A method for determining overall protein fold from NMR distance restraints

Summary We describe a simple method for determining the overall fold of a polypeptide chain from NOE-derived distance restraints. The method uses a reduced representation consisting of two particles per residue, and a force field containing pseudo-bond and pseudo-angle terms, an electrostatic term, but no van der Waals or hard shell repulsive terms. The method is fast and robust, requiring relatively few distance restraints to approximate the correct fold, and the correct mirror image is readily determined. The method is easily implemented using commercially available molecular modeling software.     

Fractionated extraction and speciation of macronutrients from Scots pine and Norway spruce needles of different ages

Summary The relationship between proteins and the macroelements potassium, magnesium, calcium, sulphur and phosphorus was studied in homogenates of needles of different ages from Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.]. Complete extractions by acid digestion, protein extractions by a buffer-detergent and non-protein extractions by a buffer alone showed that most of the potassium and magnesium of the needles was soluble independent of the proteins. Only a minor part (50–60 ppm, dry weight) of the magnesium could be referred to the chlorophyll content of the needles. Both potassium and magnesium appeared independent of the protein gel filtration. This was also valid for the minor fraction of the total calcium content, which was extractable in a buffer. Heterogeneous calcium deposits are also suggested by extraction using a chelator (EGTA). Part of the buffer-soluble sulphur and phosphorus compounds occurred independently of solubilized proteins, and had complex distribution patterns after gel filtration, even outside the high molecular separation range. It is suggested that further analyses along the present lines, compared with conventional analyses on total extracts, may extend the usefulness of mineral nutrient analyses in plants.     

利用带有原核增强子样序列的新型载体在大肠杆菌中高效表达人α肿瘤坏死因子

将去除信号肽的人肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)cDNA插入到带有原核增强子样序列Px的新型表达载体pBV320中,使TNF cDNA 5′端直接置于大肠杆菌trp启动子下游,采用37℃恒温培养,使TNF在大肠杆菌中获得了高效表达,表达活性达1.35(±0.17)×10~6U/L菌液。表达的TNF-α对L929细胞的毒性作用可被抗人肿瘤坏死因子-α的单克隆抗体所中和。表达菌裂解液作SDS-聚丙烯酰胺凝胶电泳,显示有一条分子量与TNF分子量吻合、约为17000道尔顿的蛋白带。利用DEAE-Sepharose阴离子交换层析及Sephacryl S-200凝胶过滤对上述重组人TNF-α进行纯化,获得电泳纯产品,比活性为1.48×10~6U/mg。     

Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

Summary In the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.     

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity

Summary Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.