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At least 55 records · Page 3Linked to original sources
PubMed1986

Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms.

When outer-row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S alpha- and beta-heavy chains and the 83,000-dalton and 70,000-dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the alpha-chain and a 16,000-dalton light chain and between the two intermediate chains. Additional close associations of the beta-heavy chain with an 18,000-dalton light chain and 70,000-dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.

Adenosine Triphosphatases↗
PubMed2001

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.

Binding Sites↗
PubMedDate not supplied

[Low-reactive free radicals inhibiting nonbranched chain processes of addition].

The kinetics of inhibition of nonbranched chain processes of addition of reactive free radicals at double bonds O=O, C=O, and C=C of molecules in liquid homogeneous binar systems from saturated and unsaturated components by low-reactive free radicals (o-CH3C6H4CH2O*4, HO*4; HC=O; CH2=C(CH3)CH2, and CH2=CHCHOH) was studied. It was assumed that as the concentration of the unsaturated component (as a source of low-reactive free radicals) increases (after the optimum concentration is attained, which corresponds to the maximum rate of the process) the phenomena of increasing inhibition of these processes may act as the elements of self-regulation of similar processes in nature, which returns them into the stationary state. The energetics of the key radical-molecular gas phase reactions was analyzed. The thermal effects of total dissociation reactions of simple alkylperoxyl (exo effect) and alkoxyl (endo effect) reactive free radicals in the gas phase were compared. The kinetic equations (with one to three parameters being directly determined), obtained by the quasi-steady-state treatment, are presented, which make it possible to describe nonmonotonic (with a maximum) dependencies of the rates of formation of molecular 1:1-adducts on the concentration of the unsaturated component.

Free Radicals↗
PubMed1990

A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity.

A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.

Amino Acid Sequence↗
PubMed2002

Coupling of the antitumoral enzyme bovine seminal ribonuclease to polyethylene glycol chains increases its systemic efficacy in mice.

Bovine seminal ribonuclease (BS-RNase) is an antitumoral active enzyme exhibiting specific antitumoral action against a number of different cancer cell lines. However, its systemic use is limited by its pharmacokinetic properties and antigenicity. Therefore, it was conjugated to polyethylene glycol (PEG) chains to overcome these problems. Measurement of aspermatogenic effects of the preparation after s.c. injection and injection into the scrotum was chosen as a model for the distribution of the enzyme in the body mediated by the linkage to PEG chains. Additionally, the antigenicity of BS-RNase coupled to PEG chains (BS-RNase-PEG) was compared to that of free BS-RNase, as antigenicity is known to be one of the main obstacles in the use of protein-based drugs. BS-RNase-PEG caused aspermatogenic effects after systemic administration to mice in very low concentrations at which free BS-RNase is not effective. Moreover, BS-RNase possessed a very low antigenicity as long as it was coupled to the PEG chains. In order to investigate the antitumoral efficacy of BS-RNase-PEG in vivo, preliminary experiments on the effect of the conjugate on neuroblastoma growth in mice were performed in a UKF-NB-3 xeno-transplantate model, demonstrating a drastically increased anti-tumoral activity of the conjugate compared to the free enzyme.

Animals↗
PubMed2000

Synthesis, structure, and bonding of A5Cd2Tl11, A = Cs, Rb. Naked pentagonal antiprismatic columns centered by cadmium.

A new anionic thallium cluster chain 1 infinity[Cd2Tl11(5-)] has been discovered in the A-Cd-Tl systems for A = Cs, Rb. The compounds are synthesized by direct fusion of the elements at 700 degrees C and equilibration of the quenched product at 200 degrees C for 1 month. The thallides crystallize in the orthorhombic space group Amm2, Z = 2, a = 56107(7) and 55999(6) A, b = 18090(3) and 17603(3) A, c = 13203(3) and 12896(2) A for A = Cs and Rb, respectively, and contain chains of face-sharing pentagonal Tl10 antiprisms embedded in a matrix of alkali metal cations. Cadmium atoms occupy the center of the antiprisms and donate electrons to the anionic chain. Additional four-bonded Tl atoms on one side of the chain make the structure acentric. The compounds are diamagnetic (chi 296 = -08, -40 (x 10(-4) emu/mol, respectively) and metallic (10-20 mu omega cm at 275 K), and the indirect band gap energy of both compounds is close to zero according to extended Hückel calculations on the isolated chain.

Journal Article↗
PubMed1994

Amino acid sequence of porcine antithrombin III.

