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Biomedical subjects

E Margoliash

Publications and source records attributed to E Margoliash.

At least 91 records · Page 5Linked to original sources
PubMed1980

Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues.

The carbonate binding site on horse cytochrome c was mapped by comparing the yields of carboxydinitrophenyl-cytochromes c, each with a single carboxydinitrophenyl-substituted lysine residue per molecule, when the modification reaction was carried out in the presence and absence of carbonate. The site is located on the "left surface" of the protein and consists of lysine residues 72 and/or 73 as well as 86 and/or 87 (Carbonate Site). Although one of the binding sites for phosphate on cytochrome c (Phosphat Site I) is located near the carbonate site, the sites are distinctly different since carbonate does not displace bound phosphate, as monitored by 31P NMR. Furthermore, citrate interacts with Phosphate Site I with high affinity, whereas chloride, acetate, borate, and cacodylate have a much lower affinity for this site, if they bind to it at all. The affinity of phosphate for Phosphate Site I (KD = 2 X 10(-4) M) is at least 1 order of magnitude higher than it is for other sites of interaction.

Animals↗
PubMed1979

Topographic antigenic determinants on cytochrome c. Immunoadsorbent separation of the rabbit antibody populations directed against horse cytochrome.

Seven populations of site-specific antibodies were isolated from each of three sera of rabbits immunized against glutaraldehyde-polymerized horse cytochrome c. The antibodies were separated using an immunoadsorption scheme which employed the following cytochromes c: horse, beef, guanaco, rabbit, mouse testicular, pigeon, and the cyanogen-bromide cleaved fragment of the rabbit protein containing residues 1 to 65. The monovalent, antigen-binding fragments of the antibodies (Fab') gave 1:1 stoichiometries with native horse cytochrome c in fluorescence quenching assays. Cross-reactivities with heterologous cytochromes c using fluorescence quenching and a modified Farr assay demonstrated that the antigenic determinants are situated around residues 44, 60, and 89/92, four of the six amino acid sequence positions where horse and rabbit cytochromes c differ. The remaining two differences occur at residues 47 and 62. The apparent lack of immunogenicity of these two substitutions may result from the presence of the more immunogenic residues 44 and 60 nearby. Of the seven antibody populations isolated, four were shown to bind in the region of residues 89 and 92. Since several cytochromes c have amino acid sequence differences from the horse protein at either of these two residue positions, it was possible to fractionate the antibodies directed against this complex site on the basis of subtle specificity differences between them. Two antibody populations bind in the region of residue 44. One of these is specific for proline at that position, while the other antibody population also binds to cytochrome c containing glutamic acid at position 44. The remaining antibody population binds in the region of the lysine residue at position 60. Each of the seven site-specific antibody populations binds effectively to any cytochrome c having a suitable amino acid sequence in the antigenic determinant regardless of any residue differences from the immunogen outside of that area. It was also demonstrated that these seven antibody populations represent the totality of the antibodies elicited in rabbits against horse cytochrome c, since the immunoadsorbants bound all the antibodies specific for the native protein. Furthermore, the rabbit antisera contained no other antibody population that could bind to the conformationally disturbed, cyanogen bromide-cleaved fragment of horse cytochrome c containing residues 1 to 65, making it appear that there were no antibodies elicited against a "processed" form of cytochrome c.

Animals↗
PubMed1979

Specificity of the antibody response of rabbits to a self-antigen.

The demonstration that the antigenic determinants on rabbit cytochrome c that elicit antibodies in rabbits occur in regions of variability among mammalian cytochromes c suggests the existence of an evolutionary process which eliminates genetic specificities for self-determinants.

Amino Acid Sequence↗
PubMed1979

The isolation of bovine-heart cytochrome c oxidase subunits. Dependence on phospholipid and cholate-content.

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate. The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated. Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS. Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts. The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.

Amino Acids↗
PubMed1979

T-lymphocyte response to cytochrome c. I. Demonstration of a T-cell heteroclitic proliferative response and identification of a topographic antigenic determinant on pigeon cytochrome c whose immune recognition requires two complementing major histocompatibility complex-linked immune response genes.

The T-lymphocyte proliferative response to pigeon cytochrome c was studied in the mouse. H-2a and H-2k strains were responders to this antigen whereas H-2b, H-2d, H-2f, H-2ja, H-2p, H-2q, H-2r, H-2s, and H-2u strains were low or nonresponders. Genetic mapping demonstrated that two major histocompatibility complex (MHC)-linked Ir genes control the response, one in I-A, the other in I-E/I-C. The major antigenic determinant recognized in this response was localized by cross-stimulations with species variants and cyanogen bromide cleavage fragments of cytochrome c. It was found to be a topographic surface determinant composed of an isoleucine for valine substitution at residue 3, a glutamine for lysine substitution at residue 100 and a lysine for glutamic acid substitution at residue 104. Tobacco hornworm moth cytochrome c, which contains a glutamine at residue 100 but a terminal lysine at residue 103 (one amino acid closer to the glutamine), stimulated pigeon cytochrome c immune T cells better than the immunogen. This result demonstrates for the first time a functional T-cell heteroclitic proliferative response in a system under Ir gene control. Immunization with the cyanogen bromide cleavage fragments revealed that only pigeon cytochrome c fragment 81-104 was immunogenic. This fragment primed for a T-cell proliferative response whose specificity was nearly identical to that of the T-cell response primed for by the whole molecule, suggesting that the glutamine at 100 and the lysine at 104 form the immunodominant portion of the antigenic site. Furthermore, mixing experiments using the two cross-reacting antigens, hippopotamus cytochrome c and Pekin duck or chicken cytochrome c fragment (81-104), each of which contains only one of the two immunodominant substitutions, demonstrated that the T lymphocytes responding to the major antigenic determinant comprise a single family of clones that recognize both amino acids as part of the same determinant. Thus, two complementing MHC-linked Ir genes can control the immune response to a single antigenic determinant.

