Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc,Haliotis rufescens,as a function of development

Summary Conchiolin peptides of the molluscan shell are believed to determine structural organization and facilitate calcification during shell formation. Changes in patterns of conchiolin synthesis during development, and the possible contribution of these peptides to shell formation, have been investigated by purification and characterization of the soluble peptides extracted from the shell of the gastropod mollusc,Haliotis rufescens (red abalone), at various stages of development. Shell peptides were purified from young post-larvae, juveniles and adults by gel-filtration column chromatography in aggregation-reducing bicarbonate buffers. Calcium-binding domains were detected spectrophotometrically after reaction with a cationic carbocyanine dye. Juvenile and adult shell peptides were found to be heterogeneous, and rich in aspartic acid and glycine residues; in contrast, post-larval shells were found to contain one major glycine-rich component. The juvenile shell peptide population shares components from each of the other two populations, suggesting that the synthesis of the different shell peptides results from the differential expression of a multi-gene family, in a developmentally controlled progression. Enzymatic analyses suggest that calcium binds to the aspartic acid residues of the peptide core, rather than to satellite groups such as phosphate, sulfate or carbohydrate. The possibility is discussed that the aspartic acid residues found in shell peptides may play an important role in the calcification of the abalone shell matrix. The methods demonstrated here also should prove useful for the purification, characterization, and comparative analysis of calcium-binding proteins of connective tissues, extracellular matrices and support structures in many other systems.Abbreviations Asp aspartic acid - BSA bovine serum albumin - Da daltons - EDTA ethylenediaminetetraacetic acid - GABA -aminobutyric acid - HPLC high-pressure liquid chromatography - ODS octadecylsilane - OPA o-phthaldialdehyde - SDS sodium dodecyl sulfate     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

Purification and characterization of hyaluronic acid from the mollusc bivalve Mytilus galloprovincialis

Hyaluronan (hyaluronic acid, HA) was for the first time extracted, purified and characterized from the species of mollusc bivalve Mytilus galloprovincialis. HA was characterized by agarose-gel electrophoresis, 13C-NMR, HPLC and normal polarity capillary electrophoresis by evaluating the unsaturated disaccharide, DeltaDiHA (Delta-hexuronic acid-N-acetyl-glucosamine) after treatment with chondroitin ABC lyase, and by separating Delta-tetrasaccharide and Delta-hexasaccharide generated by the specific action of hyaluronate lyase from Streptomyces hyalurolyticus. The weight average molecular weight (M(w)) was found to be about 200 kDa as determined by HPSEC. HA from M. galloprovincialis was not able to interact with aggrecan from bovine cartilage to form high molecular mass aggregate and also had a very low specific viscosity, but it showed the same capacity to inhibit cell proliferation (50 microg per 10(3) human fibroblasts inhibit cell proliferation by about 50%) than high molecular mass HA. HA of M. galloprovincialis could have a physiological role in the regulation of cell functions.     

Purification and characterization of two phospholipid exchange proteins from bovine heart.

Two phospholipid exchange proteins from bovine heart have been purified approximately 2000-fold and judged greater than 90% pure. The proteins are similar in molecular weight (both 33,400 by polyacrylamide gel electrophoresis and 23,500 by gel filtration), in amino acid composition, and in specificity, although they differ in isoelectric points, 5.3 and 5.6. The transfer of phospholipids between artificial membranes is catalyzed by these proteins at the following relative rates: 100 for phosphatidylinositol, 35 for phosphatidylcholine, 5 for sphingomyelin, and 0.1 for phosphatidylethanolamine. The use of these exchange proteins in the study of mixed phospholipid vesicle structure is demonstrated. The purified proteins catalyze the substitution of one membrane phospholipid species for another at a rate comparable to true exchange. The phospholipid exchange activity is inhibited by the presence of sphingomyelin, and also by reagents which react with sulfhydryl groups. Evidence is presented for two sites of N-ethylmaleimide binding on these exchange proteins. Reaction with one site has little effect on activity and occurs in the absence of membranes. Reaction with the second site occurs in the presence of phospholipid vesicles and leads to complete, irreversible inhibition of exchange activity.     

Purification and characterization of two basic spermatid-specific proteins isolated from the dog-fish Scylliorhinus caniculus

In dog-fish spermatid nuclei two intermediate proteins S1 and S2 replace histones before the setting down of protamines. These spermatid-specific proteins were isolated by carboxymethyl-cellulose chromatography and purified by high pressure liquid chromatography. S1 and S2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N- and C-terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.     

Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

Newly absorbed retinol is transported in association with chylomicrons and their remnants. In addition, after intake of high doses of retinol, significant amounts are also found in low-density lipoprotein (LDL). As both chylomicron remnants and LDL may be taken up by cells via the LDL receptor, and retinoids inhibit proliferation of some leukaemic cells, we have studied the uptake of retinol in leukaemic cells via the LDL-receptor pathway. HL-60 cells contain saturable binding sites for LDL. The binding of LDL to its receptor has a dissociation constant of about 3.2 x 10(-9) M, and the number of receptors per cell was calculated to be about 2700. Uptake of 125I-LDL by HL-60 cells was increased 2-fold by preincubating the cells with mevinolin. The presence of specific receptors for LDL on HL-60 cells was further confirmed by the finding that exogenous LDL cholesterol was able to up-regulate the ACAT (acyl-CoA: cholesterol acyltransferase) activity of HL-60 cells. We then tested the uptake of retinyl ester in leukaemic cells via the LDL-receptor pathway. HL-60 cells were incubated with LDL or chylomicron remnants labelled with [3H]retinyl palmitate. Uptake of retinyl ester associated with both LDL and chylomicron remnants was observed. Furthermore, the presence of excess LDL decreased the uptake by 75-100%, supporting the hypothesis that the uptake of retinyl ester occurred via the LDL receptor in HL-60 cells.     

Purification and characterization of PSP-I and PSP-II, two major proteins from porcine seminal plasma.

Two major glycoproteins, designated PSP-I and PSP-II, were purified from porcine seminal plasma by ammonium sulfate fractionation, CM-cellulose chromatography, gel filtration on Sephadex G-75 (superfine), and reverse phase high performance liquid chromatography. These two proteins exist in several forms differing mainly in the carbohydrate moiety. The complete amino acid sequence of PSP-I has been determined by automated Edman degradation of peptides generated by proteolytic digestion and cyanogen bromide cleavage. The protein is 109 residues long and has a single glycosylation site at the asparagine residue at position 47. In addition, the N-terminal sequence of PSP-II has also been determined. PSP-I is a unique protein; a sequence homology search using the protein data base did not reveal any significant homology with other proteins. PSP-II shares 50% sequence homology with a family of zona pellucida-binding glycoproteins at the N-terminus.     

Purification and characterization of two new allergens from the venom of Vespa magnifica

Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.     

Purification, characterization and molecular modelling of two toxin-like proteins from the Androctonus australis Hector venom.

Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.     

Purification and partial characterization of two basic proteins from human peripheral nerve.

Two basic proteins, denoted P1 and P2 protein, were purified from human sciatic nerve. The isolation was achieved by the following steps: delipidation with chloroform/methanol mixtures, dry acetone and dry ether; acid extraction at pH 2; ion exchange chromatography on QAE-Sephadex A-25 and gel filtration on Sephadex G-100. P1, P2 proteins and the basic protein of the central nervous system have been shown to have different electrophoretic mobility, and each of the two peripheral basic proteins was shown to be homogeneous by disc electrophoresis. The molecular weight of P1 protein is around 14 100 and that of P2 protein is around 12 200, as determined by ultracentrifugal analysis. There was some difference in the amino acid composition of human P1 and P2 protein, and a marked difference between their composition and the composition of central basic protein and bovine peripheral P1 and P2 proteins which were described previously. When injected to animals, P1 protein induced only experimental allergic neuritis while P2 protein induced both mild experimental allergic neuritis and experimental allergic encephalomyelitis. Thus, the human P1 protein is similar to the bovine P1 protein and human P2 protein is similar to bovine P2 protein, concerning their electrophoretic mobilities, molecular weights and biological properties.     

Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris: rubrerythrin and a new, highly active, enzyme

The inorganic pyrophosphatase activity of a soluble extract from the strict anaerobe, sulfate-reducing, Desulfovibrio vulgaris, is readily resolved into two peaks. After purification, two active proteins with very dissimilar properties are obtained. One is the non-heme iron-containing rubrerythrin, with a specific activity of 350 pyrophosphate hydrolyzed, min-1, mg protein-1. The other, a protein of Mr = 39,000, with a specific activity of 12,000.     

Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis

In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.     