Antithrombin III (ATIII) is a member of the serine protease inhibitor (serpin) family. As a step towards a better understanding of the heparin-binding mechanism of mammalian ATIIIs, the amino acid sequence of porcine ATIII was established by sequence analysis of the peptides derived from cyanogen bromide cleavage and enzymatic digestion with lysyl endopeptidase, V8 protease, and trypsin. Porcine ATIII was found to consist of 431 amino acid residues, with a calculated molecular weight of 48,930 without carbohydrate. Its molecular weight with 16.4% carbohydrate was estimated as 56,955, which is in good agreement with the value determined by SDS-PAGE. Porcine ATIII showed high sequence similarity to other mammalian ATIIIs, including the reactive site, heparin-binding basic amino acid residues, and disulfide bonds. The most notable feature of porcine ATIII was that it possesses only three carbohydrate chains, at Asn136, 156, and 193, whereas other mammalian ATIIIs have four, additional chain being at Asn97; this is replaced by Asp in porcine ATIII. In the case of human ATIII, the chains are at Asn96, 135, 155, and 192. The heparin-binding affinities of human and porcine ATIIIs were compared using an immobilized heparin column. Porcine ATIII eluted from the column with a peak at an NaCl concentration of 924 mM while human ATIII eluted at 838 mM NaCl. Neuraminidase treatment of each ATIII enhanced the heparin-affinity to the same extent. These results suggest that in spite of the high degree of amino acid sequence identity between porcine and human ATIIIs (91% identical), porcine ATIII has a higher heparin-binding affinity than human, because it lacks a carbohydrate chain at Asp97.

Amino Acid Sequence↗
PubMed1990

IL-2 receptors on rabbit T-cell lines and their transfectants expressing the human IL-2 receptor alpha chain.

Low-affinity (dissociation constant: Kd = 7 nM) and high-affinity (Kd = 27 pM) interleukin-2 receptors (IL-2R) were detected on rabbit T-cell lines by IL-2 binding studies. Chemical cross-linking studies using 125I-labelled IL-2 showed that rabbit low-affinity IL-2R was singly expressed alpha-chain (MW 55,000) and that high-affinity IL-2R was composed of at least alpha- and beta- (MW 75,000) chains, similar to the human and murine counterparts. The existence of an additional chain (MW 25,000) was suggested in the rabbit IL-2R. Rabbit T-cell transfectant lines were established by human IL-2R alpha-chain (IL-2R alpha) cDNA transfection. These transfectant lines possessed not only extremely large numbers of human IL-2R alpha (over 10 times more than endogenous rabbit alpha-chain) but also twice as many high-affinity sites as their parental lines. The number of high-affinity sites on the transfectants significantly decreased when human alpha-chains were blocked, indicating that these transfectants expressed high-affinity receptor consisting of the exogenous human alpha-chain and rabbit beta-chain. This was confirmed by cross-linking experiments. The observation that expression of extremely large numbers of exogenous alpha-chains lead to an increase of the total number of high-affinity sites in the apparent absence of an increase of beta-chain expression raises the possibility that not only the beta-chain but also the alpha-chain may play an important role in regulating the number of high-affinity receptors.

Animals↗
PubMed1985

A case of multiple myeloma with two subclones producing separately kappa and defective gamma chains.

A case of multiple myeloma is described that contained two morphologically identical but immunologically diverse subpopulations. One of the subclones produced only kappa chains, whereas the other contained only gamma chains. Additional immunohistologic investigations revealed production by one of the subpopulations of defective gamma chains that were devoid of demonstrable Fd parts. It could not be decided whether this was due to a structural or an antigenetical defect. Furthermore, only the kappa-producing subclone exhibited J-chains. Based upon these findings and their possible cause and on respective findings concerning other B-cell neoplasias, it is concluded that the consequent application of an ever-increasing number of different immunologic markers, in order to better classify malignant lymphomas, will eventually result in the reverse, namely, in a complete breakup of this group of malignancies into individual cases.

Humans↗
PubMed1986

The HLA-D-associated invariant chain binds palmitic acid at the cysteine adjacent to the membrane segment.

The highly polymorphic HLA-D antigens are associated with a nonpolymorphic polypeptide chain, designated invariant chain. This invariant chain is shown to incorporate fatty acid. Invariant chain metabolically labeled with [3H]palmitic acid releases its label after treatment with hydroxylamine indicating an ester linkage of the palmitic acid. The binding of fatty acid to the invariant chain inhibits the formation of S-S-linked dimers. This suggests that the sole cysteine residue of the invariant chain is blocked by binding of fatty acid. A peptide shared by [3H]palmitic acid- or [35S]cysteine-labeled invariant chain digests supports the hypothesis that the palmitic acid binds to the cysteine which is located close to the membrane-spanning domain on the cytoplasmic site. Inhibition of N-glycosylation with tunicamycin demonstrates binding of the fatty acid to the nonglycosylated precursor of the invariant chain. Additionally, blocking of fatty acylation by cerulenin inhibits further maturation of the invariant chain, as sialylation.

Antibodies, Monoclonal↗
PubMed1999

Direct analysis of viral-specific CD8+ T cells with soluble HLA-A2/Tax11-19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy.

