-bridged Mr = 40,000 fragment set. The fragments are located in the COOH-terminal part of alpha 2-macroglobulin (CB18, residues 776-936; CB23, residues 1292-1355; and CB26, residues 1376-1451). CB18 contains one glucosamine-based carbohydrate group attached to Asn-71 and two internal disulfide bridges (Cys-23 bound to Cys-51 and Cys-49 bound to Cys-85, respectively). Cys-123 is disulfide bridged to Cys-7 of CB23. Cys-38 of CB23 is disulfide bridged to Cys-69 of CB26. CB26, which contains one glucosamine-based carbohydrate group attached to Asn-26, is the COOH-terminal CNBr fragment. CB18, CB23, and CB26 account for 301 of the 1451 residues of the subunit of alpha 2-macroglobulin.

Amino Acid Sequence↗
PubMed1984

Primary structure of human alpha 2-macroglobulin. V. The complete structure.

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.

Amino Acid Sequence↗
PubMed1983

Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study.

Comparison of the fluorescence spectra and the effect of temperature on the quantum yields of fluorescence of Azurin (from Pseudomonas fluorescens ATCC-13525-2) and 3-methylindole (in methylcyclohexane solution) provides substantive evidence that the tryptophan residue in azurin is completely inaccessible to solvent molecules. The quantum yields of azurin (CuII), azurin (CuI), and apoazurin (lambda ex = 291 nm) were 0.052, 0.054, and 0.31, respectively. Other evidence indicates that there is no energy transfer from tyrosine to tryptophan in any of these proteins. The fluorescence decay behavior of each of the azurin samples was found to be invariant with emission wavelength. The fluorescences of azurin (CuII) and azurin (CuI) decay with dual exponential kinetics (tau 1 = 4.80 ns, tau 2 = 0.18 ns) while that of apoazurin obeys single exponential decay kinetics (tau = 4.90). The ratio of pre-exponentials of azurin (CuII), alpha 1/alpha 2, is found to be 0.25, and this ratio increases to 0.36 on reduction to azurin (CuI). The results are interpreted as originating from different interactions of the tryptophan with two conformers of the copper-ligand complex in azurin.

Azurin↗