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Biomedical subjects

F Hucho

Publications and source records attributed to F Hucho.

At least 73 records · Page 4Linked to original sources
PubMed1993

Secondary structure and temperature behaviour of acetylcholinesterase. Studies by Fourier-transform infrared spectroscopy.

The secondary structure of the acetylcholinesterase and its temperature behaviour have been investigated using Fourier-transform infrared (FTIR) spectroscopy. The data are compared to the structure obtained by X-ray analysis of the crystalline enzyme. The secondary structure was determined using the spectral features observed in the amide-I band (H2O buffer) and amide-I' band (D2O buffer) at 1600-1700 cm-1, taking advantage of resolution-enhancement techniques along with least-squares band-fitting procedures. The relative amounts of different secondary-structure elements, 34-36% for alpha-helices, 19-25% for beta-sheets, 15-16% for turns and 13-17% for irregular structures, were estimated. These data, obtained with the enzyme in solution, correlate well with X-ray data of the crystalline protein [Sussman, J. L., Hard, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I. (1991) Science 253, 872-879]. These results are also in good agreement with those obtained by computing the psi and phi angles of the peptide backbone using the Kabsch and Sanders method [Kabsch, W. & Sanders, C. (1983) Biopolymers 22, 2577-2637]. In conjunction with the X-ray data, two bands in the FTIR spectra were assigned to different populations of long and short alpha-helices. Until now this phenomenon has only been described by theoretical calculations [Nevskaya, N. A. & Chirgadze, Yu. N. (1976) Biopolymers 15, 637-648]. The relationship between the thermally induced loss of enzyme activity and secondary-structure changes has also been investigated. The decrease in enzyme activity to zero at 30-40 degrees C was accompanied only by minor changes in the secondary structure. At 55-60 degrees C, denaturation of AChE occurs. In this temperature range, all bands assigned to the various secondary-structure elements abruptly disappear in a co-operative and irreversible manner, whereas the beta-aggregation bands (at 1622 cm-1 and the corresponding high-frequency band) increase in intensity at the same rate.

Acetylcholinesterase↗
PubMed1993

Secondary structure and temperature behavior of the acetylcholine receptor by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) was used to test the secondary structure of purified acetylcholine receptor membranes from Torpedo californica. The secondary structure was estimated using the spectral features observed in the structure sensitive region of amide I and amide I' (between 1600 and 1700 cm-1), taking advantage of Fourier self-deconvolution and second-derivative techniques along with least-squares band fitting procedures. At least six different amide I' band components could be resolved in D2O and were tentatively assigned to beta-structures (1680 and 1636 cm-1), alpha-helices (1657 cm-1), aperiodic structures and/or distorted helices (1646-1648 cm-1), and turns (1690 and 1668 cm-1), respectively. The beta-band around 1637 cm-1, in particular, turned out to be complex since it reproducibly exhibited weak features near 1630 and 1627 cm-1, thereby suggesting the presence of different chain interacting beta-structures. The band near 1657 cm-1 was assigned to alpha-helices which transverse the membrane bilayers, while 1646-1648-cm-1 component was tentatively attributed to aperiodic structures and alpha-helices localized within the "globular head" of the receptor protein protruding from the membrane surface into the surrounding water. Least-squares band fitting procedures were applied in order to estimate relative amounts of secondary structures. The results suggest 36-43%, 32-33%, 14-24%, and 18-19% for beta-, alpha-helical, turn, and "rest" structures, respectively. Additionally, the temperature- and time-dependent variations of the secondary structure was tested by evaluating the changes of amide I and amide II band components of receptor membranes dispersed in H2O and D2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Nuclear substrates of protein kinase C.

Starting from the finding that, for neuronal cells, the nuclear-membrane-associated protein kinase C (PKC) is the so-called 'membrane inserted', constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in-vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two-dimensional gel electrophoresis, in-situ digestion, reverse-phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.

Amino Acid Sequence↗
PubMed1992

Ganglioside-induced CD4 endocytosis occurs independent of serine phosphorylation and is accompanied by dissociation of P56lck.

