Ca2+ ionophoretic activity of oligomers of prostaglandin B1 with isolated hepatocytes

Chemically synthesized oligomers (dimers, trimers and tetramers) of 15-dehydroprostaglandin B1 and 16,16'dimethyl-15-dehydroprostaglandin B1 (16,16'diMePGB1) are effective Ca2+ ionophores with isolated mitochondria and in artificial systems. The trimer of 16, 16'diMePGB1 mediated a dose dependent Ca2+ efflux from intact rat hepatocytes; at 9.2 microM oligomer, Ca2+ was released primarily from the mitochondrial pool but at higher concentrations from other cellular pools. The 16, 16'diMePGB1 trimer did not alter Ca2+ release mediated by epinephrine suggesting that the PGB1 oligomer interacts at a different site. The oligomer also caused an activation of phosphorylase similar to that mediated by epinephrine.     

Calcium ionophoretic activity of chemically synthesized oligomeric derivatives of prostaglandin B1

Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

Properties of the interspecies hybrids between haptoglobin alpha and beta subunits

1. Subunits alpha isolated from human haptoglobin were recombined with beta subunits of equine haptoglobin, and vice versa. Both hybrid proteins were separated on electrophoresis in polyacrylamide gel into four bands with mobilities corresponding to tetramers 2alpha.2beta, trimers 2alpha.beta, and dimers alpha.beta, in addition to free subunits beta. 2. The binding ability of haemoglobin and the antigenic specificity of tetramers depended on the origin of beta subunit. 3. Reduction of native and hybrid proteins with 2-mercaptoethanol led to gradual formation of alpha.beta, alpha, and beta; the components 2alpha.beta and 2alpha appeared in trace amounts.     

Letter: Cytoplasmic DNA polymerase: polymeric forms and their conversion into an active monomer resembling nuclear DNA polymerase

DNA polymerase isolated from the cytoplasm is an aggregate enzyme which can exist in forms of dimers, trimers and tetramers of a monomer of 54,000 molecular weight. Trimers and tetramers could be quantitatively converted into dimers and monomers by sedimentation in sucrose gradients at high ionic strength (0.45 m). Molecular properties of the monomer resembled nuclear DNA polymerase: sedimentation coefficient (3.5 S), template specificity and resistance to inhibition by N-ethylmaleimide and heparin.     

Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.     

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.     

Conformation of Lariat Structures Formed in the Splicing of Pre-mRNA by NMR Spectroscopy

Abstract

Temperature dependent ¹H- and 31p-NMR studies have shown that lariat (branched) trimers show a preferential 2′ → 5′ stacking, while the branched tetramers resemble 3′ → 5′ linked linear trimers, reminiscent of a single stranded A-RNA helix.     

Purification and properties of a lectin from ascomycete mushroom,Ciborinia camelliae

A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-D-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of approximately 17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found.     

Active oligomeric ATP synthases in mammalian mitochondria

Recently, by analysis of mildly solubilized mitochondrial membranes new biochemical evidences were obtained for the occurrence of ATP synthase dimers in mitochondria of different eukaryotes from yeast to mammals. In the case of yeast even higher ATP synthase oligomers could be found. Here, we analysed by BN- and CN-PAGE mammalian (bovine and rat) mitochondria from five different tissues, which were efficiently but very mildly solubilized with digitonin. In mitochondria from all investigated tissues besides ATP synthase monomers (V(1)) not only dimeric ATP synthase (V(2)) but for the first time also higher oligomers, at least trimers (V(3)) and tetramers (V(4)), were separated. Compared with BN-PAGE, by CN-PAGE analysis the yields of preserved respiratory supercomplexes as well as of oligomeric ATP synthases (V(2-4)) were significantly increased. The latter represent the majority of total ATP synthases in all cases. Importantly, all different ATP synthase species from the five tissues displayed in-gel ATP hydrolase activity, suggesting that homooligomeric ATP synthases are the constitutive, enzymatically competent organization of mammalian ATP synthases in the inner mitochondrial membrane.     

