A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'.
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We have constructed a plasmid-borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: tau, gamma, delta, delta', chi and psi. Induction of this operon followed by assembly in vivo produced two taugamma mixed DnaX complexes with stoichiometries of tau(1)gamma(2)deltadelta'chipsi and tau(2)gamma(1)deltadelta'chipsi rather than the expected gamma(2)tau(2)deltadelta'chipsi. We observed the same heterogeneity when taugamma mixed DnaX complexes were reconstituted in vitro. Re-examination of homomeric DnaX tau and gamma complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX(3)deltadelta'chipsi. Equilibrium sedimentation analysis showed that free DnaX is a tetramer in equilibrium with a free monomer. An assembly mechanism, in which the association of heterologous subunits with a homomeric complex alters the stoichiometry of the homomeric assembly, is without precedent. The significance of our findings to the architecture of the holoenzyme and the clamp-assembly apparatus of all other organisms is discussed. (+info)
Nucleotide effects on liver and muscle mitochondrial non-phosphorylating respiration and membrane potential.
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Uncoupling protein-1 homologs are hypothesized to mediate mitochondrial proton leak. To test this hypothesis, we determined the effects of ATP and other nucleotides on liver and skeletal muscle mitochondrial non-phosphorylating respiration (VO(2)), membrane potential, FCCP-stimulated respiratory control ratios, and swelling. Neither ATP nor CTP affected liver or muscle proton leak, but both inhibited the respiratory chain. Unexpectedly, CMP stimulated liver proton leak (EC(50) approximately 4.4+/-0.5 mM). Using CMP chromatography, we identified two proteins (M(r)=31.2 and 32.6 kDa) from liver mitochondria that are similar in size to members of the mitochondrial carrier protein family. We conclude (a) liver and muscle mitochondrial proton leak is insensitive to ATP and CTP, and (b) CMP activates a leak in liver mitochondria. The CMP-inducible leak may be mediated by a 30-32 kDa protein. Based on the high concentrations required, CMP is unlikely to be a physiologically important leak regulator. Nonetheless, our results show that tissues other than brown fat have inducible leaks that may be protein-mediated. (+info)
Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation.
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The transferrin receptor (TfR) of reticulocytes is released in vesicular form (exosomes) during their maturation to erythrocytes. The heat shock cognate 70-kDa protein (Hsc70) has been demonstrated to interact with the cytosolic domain of the TfR and could thus trigger the receptor toward this secretion pathway. We investigated the characteristics of the interaction between Hsc70 and the TfR in exosomes with an in vitro binding assay using TfR immobilized on Sepharose beads and purified Hsc70. The results show that Hsc70 binds to exosomal TfR with characteristics expected of a chaperone/peptide interaction. We demonstrated that heat-denatured luciferase competed for in vitro binding, dependent on the nucleotide bound to Hsc70, and that this interaction activates the ATPase activity of Hsc70. Moreover, we used immunosuppressive agents that interact with Hsc70, thus decreasing Hsc70 binding to TfR in our in vitro binding assay and enabling us to assess the role of this interaction in vivo during reticulocyte maturation. (+info)
Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain.
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Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser/Thr residues is ubiquitous in higher eukaryotes and is analogous to protein phosphorylation. The enzyme for the addition of this modification, O-GlcNAc transferase, has been cloned from several species. Here, we have cloned a human brain O-GlcNAcase that cleaves O-GlcNAc off proteins. The cloned cDNA encodes a polypeptide of 916 amino acids with a predicted molecular mass of 103 kDa and a pI value of 4.63, but the protein migrates as a 130-kDa band on SDS-polyacrylamide gel electrophoresis. The cloned O-GlcNAcase has a pH optimum of 5.5-7.0 and is inhibited by GlcNAc but not by GalNAc. p-Nitrophenyl (pNP)-beta-GlcNAc, but not pNP-beta-GalNAc or pNP-alpha-GlcNAc, is a substrate. The cloned enzyme cleaves GlcNAc, but not GalNAc, from glycopeptides. Cell fractionation suggests that the overexpressed protein is mostly localized in the cytoplasm. It therefore has all the expected characteristics of O-GlcNAcase and is distinct from lysosomal hexosaminidases. Northern blots show that the transcript is expressed in every human tissue examined but is the highest in the brain, placenta, and pancreas. An understanding of O-GlcNAc dynamics and O-GlcNAcase may be key to elucidating the relationships between O-phosphate and O-GlcNAc and to the understanding of the molecular mechanisms of diseases such as diabetes, cancer, and neurodegeneration. (+info)
On the molecular weights of the three nonidentical subunits of citrate lyase from Klebsiella aerogenes.