Animals↗
PubMed1979

Definition of cytochrome c binding domains by chemical modification: kinetics of reaction with beef mitochondrial reductase and functional organization of the respiratory chain.

An assay has been developed to study the steady-state kinetics of the reduction of cytochrome c by purified beef heart mitochondrial cytochrome c reductase (cytochrome bc(1) complex, complex III). An analogue of coenzyme Q(2) (2,3-dimethoxy-5-methyl-6-decylhydroquinone) was employed as an antimycin-sensitive reductant. The kinetics of reaction of ten different mono(4-carboxy-2,6-dinitrophenyl) derivatives of horse cytochrome c were determined. The modified proteins showed higher apparent K(m) values than the native protein and greater sensitivity to ionic strength, defining an interaction domain on cytochrome c for purified cytochrome c reductase. This interaction site is located on the front surface of the molecule (which contains the exposed heme edge) and surrounds the point at which the positive end of the dipole axis crosses the surface of the protein. The site is similar to that previously determined for mitochondrial cytochrome c oxidase and yeast cytochrome c peroxidase, suggesting that the primary interaction with redox partners is directed by the dipolar charge distribution on cytochrome c. The extensive overlapping of the interaction domains for the mitochondrial cytochrome c oxidase and reductase indicates that cytochrome c must be mobile in order to transfer electrons between them, depending on their relative positions in the membrane. Whether such mobility is necessary in intact mitochondria depends on whether the interactions with the complete membrane-bound system are the same as with the purified components.

Animals↗
PubMed1979

Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster.

The results of electron-nuclear double resonance and electron paramagnetic resonance (EPR) studies on the hydrogen peroxide compound of yeast cytochrome c peroxidase are inconsistent with previous proposals for the source of the EPR signal in this compound, in particular with its identification with an aromatic amino acid radical such as would arise by oxidation of a tryptophanyl side chain. The present observations lead us to propose that the EPR signal is associated with a cluster containing at least one methionine and in which proximate side chains share the charge created by loss of one electron.

Binding Sites↗
PubMed1978

Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

Amino Acid Sequence↗
PubMed1978

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.

Animals↗
PubMed1978

Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99.

Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.

Binding Sites↗
PubMed1978

Genetic control of the T-lymphocyte proliferative response to cytochrome c.

Cytochromes c have been used as antigens in a murine T-lymphocyte proliferation assay in order to characterize the nature of determinants whose recognition is under immune response (Ir) gene control. The cytochromes are advantagous as antigens because 1) they have well-characterized primary and tertiary structures, 2) they are antigenically simple, differing from mouse cytochrome c at only a small number of amino acid residues, and 3) there exist a large number of evolutionary variants which can be used to locate antigenic sites by cross-stimulation. In the present studies, the T-lymphocyte proliferative response to pigeon cytochrome c was shown to be under the control of two complementing major histocompatibility (MHC)-linked Ir genes in mice of the H-2a and H-2k haplotypes. Mice of the H-2b, H-2d, H-2p, H-2q, H-2s, and H-2u haplotypes were low or nonresponders. Complementation was demonstrated by showing that an F1 hybrid between two nonresponder recombinant strains, B10.A(4R) and B10.A(5R), could respond to pigeon cytochrome c. The determinant on the cytochrome recognized in this immune response was located to the C-terminal portion of the molecule around residues 89 and/or 100. This was shown by the failure of closely related cytochromes from the Pekin duck and chicken to cross-stimulate T lymphocytes immune to pigeon cytochrome; position 89 and 100 carry the only residues different from those in mouse cytochrome c that are unique to pigeon cytochrome among the three bird cytochromes tested. This localization was further substantiated by demonstrating that the cyanogen bromide cleavage-fragment (residues 81-104) from pigeon cytochrome, but not the same fragment from Pekin duck cytochrome, was as good a stimulant of T cells immune to the whole molecule as the intact cytochrome. These results identify the immunogenic site on the molecule as one which differs from mouse cytochrome c by only one or two amino-acid residues. Thus, T-cell immune responses, which are under MHC-linked Ir gene control, are as capable as antibody responses of recognizing subtle differences in protein structure. However, the ability of T cells to respond equally well to stimulation with polypeptide fragments or with the whole molecule suggests either that T-cell recognition involves certain differences from B cell recognition or that in some cases the fragments possess a similar spatial structure to that of the corresponding segment in the native protein.

Animals↗