Identification of acetaminophen polymerization products catalyzed by horseradish peroxidase

Horseradish peroxidase catalyzed the H2O2-dependent oxidation and polymerization of acetaminophen. Six acetaminophen polymers were isolated from horseradish peroxidase reaction mixtures by semipreparative high pressure liquid chromatography. Chemical structures were determined by a combination of electron impact and chemical ionization mass spectrometry and 500-MHz proton magnetic resonance spectroscopy. Two dimers, three trimers, and one tetramer were identified. The polymers formed primarily through a covalent bond between carbons ortho to the hydroxyl group, and to a lesser extent, between the carbon ortho to the hydroxyl group and the amino group of another acetaminophen molecule. Greater than 99% of the polymerization reaction products were quenched by the addition of 2.0 mM ascorbate. High acetaminophen concentration favored dimer formation, whereas low acetaminophen concentration favored formation of trimers and tetramers. Since approximately 1 mol of H2O2 was consumed per mol of covalent ligand formed between acetaminophen molecules, these products probably result from free radical termination reactions.     

Factors affecting gamma-chain multimer formation in cross-linked fibrin.

The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy-terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma-chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Green plant photosystem I binds light-harvesting complex I on one side of the complex

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.     

Assembly of F1-ATPase in isolated mitochondria

The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system. Following such an incubation, the ATPase complex (F1F0) was isolated. Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive. Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria. Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors. These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria.     

Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of beta-lactam antibiotics

Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of beta-lactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the 'random addition' model for multiple cross-link formation and not at all those predicted by the 'monomer addition' model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.     

Chemistry and occurrence of hydroxycinnamate oligomers

Hydroxycinnamates such as ferulic acid, sinapic acid and p-coumaric acid ester-linked to plant cell wall polymers may act as cross-links between polysaccharides to each other, but also to proteins and lignin. Although sinapates and p-coumarates also form cell wall cross-links by the formation of radically or photochemically formed dimers, ferulate derivatives are the quantitatively most important cross-links in the plant cell wall. While the first radically generated ferulate dimer was already identified almost 40 years ago, the spectrum of known ferulate dimers was considerably broadened within the last 15 years. Higher ferulate oligomers were generated in model systems, but also isolated from plant materials. Different model systems using either free hydroxycinnamic acids or their esters are reviewed, highlighting a discussion of the relevance of these models for the plant cell wall. The first ferulate trimer from plant material was discovered in 2003 and seven dehydrotrimers of ferulic acid were isolated from maize bran since. Some of these trimers were also identified in other plant materials such as wheat and rye grains, corn stover, sugar beet and asparagus. Formation mechanisms of ferulate trimers and implications for the plant cell wall are discussed. Ferulate tetramers are the highest oligomers isolated from plant materials so far. These compounds can theoretically cross-link up to four polysaccharide chains, assuming all cross-links are formed intermolecularly. Formation of intramolecular versus intermolecular polysaccharide cross-links is a key question to be answered in the future if we want to judge properly the importance of hydroxycinnamate cross-links in the plant cell wall.     

Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes

Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.     

Evidence for an endogenous ATPase inhibitor protein in plant mitochondria. Purification and characterization

An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria. The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity. The isolated inhibitor is a heat-stable, trypsin-sensitive, basic protein, with a molecular mass approximately 8.3 kDa. Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline; threonine and leucine. The interaction of the inhibitor with F1-ATPase requires the presence of Mg2(+)-ATP in the incubation medium. The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 micrograms inhibitor/mg F1. A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value. The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1. The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, epsilon subunit, of chloroplast ATPase.     

The increased potency of cross-linked lymphocyte function-associated antigen-3 (LFA-3) multimers is a direct consequence of changes in valency.

We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA-3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions.