Biomedical subjects

L Moens

Publications and source records attributed to L Moens.

At least 55 records · Page 3Linked to original sources

PubMed1997

Biochemical characterization of cardiotin, a sarcoplasmic reticulum associated protein.

The further biochemical characterization and subcellular localization of cardiotin, a high molecular weight (300 kDa) constituent of cardiac muscle, is described. Immunofluorescence assays revealed a colocalization of cardiotin and the Ca2+ pump SERCA2a in the longitudinal sarcoplasmic reticulum (SR). However, in contrast to SERCA2a, cardiotin is not detected in the junctional SR. Differential centrifugation experiments show that cardiotin cosediments with the microsomal fraction of swine heart, while differential extraction demonstrates that cardiotin is associated with the SR membranes. In the SR enriched cell fraction a 60 and a 100 kDa protein band are detected. Microsequence analyses of these two fragments showed a common amino-terminus of 14 amino acids, with great homology to amino acid positions 11-24 of human skeletal muscle alpha-actinin. Second generation antibodies directed to these specific fragments show the typical cardiotin pattern in cardiomyocytes and cross-reactivity amongst the respective antigens. Cardiotin did not colocalize with alpha-actinin, and alpha-actinin could not be detected in the microsomal SR fraction. Cardiotin therefore represents a new SR associated constituent.

Actinin↗

PubMed1997

Trematode myoglobins, functional molecules with a distal tyrosine.

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Amino Acid Sequence↗

PubMed1997

Solution of 1H NMR structure of the heme cavity in the oxygen-avid myoglobin from the trematode Paramphistomum epiclitum.

A two-dimensional 1H NMR study has been carried out on the heme cavity of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr, and 2 upfield ring current aliphatic side chains. Based solely on the conserved myoglobin folding topology that places the E helix-heme crossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix alignment were unambiguously established that place Tyr66 at position E7. Moreover, all eight aromatic and the two aliphatic side chains were shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr32(B10) and Tyr66(E7) rings indicate that both residues are oriented into the heme cavity, which is unprecedented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordinated oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autoxidation resistance of this myoglobin.

Amino Acid Sequence↗

PubMed1997

Computer analysis of two-dimensional electrophoresis gels: a new segmentation and modeling algorithm.

The complexity of the spot patterns of two-dimensional electrophoresis gels made it necessary to use image processing techniques to analyze the gels. An important issue in the analysis is the detection and quantification of the protein spots. In this paper we describe a new technique to segment and model the different spots. For the segmentation of a gel into its different spot regions we apply a watershed technique, which is robust and efficient. For the quantification of the spots, a new spot model is constructed, based on diffusion principles. Besides the advantage of having a physical interpretation, the model is demonstrated to be superior to the commonly used Gaussian models.

Algorithms↗

PubMed1996

Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata.

The globin of the nerve cord of the polychaete annelid Aphrodite aculeata was isolated and purified to homogeneity. The native molecule has a pI of 6.3 and acts as a dimer of two identical Mr 15, 644.5 polypeptide chains as determined by electrospray mass spectrometry. It has an average affinity for oxygen (P50 = 1.24 torr) resulting from fast association (kon = 170 X 10(6) M-1 . s-1) and dissociation rates (koff = 360 s-1). The partial primary structure of this nerve globin was determined at the protein level and completed and confirmed by translation of the cDNA sequence. The globin chain has 150 amino acid residues and a calculated Mr of 15, 602.69 strongly suggesting that the amino terminus is acetylated. The absence of a leader sequence and the lack of Cys at the positions NA2 and H9 needed for the formation of the high Mr complexes found in extracellular annelid globins classify the Aphrodite globin with the cellular globin species. The Aphrodite nerve globin is unlikely to represent a separate globin family, as cDNA derived primers detect globin messenger RNA in muscle, gut, and pharynx tissue as well. The gene encoding this globin species is interrupted by a single intron, inserted at position G7.0. Comparison to other globin gene structures strongly suggest that introns can be lost independently, rather than simultaneously as a result of a single conversion event as suggested previously (Lewin, R. (1984) Science 226, 328).

Amino Acid Sequence↗

PubMed1996

Time-dependent pseudo-activation of hepatic glycogen synthase b by glucose 6-phosphate without involvement of protein phosphatases.

During a 30 min incubation at 25 degrees C in the presence of 5-10 mM glucose 6-phosphate, pure glycogen-bound glycogen synthase b from dog liver was progressively converted into a form that was fully catalytically active in the presence of 10 mM Na2SO4 plus 0.5 mM glucose 6-phosphate. The latter enzyme was unlike synthase a (which does not require glucose 6-phosphate for activity), and unlike synthase b (which is strongly inhibited by sulphate). The conversion was insensitive to various inhibitors of Ser/Thr-protein phosphatases and alkaline phosphatases, and was therefore termed 'pseudo-activation'. Kinetically, pseudo-activation increased the V(max) 4-fold without affecting the K(m) for the substrate UDP-glucose. Pseudo-activation appeared to be an irreversible process, but several lines of evidence argue against a limited proteolysis. Pseudo-activation of glycogen synthase occurred also readily in a rat liver cytosol, but it was not observed with purified synthase from skeletal muscle. These observations have important implications for the assay of liver gycogen-synthase phosphatase; the possible physiological implications remain to be explored.

