Complete Denaturation Research Articles

Complete Unfolding of the Titin Molecule under External Force

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch–release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.

γ-Radiation Effects on the Thermal Stability of Bovine γ-Globulin

Low-dose γ-radiation produces subtle effects on bovine γ-globulin, as revealed by calorimetric investigations carried out on concentrated aqueous solutions. The irradiation rate used seems below the threshold that allows complete protein denaturation; the denaturation temperature, Td, remained unaffected; nonetheless, modifications of the shape of the calorimetric signal reveal that other changes, such as the dissociation of protein oligomers, could take place.

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.

Basic thermoanalytical studies of insoluble collagen matrices

Insoluble collagen has been utilized as a base material for parenteral drug carrier systems. Information on its physicochemical properties was obtained by focussing on thermoanalytical methods. On the way from the raw material to the matrices, the acidic aqueous dispersion represents an important intermediate state. DSC and FTIR revealed its complete denaturation at 43 °C. Dense homogeneous collagen matrices were prepared by air-drying at 25 °C and became denatured at 103.5 °C, far above normal storage temperatures. Dielectrical Thermal Analysis demonstrated transitions in the dielectrical storage and loss moduli, reflecting the dissipation of electrical energy and increased molecular mobility caused by collapse of the triple helical structure. Cross-linking of the collagen dispersion with glutaraldehyde induced no alteration in the thermoanalytical properties of dry matrices. However, in the swollen state, after incubation of the devices in phosphate buffer the transition temperature increased from 50 to 70 °C as cross-linking was intensified. This indicated stronger interactions between the collagen fibre structures. Dissolution tests with cytochrome c-loaded matrices showed that higher amounts of the model protein were trapped inside the matrices as more glutaraldehyde was added.

Molecular basis for amyloidosis related to hereditary brain hemorrhage

The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.

Kinetically controlled folding of the serpin plasminogen activator inhibitor 1.

The serpin plasminogen activator inhibitor 1 (PAI-1) folds into an active structure and then converts slowly to a more stable, but low-activity, "latent" conformation [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. Thus, the folding of PAI-1 is apparently under kinetic control. We have determined the urea denaturation and refolding transitions of both latent and active PAI-1 proteins by using intrinsic tryptophan fluorescence. While folding of active PAI-1 is reversible, the denaturation and refolding of latent PAI-1 are not. Instead, denatured latent PAI-1 refolds in lower concentrations of urea to give the active protein. Thus, the high-stability latent conformation is kinetically inaccessible over a range of urea concentrations. Complete denaturation of latent PAI-1 occurs at 5.5 M urea [delta G(H2O) approximately 21 kcal] whereas active PAI-1 denatures in only 3.8 M urea [delta G(H2O) approximately 12 kcal]. The fluorescence emission profile, as a function of urea of both the active and latent forms of the protein, reveals intermediates with partial structure. Circular dichroism measurements and limited protease digestion with Lys-C suggest that the intermediate in the denaturation of latent PAI-1 retains most of the secondary structure of the fully folded protein, whereas the intermediate in the denaturation of active PAI-1 exhibits significant loss of secondary structure. The Lys-C digestion patterns show that the active protein is more susceptible to proteolysis near sheet A than is the latent form. The studies suggest a model for the kinetically controlled folding pathway of PAI-1.

CHARACTERISATION OF A COMMERCIAL SOY ISOLATE BY PHYSICAL TECHNIQUES

ABSTRACTThe mechanical features of a partially denatured commercial soy isolate (PP 500E) have been explored for comparison with data available on the gelation characteristics of native globular proteins and to improve the understanding of its textural properties as a structuring ingredient in the production of low fat products. The soy sample was reconstituted at 30C and networks were developed either during cooling to 5C or on heating to 90C (complete denaturation of the protein) followed by cooling to 5C. Throughout the course of experimentation, dynamic oscillatory (time, temperature, frequency and strain sweeps) and creep testing (in aqueous or urea solutions) measurements were recorded. Reduction in the thermal energy of the system causes a monotonic increase in storage modulus (G′) whereas the temperature rise results in equilibrium G′ values well below the elastic response observed at 30C. The absence of a positive thermal transition, observed in the gelation of native globular proteins, indicates a different mechanism for structure formation in commercial soy isolates. Application of the cascade treatment to the concentration dependence of the storage modulus argues that the heated and cooled networks possess a higher degree of bond permanency than their cooled‐only counterparts. Mechanical spectra in combination with the pattern of network breakage at high deformations suggests that disulphide bonds participate in the network formed by totally denatured soy protein (heated and cooled samples). Inclusion of urea in the aqueous preparations destabilises the predominantly physical forces of attraction in the unheated gels. By contrast, the heated and cooled samples achieve an equilibrium deformation whose storage modulus can be employed in the constitutive equation of rubber elasticity theory. On that basis the number of disulphide bridges per molecule was found to vary between 2.0 and 2.03. This result is consistent with the “string of beads” model proposed for the three‐dimensional structure of globular protein gels, where a dendric network is built by the occasional cross‐linking of corpuscular strands.

Chemical denaturation of ovalbumin abrogates the induction of oral tolerance of specific IgG antibody and DTH responses in mice.

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA, for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.

The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin

The outer membranes of invasive spirochetes contain unusually small amounts of transmembrane proteins. Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has a deduced amino acid sequence predictive of multiple transmembrane beta-strands. Studies were conducted to characterize the structure and function of this protein. Alkali, high-salt, and urea fractionation of leptospiral membranes demonstrated that OmpL1 is an integral membrane protein. The electrophoretic mobility of monomeric OmpL1 was modifiable by heat and reduction; complete denaturation of OmpL1 required prolonged boiling in sodium dodecyl sulfate (SDS), 8 M urea, and 2-mercaptoethanol. When solubilized in SDS at low temperature, a small proportion of OmpL1 exhibited an apparent molecular mass of approximately 90 kDa, indicating the existence of an SDS-unstable oligomer. OmpL1 dimers and trimers were demonstrated by nearest neighbor chemical cross-linking. In order to generate purified protein for functional studies, the ompL1 gene was ligated into the pMMB66 expression plasmid under control of the tac promoter. Although expression in Escherichia coli was toxic, most of the OmpL1 produced was found in the outer membrane, as determined by subcellular fractionation. Purified recombinant OmpL1 was reconstituted into planar lipid bilayers, demonstrating an average single channel conductance of 1.1 nS, similar to the major porin activity of native leptospiral membranes. These findings indicate that OmpL1 spans the leptospiral outer membrane and functions as a porin.

Atypical Porcine Type I Interferon. Biochemical and Biological Characterization of the Recombinant Protein Expressed in Insect Cells

A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefevre, F. & Boulay, V. C. (1993) J. Biol. Chem. 268, 19760–19768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7×107IU/mg, is an N-glycosylated monomer of 19kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1 % SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-α. A major difference with porcine type 1 interferon-α was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-α. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.

Atypical porcine type I interferon. Biochemical and biological characterization of the recombinant protein expressed in insect cells.

A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.

Topology of the Na,K-ATPase: EVIDENCE FOR EXTERNALIZATION OF A LABILE TRANSMEMBRANE STRUCTURE DURING HEATING

The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.

Production and characterization of monoclonal antibodies against Panulirus interruptus hemocyanin.

Since the primary and higher-order structures of hemocyanin from the crustacean arthropod Panulirus interruptus have been elucidated completely, it should be possible to determine which regions of this immunogenic molecule are recognized most often by antibodies. Monoclonal antibodies were raised against subunits a and b of this hemocyanin, and fourteen of them were further characterized. The produced antibodies were of class IgG, subclasses 1 or 2a. Most of them had dissociation constants on the order of magnitude 10(-8)-10(-10), a few had lower affinities. Most clones showed no or negligible cross-reactivity with other crustacean hemocyanins. The reactivity of most other clones diminished with increasing sequence difference between the investigated hemocyanins. However, in a few instances a stronger reactivity with other hemocyanins was observed than with that from Panulirus interruptus. After complete denaturation of the hemocyanin there was no reaction with the monoclonal antibodies, indicating that the latter recognize conformational epitopes. Only one monoclonal antibody reacted with denatured hemocyanin. This antibody was also the only one which reacted with a CNBr digest, which means that it recognizes a sequential epitope. Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure. Limited proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment representing domains 2 and 3. It was determined on Western blots of the digest on which fragment epitopes for eleven of the monoclonal antibodies were located.

