A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase.

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by 1H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.

Mass spectrometry of a human tumor glycolipid antigen being defined by mouse monoclonal antibody NS-19-9.

With an antibody-to-chromatogram binding assay to follow the preparation a glycolipid was isolated from human pancreatic carcinoma using a mouse monoclonal antibody of apparent specificity for gastrointestinal tumors. Direct probe mass spectrometry of three derivatives established the sugar sequence as NeuAc-hexose-(fucose)N-acetylhexosamine-hexose-hexose and the ceramide to be composed mainly of phytosphingosine and 16-24 carbon 2-hydroxy fatty acids. NMR spectroscopy of two of the derivatives made likely the presence of the sequence Gal beta 1 leads to 3GlcNAc(4 comes from 1 alpha Fuc)beta 1 leads to, which is the blood group Lewis a determinant. This is in agreement with recent results from degradation studies.

Chemical fingerprinting of non-acid glycosphingolipids in meconium of a human individual of blood group B Le(a-b+) and secretor.

In an attempt to elucidate blood group polymorphism systematically in human intestinal tissue, glycosphingolipids of meconia of single individuals have been studied. In this report we describe the major glycosphingolipids in meconium of a newborn typed as a B Le(a-b+) secretor. The glycolipids were analysed as four subfractions obtained after deacetylation of fractions from silicic acid column chromatography of the peracetylated total non-acid glycolipid extract. Chemical characterization (mass spectrometry and 1H-NMR spectroscopy of permethylated and LiA1H4-reduced permethylated derivatives) and immunological characterization (haemagglutination-inhibition of native glycolipids in the liposome form) revealed a very complex pattern of fucosyl-containing glycolipids, probably the result of several blood group glycosyltransferase activities. The major fucolipids, all based on lactotetraosylceramide, had a characteristic ceramide of mainly phytosphingosine and 2-hydroxy fatty acids with 16 and 20-24 carbon atoms.

Chemical fingerprinting of glycosphingolipids in meconium of a human blood group O Le(a-b+) secretor.

Studying blood group polymorphism, as expressed in intestinal tissue of single individuals, total non-acid glycosphingolipids of meconium of individual human newborns have been prepared. Silicic acid column chromatography of the acetylated derivatives were used for a stepwise separation into four groups of glycolipids from each individual meconium. By the combined use of mass spectrometry and NMR spectroscopy of permethylated and LiA1H4-reduced, permethylated derivatives and by immunology of the native fractions all the major glycolipids were identified, although in mixtures. The interest was focused on fucolipids known to be strictly regulated by the ABO, H, Le and Se genes. The fucosylated glycolipids of an O Le (a-b+) secretor child were dominated by blood group H-active and Lewis-active mon- and difucosyl compounds with 5-6 sugar residues and having a core lactotetraosyl structure. The lipophilic part was dominated by 2-hydroxy fatty acids with 16 and 20-24 carbon atoms bounds to either sphingosine of phytosphingosine.

Structural identification of two ten-sugar branched chain glycosphingolipids of blood group H type present in epithelial cells of rat small intestine.

Two novel blood group H-type decaglycosylceramides with a branched core saccharide have been identified in mixture in a fraction isolated from rat small intestine. They were present exclusively in the epithelial cells. The number and sequence of sugars were established by direct inlet mass spectrometry of the permethylated and LiAlH4-reduced permethylated derivatives. Gas-liquid chromatography of the products after degradation of the native and permethylated glycolipids gave the type of sugars and the binding positions. A di- and a trisaccharide were identified by mass spectrometry after degradation of the permethylated-reduced derivative. One trisaccharide had the structure (formula see text) and was therefore additional evidence for a branched structure. Treatment of the decaglycosylceramide fraction with alpha-L-fucosidase gave free fucose and an octaglycosylceramide identified by mass spectrometry. Proton NMR spectra of the permethylated and permethylated-reduced octa- and decaglycosylceramides provided evidence for the binding configurations and the localization of type 1 and type 2 sequences in the two branches. The 3-linked branch was homogeneous with a type 1 saccharide (Gal beta 1 leads to 3GlcNAc) but the 6-linked branch had both type 1 and type 2 (Gal beta 1 leads to 4GlcNAc) sequences. Two glycolipids with the following probable structures were therefore present, making up 60 and 40% of the mixture, respectively: (formula see text) The lipophilic part contained mainly trihydroxy 18:0 long chain base (phytosphingosine) and 16:0 to 24:0 nonhydroxy fatty acids.

Chemical characterization of penta-, hexa-, hepta-, octa-, and nonaglycosylceramides of rat small intestine having a globoside-like terminus.

A novel series of glycosphingolipids has been isolated from the nonepithelial part of rat small intestine. A mixed fraction containing 3 major components corresponding to glycolipids with 5, 6, and 7 sugars and 2 minor components with 8 and 9 sugars was characterized. The structure of the major components was deduced by mass spectrometry and proton NMR spectroscopy of nondegraded permethylated and permethylated-reduced (LiAlH4) derivatives and gas-liquid chromatography of degradation products of native, permethylated, and permethylated-reduced glycolipids. The structures of the penta-, hexa-, and hepta-glycosylceramides were found to be GalNAcp beta 1 leads to (3Galp alpha 1 leads to)2-44Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. By analogy reasoning, supported by mass spectrometry, the octa- and nonaglycosylceramides were concluded to have 1 and 2 additional internal leads to 3Galp alpha 1 leads to 3 structures, respectively. A pentaglycosylceramide fraction from another rat strain was also isolated. The NMR spectra were in agreement with 2 isomeric structures of which 1 had the internal alpha 1 leads to 4 linkage replaced by an alpha 1 leads to 3 linkage. The fatty acids of all components were nonhydroxy 16:0 to 24:0 acids with the 18:0 homologue as dominating species. The major base was sphingosine and possibly monohydroxy 18:1 base in the larger glycolipids. This is a novel series of structures with a terminal saccharide identical with isoglobotetraoxylceramide (cytolipin R). The glycosyltransferase for the terminal GalNAc beta 1 leads to 3 of cytolipin R may possibly be identical with the enzyme adding the terminal sugar of this novel series. This is supported by the presence in the same tissue of probable precursor glycolipids with 4 to 8 hexoses.