Gangliosides induce a selective and complete modulation of CD4 from the surface of T cells. CD4 down-modulation occurs by CD4 endocytosis. This process is independent of serine phosphorylation of the cytoplasmic tail of CD4 and does not require the association between the tyrosine protein kinase p56lck and the cytoplasmic tail of CD4. Ganglioside-induced CD4 endocytosis is accompanied by the loss of p56lck activity associated with CD4. Sequential immunoprecipitation analysis using an anti-CD4 antibody and an anti-p56lck antiserum showed that this is caused by the dissociation of the enzyme from the cytoplasmic tail of CD4. The kinetics of p56lck dissociation after ganglioside treatment is identical to that of CD4 endocytosis, suggesting that p56lck is displaced in the process of endosome formation. The results indicate that CD4 endocytosis alone can cause the dissociation of the p56lck complex without the requirement for CD4 phosphorylation.

CD4 Antigens↗
PubMed1992

Investigation of ligand-binding sites of the acetylcholine receptor using photoactivatable derivatives of neurotoxin II from Naja naja oxiana.

Several photoaffinity derivatives of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana have been prepared. After reaction of the 125I-labeled derivatives with the nicotinic acetylcholine receptor from electric organ, the alpha-subunit of the nAChR is almost exclusively labeled by the derivative carrying the photoactivatable group in position Lys46. In contrast to this, a reactive group at Lys26 predominantly labels the gamma- and delta-subunits, while the alpha- and beta-subunits incorporate much less radioactivity. Competition experiments with d-tubocurarine show that the gamma-subunit is labeled when this derivative occupies the high affinity d-tubocurarine-binding site, while the delta-subunit is labeled by the toxin bound at the low-affinity d-tubocurarine site. A model is discussed for the orientation of different loops of the toxin molecules in the binding site for agonists and competitive antagonists.

Animals↗
PubMed1992

Fourier transform infrared (FTIR) spectroscopic investigation of the nicotinic acetylcholine receptor (nAChR). Investigation of agonist binding and receptor conformational changes by flash-induced release of 'caged' carbamoylcholine.

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically released carbamoylcholine ('caged' carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N-[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N-(alpha-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm-1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.

Animals↗
PubMed1992

Properties of protein kinase C associated with nuclear membranes.

To study signal transduction directed towards the cell nucleus and at the nuclear membranes, we investigated the association of protein kinase C (PKC) with nuclear membranes obtained from nuclei isolated from bovine brain. By use of phorbol-ester-binding assays, significant amounts of PKC could be demonstrated in nuclei and nuclear membranes. Nuclear membranes are shown to be able to activate purified PKC. The PKC endogenously present in nuclear membranes appears to be a so-called 'membrane-inserted' form: it is permanently active, still binds phorbol ester, but its activity is no longer dependent on Ca2+ and cannot be activated by phorbol ester. On the other hand, this form of PKC can be inhibited by specific PKC inhibitors. By using histone HIIIS and a specific peptide substrate, it could be shown that after extraction with Triton X-100 the PKC can be stimulated by phospholipid again. Immunoblot analysis with isoenzyme-specific antibodies revealed that the alpha- and gamma-isoenzymes, but not the beta-isoenzyme, are associated with membranes derived from brain nuclei.

Amino Acid Sequence↗
PubMed1992

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.

Adenosine Diphosphate Ribose↗
PubMed1992

The HIV-1 surface protein gp120 has no effect on transmembrane signal transduction in T cells.

The ability of HIV-1 envelope glycoprotein gp120 to induce transmembrane signaling processes in human T cells and tumor T-cell lines was investigated. Differently glycosylated gp120 preparations were characterized with respect to their purity, the fraction of native gp120, and the affinity of the gp120-CD4 interaction. These data were used to establish experimental conditions that allow a substantial fraction of the CD4 receptor to be complexed with gp120 in the course of the experiments. The results are in contrast to several previous studies since no effect of gp120 on the intracellular Ca2+ concentration, the metabolism of inositol phosphates and arachidonic acid, protein kinase C translocation, and tyrosine phosphorylation was found. Cross-linking of the gp120:CD4 complex by anti-gp120 antibodies did not elicit additional effects.

Arachidonic Acids↗
PubMed1991

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.

Amino Acid Sequence↗
PubMed1991

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.

Acetylcholinesterase↗
PubMed1991

Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362.

The delta-subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue isolated form receptor purified in the absence of protein phosphatase inhibitors contains a total of four phosphate groups. Three of these are shown to represent phosphoserine groups. The fourth possible represents phosphotyrosine. The phosphate groups are localized within the primary structure: We found phosphoserine in positions delta S361 and delta S377, the predicted sites phosphorylated by PKA and PKC, respectively. In addition, we found that position delta S362 is also phosphorylated. Phosphorylation experiments with the synthetic peptide delta L357-delta K368 show that phosphorylation of this novel site can be catalyzed by PKA and by PKC. It is concluded that the delat-subunit of the acetylcholine receptor is stably and not transiently phosphorylated. Implications for the physiological functions of receptor phosphorylation are discussed.

