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Biomedical subjects

A Seelig

Publications and source records attributed to A Seelig.

At least 37 records · Page 2Linked to original sources
PubMed1994

20 S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16 S preproteasome complexes.

The non-essential mouse proteasome beta-type subunits LMP2 and LMP7 are thought to connect proteasomes to the MHC class I antigen processing pathway. Both subunits are synthesized as proproteins. We have studied the processing of both subunits, correlated with the maturation of 20 S proteasomes in mouse T cells. Our data show that proteasome assembly occurs via 13-16 S precursor complexes which possess a protein pattern distinct from that of 20 S proteasomes. Both LMP2 and LMP7 proproteins are processed within these preproteasome complexes and only their processed forms become part of active 20 S proteasomes. Our data show that the maturation and assembly of 20 S proteasomes via precursor particles is a translation-dependent gradual process, that processing of subunit proproteins takes place in these 13-16 S complexes and that subunit processing and proteasome formation occur together.

Animals↗
PubMed1994

A method to determine the ability of drugs to diffuse through the blood-brain barrier.

A method has been devised for predicting the ability of drugs to cross the blood-brain barrier. The criteria depend on the amphiphilic properties of a drug as reflected in its surface activity. The assessment was made with various drugs that either penetrate or do not penetrate the blood-brain barrier. The surface activity of these drugs was quantified by their Gibbs adsorption isotherms in terms of three parameters: (i) the onset of surface activity, (ii) the critical micelle concentration, and (iii) the surface area requirement of the drug at the air/water interface. A calibration diagram is proposed in which the critical micelle concentration is plotted against the concentration required for the onset of surface activity. Three different regions are easily distinguished in this diagram: a region of very hydrophobic drugs which fail to enter the central nervous system because they remain adsorbed to the membrane, a central area of less hydrophobic drugs which can cross the blood-brain barrier, and a region of relatively hydrophilic drugs which do not cross the blood-brain barrier unless applied at high concentrations. This diagram can be used to predict reliably the central nervous system permeability of an unknown compound from a simple measurement of its Gibbs adsorption isotherm.

Blood-Brain Barrier↗
PubMed1993

Drosophila proteasome Dm25 subunit substitutes the mouse MC3 subunit in hybrid proteasomes. The N-terminal domain is essential for subunit incorporation.

The proteasome is a multisubunit 20 S proteinase complex involved in ubiquitin-dependent and -independent intracellular protein metabolism. Individual subunits of the alpha- and beta-type share extensive sequence homology and are encoded as members of two related and evolutionarily conserved gene families. Due to the lack of viable deletion mutants of essential alpha-type proteasome subunits in higher eukaryotes, an identification and analysis of potentially homologous subunits of different species was so far not possible. It is shown here that the novel Drosophila alpha-type Dm25 subunit can be incorporated into mouse proteasomes of stably transfected NIH 3T3 cells. The Dm25 subunit is able to substitute the mouse MC3 alpha-type subunit in proteasomes, indicating a high structural and possibly also functional homology of the two subunits. In contrast and pointing at the importance of the slightly hydrophobic N-terminal region stabile expression of a Dm25 subunit, which is truncated at its N terminus and lacks PROS box I, results in a subunit which cannot be incorporated into mouse proteasomes. The ability to form hybrid proteasomes involving essential nondeletable subunits now opens the possibility for structural and also functional analysis of such subunits by mutagenesis in higher eukaryotes.

3T3 Cells↗
PubMed1993

Sequence and genomic organization of the Drosophila proteasome PROS-Dm25 gene.

The genomic region encoding the Drosophila proteasome alpha-type subunit Dm25 has been isolated and analysed with regard to its nucleotide sequence and structure. Our data show that the Dm25 coding region is interrupted by four introns and that the 5' upstream region contains no sequence motifs common with the two previously described proteasome genes.

Amino Acid Sequence↗
PubMed1993

The major-histocompatibility-complex-encoded beta-type proteasome subunits LMP2 and LMP7. Evidence that LMP2 and LMP7 are synthesized as proproteins and that cellular levels of both mRNA and LMP-containing 20S proteasomes are differentially regulated.

