Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle

The acetylcholine receptor (AChR) content and the autorphosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rat soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [¹²⁵I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro³²P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the³²P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the³²P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.Abbreviations ACh acetylcholine - AChR acetylcholine receptor(s) - BUTX alpha-bungarotoxin - Kd kilodalton - PAGE polyacrylamide gel electrophoresis - R-II regulatory subunit of cyclic AMP-dependent protein kinase type II - SDS sodium dodecyl sulfate     

A flexible method to align large numbers of biological sequences

Summary A method for the alignment of two or more biological sequences is described. The method is a direct extension of the method of Taylor (1987) incorporating a consensus sequence approach and allows considerable freedom in the control of the clustering of the sequences. At one extreme this is equivalent to the earlier method (Taylor 1987), whereas at the other, the clustering approaches the binary method of Feng and Doolittle (1987). Such freedom allows the program to be adapted to particular problems, which has the important advantage of resulting in considerable savings in computer time, allowing very large problems to be tackled. Besides a detailed analysis of the alignment of the cytochrome c superfamily, the clustering and alignment of the PIR sequence data bank (3500 sequences approx.) is described.     

Immunogold localization of acyl carrier protein in plants and Escherichia coli: Evidence for membrane association in plants

Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5–8_. In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25–30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.Abbreviations ACC acetyl CoA carboxylase - ACP acyl carrier protein - FAS fatty-acid synthetase     

Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.     

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.