Distribution and characterization of mineral-binding phosphoprotein particles in Bivalvia

Representative species of four bivalve subclasses were examined for the presence of mineral-binding phosphoprotein particles in the physiological fluids. The particles were identified in Heterodont bivalves only, and particles from nine different Heterodont species were isolated and characterized. All phosphoprotein particles are internally cross-linked via histidinoalanine residues. In all species over 80% of the amino acid residues in the particles are aspartic acid, phosphoserine (and/or phosphothreonine), and histidine. These amino acids are probably the only residues directly related to mineral ion binding, since all phosphoprotein particles bind mineral irrespective of the minor amino acid content, which is species dependent. In their native state the phosphoprotein particles contain large amounts of calcium, magnesium, and inorganic phosphate ions (up to 45 metal ions and 8 phosphate ions per 100 amino acid residues) and trace amounts of transition elements. Evidence for the presence of calcium phosphate complexes in the native phosphoprotein particles was obtained by observing a concomitant increase in the inorganic phosphate and calcium ion content of the particles with pH in vivo.     

Opening and refolding of simian virus 40 and in vitro packaging of foreign DNA.

Simian virus 40 (SV40) can be disassembled under mild conditions by reducing disulfide bonds in the capsid and removing calcium ions. The nucleoprotein complexes formed, analyzed by electron microscopy, were circular and made up of 59 +/- 4 subunits, each with a diameter of about 10 nm. The complexes contained the viral DNA, histones, and the viral capsid proteins. The complexes had much-reduced infectivities compared with intact SV40. Addition of calcium ions to the disrupted virus caused the nucleoprotein complexes to refold into virus-like structures which sedimented at the same rate as intact SV40 and regained infectivity. Treatment of the disrupted SV40 with a high concentration of salt dissociated the viral proteins from the DNA. Lowering stepwise the salt concentration, removing the reducing agent, and adding calcium ions allowed structures to be reformed, and these structures sedimented, like SV40, at 240S and were infectious. The plaque-forming ability of the reconstituted particles was between that of the dissociated components and that of intact SV40. The addition of purified DNA of polyomavirus to the dissociated SV40 before the lowering of the salt concentration showed that virus-like structures could be formed from SV40 proteins and a foreign DNA.     

Protein dynamics of bovine dentin phosphophoryn.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and o-phosphoseryl [Ser(P)] residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentin. The elucidation of the structure-function relationship of phosphophoryn has been a challenge because of its high-molecular weight, high negative charge, repetitive sequence, and lability. We have used the dynamic behavior of the (1)H NMR signal at 600 MHz to provide insight into the molecular dynamics of phosphophoryn. Our results indicate that phosphophoryn is a molecule of uniformly high mobility, thus belonging to a recently identified class of intrinsically disordered proteins that are characterized by sequences of low complexity and rich in polar and charged residues. The significance of our results is that phosphophoryn, because of its uniform nature has the potential to be replaced by biomimetic synthetic peptide analogs that together with amorphous calcium phosphate may lead to the development of novel, nontoxic, apatite-based dental restorative materials.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Effect of calcium ions on creatine kinase systems of myocardial cells

The kinetics of the creatine phosphokinase reaction catalyzed by different creatine phosphokinase (CPK) isoenzymes from the heart and the mechanism of the regulatory action of calcium ions on this reaction have been studied. It has been shown that the kinetic parameters of the reaction in the presence of calcium are similar to those in the presence of magnesium with the exception of the maximal rate of the reverse reaction, which is about three times lower in the presence of calcium. Calcium ions are able to exert a significant regulatory action on the CPK reaction. Simultaneous regulation of the CPK reaction by calcium and magnesium ions can be quantitatively described by a kinetic model, which takes into account the formation of complexes of adenine nucleotides with calcium and magnesium. The mechanism of the CPK reaction regulation by metal ions involves changes in concentrations of the metal -- adenine nucleotide compexes. The regulatory action of calcium on the creatine kinase reaction is the same for all CPK isoenzymes studied.     

Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B

Apolipoproteins play a central role in lipoprotein metabolism, and are directly implicated in cardiovascular diseases, but their structural characterization has been complicated by their structural flexibility and heterogeneity. Here we describe the structural characterization of the N-terminal region of apolipoprotein B (apoB), the major protein component of very low-density lipoprotein and low-density lipoprotein, in the presence of phospholipids. Specifically, we focus on the N-terminal 6.4-17% of apoB (B6.4-17) complexed with the phospholipid dimyristoylphosphatidylcholine in vitro. In addition to circular dichroism spectroscopy and limited proteolysis, our strategy incorporates nanogold-labeling of the protein in the reconstituted lipoprotein complex followed by visualization and molecular weight determination with scanning transmission electron microscopy imaging. Based on the scanning transmission electron microscopy imaging analysis of approximately 1300 individual particles where the B6.4-17 is labeled with nanogold through a six-His tag, most complexes contain either two or three B6.4-17 molecules. Circular dichroism spectroscopy and limited proteolysis of these reconstituted particles indicate that there are no large conformational changes in B6.4-17 upon lipoprotein complex formation. This is in contrast to the large structural changes that occur during apolipoprotein A-I-lipid interactions. The method described here allows a direct measurement of the stoichiometry and molecular weight of individual particles, rather than the average of the entire sample. Thus, it represents a useful strategy to characterize the structure of lipoproteins, which are not structurally uniform, but can still be defined by an ensemble of related patterns.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

DNA contained by two densonucleosis viruses.