The proteasome (high-molecular-mass multicatalytic proteinase complex) is composed of a large number of non-identical protein subunits of the alpha and beta types. The mouse beta-type subunits LMP2 and LMP7 (LMP, low-molecular-mass protein) are encoded within the mouse major histocompatibility complex (MHC II) region, and are thought to connect the proteasome to the MHC class-I antigen-processing pathway. In the present communication, we have analysed the two proteasome subunits with regard to their identity within the proteasome complex, their protein levels, their amounts of mRNA in different mouse tissues and cell lines, and have investigated the intracellular localization of LMP2 and LMP7 subunits in thymus and liver by immunocytology. Our experiments indicate that LMP2 and LMP7 subunits are synthesized as precursor proteins of 24 kDa and 30 kDa, respectively, and that only the processed 21-kDa and 23-kDa subunits are part of the 20S proteasome complex. The proportion of LMP2-subunit-containing and LMP7-subunit-containing proteasome complexes, as well as LMP2 and LMP7 mRNA levels, vary strongly and are shown to be dependent on the tissues or cell lines analysed. Furthermore, high LMP2 and LMP7 mRNA levels do not always correlate with high protein levels, suggesting a specific translational mechanism which controls proteasome subunit synthesis. Generally, mRNA levels appear to be particularly high in those tissues which are known to be involved in MHC class-I antigen presentation. Immunocytological analysis shows a strong nuclear localization of the subunits in cells of the thymus, while in the liver they appear to be evenly distributed between the two cellular compartments. Our data support the idea that both LMP2 and LMP7 proteins are non-essential proteasome subunits which are probably involved in the regulation of proteasome activities. The function of the two subunits, however, may not be restricted to the proposed role of proteasomes in antigen presentation.

Animals↗
PubMed1993

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Characterization of mouse proteasome subunit MC3 and identification of proteasome subtypes with different cleavage characteristics. Proteasome subunits, proteasome subpopulations.

We have isolated and characterized a cDNA encoding the mouse proteasome subunit MC3 and identified four proteasome subtypes which differ in their peptide-hydrolyzing and polypeptide-cleavage properties. Immunoblotting data show that the 25-kD MC3 subunit is a constitutive proteasome subunit which exists in several isoforms. In addition, by immunoprecipitation of proteasomes with AbMC3, a subset of enzyme complexes could be recognized which differ in their relative subunit composition from the bulk of proteasomes. Using DEAE-column chromatography we identified three different proteasome subtypes in sol-80 mouse liver extracts and, by Trition X-100 extraction, a distinct membrane-bound subtype. The four proteasome subtypes are shown to differ in their trypsin- and chymotrypsin-like hydrolyzing activities as well as in their ability to cleave a 25mer polypeptide substrate derived from the MCMV IE pp89. Our data indicate that the enzymatic properties observed for the total proteasome population may be the summary of cleavage properties of different types of proteasome complexes.

3T3 Cells↗
PubMed1992

Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1991

Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex.

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.

Amino Acid Sequence↗
PubMed1991

Subunit of the '20S' proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex.

Cytotoxic T lymphocytes recognize fragments (peptides) of protein antigens presented by major histocompatibility complex (MHC) class I molecules. In general, the peptides are derived from cytosolic proteins and are then transported to the endoplasmic reticulum where they assemble with the MHC class I heavy chains and beta 2-microglobulin to form stable and functional class I molecules. The proteases involved in the generation of these peptides are unknown. One candidate is the proteasome, a nonlysosomal proteinase complex abundantly present in the cytosol. Proteasomes have several proteolytically active sites and are complexes of high relative molecular mass (Mr about 600K), consisting of about 20-30 subunits with Mrs between 15 and 30K. Here we show that at least one of these subunits is encoded by the mouse MHC in the region between the K locus and the MHC class II region, and inducible by interferon-gamma. This raises the intriguing possibility that the MHC encodes not only the MHC class I molecules themselves but also proteases involved in the formation of MHC-binding peptides.

Animals↗
PubMed1990

Substance P and antagonists. Surface activity and molecular shapes.

