Osmium (VI) complexes of the 3', 5'-dinucleoside monophosphates, ApU and UpA.

The dinucleoside monophosphates, ApU and UpA, react with potassium osmate (VI) and 2,2'-bipyridyl to form the corresponding oxo-osmium (VI) bipyridyl sugar ester in which the osmate group is bonded to the terminal 2',3'-glycol. Osmium (VIII) tetroxide and 2,2'-bipyridyl react with the dinucleosides to form the corresponding oxo-osmium (VI) bipyridyl heterocyclic esters which result from addition of the tetroxide to the 5,6-double bond of the uracil residue. Although capable of transesterification reactions, these heterocyclic esters are exceptionally stable toward exchange reactions in solution. No apparent exchange was observed after 1 month. This reaction thus seems promising for single-site osmium labeling in polynucleotides.     

Chemical aspects of glycogen contrast-staining by potassium osmate

Summary To obtain contrast-staining of glycogen in electron microscopy, various contrast-enhancing additives can be used in combination with potassium osmate. Examples are potassium ferrocyanide and certain nitrogen heterocyclic compounds, such as triazoles. In the reaction sequence leading to contrast-stained glycogen, a primary reaction is the formation of glycogen osmate. This reaction was studied with isolated glycogen. On the basis of the stoichiometric findings, a molecular structure of the reaction product is proposed; apparently, osmate is bound by glycogen because of the presence of suitably located hydroxyl groups. The resulting compound is not itself sufficiently electron dense, but it binds 1,2,4-triazole as an additional ligand. Secondary reactions can result in additional osmium binding, and finally to osmium (IV) deposits, leading to contrast.     

Osmium-labeled polynucleotides. Incorporation of additional heavy atoms (mercury) via ligand substitution reactions

The reaction of osmium tetroxide with biological materials, particularly nucleic acids, has considerable utility in electron microscopy and X-ray crystallography. This heavymetal label introduces an electrophilic center which may serve as a means of attachment of nucleophiles. Nucleophilic ligand substitution reactions have been exploited as a means of adding more heavy metals (mercury) onto the osmium label. Furthermore, the technique is quite general and could be used to modify the osmium label with, for example, fluorescent groups. The nucleophilic exchange reactions have also been studied using ¹H nmr spectroscopy with a representative heterocyclic osmate ester derivative, the bis(pyridine) osmate ester derivative of TMP. These studies have defined the nature of the ligands which lead to stable osmium labels.     

The chemical nature of osmium tetroxide fixation and staining of membranes by X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at ?100 °C, began to react at ?70 °C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Os(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, resulting in a final mixture which was 50–60% Os(III).The results support a scheme for the reaction of osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.     

The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at -100 degrees C, began to react at -70 degrees C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Ss(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, rpesulting in a final mixture which was 50-60% Os(III). The results support a scheme for the reaction os osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.     

Conformational properties of the osmium tetraoxide bispyridine ester of 1-methylthymine and a comment on the linearity of the trans O=Os=O group.

The preparation and crystal and molecular structure of the osmium tetraoxide bispyridine ester of 1-methylthymine are reported. The complex crystallizes in the triclinic system, space group P1, with a = 11.493(6)A, b = 16.655(7)A, c = 6.082(2)A, alpha = 92.07(3) degrees, beta = 90.58(3) degrees, gamma = 71.36(4) degrees, V = 1102.4 A3, Dm = 1.85(1) g cm-3, DC = 1.84 g cm-3. The unit cell contains 2 osmium tetraoxide bispyridine esters of 1-methylthymine, 2 waters of crystallization and 1 disordered pyridine of solvation. Intensities for 3814 independent reflections were collected by counter methods. The structure was solved by standard heavy-atom techniques and has been refined by full-matrix least squares, based on F, to a final R value of 0.065. The osmium complex binds as a cis osmate ester to the C(5)-C(6) bond of the methylated pyrimidine in a fashion which is expected to be similar to the binding of the complex to thymidine residues in nucleic acids. The conformation of the 1-methylthymine ester is that of a half chair with C(6) showing a substantial deviation, 0.55 A, from the best mean plane of the thymine moiety. The primary coordination sphere about the Os(VI) atom is completed by 2 axial Os=O bonds and the binding of the 2 pyridine ligands in cis positions in the equatorial plane containing the ester linkages. The O=Os=O group is substantially nonlinear, 164.0(5) degrees, and this nonlinearity is attributed to intracomplex electronic effects.     

The reactions of oxo-osmium ligand complexes with isopentenyl adenine and its nucleoside.

We report syntheses of oxo-osmium(VI)bis(ligand) esters of N6-(delta2-isopentenyl) adenine (6-ipAde) and its nucleoside (IPA) which result from the addition of OsO4 to the double bond of the isopentenyl group. A study of the kinetics of these reactions shows that under typical conditions the rates of reaction relative to thymidine are as follows: for OsO4-pyridine: thymidine = 1; 6-ipAde = 4600: for OsO4-2,2'-bipyridyl: thymidine = 380; 6-ipAde = 8600; IPA = 8600. We also report syntheses of osmate esters of IPA in which the osmium is bonded through the 2'-and 3'-hydroxyl groups of the ribose residue.     