The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.     

Binding and cross-linking of rabbit fibronectin by rabbit hepatocytes in suspension

Fibronectin purified from rabbit plasma was radioiodinated, and its interaction with rabbit hepatocytes in suspension was studied. Iodinated fibronectin interacted in a time-dependent fashion reaching plateau at 3 h. The interaction was greater in the presence of calcium than in the presence of magnesium or EDTA. Saturation occurred at about 140 nM fibronectin with about 1,400,000 molecules bound per cell. The interaction could be inhibited by unlabeled fibronectin or fibrinogen but not by the tetrapeptide Arg-Gly-Asp-Ser or by albumin, transferrin, or fetuin. About 50% of the bound iodinated fibronectin was incorporated, in a calcium-dependent fashion, into cross-linked high molecular weight complexes at the cell surface through a mechanism consistent with a cellular transglutaminase-mediated reaction. Iodinated fibronectin which could be displaced from the cell was monomeric in nature, while the cell-associated material remained in high molecular weight complexes. The role of the interaction is currently under investigation, but it is possible that the binding may promote cellular adhesion or facilitate intercellular interaction.     

Bovine dentin phosphophoryn: composition and molecular weight

The molecular weight of phosphophoryn, an acidic phosphoprotein unique to dentin matrix, has been difficult to determine because of a combination of neutral protease activities in this tissue and the intrinsic high charge density of the molecule. In this study, bovine dentin phosphophoryn (BDPP) was isolated by a procedure designed to prevent proteolysis. Bovine unerupted third molar powder was demineralized by ethylenediaminetetraacetic acid (EDTA). The EDTA-soluble phosphophoryn fraction was isolated and purified by sequential calcium chloride precipitation, gel filtration in sodium dodecyl sulfate (NaDodSO4) containing buffer, anion-exchange chromatography, and finally gel filtration in 4 M guanidine hydrochloride (4 M Gdn.HCl) buffer. Sedimentation equilibrium, sedimentation velocity, and diffusion coefficient data, viscosity studies in a high ionic strength buffer, and NaDodSO4 gradient gel electrophoresis data gave consistent results for the molecular weight of BDPP, all being in the range of 151 000-167 000. This range is much higher than any previously reported value. An anomalous behavior was observed in nongradient NaDodSO4 gel electrophoresis. Dissociative analytical gel filtration chromatography in 4 M Gdn.HCl gave a molecular weight value of 100 000. This discrepancy was resolved by studying the viscosity of BDPP in 4 M Gdn.HCl which showed BDPP does not assume a true random-chain conformation in this solvent.     

Structure of the complexes of distamycin type antibiotics and actinomycin D with DNA: new data on the localization of these antibiotics within the DNA narrow groove]

It is shown that antibiotics actinomycin D (AM), netropsin (Nt), distamycin A (DM) and the propyl analogue of distamycin A (pDM) being complexed with DNA are located within the narrow groove of DNA. A comparative investigation of the 3H-dimethyl sulphate methylation extent of free calf thymus DNA and its complexes with AM, Nt, DM and pDM reveals that upon DNA saturation these antibiotics decrease the methylation level of the narrow groove (AM by 30%, pDM by 50%, DM by 65% and Nt by 70%). In the triple complex of DNA+AM+DM the methylation level of the narrow groove drops by 80%. The large groove is not shielded by these antibiotics at all. However, the methylation level of the large groove decreases by 50% for T6 phage DNA due to the presence of glucosyl residues linked to 5-hydroxymethylcytosine within the large groove. The binding of AM to DNA saturated with Nt or with the analogue of distamycin A (DM2) containing the 2 N-methylpyrrole residues has been investigated by spectrophotometry. The apparent number of binding sites for AM in these 2 complexes is about half as much as observed for free DNA while the saturation level of the binding decreased only by about 20%. This proves simultaneous presence of AM and Nt (DM2) within the narrow groove of DNA.     

The temperature hysteresis of water-salt metabolism in the frog Rana temporaria]

Frogs acclimated to 4 degrees C were transported to a medium with temperature 20 degrees C, which caused polyuria; recovery of normal diuresis took about 24 h. During this period, hypernatremia was observed together with the increase in natriuresis, the rate of renal excretion of potassium ions with urea remaining constant. Water content of skeletal muscles decreased. Transportation of frogs acclimated to 20 degrees C into a medium with a temperature 4 degrees C decreased their diuresis. Renal excretion of sodium, calcium and magnesium ions remained unchanged, whereas that of potassium ions significantly decreased. The content of potassium and magnesium in the blood serum increased, that of sodium--decreased. Hydratation of muscled and kidneys was accompanied by the decrease of calcium, potassium and magnesium ions calculated per wet weight of the tissues, the level of sodium remaining unaffected. The data obtained indicate significant changes in the pattern of water and salt metabolism in frogs during temperature hysteresis.     

