Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80–100nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.     

Association of Nuclear Matrix Proteins with Granular and Threaded Nuclear Bodies in Cell Lines Undergoing Apoptosis

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyeret al., Exp. Cell Res.221, 27–40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-α, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequencesin vitro,thus mediating the formation of looped DNA structuresin vivo.Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.     

Partial inactivation of the mosquitocidal activity of Clostridium bifermentans serovar malaysia by extracellular proteinases

Summary Clostridium bifermentans serovar malaysia is toxic to mosquito larvae. During large-scale preparation in a fermentor, the bacteria enter the sporulation stage after 5 h culture, whereupon high larvicidal activity is obtained (LC₅₀ 48 h on Anopheles stephensi = 3.1 × 10^–5). The toxicity becomes maximal around 3–5 h later (LC₅₀ 48 h = 1.3 × 10^–5) and remains unchanged until sporangium lysis. An important loss of toxicity is then observed when the cells lyse. This loss appears to be due to the fact that C. bifermentans serovar malaysia synthesizes and excretes, mainly during vegetative growth, metallo- and/or cystein-proteinases, which are active between pH 6.0 and pH 8.0. Extracellular proteinases are most likely responsible in large part for the decrease in toxic activity concomitant with cell lysis. Lysis is however prevented by addition of 10 mM ethylenediaminetetraacetic acid to the culture medium before forespore formation, and under these conditions the larvicidal activity can be maximized. Offprint requests to: L. Nicolas     

In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Purified preparations of pancreatic zymogen granules have the peculiar property of lysing instantaneously at neutral pH, a property clearly irreconcilable with the cytoplasmic pH of the acinar cell. Two important factors known for regulating the stability of secretory granules are calcium and pH. Fluorescence microscopy of acinar cells in the presence of weak bases showed that zymogen granules have an acidic pH. In vivo, abolition of the delta pH by NH4Cl did not induce any lysis of the granules. In vitro, with purified granules, an acidic intragranular pH was measured. This delta pH was produced by a Donnan potential. The importance for granule stability of keeping the intragranular pH acidic has been confirmed in vitro by addition of K+ and nigericin to the suspension medium. These conditions produced alkalinization of the granule matrix and caused instantaneous solubilization of the granules. Concentrations of 15 mM total, and 10 mM free calcium were measured in purified granules. The importance of intragranular Ca2+ was evaluated by means of the ionophore A23187 which induced calcium efflux and granule lysis. The lysis induced by the calcium ionophore was in direct relation with the calcium efflux, since addition of Ca2+ to the medium, at concentrations corresponding to that measured in the granule, relieved the effect. The role of calcium-binding sites on the cytoplasmic surface of the granules was investigated with Ca2+, EGTA, and La3+. Calcium did not have any damaging effects; EGTA induced a slight lysis, while lanthanum yielded a strong and spontaneous lysis at micromolar concentrations. In addition to calcium-binding sites, La3+ would bind to specific sites on the granule that would be directly coupled to maintenance of its stability. These findings suggest that the intragranular acidic pH and calcium are both important for the in vitro stability of the zymogen granule and that purified granules have lost, in the course of purification, some cytoplasmic factors that in vivo, control the permeability of the membrane to protons, and chloride more particularly. Calcium-binding sites and other specific sites probed with La3+, presumably on proteins at the surface of the granule, are also believed to have key roles in preserving the integrity of the membrane and the resulting stability of the granule.     

Calcium-induced aggregation of neuroendocrine protein 7B2in vitro and its modulation by ATP

To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca²⁺ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2_156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.     

Improved culture conditions forCowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants

The causal agent of heartwater disease of domestic ruminants,Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0–6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.     

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm^–1 using cells at 30 g l^–1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.     

