Inhibition of Bacillus thuringiensis proteases and their effects on crystal toxin proteins and cell-free translations

1988 ◽  
Vol 34 (6) ◽  
pp. 740-747 ◽  
Author(s):  
Margaret Bibilos ◽  
Robert E. Andrews Jr.
Keyword(s):  
Molecular Weight ◽  
Bacillus Thuringiensis ◽  
Proteolytic Activity ◽  
Exponential Phase ◽  
Late Exponential Phase ◽  
Stages Of Growth ◽  
Translational Activity ◽  
Low Levels ◽  
Crystal Toxin ◽  
Neutral Metal

Proteases produced during growth and sporulation of four strains of Bacillus thuringiensis were examined. Low levels of proteolytic activity were detected during the late exponential phase of growth in all four strains: two strains of B. thuringiensis subsp. kurstaki and one strain each of subsp. israelensis and berliner. In all strains, protease activities increased dramatically at the onset of sporulation. The principal proteases, both extra- and intra-cellular, were neutral, metal-loproteases. The pH optima and substrate specificities of proteases extracted from cells in various stages of growth and sporulation indicated that substantial diversity existed among the strains. Intracellular proteases from all four strains converted the 135 000 molecular weight protoxin of strain HD251, an isolate previously shown to have reduced intracellular proteolytic activity and which normally does not contain a protein of the lower molecular weight, to a 68 000 molecular weight toxin. Cell-free translational activity of extracts from strain HD251 were approximately fivefold more active than were equivalent extracts from strain LB1 presumably because of the reduced intracellular proteolytic activity of this strain.

Alkaline extraction of toxin from spores of the mosquito pathogen, Bacillus sphaericus strain 1593

1983 ◽  
Vol 29 (2) ◽  
pp. 271-275 ◽  
Author(s):  
Elizabeth West Davidson
Keyword(s):  
Molecular Weight ◽  
Bacillus Thuringiensis ◽  
Bacillus Sphaericus ◽  
Alkaline Extraction ◽  
Toxic Activity ◽  
Crystal Toxin ◽  
Mosquito Pathogen

Toxin was extracted from spores of the mosquito pathogen Bacillus sphaericus strain 1593 using 0.05 M NaOH. The molecular weight of this toxin was 35 000–54 000. Toxic activity of this extract was resistant to a variety of enzymes including subtilisin, but was degraded by pronase. Antiserum produced to 1593 spore toxin neutralized spore toxin and cytoplasmic toxin activity, but did not react with Bacillus thuringiensis var. israelensis crystal toxin, nor did var. israelensis toxin antiserum react with B. sphaericus toxin. Crystallike parasporal inclusions accompanying the B. sphaericus 1593 spores were removed by NaOH extraction.

scholarly journals Characterization of Trypsin Inhibitor in Florunner Peanut Seeds (Arachis hypogaea L.)1

1988 ◽  
Vol 15 (2) ◽  
pp. 81-84 ◽  
Author(s):  
E. M. Ahmed ◽  
J. A. Applewhite
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Arachis Hypogaea ◽  
Trypsin Inhibitor ◽  
Low Molecular Weight ◽  
Arachis Hypogaea L ◽  
Amino Acid Profiles ◽  
Low Levels

Abstract Florunner peanut seeds contained five trypsin isoinhibitors. Amino acid profiles of the trypsin inhibitors fraction showed high levels of aspartic acid, half-cystine and serine and low levels of histidine and tyrosine. The molecular weight of the inhibitor was 8.3 KDa. The presence of multiforms of this inhibitor, its low molecular weight and the high amount of half-cystine indicate that peanut trypsin inhibitor is of the Bowman-Birk type.

scholarly journals The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying α1,4-N-acetylgalactosaminyltransferase

2004 ◽  
Vol 382 (1) ◽  
pp. 67-74 ◽  
Author(s):  
Ján MUCHA ◽  
Jiří DOMLATIL ◽  
Günter LOCHNIT ◽  
Dubravko RENDIĆ ◽  
Katharina PASCHINGER ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Bacillus Thuringiensis ◽  
Pichia Pastoris ◽  
Genetic Basis ◽  
Methylation Analysis ◽  
Yeast Pichia Pastoris ◽  
Glycan Receptors ◽  
Cloned Cdna ◽  
Crystal Toxin ◽  
Culture Supernatants

Insects express arthro-series glycosphingolipids, which contain an α1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian α1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcβ1,4GlcNAcβ1-R α-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal β-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an α1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.

Study of the Proteolytic Activity of the Tropical Legume Crotalaria spectabilis

2012 ◽  
Vol 67 (9-10) ◽  
pp. 495-509 ◽  
Author(s):  
Juliana da Silva Pacheco ◽  
Raquel Elisa da Silva-Lopez
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
Ph Value ◽  
Cysteine Proteases ◽  
Leaf Extracts ◽  
Serine Protease Activity ◽  
Proteolytic Activities ◽  
Low Levels ◽  
Crotalaria Spectabilis

The characterization of legume proteases contributes to the understanding of the physiology of plants and their interaction with the environment. Thirteen extracts from various parts of Crotalaria spectabilis were made using different extraction systems. The highest protein content was found in seeds, and the most pronounced proteolytic activity was observed in leaf extracts, with an optimal pH value in the alkaline range. Proteases in extracts from roots, stems, and fl owers were active in various pH ranges. Proteases in all extracts were maximally active between 30 °C and 60 °C and were thermostable (24 h, 60 °C). Hemoglobin, bovine serum albumin, casein, and gelatin were hydrolyzed by C. spectabilis extracts in different ways. The highest serine protease activity was found in leaves. Seeds contained high levels of serine proteases and low levels of cysteine proteases. Flowers, roots, and stems contained different levels of serine, aspartic, and metalloproteases, respectively. The proteolytic activities in extracts were modulated by cations and oxidants to various degrees. C. spectabilis proteases are differentially expressed in distinctive organs, and their stability against heat and oxidants makes this plant an important source of stable proteases

scholarly journals Ca2+-Induced Cleavage of Human Platelet Polypeptides Mediated by the Calcium Ionophore A-23187

1977 ◽  
Author(s):  
Milica Jakábová ◽  
David R. Phillips
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteolytic Activity ◽  
Platelet Activation ◽  
Human Platelet ◽  
Calcium Ionophore ◽  
Lactic Dehydrogenase ◽  
Actin Binding ◽  
A 23187 ◽  
Hydrolysis Of

The effect of calcium on human platelet polypeptides was investigated. When lysed platelets were incubated with mM Ca++, two major intracellular polypeptides (Mr = 255,000 and 230,000) were found to rapidly disappear. A similar phenomenon was also observed when intact platelets were treated with the calcium ionophore A-23187 in the presence of mM Ca++. Determinations of lactic dehydrogenase activity in supernatant fractions demonstrated that these losses occurred before platelet lysis. Investigations into the identity of the high molecular weight polypeptides revealed that one (Mr = 255,000) had similar properties to actin binding protein. The loss of the high molecular weight polypeptides was accompanied by formation of lower molecular weight polypeptides (Mr = 135,000, 93,000 and 48,000), indicating that Ca++ activates a polypeptide cleavage mechanism. The Ca++-activated polypeptide cleavages were rapid, with significant changes being observed within the first 0.5 min of incubation. An obvious explanation for these effects is. that there is Ca++-activated proteolytic activity within platelets. The Ca++-activated proteolytic activity was determined by the hydrolysis of the artificial substrate azocasein. We found that more than 90% of the proteolytic activity in lysed platelets was due to Ca++-activated proteases. These studies show that Ca++-activated proteases may play an important role in platelet activation.

Response of Liverwort Cells to Peroxidizing Herbicides

1992 ◽  
Vol 47 (5-6) ◽  
pp. 394-399
Author(s):  
Shuji Iwata ◽  
Naoko Nakayama ◽  
Shunji Nakagawara ◽  
Yoshimoto Ohta ◽  
Takaharu Tanaka ◽  
...  
Keyword(s):  
Cell Growth ◽  
Oxidative Damage ◽  
Early Treatment ◽  
Protoporphyrin Ix ◽  
Cell Suspension Cultures ◽  
Exponential Phase ◽  
Marchantia Polymorpha ◽  
Late Exponential Phase ◽  
Kinetics Of

Cell suspension cultures of the liverwort, Marchantia polymorpha L. were found useful to study the influence of peroxidizing herbicides either on the greening process or on the fully green cells. The cells of both physiological stages exhibit a characteristic sensitivity to the herbicides. The sensitivity increased rapidly during the exponential phase of growth, reached a maximum during the late exponential phase, and then decreased in the stationary phase. We investigated the kinetics of accumulation of protoporphyrin IX (PPIX) in Marchantia cells treated with several peroxidizing herbicides at various stages of cell growth, and observed a correlation between accumulation of PPIX and herbicidal damage. The glutathione (GSH) content in the cell was also investigated to examine the role of GSH against herbicide treatment. In the light, GSH levels in the cells treated with AFM rose rapidly reaching a peak after 8 h, and rapidly decreased subsequently. The beginning of PPIX accumulation coincided with the decline of GSH after 8 h of treatment. Obviously, GSH plays a key role in protection against oxidative damage caused by AFM in the early treatment period. In the dark, AFM also induced an accumulation of GSH and PPIX, followed by a decline in GSH and PPIX contents during a 20 h incubation. The decline of PPIX was observed several hours after GSH starts to decrease, remaining at a constant level for the following 40 h, leading to accumulation of an other fluorescent still-unknown pigment.

scholarly journals The mannophosphoinositides of Corynebacterium aquaticum

1975 ◽  
Vol 148 (2) ◽  
pp. 253-258 ◽  
Author(s):  
J A Hackett ◽  
P J Brennan
Keyword(s):  
Stationary Phase ◽  
Exponential Growth ◽  
Growth Cycle ◽  
Exponential Phase ◽  
Late Stationary Phase ◽  
Late Exponential Phase

Besides the monomannophosphoinositide previously reported in Corynebacterium aquaticum small amounts of other, apparently more glycosylated, mannophosphoinositides have been identified in stationary phase cells. Moreover, by labelling cells with [32P]Pi, phosphatidylinositol was found, comprising about 1.5% of the stationary-phase phospholipids. 2. Pulse-chase experiments performed on cells in the late exponential phase of growth further suggested the sequence phosphatidylinositol leads to monomannophosphoinositide as the first step in the biosynthesis of the mannophosphoinositides. 3. Di-and tri-mannophosphoinositides are apparently the main mannophosphoinositides present during exponential growth. Monomannophosphoinositide predominates only in late stationary phase; in the earlier stationary phase, phosphatidylinositol comprises 50% of the phosphoinositide lipid, and tetramannophosphoinositide constitutes much of the remainder. 4. The metabolism and functions of the mannophosphoinositides are discussed, particularly in relation to changes in their composition throughout the growth cycle.

Characteristic Ratio and Plateau Modulus of 1,2-Polybutadiene. A Comparison with Other Rubbers

1990 ◽  
Vol 63 (5) ◽  
pp. 734-746 ◽  
Author(s):  
Jacques Roovers ◽  
Paul M. Toporowski
Keyword(s):  
Molecular Weight ◽  
Star Polymer ◽  
Dilute Solution ◽  
Plateau Modulus ◽  
Characteristic Ratio ◽  
Polymer Systems ◽  
Dilute Solution Properties ◽  
Linear Viscoelastic ◽  
Low Levels

Abstract In the course of work on linear and ring polybutadienes with 62% 1,2 units, a number of discrepancies were noted with data on polybutadienes of various microstructure available in the literature. For example, GNο=870 kPa for our 62% 1,2-polybutadiene. This is larger than GNο=730 kPa for a 56% 1,2-polybutadiene and GNο=550 kPa for a 78% 1,2-polybutadiene sample. The cis : trans ratio of our 62% 1,2-polybutadiene, prepared with potassium counterion, is 1 : 4, On the other hand, the cis : trans ratio of 62% 1,2-polybutadiene prepared with a modified Li catalyst is estimated to be 1 : 2. It is conceivable that the different cis : trans ratio leads to different properties at constant 1,2 content. Nevertheless, the low levels of both the cis and the trans units are not expected to cause more than minor differences in the properties of the polybutadienes. Correct values for GNο of model polymers are important for the study of the influence of the chemical structure on the melt characteristics of a polymer. For this reason, it was thought useful to reinvestigate 1,2-polybutadiene itself in some detail. The synthesis of narrow molecular-weight distribution 1,2-polybutadiene by anionic polymerization techniques has been described recently. The dilute-solution properties of 1,2-polybutadiene has been investigated. The melt rheology of two 1,2-polybutadiene samples have been studied, but no systematic study of the molecular-weight dependence of the melt properties was made. 1,2-Polybutadiene has been used as a component in block copolymers with 1,4-polybutadiene. These studies have permitted an investigation of the phase behavior of two rubbery blocks at room temperature. Poly(l,4-butadiene-graft-l,2-butadiene)s with well-defined composition and architecture have also been prepared. Hydrosilylated 1,2-polybutadiene has found use as the coupling agent for multiarm star polymer, and this method can easily be extended to the preparation of poly( l,2-butadiene-graft-l,4-butadiene). Hydrogenated 1,2-polybutadienes are prepared as model polymers for poly(l-butene). The synthesis and characterization of a series of 1,2-polybutadienes are described here. Special attention is given to low-molecular-weight polymers. The linear viscoelastic properties of the melts are also described. In the discussion, the relation between the characteristic ratio, C∞, and the plateau modulus, GNο, of a number of model polymer systems is explored.

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