The binding of human lactoferrin to mouse peritoneal cells.
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Human iron-saturated Lf (FeLf), which was labeled with 125I or 50Fe, was found to combine with the membrane of mouse peritoneal cells (MPC) which consisted of 70% macrophages. The following experimental data suggested the involvement of a specific receptor. (a) The binding of FeLf to MPC reached a saturation point. (b) The binding of radioactive FeLf was inhibited by preincubating the cells with cold FeLf but not with human Tf, human aggregated and nonaggregated IgG, or beef heart cytochrome c (c) Succinylation and carbamylation of FeLf resulted in a loss of its inhibiting activity on the binding of radioactive FeLf. Removal of neuraminic acid from FeLf increased its inhibitory activity. (d) The ability of apoLf to inhibit the binding of FeLf to MPC was significantly lower than that of FeLf. The existence of a Lf receptor capable of concentrating Lf released from neutrophils on the membrane of macrophages could explain the apparent blockade of the release of iron from the reticuloendothelial system, which accounts for the hyposideremia of inflammation. A receptor for FeLf was also found on mouse peritoneal lymphocytes. The affinity constant of FeLf for both lymphocytes and macrophages was 0.9 X 12(6) liter/mol. Howerver, macrophages bound three times more FeLf molecules (20 X 10(6)) per cell than did lymphocytes (7 X 10(6)). (+info)
Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor.
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The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels. (+info)
Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. A study of structure and function.
(19/32454)
Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented. To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site. More specifically, beta-Ala348 succeeds betaB. The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds. beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase. (+info)
Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells.
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This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-lactalbumin showed that the active fraction contains oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-lactalbumin. (+info)
The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase.
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The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 A. Arginine and asparagine could provide only one hydrogen bond of 2. 52 and 3.02 A, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 +/- 7. 0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 +/- 8.0 and 129 +/- 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 +/- 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 +/- 71. 2 and 2960 +/- 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal. (+info)
Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners.
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P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4 presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP. (+info)
Structure-function analysis of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold.
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Mucin-type O-glycosylation is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases). Based on sequence relationships with divergent proteins, the ppGaNTases can be subdivided into three putative domains: each putative domain contains a characteristic sequence motif. The 112-amino acid glycosyltransferase 1 (GT1) motif represents the first half of the catalytic unit and contains a short aspartate-any residue-histidine (DXH) or aspartate-any residue-aspartate (DXD)-like sequence. Secondary structure predictions and structural threading suggest that the GT1 motif forms a 5-stranded parallel beta-sheet flanked by 4 alpha-helices, which resembles the first domain of the lactose repressor. Four invariant carboxylates and a histidine residue are predicted to lie at the C-terminal end of three beta-strands and line the active site cleft. Site-directed mutagenesis of murine ppGaNTase-T1 reveals that conservative mutations at these 5 positions result in products with no detectable enzyme activity (D156Q, D209N, and H211D) or <1% activity (E127Q and E213Q). The second half of the catalytic unit contains a DXXXXXWGGENXE motif (positions 310-322) which is also found in beta1,4-galactosyltransferases (termed the Gal/GalNAc-T motif). Mutants of carboxylates within this motif express either no detectable activity, 1% or 2% activity (E319Q, E322Q, and D310N, respectively). Mutagenesis of highly conserved (but not invariant) carboxylates produces only modest alterations in enzyme activity. Mutations in the C-terminal 128-amino acid ricin-like lectin motif do not alter the enzyme's catalytic properties. (+info)
Quantitative structure-activity relationships for nasal pungency thresholds of volatile organic compounds.
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A model was developed for describing the triggering of nasal pungency in humans, based on the partition of volatile organic compounds (VOCs) between the air phase and the biophase. Two partition parameters are used in the model: the water-air partition coefficient and the octanol-water partition coefficient. The model was validated using data from the literature, principally on alcohols, acetates and ketones. The model suggests that all test compounds, regardless of their chemical functional groups, bind to a common receptor site within the hydrophobic interior of the bilayer membrane of the trigeminal nerve endings. There is probably only a slight, non-specific interaction between the VOC molecule and the receptor molecule, whereas this type of non-specific interaction for the detection of odor is much stronger. In practical terms, the suggestion that all VOCs share a common irritation receptor site implies that nasal-pungency thresholds of individual VOCs may be additive. Quantitative structure-activity relationships (QSARs) for nasal-pungency thresholds were also developed from the model, which can be used to predict nasal-pungency thresholds of common VOCs. Although the present model does not offer additional precision over that of M.H. Abraham et al., 1996, Fundam. Appl. Toxicol. 31, 71-76, it requires fewer descriptors and offers a physiological basis to the QSAR. Another advantage of the present model is that it also provides a basis for comparison between the olfactory process and nasal pungency. (+info)