Animals↗
PubMed1991

Biotinylation of transducin and G0 from bovine brain.

A method is described for modifying G proteins with biotin. With transducin, better results were obtained with the amino group-specific derivative BXNHS (biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester) as compared to the -SH-group specific reagent MBB (maleimidobutyrylbiocytin). Modification occurred under conditions preserving functional activity: Interaction of the biotinylated transducin with rod outer segment membranes was shown by its light-dependent association and by a GTP gamma S-binding assay. G0 from bovine brain was also biotinylated under conditions preserving its activity. Biotinyl-alpha 0 was shown to bind to a streptavidin Sepharose matrix. Biotinyl-G proteins, therefore, are proposed as tools for extracting proteins (receptors and effector systems), which interact under specific conditions with G proteins.

Animals↗
PubMed1991

Substrate-binding sites in acetylcholinesterase.

Acetylcholinesterase is among the most efficient enzymes known. In order to provide an explanation for its catalytic and regulatory mechanisms, including the high turnover rate, the specific amino acid residues involved in substrate binding and hydrolysis need to be identified. In this article, Ferdinand Hucho, Jaak Järv and Christoph Weise describe the topography of the enzyme as deduced from protein chemistry studies. One result of this approach is the finding that the binding pocket for the substrate's cationic cholinium group appears to be hydrophobic rather than anionic.

Acetylcholinesterase↗
PubMed1991

The phospholipid analogue, hexadecylphosphocholine, inhibits protein kinase C in vitro and antagonises phorbol ester-stimulated cell proliferation.

The antineoplastic agent, hexadecylphosphocholine, a phospholipid analogue, inhibited phosphatidylserine-activated protein kinase C in vitro at concentrations higher than 40 mumol/l. The half-inhibitory concentration (IC50) was 62 mumol/l. Another alkylphosphocholine, dodecylphosphocholine, did not have an inhibitory effect on protein kinase C. At the same concentrations, hexadecylphosphocholine antagonised the phorbol ester-stimulated proliferation of Madin-Darby canine kidney cells whereas dodecylphosphocholine had no effect. In addition, phorbol ester-induced morphological changes of these epithelial cells were antagonised by hexadecylphosphocholine. Both effects of hexadecylphosphocholine, the inhibition of protein kinase C and the antagonisation of the altered cell morphology induced by phorbol ester, were comparable to those observed after treatment with sphingosine, a known protein kinase C inhibitor. We conclude that one possible mechanism of the antineoplastic action of hexadecylphosphocholine is mediated by inhibition of protein kinase C.

Animals↗
PubMed1990

Identification of phosphopeptides by mass spectrometry.

Phosphopeptides can be identified by ion spray mass spectrometry. The method was tested with phosphokemptide and with a proteolytic digest of one subunit (delta-subunit) of the nicotinic acetylcholine receptor. In the latter one peptide containing tyrosine phosphate, one with two serine phosphates, and two different peptides each containing one serine phosphate were unambiguously identified. Thus it is proven that ion spray mass spectrometry can be applied for the localization of phosphorylation sites in a known primary structure.

Chromatography, High Pressure Liquid↗
PubMed1990

Characterization of a 17 kDa protein gene upstream from the small cytoplasmic RNA gene of Bacillus subtilis.

The Bacillus subtilis small cytoplasmic RNA (scRNA) is the structural homologue of both the RNA component of the eukaryotic signal recognition particle (SRP) and the Escherichia coli 4.5S RNA, and it can complement the essential function of the latter RNA in vivo. In the course of characterization of the single-copy scRNA gene locus (scr) we identified an open reading frame, termed ORF17, upstream from scr that encodes an acidic 17 kDa protein of unknown function. This analysis involved DNA sequencing, monitoring expression of transcriptional and translational ORF17-cat and ORF17-lacZ fusions, respectively, and purification and sequencing of the ORF17-lacZ fusion protein. Apparently, transcription of ORF17 proceeds into scr. A small portion of the 17 kDa protein shows homology to deoxycytidylate (DCMP) deaminase of bacteriophagphage T2, but no similarity exists to the sequenced SRP-polypeptides or any other known protein sequences.

Amino Acid Sequence↗