Tetrabrachion: A Filamentous Archaebacterial Surface Protein Assembly of Unusual Structure and Extreme Stability

The surface (S-) layer of the hyperthermophilic archaebacterium Staphylothermus marinuswas isolated, dissected into separate domains cy chemical and proteolytic methods, and analyzed by spectroscopic, electron microscopic and biochemical techniques. The S-layer is formed by a poorly ordered meshwork of branched, filiform morphological subunits resembling dandelion seed-heads. A morphological subunit (christened by us tetrabrachion) consists of a 70 nm long, almost perfectly straight stalk ending in four straight arms of 24 nm length that provide lateral connectivity by end-to-end contacts. At 32 nm from the branching point, tetrabrachion carries two globular particles of 10 nm diameter that have both tryptic and chymotryptic protease activity. Tetrabrachion is built by a tetramer of M r92,000 polypeptides that form a parallel, four-stranded α-helical rod and separate at one end into four strands. These strands interact in a 1:1 stoichiometry with polypeptides of M r85,000 to form the arms. The arms are composed entirely of β-sheets. All S-layer components contain bound carbohydrates (glucose, mannose, and glucosamine) at a ratio of 38 g/100 g protein for the complete tetrabrachion-protease complex. The unique structure of tetrabrachion is reflected in an extreme thermal stability in the presence of strong denaturants (1% (w/v) SDS of 6M guanidine): the arms, which are stabilized by intramolecular disulphide bridge, melt around 115°C under non-reducing conditions, whereas the stalk sustains heating up to about 130°C. Complete denaturation of the stalk domain requires treatment with 70% (v/v) sulfuric acid or with fumin trifluoromethanesulfonic acid. The globular protease can be heated to 90°C in 6M guanidine and to 120°C in 1% SDS and represents one of the most stable proteases characterized to date.

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the proteins, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.

Conformational studies on δ-crystallin, the core protein of the bird eye lens

Proteins that perform other functions elsewhere appear to be recruited for structural purposes in the eye lens. The lens being a tissue with very little metabolic activity and little or no turnover, the lens proteins, crystallins, are long lived. In an effort to understand whether their recruitment might be related to their conformation and structural stability, we have examined these features of the avian lens protein δ-crystallin. The native molecule is a tetramer (molecular mass 200 kDa) that is highly α-helical in conformation, and with an unusually blue tryptophan fluorescence (315,325 nm), which is only partially quenched by conventional quenchers. We show that the fluorescence doublet arises due to Trp residues that are effectively buried inside the rigid hydrophobic core of the tetrameric aggregate. The protein is heat stable up to 91°C. Guanidinium chloride (GuHCl) effects the complete denaturation of δ-crystallin, whereas heat or urea treatment results in only partial unfolding or dissociation. The initial transition is the disruption of the quaternary structure by perturbing the intersubunit interactions, leading to exposure of hydrophobic contact surfaces (as monitored by extrinsic probe fluorescence). This initial transition is seen upon heating to 60°C as well as in 1 M GuHCl and 4 M urea. We show that in 2.2 M GuHCl the molecule is swollen but is still largely helical with the Trp residues being present in a somewhat more polar environment than in the native molecule. Beyond 4 M GuHCl there is a gradual unfolding of the molecule, which is complete in 6 M GuHCl. This structural robustness of δ-crystallin might be important in its recruitment as the core protein of the avian lens.

THERMAL STABILITY OF A NEUTRAL PROTEASE OF ASPERGILLUS ORYZAE

The thermal stability of the neutral protease of Aspergillus oryzae was measured by differential scanning calorimetry. The activation energy, preexponential factor and the reaction rate constant calculated by means of the dynamic method are similar to those obtained for denaturation of other proteins. Half life (t 1/2 ) calculated by using the above-mentioned parameters permitted estimation of the amount of native enzyme remaining after different thermal treatments. Complete denaturation occurred above 60C. The presence of substrate stabilizes the enzyme. The behavior of flour samples treated with the neutral protease was studied as well. A tendency to shift towards higher temperatures when flour was treated with the enzyme was observed for both the T p (DSC peak maximum temperature) corresponding to gelatinization of starch and the dissociation of the amylose-lipid complex