Animals↗

PubMed1996

Determination of low amounts of platinum in environmental and biological materials using thermospray nebulization inductively coupled plasma-mass spectrometry.

A description is given of the experimental arrangement for thermospray nebulization of discrete liquid samples (100 microl) into an inductively coupled plasma-mass spectrometer (ICP-MS). The determination of ultratrace levels of platinum in environmental and biological materials is described. This method comprises an off-line sample enrichment by adsorptive chromatography, elution with ethanol and subsequent analysis of the evaporated eluate. The quantification of the concentration was performed with isotope dilution. The recovery, precision, accuracy and detection limit of the method are discussed.

Journal Article↗

PubMed1996

Non-spectral interferences encountered with a commercially available high resolution ICP-mass spectrometer.

Results of a systematic study concerning non-spectral interferences observed with a commercially available high resolution ICP-mass spectrometer are reported and compared to observations made with a quadrupole-based instrument. In general, matrix effects were observed to be to a large extent comparable for both instruments used. In all cases, the matrix-induced signal suppression or enhancement was seen to depend in a regular way on the mass number of the nuclides monitored. In most cases, the ionization potential of the nuclides has little or no influence on the extent of suppression or enhancement. For As, Se and Te, the introduction of 2.5% ethanol, 0.5 mol/l H(2)SO(4), or to a lesser extent 0.5 mol/l H(3)PO(4), leads to an exceptional increase in the signal intensity for both instruments. Registration of signal behaviour plots (signal intensity as a function of the nebulizer gas flow rate) in different matrices revealed that both the height of the plot and the optimum nebulizer gas flow rate are a function of the matrix composition. Finally, no indication was found that the acceleration of the extracted ions over 8000 V with the high resolution instrument would lead to an alleviation of space charge effects when compared to a quadrupole-based ICP-mass spectrometer.

Journal Article↗

PubMed1996

Isolation, characterization and immunolocalization of a globin-like antigen from Ostertagia ostertagi adults.

Western blot analysis using an anti-globin rabbit serum Rb94 revealed a major band of 17 kDa in extracts of Ostertagia ostertagi adults and 4th-stage larvae. The adult stage globin-like antigen (OoAdGlb) was purified from total worm extracts by liquid chromatography. The protein has an estimated molecular mass of 36 kDa under non-reducing conditions, suggesting a dimeric structure containing 2 non-covalently linked 17 kDa monomers. Tryptic peptides were sequenced and showed strong similarities with the globins of free-living and parasitic nematodes. Immunolocalization revealed the presence of this globin-like antigen in the body wall musculature and/or the cuticle of O. ostertagi adults. An enzyme-linked immunosorbent assay based on the purified OoAdGlb showed no differences in response between calves infected by O. ostertagi and/or Cooperia oncophora and the negative controls.

Amino Acid Sequence↗

PubMed1996

Globins in nonvertebrate species: dispersal by horizontal gene transfer and evolution of the structure-function relationships.

Using a new template based on an alignment of 145 nonvertebrate globins we examined several recently determined sequences of putative globins and globin-like hemeproteins. We propose that all globins have evolved from a family of ancestral, approx. 17-kDa hemeproteins, which displayed the globin fold and functioned as redox proteins. Once atmospheric O2 became available the acquisition of oxygen-binding properties was initiated, culminating in the various highly specialized functions known as present. During this evolutionary process, we suggest that (1) high oxygen affinity may have been acquired repeatedly and (2) the formation of chimeric proteins containing both a globin and a flavin binding domain was an additional and distinct evolutionary trend. Furthermore, globin-like hemeproteins encompass hemeproteins produced through convergent evolution from nonglobin ancestral proteins to carry out O2-binding functions as well as hemeproteins whose sequences exhibit the loss of some or all of the structural determinants of the globin fold. We also propose that there occurred two cases of horizontal globin gene transfer, one from an ancestor common to the ciliates Paramecium and Tetrahymena and the green alga Chlamydomonas to a cyanobacterium ancestor and the other, from a eukaryote ancestor of the yeasts Saccharomyces and Candida to a bacterial ancestor of the proteobacterial genera Escherichia, Alcaligenes, and Vitreoscilla.

Amino Acid Sequence↗

PubMed1995

Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.

Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.