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The molecular weights of the three nonidentical subunits of citrate lyase of Klebsiella aerogenes have been determined by three methods: sedimentation equilibrium in 6 M guanidinium chloride, sodium dodecyl sulfate gel electrophoresis, and gel filtration on 6 percent agarose column in 6 M guanidinium chloride. The molecular weights of the subunits, names I, II, and III (or acyl carrier protein) in order of elution from the agarose column, were 54,500, 32,000, and 11,000, respectively. The agarose-guanidine column provided a nearly complete separation of the three subunits. The molecular weight of the native enzyme was found by sedimentation equilibrium to be 520,000 plus or minus 10,000. The uncertainties in the molecular weights of the enzyme and its subunits did not permit a valid postulation of the subunit composition. (+info)
Cryptococcus laurentii cell envelope glycoprotein. Evidence for separate oligosaccharide side chains of different composition and structure.
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Particulate enzyme preparations of the fungus imperfectus Cryptococcus laurentii catalyze transfer of mannosyl and galactosyl residues from GDP-[14C]mannose and UDP-[3H]-galactose to the same endogenous acceptor. After solubilization with pronase, the major portion of both labels is retarded on Sepharose columns and forms a symmetrical peak, in which 14C and 3H coincide. Label also coincides with endogenous protein and carbohydrate. Both labels bind to Sepharose-Concanavalin A (Con A) and are eluted with alpha-methylglucoside. After beta elimination with NaOH-NaBH4 only 14C label retains binding to Sepharose-Con A; 3H label representing (6-O-alpha-galactosyl)10-O-beta-galactosyl-O-mannitol as previously reported (Raizada, M. K., Kloepfer, H. G., Schutzbach, J. S., and Ankel, H. (1974) J. Biol. Chem. 249, 6080-6086) no longer binds. The [14C]mannose-containing material after beta elimination yields a pentasaccharide and a trisaccharide. Similar penta- and trisaccharides can be isolated following beta elimination of particulate preparations of the organism after pronase treatment. Analytical data suggest that the structure of the isolated pentasaccharides corresponds to that of a pentasaccharide previously synthesized de novo using cell-free enzyme preparations of the organism: 2-O-alpha-mannosyl-6-O-alpha-mannosyl-3-O-alpha-mannosyl-(2-O-beta-xylosyl)-O-man nose (Schutzbach, J. S., Raizada, M. K., and Ankel, H. (1974) J. Biol. Chem. 249, 2953-2958). The trisaccharide has the structure 2-O-alpha-mannosyl-2-O-alpha-mannosyl-O-mannitol. The data are consistent with a glycoprotein structure in which these three types of oligosaccharides are bound to a common polypeptide core through O-glycosidic linkages to threonyl and seryl residues. (+info)
SH2-B and APS are multimeric adapters that augment TrkA signaling.
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Neurotrophins influence growth and survival of sympathetic and sensory neurons through activation of their receptors, Trk receptor tyrosine kinases. Previously, we identified Src homology 2-B (SH2-B) and APS, which are structurally similar adapter proteins, as substrates of Trk kinases. In the present study, we demonstrate that both SH2-B and APS exist in cells as homopentamers and/or heteropentamers, independent of Trk receptor activation. Structure-function analyses revealed that the SH2-B multimerization domain resides within its amino terminus, which is necessary for SH2-B-mediated nerve growth factor (NGF) signaling. Overexpression of SH2-B enhances both the magnitude and duration of TrkA autophosphorylation following exposure of PC12 cells to NGF, and this effect requires the amino-terminal multimerization motif. Moreover, the amino terminus of SH2-B is necessary for TrkA/SH2-B-mediated morphological differentiation of PC12 cells. Together, these results indicate that the multimeric adapters SH2-B and APS influence neurotrophin signaling through direct modulation of Trk receptor autophosphorylation. (+info)
Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells.
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In the mechanically active environment of the artery, cells sense mechanical stimuli and regulate extracellular matrix structure. In this study, we explored the changes in synthesis of proteoglycans by vascular smooth muscle cells in response to precisely controlled mechanical strains. Strain increased mRNA for versican (3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels. Strain also increased versican, biglycan, and perlecan core proteins, with a concomitant decrease in decorin core protein. Deformation did not alter the hydrodynamic size of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycans and heparan sulfate proteoglycans (p < 0.05 for both). Using DNA microarrays, we also identified the gene for the hyaluronan-linking protein TSG6 as mechanically induced in smooth muscle cells. Northern analysis confirmed a 4.0-fold increase in steady state mRNA for TSG6 following deformation. Size exclusion chromatography under associative conditions showed that versican-hyaluronan aggregation was enhanced following deformation. These data demonstrate that mechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, indicating a highly coordinated extracellular matrix response to biomechanical stimulation. (+info)