The specific cytochemical demonstration in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups

Summary A technique is described for the localization in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups. In this technique the sugar residues are oxidized by periodic acid and the resulting aldehydes condensed with pentafluorophenylhydrazine under specified conditions. Further increase in specific electron density is achieved by treating the hydrazone end-product with ammonium sulphide followed by osmium tetroxide to form an osmium black.The technique has been applied to liver and small intestine cells in which glycogen, sialomucins and sulphated mucosubstances reacted especially strongly. A marked positive reaction has also been obtained from the interstitial cell matrix and from the Golgi apparatus and multivesicular bodies of the intestinal epithelial cells.The reaction can be prevented by the omission of the periodate oxidation and, if due to glycogen, by prior treatment with diastase.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

Complexes of amylose and amylopectins with multivalent metal salts

Metal cations [Cu(II), Fe(III), Mn(II), and Ni(II)] are ligated by amylose as well as potato, and corn amylopectins as proven by electron paramagnetic resonance spectra and conductivity measurements. The hydroxyl groups of polysaccharides are the coordination sites. Isolated starch polysaccharides did not coordinate to metal ions so well as starch did. The resulting polycenter Werner complexes were mainly square planar species. The ligation of the central metal atoms resulted in a variation of the thermal stability, pathway, and rate of thermal decomposition of starch as proven by thermogravimetric (TG, DTG) and scanning differential calorimetric measurements. Frequently, amylose and potato amylopectin willingly formed clathrates in which the water molecules were caged. The mode of the coordination of the hydroxyl groups to the central metal atom controlled the clathrate formation from amylose and in the case of potato amylopectin metal atoms bound to the phosphoric acid moiety formed cage by coordination of the hydroxyl groups to them. Coordination to selected metal salts controls pathway and products of polysaccharide ligand thermolysis.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

Coordination between manganese and nitrogen within the ligands in the manganese complexes facilitates the reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations

The water-oxidizing complex (WOC) within photosystem II (PSII) can be reconstituted with synthetic manganese complexes by a process called photoactivation; however, the key factors affecting the efficiency of synthetic manganese complexes in reconstitution of electron transport and oxygen evolution activity in manganese-depleted PSII remain unclear. In the present study, four complexes with different manganese coordination environments were used to reconstitute the WOC, and an interesting relationship was found between the coordination environment of the manganese atom in the complexes and their efficiency in restoring electron transport and oxygen evolution. If Mn(II) is coordinated to nitrogen atoms within the ligand, it can restore significant rates of electron transport and oxygen evolution; however, if the manganese atom is coordinated only to oxygen atoms instead of nitrogen atoms, it has no capability to restore electron transport and oxygen evolution. So, our results demonstrate that the capability of manganese complexes to reconstitute the WOC is mainly determined by the coordination between nitrogen atoms from ligands and the manganese atom. It is suggested from our results that the ligation between the nitrogen atom and the manganese atom within the manganese complex facilitates the photoligation of the manganese atom to histidyl residues on the apo-protein in manganese-depleted PSII during photoactivation.     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′→P5′ Phosphoramidates

Abstract

2′-Deoxyribo- and ribo-oligonucleotide N3′→P5′phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 °C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3′→5′ phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.     

Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.     

Immune reactions in polysaccharide media. The effect of hyaluronate, chondroitin sulphate and chondroitin sulphate–protein complex on the precipitin reaction

The influence of the connective-tissue polysaccharides hyaluronate, chondroitin 4-sulphate and a chondroitin 4-sulphate-protein complex (PP-L) from cartilage on the precipitin reaction was investigated. In a system consisting of (125)I-labelled human serum albumin and the immunoglobulin G fraction from rabbit anti-albumin sera, the precipitation is greatly increased in the region of antigen excess. This effect depends on the concentration, molecular weight and configuration of the polysaccharide. The increase parallels a decrease in the amount of soluble immune complexes in the supernatant. It is suggested that the effect is due to steric exclusion of the complexes from the domains of the polysaccharides. The possibility that such a mechanism might enhance precipitation of antigen-antibody complexes in certain pathological conditions is discussed.     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.     

Functional and structural analysis of the photosynthetic apparatus of Rhodobacter veldkampii

In the widely studied purple bacterium Rhodobacter sphaeroides, a small transmembrane protein, named PufX, is required for photosynthetic growth and is involved in the supramolecular dimeric organization of the core complex. We performed a structural and functional analysis of the photosynthetic apparatus of Rhodobacter veldkampii, a related species which evolved independently. Time-resolved optical spectroscopy of R. veldkampii chromatophores showed that the reaction center shares with R. sphaeroides spectral and redox properties and interacts with a cytochrome bc(1) complex through a Q-cycle mechanism. Kinetic analysis of flash-induced cytochrome b(561) reduction indicated a fast delivery of the reduced quinol produced by the reaction center to the cytochrome bc(1) complex. A core complex, along with two light-harvesting LH2 complexes significantly different in size, was purified and analyzed by sedimentation, size exclusion chromatography, mass spectroscopy, and electron microscopy. A PufX subunit identified by MALDI-TOF was found to be associated with the core complex. However, as shown by sedimentation and single-particle analysis by electron microscopy, the core complex is monomeric, suggesting that in R. veldkampii, PufX is involved in the photosynthetic growth but is unable to induce the dimerization of the core complex.