Amino Acid Sequence↗

PubMed1994

Tyrosine phosphorylation of a M(r) 38,000 A/B-type hnRNP protein selectively modulates its RNA binding.

The M(r) 38,000 RNA-binding protein (P38) is the major component of translationally repressed messenger ribonucleoproteins in cryptobiotic gastrulae of the brine shrimp Artemia. Partial elucidation of the amino acid sequence of P38 reveals that it is homologous to A/B-type hnRNP proteins. This was confirmed by immunodetection with antibodies specific for A/B-type hnRNP proteins from Drosophila melanogaster. P38 can be phosphorylated in vitro by a src-related protein tyrosine kinase on multiple tyrosine residues located predominantly in the glycine-rich domain. Tyrosine phosphorylated P38 can be efficiently dephosphorylated by a specific protein tyrosine phosphatase (1B-like) and by protein phosphatase 2A activated by the phosphotyrosyl phosphatase activator. Tyrosine phosphorylation of P38 slightly influences its subsequent phosphorylation by casein kinase II. The latter phosphorylation site is located in the glycine-rich domain of P38. Two-dimensional gel electrophoresis resolves P38 into multiple isoforms which shift to more acidic pI values after phosphorylation by protein tyrosine kinase or casein kinase II. From nitrocellulose filter binding and UV cross-linking analysis, evidence was obtained that tyrosine phosphorylation of P38 impairs its binding to poly(A) but not to poly(U). This demonstrates the involvement of tyrosine residues in polynucleotide-specific RNA binding that can be regulated by phosphorylation/dephosphorylation.

Amino Acid Sequence↗

PubMed1994

Structural characterization of an Ascaris myoglobin.

Globin was purified from the body wall of adults of the parasitic nematode Ascaris suum. Internal peptide fragments were sequenced and cDNAs encoding a polypeptide of 154 amino acids isolated by polymerase chain reaction. The polypeptide lacks a signal sequence, identifying it as a cytosolic myoglobin-like species. The native protein is a dimer. The predicted amino acid sequence shares several unusual substitutions with other nematode globins. Like the abundant pseudocoelomic A. suum hemoglobin it has a Tyr at B10 and a Gln at E7, substitutions thought to be determinants of high affinity. However, the 10-fold lower oxygen affinity of body wall globin suggests that in this molecule Tyr(B10) does not form an additional hydrogen bond with the heme bound oxygen. Evolutionary analysis of the nematode globins suggests that the monodomain myoglobin-like molecules and the two-domain hemoglobin-like molecules diverged about 500 million years ago, well before the divergence of the ascarid genera Ascaris and Pseudoterranova. The absence of introns in the A. suum myoglobin, in contrast to other nematode globin genes, is consistent with the hypothesis that during evolution intron elimination was the predominant event.

Amino Acid Sequence↗

PubMed1994

Molecular genealogy of some nematode taxa as based on cytochrome c and globin amino acid sequences.

We have begun to reconstruct the ancient history of the nematode phylum based on cytochrome c and globin amino acid sequences. The data suggest that the nematode ancestor diverged from a line leading to mammals about 1 billion years ago and that the most recent common ancestor of the extant species Caenorhabditis elegans, Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ascaris suum, and Pseudoterranova decipiens lived about 550 MY ago. The rhabditids and strongylids emerged as one offshoot of this ancestor, the ascarids as another. Rhabditids and strongylids diverged some 400 MY ago, whereas the genera Trichostrongylus and Nippostrongylus diverged slightly over 200 MY ago. A gene duplication event in the strongylid branch is predicted to have occurred around 250-335 MY ago. There are two globin genes in Nippostrongylus, expressed in anatomically distinct compartments (body and cuticle), and the single sequence from Trichostrongylus is most like the Nippostrongylus body globin gene. A strikingly different duplication event occurred within the same period in the line leading to the extant ascarid genera, creating a single polypeptide containing two globin domains. The genera Ascaris and Pseudoterranova diverged some 150-250 MY ago. Interestingly, the second globin repeat evolved at a faster rate in both species examined. This is possibly related to the acquisition of an unusual carboxyterminal extension, composed of alternating positively and negatively charged residues, that is necessary for the assembly of several monomers into the native polymeric molecules.

Amino Acid Sequence↗

PubMed1994

Interdomain linkage in the polymeric hemoglobin molecule of Artemia.

Artemia has evolved the longest known concatenation of hemoglobin domains, the alpha subunit containing nine domains and the beta subunit having a similar size. Translation of the cDNA sequence of the alpha subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit. The interdomain linker sequences were 14%-64% identical (62%-91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

The occurrence of artemocyanin in Branchiopoda (Crustacea).