Partial purification and characterization of neutral proteases in lymph nodes of rats with experimental allergic encephalomyelitis

Two kinds of neutral protease activities in lymph nodes from Lewis rats with acute experimental allergic encephalomyelitis (EAE) have been separated and partially purified and characterized. A soluble enzyme preparation enriched by gel filtration and ion-exchange chromatography hydrolyzes myelin basic protein, polylysine, and other basic proteins with an optimum pH at 6.0–6.5. It is inhibited byp-chloromercuribenzoate, and thus appears to be a mixture of thiol proteases. Another fraction containing proteolytic enzyme activity is strongly bound to the insoluble lymph node residue, and it also hydrolyzes myelin basic protein and histone, but not polylysine. It has a pH optimum above 7.5, is inhibited by phenylmethylsulfonyl fluoride, thus resembling elastase, but does not hydrolyze elastin-Congo red. The insoluble enzyme preparation hydrolyzes basic protein to 4–5 peptides in a pattern on polyacrylamide gels resembling that of the hydrolysis of basic protein by whole lymphocytes; the soluble enzyme mixture produces small fragments not retained on gels. Lymphocytes are a major component of the cells inflitrating the nervous system in experimental allergic encephalomyelitis, and neutral proteases contained in these cells may contribute to the degradation of myelin, especially of the basic protein.     

Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate

Pseudomonas aeruginosa strain PAO1 grew with the detergent sodium dodecyl sulfate (SDS). The growth started with the formation of macroscopic cell aggregates which consisted of respiring cells embedded in an extracellular matrix composed of acidic polysaccharides and DNA. Damaged and uncultivable cells accumulated in these aggregates compared to those cells that remained suspended. We investigated the response of suspended cells to SDS under different conditions. At high energy supply, the cells responded with a decrease in optical density and in viable counts, release of protein and DNA, and formation of macroscopic aggregates. This response was not observed if the energy supply was reduced by inhibiting respiration with KCN, or if cells not induced for SDS degradation were exposed to SDS. Exposure to SDS caused cell lysis without aggregation if cells were completely deprived of energy, either by applying anoxic conditions, by addition of CCCP, or by addition of KCN to a mutant defective in cyanide-insensitive respiration. Aggregated cells showed a more than 100-fold higher survival rate after exposure to SDS plus CCCP than suspended cells. Our results demonstrate that cell aggregation is an energy-dependent response of P. aeruginosa to detergent stress which might serve as a survival strategy during growth with SDS.     

Fundamental aspects of waste sewage sludge treatment: Microbial solids biodegradation in an aerobic thermophilic semi-continuous system

The solubilization and biodegradation of whole microbial cells by an aerobic thermophilic microbial population was investigated over a 72 h period. Various parameters were followed including total suspended solids reduction, changes in the dissolved organic carbon, protein and carbohydrate concentrations, and carboxylic acid production and utilisation. From the rates of removal of the various fractions a simple model for the biodegradation processes is proposed and verified with respect to acetic acid production and utilization, and total suspended solids removal. The process is initiated by enzymic degradation of the substrate microbe cell walls followed by growth on the released soluble substrates at low dissolved oxygen concentration with concommitant carboxylic acid production. Subsequent utilization of the unbranched, lower molecular weight carboxylic acids allows additional energy supply following exhaustion of the easily utilisable soluble substrate from microbial cell hydrolysis.List of Symbols Y _Xp/Xs kg/kg yield process microbes on substrate yeast cells - Y _Xp/Ac kg/kg yield process microbes on acetate - Y _Ac/Ss kg/kg yield acetate produced by process microbes growing on substrate yeast cells - Y _Ss/Xs kg/kg yield soluble substrate from lysis of yeast cells - Y _Ss/Xp kg/kg yield soluble substrate from lysis of process microbes - Y _P/Xs kg/kg yield particulates from lysis of yeast cells - Y _P/Xp kg/kg yield particulates from lysis of process microbes - _{max (Ss)} h^–1 maximum specific growth rate constant for growth of process microbes on soluble substrate - _{max (Ac)} h^–1 maximum specific growth rate constant for growth of process microbes on acetate - Ks _Ss kg/m³ saturation coefficient for growth of process microbes on soluble substrate - Ks _Ac kg/m³ saturation coefficient for growth of process microbes on acetate - K _d h^–1 death/lysis rate constant for process microbes - K _i kg/m³ inhibition constant for growth of process microbes on acetate - K _L h^–1 lysis rate constant for whole yeast cells - K _h h^–1 hydrolysis rate constant for particulate biomass     

Defective Lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128–192 nm in length and 13–21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55–59, 66–75, and 92–98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid–induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

Characterization of temperature-sensitive mutants of bacteriophage PM2: Membrane mutants

