Resonance Raman characterization of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

 Di-heme Pseudomonas stutzeri cytochrome c ₄ has been characterized by electronic absorption and resonance Raman spectroscopies in the ferric and ferrous forms at pH 7.5 and at room temperature. The data indicate that the two hemes are inequivalent. It is proposed that the N-terminal contains a more relaxed heme as a consequence of the relative orientation of the methionine and histidine ligands with respect to the N-Fe-N directions of the heme plane. This causes a weakening of the Fe-S bond with concomitant partial dissociation of the methionine and the formation of an Fe-aquo bond. Heme group relaxation is further accompanied by less distortion of the heme group than that associated with cytochrome c, expansion of the "core" and a negative shift of the redox potential. Received: 17 December 1996 / Accepted: 6 March 1997     

Resolution and determination of enantiomeric purity of the enantiomers of felodipine using chiral-AGP® as stationary phase

A direct chiral chromatographic reversed phase method for the determination of the enantiomers of felodipine is described. The influence of charged and uncharged modifiers as well as the effect of the mobile phase pH on the enantiomeric resolution is discussed. A high mobile phase pH and the addition of 2-propanol as organic modifier gave the highest separation factor (α = 1.3). The high mobile phase pH (pH = 7.6) is outside the recommended pH limit of silica based columns but was necessary to achieve baseline resolution of (R)- and (S)-felodipine. Improvement of column efficiency by increasing column temperature was utilized for optimization of the enantiomeric resolution (Rs = 1.7). The enantiomers of felodipine and three related compounds were separated within 15 min. The enantiomeric purity of (R)- and (S)-felodipine in injections and (R)-felodipine in bulk substance was higher than 99.5% and no racemization was observed after storage at accelerated conditions. A poor Chiral-AGP® column used for a long period was restored using a simple wash step together with repacking the top of the chromatographic column. © 1995 Wiley-Liss, Inc.     

The passage of orally fed proteins from mother to foetus in the rat

Pregnant rats were orally fed with a mixture of bovine IgG, bovine albumin, ovalbumin and beta-lactoglobulin. Using immunoprecipitation methods, these proteins were detected in the maternal blood serum, urine and uterine fluids, and also in the foetal blood serum and the amniotic fluid. The results imply that a variety of dietary proteins, still immunoprecipitable, are able to cross the materno-foetal barriers to be detected in the foetal circulation, where they may influence the maturation of the immune system of the foetus.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Blood group ABH-active substances.

High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH₄) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.     

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.     

Isolation and characterization of cells from rat adipose tissue developing into adipocytes.

To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.     

Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme.

Ribonucleotide reductase catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides via a radical-mediated mechanism. The enzyme from Escherichia coli consists of the two non-identical proteins, R1 and R2, the latter of which contains the necessary free radical located to a tyrosine residue. The radical scavenger hydroxyurea was found to reduce the tyrosyl radical of R2 in a second-order reaction. The rate constant (0.50 M-1 s-1 at 25 degrees C) for this process was several orders of magnitude lower than the hydroxyurea-dependent reduction of free tyrosyl radicals in solution. This difference probably reflects the fact that the R2 tyrosyl radical is buried in the interior of the protein. Formation of the R1R2 complex changed the susceptibility of the radical to hydroxyurea in a manner that reflects the regulatory state of the holoenzyme. Furthermore, binding of substrate or product to the holoenzyme complex made the R2 radical at least 10 times more susceptible to inactivation by hydroxyurea than it was in the isolated R2 protein. One active site mutation in the R1 protein was shown to affect the sensitivity of the tyrosyl radical of R2 differently than wild type protein R1 does. Our results clearly show that the susceptibility of the tyrosyl radical in R2 to inactivation by hydroxyurea can be used as an efficient probe for the regulatory state of the holoenzyme complex.     

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGc alpha-3Gal beta 4Glc beta Cer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well as thus explain the resistance of adult pigs to infection with E. coli K99.     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

Structure and evolution of the heavy chain from rat immunoglobulin E.

The nucleotide sequence of the rat epsilon-chain mRNA has been determined by sequencing cloned cDNA copies of the mRNA. The established sequence covers the coding region, the 3'-non coding region and most of the 5' non-coding region. A comparison with the nucleotide sequence of the human epsilon-chain constant region reveals that C3 and C4 are the most highly conserved domains. The rat epsilon-chain contains a C-terminal decapeptide which is not present in the human counterpart.