R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.
Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay. (+info)
Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.
The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme. (+info)
EGF precursor mRNA and membrane-associated EGF precursor protein in rat exorbital lacrimal gland.
This study was designed to demonstrate the presence of epidermal growth factor (EGF) in the rat exorbital lacrimal gland. EGF precursor gene transcription was demonstrated first by RT-PCR analysis of lacrimal gland RNA using a set of specific primers and second by Northern blot analysis of rat lacrimal gland mRNA. A rabbit polyclonal antibody (rEGF2) directed against rat submaxillary gland EGF was used to detect EGF-containing proteins by RIA. Results indicate that the rat lacrimal gland does not contain detectable soluble and mature EGF but that the EGF immunoreactivity is associated with the membrane-enriched fraction. Analysis of the detergent-solubilized membrane proteins by gel filtration shows that membrane-associated EGF immunoreactivity was present as a high-molecular-mass protein. Moreover, as shown by Western blot analysis, a specific anti-rat EGF precursor antibody (ppEGF1) can immunoprecipitate a 152-kDa EGF-containing protein. Taken together, these results demonstrate for the first time both EGF precursor gene transcription and EGF precursor protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. The demonstration that EGF appears to be stored only as its full-length membrane precursor may provide important information to study the regulation of its secretory process. (+info)
Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients.
Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs). (+info)
Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis.
We have constructed a library of DNA fragments heavily methylated in human adenocarcinomas of the lung to permit the comprehensive isolation of methylated CpG islands in cancer. Heavily methylated genomic DNA fragments from tumors of nine male patients were enriched using a methylated DNA binding column and used for construction of the library. From this library, DNA fragments having properties of CpG islands were isolated on the basis of their reduced rate of strand dissociation during denaturing gradient gel electrophoresis. Approximately 1,000 clones, corresponding to 0.3% of the library were analyzed, and nine DNA fragments were identified as being associated with CpG islands that were methylated in tumor DNA. One CpG island was methylated specifically in tumor DNA, whereas the remaining eight CpG islands were methylated both in normal and tumor DNA derived from the same patients. Our results suggest that the number of CpG islands methylated specifically in tumors is not large. The library, which contains DNA fragments from methylated CpG islands comprehensively, is expected to be valuable when elucidating epigenetic processes involved in carcinogenesis. (+info)
Deamidation of alpha-A crystallin from nuclei of cataractous and normal human lenses.
PURPOSE: To quantitate the extent of deamidation of asparagine-101, glutamine-50, and glutamine-6 of alpha-A crystallin in the nucleus from human cataractous and normal lenses. METHODS: Reverse phase chromatography was used to prepare alpha-A crystallin from total proteins of the nucleus from cataractous and age-matched normal human lenses. Synthetic peptides were made corresponding to the expected amidated and deamidated tryptic fragments containing asparagine-101, glutamine-50, and glutamine-6. The peptides were used to identify and quantitate amidated and deamidated forms of tryptic fragments from alpha-A crystallin eluting from a reverse phase column. RESULTS: Significant amounts of deamidation of asparagine-101 and glutamine-50, but not glutamine-6, were present in alpha-A crystallin from nuclear sections of both cataractous and age-matched normal lenses. Quantitative analysis of tryptic peptides containing these residues indicated no statistical difference in deamidation in cataractous versus normal lenses. CONCLUSIONS: There was no significant difference in the extent of deamidation of asparagine-101, glutamine-50, and glutamine-6 for alpha-A crystallin, purified from the nucleus of cataractous versus age-matched normal lenses. These results strongly suggest that deamidation of these residues does not play a role in the biogenesis of human nuclear cataract. (+info)
Purification and properties of bovine pituitary follitropin.
A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine. (+info)
The selective isolation of the uterine oestradiol-receptor complex by binding to oligo(dT)-cellulose. The mediation of an essential activator in the transformation of cytosol receptor.
The [3H]oestradiol-receptor complex was selectively isolated from rat uterus cytosol by column chromatography on oligo(dT)-cellulose. Optimal conditions are described for the binding of the complex to oligo(dT)-cellulose, which is shown to be similar to its binding to DNA-cellulose. The cytosol complex has an apparent mol. wt. of 50,000-60,000 in high salt concentrations, as determined by Sephadex G-100 chromatography. This corresponds to the 4S cytoplasmic oestradiol receptor. In binding to oligo(dT)-cellulose the receptor is transformed into a form with an apparent mol.wt. of 100,000-120,000, corresponding to the 5S nuclear receptor complex. This transformation mimics the conversion in vivo of the cytoplasmic oestradiol receptor into the nuclear form. The binding of the complex to oligo(dT)-cellulose as a 5S nuclear form is unequivocally demonstrated to require the mediation of an activating present in the cytosol. The requirement for an activating factor is discussed in relation to reports that nuclear binding of the oestradiol-receptor complex is not dictated solely by the availability of the cytoplasmic oestradiol receptor. (+info)