Summary In an effort to understand the genetic regulation of membrane morphogenesis, twenty-nine temperature-sensitive mutants of the membrane-containing bacteriophage PM2 were isolated. Characterization at restrictive temperature revealed groups showing no lysis (Groups I–IV), partial lysis (Groups V–VIII), and full lysis (Groups IX–XII) of the host Pseudomonas BAL-31. When the cell lysis data are considered in conjunction with data on stimulation of viral DNA synthesis, at least six mutant groups are defined. Analysis by gel electrophoresis of the pattern of viral proteins synthesized under restrictive conditions further divides the mutants into twelve groups. Temperature shift experiments delineate early, intermediate and late mutants. Complementation data support some of these groupings. The observed low levels of complementation and recombination are discussed in terms of gene product/genome restriction, bound to the membrane at the site of infection.It is of particular interest to membrane morphogenesis that under restrictive conditions late mutants in Groups II, III and IV make empty-appearing vesicles inside the cell that are the size of virus membranes as seen in thin sections of cells in the electron microscope. Mutants ts 1 (Group II) and ts 12 (Group III) show defects in their ability to incorporate into membranes viral structural proteins sp 13 and sp 6.6. The possibility is discussed that either of these proteins control the size and shape of the viral membrane.     

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes

Summary The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.     

Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105

The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively. The antagonistic action of both proteins precisely schedules lysis by formation of a non-specific lesion in the inner membrane through which the Lambda-encoded murein transglycosylase can pass. Here, we show that the main difference between lysis—effector and lysis—inhibitor is the degree by which an energized membrane inhibits either protein from hole formation. To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane-spanning region in both S proteins. Our results show that the distribution of amino-terminal charged amino acids as well as the total amino-terminal net charge of S107 and S105 influence their lethal potential. The data are interpreted in terms of a model in which the electrostatic status of the amino-terminus of both S107 and S105 is an important feature affecting their conf or mat ional change required for formation of the S-dependent hole.     

Mobility and electric charge of screening pigment granules in the superposition eye ofEphestia kuehniella Z.

Summary The mobility and the electric charge of screening pigment granules of the mealmoth superposition eye were determined electrophoretically in buffer solutions. In potassium phosphate buffer the mobility of the negatively charged granules is linearly dependent on the pH in the range from 4.8 to 7.7 (Fig. 2), and in veronal buffer from pH= 2.3 to pH=7.5 (Fig. 3). At pH=6.6 the values of the effective charge per granule vary between 9.4·10^–17 C and 2.0·10^–16 C, those of the real charge between 2.4·10^–14 C and 5.6·10^–14 C (Table 1, Appendix). For equal electric fields, the mobility of the granules decreases with increasing ionic strength, and it remains the same for > 0.075 mol/1 (Fig. 4).     

pH influence on ethanol production and retained biomass in a passively immobilizedZymomonas mobilis system

Summary A broad pH range of 4.5–7.5 for maximum ethanol productivity and ethanol yield was observed with a passively immobilizedZ. mobilis system. Total retained biomass (as suspended flocs and entrapped cells) was >50 g/l for medium pH values between 4.0–8.0. The entrapped cells to suspended flocs ratio was highest at pH 4.0, whereas at pH above 5.2 it was close to 1.0. The observed enhancement of cell immobilization on the packing support at low pH seemed to be related to formation of bacterial filaments.     

Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway.

Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.     

Involvement of Aquaporin-5 Water Channel in Osmoregulation in Parotid Secretory Granules

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.     

High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Summary We examined the possibility that rat atrial granules may contain a pro-ANF processing protease. Isolated atrial granules were lysed either by detergent, osmotic shock or sonication and incubated at 37° C. Pro-ANF processing and/or degradation were followed by radioimmunoassays and Western blotting using three antibodies which are specific either to the N-terminus, the C-terminus or the processing site (98–99) of pro-ANF. Whatever the method used for the lysis of the granules, we failed to detect any production of ANF (99–I26) and ANF (1–98). However, slight degradation of pro-ANF was recorded probably due to contamination by lysosomal proteases. The in vitro system was validated by addition of thrombin to lysed granules which resulted in a rapid disappearance of the immunoreactivity related to the processing site. These results suggest that the rat atrial granules do not contain any active processing enzyme unless adequate incubation conditions were not met to express its enzymatic activity. The atrial granules may not be directly involved in the maturation of pro-ANF.