Human T cell lymphotropic virus-I (HTLV-I)-associated myelopathy is a slowly progressive neurologic disease characterized by inflammatory infiltrates in the central nervous system accompanied by clonal expansion of HTLV-I-reactive CD8+ T-cells. In patients carrying the HLA-A2 allele, the immune response is primarily directed to the Tax11-19 peptide. The frequency, activation state, and TCR usage of HLA-A2/Tax11-19 binding T cells in patients with HTLV-I-associated myelopathy was determined using MHC class I tetramers loaded with the Tax11-19 peptide. Circulating Tax11-19-reactive T cells were found at very high frequencies, approaching 1:10 circulating CD8+ T cells. T cells binding HLA-A2/Tax11-19 consisted of heterogeneous populations expressing different chemokine receptors and the IL-2R beta-chain but not the IL-2R alpha-chain. Additionally, Tax11-19-reactive CD8+ T cells used one predominant TCR Vbeta-chain for the recognition of the HLA-A2/Tax11-19 complex. These data provide direct evidence for high frequencies of circulating Tax11-19-reactive CD8+ T cells in patients with HTLV-I-associated myelopathy.

Adult↗
PubMed1969

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease.

1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .

Ammonium Chloride↗
PubMed1987

Role of glycosylation in secretion of yeast acid phosphatase.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.

Acid Phosphatase↗
PubMed1983

[Primary structure of hemoglobins of the musk rat (Ondatra zibethica, Rodentia)].

The amino-acid sequence for the hemoglobin of the musk-rat (Ondatra zibethica) was determined. The sequence of both chains was established by automatic Edman-Begg degradation of the tryptic peptides and, in the case of the beta-chains additionally of the prolyl-peptide. The complete primary structure of the alpha-chains differs at 22, that of the beta-chains at 36 positions from the adult human hemoglobin. There are no changes in the heme-binding residues, the alpha 1--beta 2 contact positions and the allosteric regulator sites. In the heme-pocket we found beta 44 Ser leads to His and 8 positions changed within the alpha 1--beta 1 contact regions. These changes are discussed with respect to the function of the respective regions.

Amino Acid Sequence↗
PubMed1997

Preliminary crystallographic characterization of ricin agglutinin.

The quaternary structure of ricin agglutinin (RCA) has been determined by x-ray crystallography. The refined structure of ricin proved to be a successful search model using the molecular replacement method of phase determination. RCA forms an elongated molecule of dimensions 120 A x 60 A x 40 A with two A chains at the center and a B chain at each end. The A chains are covalently associated via a disulfide bridge between Cys 156 of both chains. Additional contacts at residues 114-5 stabilize the dimer interface. The covalent association of RCAA chains was confirmed by gel filtration under reducing and nonreducing conditions.

Crystallography, X-Ray↗
PubMed1987

Interaction of Cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein.

Cibacron blue F3GA, a sulfonated polyaromatic blue dye, inhibited the ability of ricin A-chain to inactivate ribosomes. Difference-spectroscopic study revealed that the dye bound to the A-chain (Kd = 0.72 microM), producing a difference spectrum with a single maximum at 688 nm and two minima at 585 and 628 nm. Such a significant difference spectrum was not observed in the presence of ricin B-chain or intact ricin, neither of which can inactivate ribosomes. Modification of arginine residues in the A-chain with phenylglyoxal showed a correlation between the loss of inhibitory activity on protein synthesis and the loss of difference absorbance produced by the dye-A-chain interaction. Both losses occurred significantly at an early stage of the modification. Furthermore, the dye protected the A-chain against a loss of its inhibitory activity resulting from the modification of arginine residues. These results suggest that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Based on these data, the dye appears to interact with the active site of the A-chain. Addition of several polynucleotides, namely rRNA, tRNA, poly(U) and DNA, to the dye-A-chain complex resulted in a marked displacement of the dye, whereas mono- and dinucleotides had little or no effect on the dye-A-chain interaction. These findings indicate the possible existence of a polynucleotide binding site in the active site of the A-chain. A combination of these and other results suggests that the A-chain recognizes and acts on some part of RNA of the 60 S ribosomal subunit.

Animals↗
PubMed2005

Highly efficient photoinitiation in the cerium(III)-catalyzed aqueous autoxidation of sulfur(IV). An example of comprehensive evaluation of photoinduced chain reactions.

The photoinitiated and cerium(III)-catalyzed aqueous reaction between sulfite ion and oxygen has been studied in a diode-array spectrophotometer using the same light beam for excitation and detection. Cerium(III) is identified as the photoactive absorbing species, and the production of cerium(IV) initiates a radical chain reaction. To interpret all the experimental findings, a simple scheme is proposed, in which the additional chain carriers are sulfite ion radical (SO3(-.)), sulfate ion radical (SO4(-.), and peroxomonosulfate ion radical (SO5(-.). The overall rate of oxidation is proportional to the square root of the light intensity per unit volume, which is readily interpreted by the second-order termination reaction of the proposed scheme. It is also shown that the reaction proceeds for an extended period of time in the dark following illumination, and a quantitative analysis is presented for this phase as well. The postulated model predicts that cerium(III) should have a cocatalytic or synergistic effect on the autoxidation of sulfite ion in the presence of other catalysts. This prediction was confirmed in the iron(III)-sulfite ion-oxygen system. The experimental method and the mathematical treatment used might be applicable to a wide range of photoinduced chain reactions.

Journal Article↗
PubMed1992

Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits.

Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.

Amino Acid Sequence↗