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins

Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, -globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (M_rs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH₂-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with M_rs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the -globulin and one of the new low-molecular-weight proteins of M_r 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH₂-terminal amino acids.Abbreviations HMW high molecular weight - IgE immunoglobulin E - LMW low molecular weight - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - RP-HPLC reversed-phase high-performance liquid chromatography - SDS sodium dodecyl sulphate We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica.     

Seascape genomics reveals adaptive divergence in a connected and commercially important mollusc,the greenlip abalone (Haliotis laevigata), along a longitudinal environmental gradient

Populations of broadcast spawning marine organisms often have large sizes and are exposed to reduced genetic drift. Under such scenarios, strong selection associated with spatial environmental heterogeneity is expected to drive localized adaptive divergence, even in the face of connectivity. We tested this hypothesis using a seascape genomics approach in the commercially important greenlip abalone (Haliotis laevigata). We assessed how its population structure has been influenced by environmental heterogeneity along a zonal coastal boundary in southern Australia linked by strong oceanographic connectivity. Our data sets include 9,109 filtered SNPs for 371 abalones from 13 localities and environmental mapping across ~800 km. Genotype–environment association analyses and outlier tests defined 8,786 putatively neutral and 323 candidate adaptive loci. From a neutral perspective, the species is better represented by a metapopulation with very low differentiation (global F_ST = 0.0081) and weak isolation by distance following a stepping‐stone model. For the candidate adaptive loci, however, model‐based and model‐free approaches indicated five divergent population clusters. After controlling for spatial distance, the distribution of putatively adaptive variation was strongly correlated to selection linked to minimum sea surface temperature and oxygen concentration. Around 80 candidates were annotated to genes with functions related to high temperature and/or low oxygen tolerance, including genes that influence the resilience of abalone species found in other biogeographic regions. Our study includes a documented example about the uptake of genomic information in fisheries management and supports the hypothesis of adaptive divergence due to coastal environmental heterogeneity in a connected metapopulation of a broadcast spawner.     

Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5'-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the alpha-subunits of which possess molecular mass values of about 40 kDa.     

Linckosides A and B, two new neuritogenic steroid glycosides from the Okinawan starfish Linckia laevigata

Two new steroid glycosides, named linckosides A and B, were isolated from the Okinawan starfish Linckia laevigata, and their stereostructures were elucidated by spectroscopic methods and chemical derivatization. Each of them possesses two monosaccharide units at C-3 and C-29 of a polyhydroxylated steroid aglycon. These steroid glycosides showed not only notable neuritogenic activity against PC12 cells but also significant synergistic effects on the NGF-induced neuronal differentiation of PC12 cells. Linckoside B showed higher activities than those of linckoside A, though the structural difference is only the kind of a sugar.     

Purification and characterization of napin-like proteins from radish

Nine 2S albumin proteins from garden and sea radish seeds (Raphanussativus and Raphanus raphanistrum, respectively) have been purified.Their molecular weights and amino acid compositions have beendetermined. Most of these proteins exhibit a molecular massof 14.5 kDa, but one component of each species shows a slightlyhigher value (15.1 kDa). The amino acid compositions obtainedare typical of napin-like proteins, although a different contentof Tyr and basic residues was observed. The isolated proteinspresent circular dichroism and fluorescence spectra nearly identicalto each other, but different from those of rapeseed or mustardnapins. The 2S albumins from radish exhibit about 53% -helix,14% ß-bend, 16% ß-turn and 17% aperiodicconformation. The microheterogeneity of these proteins has beenanalysed by high pressure liquid chromatography of the reducedmolecules, and a high degree of polymorphism has been observedin most of the obtained fractions. On the basis of the aminoacid contents of the individual chains, as well as the carboxypeptidaseY digestion data, new sites have been suggested for the processingof their polypeptide precursors to render the mature proteins. Key words: Storage proteins, Brassicaceae, spectroscopy, processing     

Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig.

1. Soluble proteins showing binding activity to 2-isobutyl-3-methoxypyrazine have been purified to homogeneity from rabbit and pig nasal tissue; their characteristics are similar to the bovine odorant-binding protein and are to be considered members of the same family. 2. The rabbit protein is a homodimer with subunits of Mr 19k and an isoelectric point of 4.7, whereas the pig protein appears to consist of a single polypeptide chain of Mr 22k and an isoelectric point of 4.2. 3. Both proteins bind 2-isobutyl-3-methoxypyrazine with dissociation constants in the micromolar range. 4. Antibodies against the bovine OBP react well with the rabbit protein, and slightly with the porcine one.