Artemocyanin, the extracellular hemolymph biliprotein of Artemia, is demonstrated in the fairy shrimp Streptocephalus, the clam shrimp Leptestheria and the water flea Daphnia. Artemocyanins can be purified from hemolymph as intact polypeptides (Mr 170-190,000), but are degraded upon homogenization of the whole animal by partial proteolysis to polypeptides with Mr 102,000 and 85,000. The aminoterminal sequence of the intact artemocyanin polypeptide was determined, but no clear-cut relationships with arthropod biliproteins or other protein families could be demonstrated.

Amino Acid Sequence↗

PubMed1993

Polar zippers.

BACKGROUND: Certain proteins are known to form leucine zippers - alpha-helical coiled-coils in which the non-polar side chains of two leucine-rich helices intermesh. We recently presented the first evidence for a polar zipper, formed by the carboxy-terminal peptides of the eight subunits of Ascaris haemoglobin. The evidence was based on the presence of pairs of acidic residues alternating with pairs of basic residues ( + + - - ) in an amino-acid sequence that has since been shown to be incomplete. The complete sequence, derived from the haemoglobin's cDNA, now shows a self-complementary polar sequence extending along the entire length of its 24-residue carboxy-terminal peptide. RESULTS: From the complete sequence, it is clear that the eight identical subunits of the haemoglobin could be held together by an eight-stranded antiparallel beta barrel made up of the carboxy-terminal 24 residues of each of the subunits, such that each strand forms 10 salt bridges with each of its neighbours. A computer search of the protein database revealed similar, but shorter, + + - - repeats in several other proteins. It also revealed long repeats of alternating arginine and aspartate residues, and long stretches of only glutamines, or only serines, suggestive of several other kinds of polar zippers. CONCLUSION: Several proteins have amino-acid sequences that suggest the formation of polar zippers made of beta strands. These could form antiparallel pleated sheets linked together by hydrogen bonds between polar side chains both above and below the plane of the sheets. Polar zippers may be important in welding together oligomeric proteins which have subunits lacking the extensive complementary surfaces necessary for stability, or in promoting the association of functionally complementary proteins.

Journal Article↗

PubMed1993

Adventitious variability? The amino acid sequences of nonvertebrate globins.

1. The more than 140 amino acid sequences of non-vertebrate hemoglobins (Hbs) and myoglobins (Mbs) that are known at present, can be divided into several distinct groups: (1) single-chain globins, containing one heme-binding domain; (2) truncated, single-chain, one-domain globins; (3) chimeric, one-domain globins; (4) chimeric, two-domain globins; and (5) chimeric multi-domain globins. 2. The crystal structures of eight nonvertebrate Hbs and Mbs are known, all of them monomeric, one-domain globin chains. Although these molecules represent plants, prokaryotes and several metazoan groups, and although the inter-subunit interactions in the dimeric and tetrameric molecules differ from the ones observed in vertebrate Hbs, the secondary structures of all seven one-domain globins retain the characteristic vertebrate "myoglobin fold". No crystal structures of globins representing the other four groups have been determined. 3. Furthermore, a number of the one-, two- and multi-domain globin chains participate in a broad variety of quaternary structures, ranging from homo- and heterodimers to highly complex, multisubunit aggregates with M(r) > 3000 kDa (S. N. Vinogradov, Comp. Biochem. Physiol. 82B, 1-15, 1985). 4. (1) The single-chain, single-domain globins are comparable in size to the vertebrate globins and exhibit the widest distribution. (A) Intracellular Hbs include: (i) the monomeric and polymeric Hbs of the polychaete Glycera; (ii) the tetrameric Hb of the echiuran Urechis; (iii) the dimeric Hbs of echinoderms such as Paracaudina and Caudina; and (iv) the dimeric and tetrameric Hbs of molluscs, the bivalves Scapharca, Anadara, Barbatia and Calyptogena. (B) Extracellular Hbs include: (i) the multiple monomeric and dimeric Hbs of the larva of the insect Chironomus; (ii) the Hbs of nematodes such as Trichostrongylus and Caenorhabditis; (iii) the globin chains forming tetramers and dodecamers and comprising approximately 2/3 of the giant (approximately 3600 kDa), hexagonal bilayer (HBL) Hbs of annelids, e.g. the oligochaete Lumbricus and the polychaete Tylorrhynchus and of the vestimentiferan Lamellibrachia; and (iv) the globin chains comprising the ca 400 kDa Hbs of Lamellibrachia and the pogonophoran Oligobrachia. (C) Cytoplasmic Hbs include: (i) the Mbs of molluscs, the gastropods Aplysia, Bursatella, Cerithedea, Nassa and Dolabella and the chiton Liolophura; (ii) the three Hb of the symbiont-harboring bivalve Lucina; (iii) the dimeric Hb of the bacterium Vitreoscilla; and (iv) plant Hbs, including the Hbs of symbiont-containing legumes (Lgbs), the Hbs of symbiont-containing non-leguminous plants and the Hbs in the roots of symbiont-free plants.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