Separation and characterization of hematosides with different sialic acids and ceramides from rat small intestine. Different composition of epithelial cells versus non-epithelial tissue and of duodenum versus jejunum-ileum.

The hematosides (sialyl-lactosylceramides) of rat small intestine were separated as their acetylated derivatives. The isolated fractions were characterized by mass spectrometry and degradative methods, and the two major fractions also by NMR spectroscopy. From these results hematosides with different sialic acid and ceramide type could be assigned to thin-layer chromatographic bands. This allowed a structural interpretation of the chromatographic patterns observed for different parts of the small intestine. Thus, epithelial cells of ileum contained only hematoside with N-glycoloylneuraminic acid. Duodenum lacked this compound and instead the epithelial cells contained hematoside with N-acetylneuraminic acid. In non-epithelial tissue or both duodenum and jejunum-ileum the major hematoside had N-acetyl-neuraminic acid. The hematosides of epithelial cells had ceramide containing 18 : 0 trihydroxy base combined with 16, 20, 22, 24 : 0, and 24 : 1 hydroxy fatty acids (major part) or non-hydroxy fatty acids. In the non-epithelial hematosides the ceramide consisted of 18 : 1 dihydroxy base combined with 16, 18, 20, 22, 24 : 0, and 24 : 1 non-hydroxy fatty acids.

N.m.r. and e.p.r. characterization of [4-carboxy-2,6-dinitrophenyl-lysine]cytochromes c.

Monosubstituted [4-carboxy-2,6-dinitrophenyl-lysine]cytochromes c were investigated by n.m.r. and e.p.r. Modification of Lys-13 or Lys-72 in ferricytochrome c by 4-chloro-3,5-dinitrobenzoate yields either of two different conformers that are rapidly exchanging in the native form. The equilibrium involves small local changes in the conformation of Met-80 (the sixth ligand) and Phe-82, as a result of whether Lys-13 is the 'on' or 'off' position in the Lys-13--Glu-90 salt bridge.

Perchlorate binding to cytochrome c: a magnetic and optical study.

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy. 2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4(-) concentration below 0.1 M. 3. Competition experiments between perchlorate and chloride show that ClO4(-) binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4(-) in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice. 4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high-spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form. The effects of perchlorate on cytochrome c are due to specific binding of this ion.

Structural analysis by mass spectrometry and NMR spectroscopy of the glycolipid sulfate from Halobacterium salinarium and a note on its possible function.

The major glycolipid sulfate of the extreme halophile Halobacterium salinarium was isolated and characterised mainly by mass spectrometry and NMR spectroscopy. The mass spectrum of the permethylated, desulfated and trimethylsilylated derivative showed the molecule to be a trihexosyl glycerol C20-diether with the sulfate group on the terminal hexose. A 3-position of the sulfate was indicated by the mass spectrum obtained after acetylation and trimethylsilylation (solvolysis of sulfate and replacement by a trimethylsilyl group). The NMR spectrum of the desulfated permethylated glycolipid gave conclusive evidence for the presence of one beta and two alpha anomeric protons. With the knowledge of degradation data it was possible to assign the beta signal to galactose (terminal hexose), and the alpha signals to glucose and mannose. These data together make it likely that the glycolipid sulfate is identical in structure with the glycolipid from Halobacterium cutirubrum characterised previously (M. Kates and P.W. Deroo, J. Lipid Res., 14 (1973) 438). On the basis of a suggested function of cerebroside sulfate of animal origin (identical polar end with the bacterial glycolipid: beta-galactopyranose-3-sulfate) and the present knowledge of ion transport in Halobacteria, it is proposed that the bacterial glycolipid may function as a selective K+ receptor for the K+ transport from a high-Na+ and low-K+ outside medium.

High-resolution proton magnetic resonance study of porcine colipase and its interactions with taurodeoxycholate.

A high-resolution 270-MHz proton NMR study of procine colipase I has been performed, and the resonances in the aromatic region of the spectrum have been assigned to amino acid residues by pH titration and decoupling experiments. The apparent pKa values of the three tyrosines were calculated to be 10.2, 10.3, and 11.8 with one of the tyrosines having properties of a "buried" residue. A tentative assignment to the amino acid residues in the primary seuqence of colipase will be discussed. The effects of taurodeoxycholate (TDC) and a positively charged deoxycholate derivative on the aromatic region of the colipase NMR spectrum indicate that all tyrosines and one histidine are affected by the bile-salt binding, suggesting that the TDC molecules bind near these residues to a hydrophobic region on colipase. Measurements and calculations on the line width of the C(18) methyl group resonance suggest that the line-width increase of this resonance upon interaction of TDC with colipase to a large extent can be explained as due to the slower tumbling of the TDC molecules bound to colipase.