Thermal stability of dried photosynthetic membrane film for photoelectrodes

Thermal stability was examined on a photoelectrode made from dried film of a photosynthetic membrane (chromatophore) isolated from a photosynthetic bacterium Rhodopseudomonas viridis. Below 60°C, neither the optical absorbance of the film nor its photoelectric response were affected when heated for 1 h. When the temperature was raised over 120 °C, both the absorbance and the response were lost. The temperature of half-denaturation of the chromatophore was 90 °C. X-ray diffraction of the film showed that heating did not cause the complete denaturation of the proteins of chromatophores. Heating did not destroy the secondary structure ( α-helix) of the proteins but the bundles of α-helices were distorted.

Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices.

Macrophage scavenger receptors exhibit unusually broad, but circumscribed, polyanionic ligand-binding specificity. For example, the polyribonucleotides poly(I) and poly(G) are ligands but poly(A) and poly(C) are not. To further investigate the molecular basis of this polynucleotide-binding specificity, we tested the capacity of various oligodeoxyribonucleic acids to inhibit the scavenger receptor-mediated degradation of 125I-labeled acetylated low density lipoprotein by Chinese hamster ovary cells expressing the type I bovine scavenger receptor. A series of short oligodeoxyriboguanines (dGn, where 5 < or = n < or = 37) were effective inhibitors. The dG6, dG12, and dA5G37 members of this series were shown by circular dichroism and UV spectroscopy to be assembled into four-stranded helices stabilized by G-quartets. [32P]dA5G37 bound directly to scavenger receptors. Partial or complete denaturation of the quadruplex structures of these oligonucleotides by boiling destroyed their inhibitory activity. Receptor activity was also inhibited by d(T4G4)4, a telomere-like oligonucleotide which forms an intramolecular quadruplex. In addition, conversion of the four-stranded potassium salt of poly(I) to the single-stranded lithium salt dramatically reduced its inhibitory activity. Addition of KCl to the Li+ salt resulted in the reformation of poly(I)'s quadruplex structure and restoration of its inhibitory activity. A variety of single-stranded and double-stranded oligo- and polydeoxyribonucleotides (e.g. dA37, HaeIII restriction fragments of phi X174) exhibited very little or no inhibitory activity. Thus, a base-quartet-stabilized four-stranded helix appears to be a necessary structural determinant for polynucleotide binding to and inhibition of scavenger receptors. This conformational requirement accounts for the previously unexplained polyribonucleotide-binding specificity of scavenger receptors. The spatial distribution of the negatively charged phosphates in polynucleotide quadruplexes may form a charged surface which is complementary to the positively charged surface of the collagenous ligand-binding domain of the scavenger receptor.

Diffusion of Sugar in Microwave Denatured Sugar Beet Tissues

Microwave-enhanced sugar beet denaturation was investigated in terms of the effective diffusion coefficient of sugar in sugar beet tissues (De), the extraction coefficient (k), and the inhibition coefficient (q) of sugar beet tissues for sugar beet disks denatured by microwave heating at the energy intensities of 216, 390, 540, 768, and 1170 kJ/kg, and by such chemicals as chloroform or carbon tetrachloride saturated at 20 C, respectively. A one-dimensional diffusion method was followed in this study. The De, k, and q values were from 8.62 1012 to 1.72 1010 m2/s, 5.67 104 to 1.13 102 1/min, and 0.02 to 0.40, respectively, for microwave energy intensities ranging from 216 to 1170 kJ/kg. The effect of microwaves on beet denaturation at the intensity of 540 kJ/kg was statistically equivalent to scalding at 75 C for 30 min or to the 8-h treatment with chloroform or carbon tetrachloride saturated in 15% sugar solution at 20 C. The diffusion coefficient of sugar in beet tissues increased only 30% at the maximum for microwave energy intensity increasing from 540 to 1170 kJ/kg. When intensity was below 540 kJ/kg, the sugar beet tissues were incompletely denatured. Thus, the microwave intensity around 540 kJ/kg could be regarded as the minimum dosage for complete denaturation.