Characterization of the region on protein L7/L12 involved in binding to ribosomal particles

Tryptic digestion of reductively methylated protein L7/L12 yields a large tryptic fragment, which comprises amino acids 1-59. At the most, two molecules of this fragment can bind to a 50-S ribosomal particle, deprived of protein L7/L12. Besides, binding of each single 1-59 fragment competes with binding of one dimeric L7/L12 molecule. Molecular weight studies on the fragment reveal a monomeric structure. Digestion of the 1-59 fragment with carboxypeptidase Y leads to the formation of a 1-55 fragment. The binding characteristics of the latter fragment are similar to those of the 1-59 fragment. The results suggest that a monomeric stretch of L7/L12, comprising the first 55 amino acids, is sufficient for attaching L7/L12 to the ribosome.     

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

Synthesis and application of two reagents for the introduction of sulfhydryl groups into proteins

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate-oxidized N termini of proteins, provided that the N-terminal residue is serine or threonine. 3-(Phenyldithio)propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.     

Blood osmolality in vitro: dependence on PCO2, lactic acid concentration, and O2 saturation

Changes of osmolality (Osm) were measured by freezing-point determination in true plasma of 10 healthy subjects. This was done after equilibration with CO2 (0.5-10.0%), after the addition of lactic acid (10 and 20 mmol/l), and after deoxygenation. The graph for the dependence of Osm on CO2 partial pressure (PCO2) in oxygenated blood resembles the classical CO2 absorption curve. The increase of Osm with PCO2 (approximately 0.2 mosmol . kg H2O-1 . Torr-1) is almost as great as the increase in dissolved CO2 plus bicarbonate (HCO-3). Addition of lactic acid shifts the curve upward by only 0.6 mosmol/mmol because of displacement of HCO-3. Deoxygenation has no significant effect at constant PCO2 despite an increase in [HCO-3]. This is probably due to the binding of 2,3-diphosphoglycerate to hemoglobin. It can be seen in the Osm-pH diagram that differences between CO2 and lactic acid titration largely disappear. For each lactic acid concentration there is a linear dependence corresponding to the linear [HCO-3]-pH relation in plasma. At constant pH, Osm increases after deoxygenation. The observed in vitro relation might explain part of the osmolality increase during physical exercise.     

Localization of Lys-51 of L7/L12 on 50S ribosomes from Escherichia coli

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys-51 with 4-(6-formyl-3-azido-phenoxy)butyrimidate. Reconstitution of ribosomal cores, lacking L7/L12, with imidate-modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation-factor-dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys-51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L12 stalk in such a way that Lys-51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.     

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.     

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Orcein and Litmus

Orcein was separated into 14 dyes by partition chromatography. Their constitutions were determined mainly by spectroscopy and led to formulae that are derived from 7-amino-2-phenoxazone, 7-hydroxy-2-phenoxazone, and 7-amino-2-phenoxazime, and that were confirmed by syntheses. The major constituent of litmus is assembled polymerically from 7-hydroxy-2-phenoxazone chromophores. The mechanism of formation is elucidated.