Structural investigations of influenza B virus

Purified preparations of influenza B/Hong Kong/5/72 have been characterized by hydrodynamical measurements, electron microscopy and small-angle neutron scattering. As judged by these techniques the preparations are highly monodisperse, the virus particles being spherical and of molecular weight about 200 × 10⁶. The lipid bilayer is located at a radius of 425 Å and its molecular weight is estimated to be 60 × 10⁶, constituting about 30% of the total virus mass. The external radius is about 580 Å.     

Structural organization of mRNA complexes with major core mRNP protein YB-1

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA–YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600–700 nt mRNA segment on its surface.     

Reassembled plasma low density lipoproteins. Phospholipid-cholesterol ester-apoprotein B complexes

Reassembled low density lipoprotein (LDL) complexes have been prepared by the interaction of lipid-free sodium deoxycholate-solubilized apoprotein B (apoB) of native human LDL with preformed, 200 A in diameter, microemulsions of cholesteryl oleate (CO), surface-stabilized by either egg yolk phosphatidylcholine ( EYPC ) or dimyristoyl phosphatidylcholine (DMPC). Gel chromatography of PC/CO/apoB complexes shows co-elution of the complex at 43% PC, 43% CO, and 14% apoB. Negative stain electron microscopy shows the particles to be circular, homogeneous, and approximately 200 A in diameter. PC/CO/apoB complexes exhibit beta-migration on agarose gels and show one high molecular weight protein band on 3.0% sodium dodecyl sulfate-polyacrylamide gels. Differential scanning calorimetry and x-ray scattering show the lipids in the complexes to undergo at least two specific thermal transitions depending on lipid composition, one associated with the core-located cholesterol esters similar to LDL and the protein-free microemulsions and the other from the phospholipid forming the surface monolayer. In addition, particle disruption-protein unfolding/denaturation occur irreversibly at 80-85 degrees C. At 4 degrees C, the secondary structure of apoB on complexes of EYPC /CO/apoB is similar to that of native LDL. For complexes of DMPC/CO/apoB, the secondary structure shows less alpha-helix which correlates with the difference in surface lipid environment. The reassembled complexes of PC/CO/apoB provide a defined system in which the components may be varied systematically in order to study the molecular organization, molecular interactions, and metabolism of LDL.     

Semliki Forest virus membrane proteins, preparation and characterization of spike complexes soluble in detergent-free medium

After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95 · 10⁴ and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as “rosette”-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.     

The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration

Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.     

Isolation and localization of phosphoprotein pp 135 in the nucleoli of various cell lines

Phosphoprotein pp 135 is one of the dominant proteins endogenously phosphorylated in cellular sonicates during short-time exposure to [gamma-32P]ATP. Mouse cells growing exponentially show the highest pp 135 level as determined by endogenous phosphorylation and immunobinding assays. Disruption of cells in the absence of calcium at low magnesium concentration renders more than 90% pp 135 into the cytosolic fraction. A five-step purification yields greater than 95% pure pp 135. The cellular location of pp 135 was determined with a rat anti-(mouse pp 135) serum by immunofluorescence in mouse cell lines and cryostat sections of normal mouse tissue. We observed fluorescence predominantly of nucleolar structures, confirmed by studies of isolated nuclei and nucleoli. Cross-reacting nucleolar phosphoproteins were identified in cell lines of other species with molecular masses of 128 kDa (human), 135 kDa (hamster) and 118 kDa (Drosophila). Endogenous phosphorylation of pp 135 investigated with purified mouse nucleoli showed optimal activity at isotonicity, pH 7.3, in the presence of 10 mM magnesium ions.     

Determining molecular weight distributions of antigen-antibody complexes by quasi-elastic light scattering.

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.     

Extended sensitivity for the calcium selective electrode

Sensitivity of calcium-selective electrodes heretofore has been limited to calcium concentrations above 10(-8) M in the absence of competing ions. We describe the use of calcium buffers to stabilize the free calcium in the reference electrode. Electrode calibration is linear to 10(-8) M and is curvilinear to 10(-11) M in the presence of 0.1 M ionic strength. Selectivity with respect to competing cations, magnesium, potassium, sodium, and hydrogen is preserved. Electrode response time is less than 2 s for small changes in calcium activity. Response range is linear over 9 log units of calcium activity. Potential-time stability is less than 10 mV/h at saturation currents. Although the silver-silver chloride terminals are photosensitive throughout the visible and near-ultraviolet regions, housing the reference and indifferent in opaque barrels avoids false photovoltaic response.