High-affinity binding of the AP-1 adaptor complex to trans-golgi network membranes devoid of mannose 6-phosphate receptors.
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The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPgammaS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1.GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF.GTP hydrolysis. (+info)
OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication.
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Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of approximately 92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication. (+info)
Chemical transformations in individual ultrasmall biomimetic containers.
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Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes. (+info)
Attracting and training more chemical pathologists in the United Kingdom.
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I have attempted to define the function of the medical graduate in the clinical biochemistry laboratory and have examined data on recrutiment in the United Kingdom into clinical biochemistry. If trainee pathologists were encouraged to become proficient in both a branch of clinical medicine and in research techniques, the resulting chemical pathologists should be able to improve the consultative and investigative functions of the laboratory. To this end I have suggested some changes in the training regulations and in the role of the chemical pathologists. (+info)
Binding of cholera toxin B-subunits to derivatives of the natural ganglioside receptor, GM1.
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In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C(1) derivatives of N-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide ( approximately 0.01 relative to native GM1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C(1)-amide group, aminopropylamide, was not bound by the toxin. Toxin binding to C(7) derivatives was reduced by about 50% compared with the native ganglioside. Molecular modeling of C(1) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses. (+info)
Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence-based prediction and 65Zn blotting.
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The availability of repeating 'Cys' and/or 'His' units in a particular order prompts the prediction of Zn(II) finger motifs in a protein. Escherichia coli RNA polymerase has two tightly bound Zn(II) per molecule of the enzyme as detected by atomic absorption spectroscopy. One Zn(II) was identified to be at the beta subunit, whereas the other putative Zn(II) binding site has recently been predicted to be at the N-terminal half of the beta' subunit, from primary sequence analysis. We show here that the beta' subunit has no ability to bind 65Zn(II). On the other hand, the N-terminal domain of the alpha subunit has strong Zn(II) binding ability with no obvious functional implications. (+info)
Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides.
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Activation of human platelets with thrombin transiently increases phosphorylation at (558)threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, approximately 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane. (+info)
Oligonucleotide-peptide conjugates as potential antisense agents.
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Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support. (+info)