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, ANT I; [D-Arg1,D-Trp7,9,Leu11]SP, ANT II and [D-Pro2,D-Trp7,9]SP, ANT III. SP is only moderately surface active. The amino acid substitutions lead, however, to an increased surface activity of the antagonists. From the concentration dependence of the surface activity it was possible to quantify the packing characteristics of the individual neuropeptides. SP shows cross-sectional areas of 300 +/- 5 A2 to 240 +/- 5 A2 (pH 5 to 8, 154 mM NaCl) at concentrations below 10(-5) M, i.e., in the physiological concentration range, indicating a folded SP conformation. Upon increasing the packing density to concentrations larger than 10(-5) M the surface area was only half as large (148 +/- 5 A2 to 124 +/- 3 A2) suggesting now a relatively extended conformation of the SP molecule with its long molecular axis perpendicular to the air/water interface. In contrast, the three antagonists were characterized by surface areas of 147 +/- 3 A2 to 126 +/- 3 A2 which were almost independent of concentration. The antagonists thus adopt a relatively extended conformation in the whole concentration range measured. This is further supported by computer modelling which shows that the antagonists are motionally restricted and can adopt neither a bent nor a alpha-helical conformation. The surface activity of the neuropeptides was dependent on the pH of the solution. At low peptide concentrations (about 10(-6) M) it was possible to resolve and determine the pK values of all individual charged amino acid side chains. The pK values observed for the neuropeptides were about two pK units lower than those of the free amino acids in solution. The pK shifts of the neuropeptides at the air/water interface are explained in terms of the Gouy-Chapman theory. SP and its antagonists bind to lipid bilayers in the order of their surface activity. While the binding of SP is mainly due to electrostatic interactions, hydrophobic peptide-lipid interactions contribute to the binding of the antagonists.

Amino Acid Sequence↗
PubMed1990

The use of monolayers for simple and quantitative analysis of lipid-drug interactions exemplified with dibucaine and substance P.

The interaction between lipids and water soluble amphiphiles was investigated by means of a monolayer technique, monitoring the area increase at constant surface pressure. The area increase could be quantitated and binding isotherms at different surface pressures were measured. A comparison of dibucaine binding to monolayers and bilayers showed that a surface pressure of 32 mN/m best represents the packing density in a lipid bilayer (Seelig, 1987). Binding isotherms measured for charged dibucaine and substance P (SP) were analyzed by means of two different models. If electrostatic effects were ignored the binding of dibucaine and SP showed biphasic Scatchard plots. If, however, electrostatic effects were taken into account by means of the Gouy-Chapman theory, the insertion of both amphiphiles was best described in terms of a partitioning into the monolayer lipids. The hydrophobic binding constant was Kp = 660 +/- 80 M-1 for charged dibucaine inserting into coarse liposomes or monolayers at 32 mN/m (Seelig et al., 1986) and 1-1.8 M-1 for SP inserting into monolayers at 32 mN/m (Seelig and Macdonald, 1989).

Dibucaine↗
PubMed1989

Binding of a neuropeptide, substance P, to neutral and negatively charged lipids.

The binding of substance P (SP), a positively charged neurotransmitter peptide, to neutral and to negatively charged phospholipids has been investigated by means of a monolayer technique. Monolayers formed at room temperature from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or mixtures of the two, were maintained throughout the course of a binding experiment at a constant surface pressure while the monolayer surface area was monitored. Injection of SP into the aqueous subphase (154 mM NaCl, 10 mM Tris adjusted to pH 7.4) led to an expansion of the monolayer surface area that was attributed to a spontaneous insertion of SP between the lipid molecules. A quantitative evaluation of the area increase at constant pressure yielded SP insertion isotherms that showed that levels of SP insertion increased directly with the monolayer POPG content and decreased to negligible levels at surface pressures above 35 +/- 1 mN/m. If electrostatic effects were ignored, these data showed biphasic behavior in Scatchard plots. The apparent binding constants ranged, at 20 mN/m, from (3.2 +/- 0.3) X 10(4) M-1 for 100% POPG monolayers to (2.0 +/- 0.05) X 10(3) M-1 for 25% POPG/75% POPC monolayers. At 32 mN/m, a monolayer surface pressure that mimics bilayer conditions, the apparent binding constant for a 100% POPG monolayer was measured to be (1.1 +/- 0.05) X 10(3) M-1. However, for a monolayer containing only 25% charged lipids, corresponding to a natural membrane composition, K app at 32 mN/m was estimated to be at most 41 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerophosphates↗
PubMed1988

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group.

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.

Acetanilides↗