Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

Angiotensinase activity in hypothalamus and pituitary of hypothyroid, euthyroid and hyperthyroid adult male rats.

A local renin-angiotensin system (RAS) that may be involved in their regulatory functions has been identified in hypothalamus and pituitary. Altered thyroid status induces modifications in the secretory function of hypothalamus and pituitary. However, few studies have analyzed the role of the RAS in hypothalamus and, to our knowledge, there is no data on the pituitary RAS during thyroid dysfunction. In the present study, angiotensinase activities (glutamyl, aspartyl and alanyl aminopeptidase: GluAP, AspAP and AlaAP, respectively) were studied in hypothalamus and in the anterior and posterior lobes of pituitary of euthyroid, hypothyroid and hyperthyroid adult male rats. In the anterior pituitary, compared with euthyroid and hyperthyroid rats, hypothyroid animals showed a highly significant increase of GluAP and AspAP activities; the percentage increase in GluAP was markedly higher than the percentage increase in AspAP. This suggests an increased metabolism of angiotensin (Ang) I and Ang II to des-Asp 1-Ang I and Ang III, respectively. We also observed an increase of Ang III-degrading activity (AlaAP) in the hypothalamus of hyperthyroid rats in soluble fraction. Increased Ang I and Ang II metabolism in the anterior pituitary of hypothyroid rats and increased metabolism of Ang III in the hypothalamus of hyperthyroid animals may be related to alterations in the secretory function of hypothalamus and pituitary in these thyroid dysfunctions.     

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.     

Social Behaviour of the Carpenter Bee Xylocopa pubescens (Spinola)

The development of about 20 relatively small nests of Xylocopa pubescens was studied. After the first offspring had become adult, these nests reached a social stage in which there was only one egg-layer per nest. Freshly emerged (teneral) adults eat a lot of food, collected by a forager, before they fly out of the nest. This food appears to be of major importance to these bees in that it makes them fully agile. It seems therefore, that part of the food needed for the development of a young bee is not given at the larval stage, via the beebread, but at the teneral adult stage. As a result of this, a necessary overlap of generations is accomplished. Besides, less pollen has to be collected for the provision of a brood cell, so more cells can be constructed within a short period of time. There is a high degree of nest competition. Many nests were taken over by conspecific individuals (or by females of X. sulcatipes) that were searching for nesting sites within the study area. However, although more than 50% of the solitary nests were taken over sooner or later, strangers hardly ever intruded into social nests, with more than one adult. This illustrates how important it is for a reproducing female to tolerate the presence of nestmates which guard the nest in her absence. Although in most cases the foundress of a nest proceeded to produce new brood in the presence of her offspring, it happened that a daughter took over reproduction from her mother. In two of these cases, it could be observed that a mother started a new nest elsewhere after having been thrown out by her own daughter. At least in these cases, nest competition between mother and daughter started long before the mother had reached the end of her reproductive capacities. Since nesting possibilities are scarce, it seems a logical strategy to stay in the maternal nest and wait for a chance to become the egg-layer if the mother dies or if she loses her dominance. The occurrence of several social interactions among nestmates is discussed: — trophallaxis was observed often, not only under ‘forced’ conditions, but also as a form of ‘voluntary’ feeding; — nestmates were observed to groom each other.     

Restoration of eutrophied shallow softwater lakes based upon carbon and phosphorus limitation

Plant communities from oligotrophic, poorly buffered waters are seriously threatened by both, acidification and eutrophication/alkalinization. Acidification is mainly caused by atmospheric deposition of acidifying substances while eutrophication is often the result of inlet of nutrient enriched, calcareous brook- or groundwater. The plant production in very soft waters is often limited by low levels of inorganic carbon, nitrogen and/or phosphorus. This paper deals with the possibilities for restoration of formerly oligotrophic but now eutrophied and alkalinized softwater systems. Restoration based upon nitrogen limitation is not likely to be successful as the atmospheric deposition of nitrogen in The Netherlands is very high. Phosphorus limitation can also be a problem. One can stop the input of phosphorus and remove the mud layer, but the problem remains that also the deeper mineral sandy sediments are saturated with phosphate. A possible remedy, however, is a combination of carbon- and phosphorus limitation. Many plants from eutrophic environments never occur in very soft waters, probably as a result of carbon limitation. In addition, mobilisation of phosphate is much lower in waters with very low bicarbonate levels. Restoration of a former oligotrophic softwater lake by reducing the inlet of calcareous surface water, in combination with removal of the organic sediment layer, appeared to be very successful. Many endangered plant species such asIsoetes echinospora, Luronium natans, Deschampsia setacea andEchinodorus repens developed spontaneously from the still viable seedbank.     

Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.This work was supported by grants from CICYT and Pharmacia-Upjohn.     

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

Entamoeba histolytica: gene linkage groups and relevant features of its karyotype

We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25?S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.     

Short communication: Effect of water activity on cell growth and phenol oxidase production by Streptomyces cyaneus var. viridochromogenes in surface culture

In surface cultures of Streptomyces cyaneus var. viridochromogenes, NaCl depressed water activity (a _w) without supporting growth. Reducing a _w from 0.987 to 0.951 led to 3- and 4-fold increases in intracellular and extracellular phenol oxidase activities, respectively.