scholarly journals The effects of Triton X-100 and chlorpromazine on the Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities of rat lung

1989 ◽  
Vol 261 (2) ◽  
pp. 673-678 ◽  
Author(s):  
P A Walton ◽  
F Possmayer
Keyword(s):  
Heat Treatment ◽  
Enzymic Activity ◽  
Rat Lung ◽  
Independent Manner ◽  
Heat Stable ◽  
Amphiphilic Drug ◽  
Dependent Activity ◽  
Phosphatidate Phosphohydrolase ◽  
Triton X ◽  
Positively Charged

Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

1981 ◽  
Vol 59 (7) ◽  
pp. 500-510 ◽  
Author(s):  
Paul G. Casola ◽  
Fred Possmayer
Keyword(s):  
Phosphatidic Acid ◽  
Initial Period ◽  
The Other ◽  
Rat Lung ◽  
Dependent Activity ◽  
Order Of Magnitude ◽  
Membrane Bound ◽  
High Concentrations ◽  
Triton X ◽  
Hydrolysis Of

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5–10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10–20 min), the rates were comparable.

scholarly journals Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner

2009 ◽  
Vol 46 (3) ◽  
pp. 159-165 ◽  
Author(s):  
Dhammika N. Atapattu ◽  
Nicole A. Aulik ◽  
Darrell R. McCaslin ◽  
Charles J. Czuprynski
Keyword(s):  
Heat Treatment ◽  
Mannheimia Haemolytica ◽  
Independent Manner

scholarly journals Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia.

1981 ◽  
Vol 91 (3) ◽  
pp. 860-865 ◽  
Author(s):  
J J Rauh ◽  
D L Nelson
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
The Other ◽  
Paramecium Tetraurelia ◽  
Whole Cells ◽  
Molecular Weights ◽  
Heat Stable ◽  
Composition Characteristic

Extruded trichocysts are composed of a family of proteins with molecular weights between 15,000 and 20,000. We have used heat treatment and affinity chromatography on fluphenazine-Sepharose to purify calmodulinlike proteins from whole cells and from extruded trichocysts. The purified protein from trichocysts is indistinguishable from that of whole cells; it is heat-stable, activates brain phosphodiesterase in a Ca++-dependent fashion, changes mobility on SDS polyacrylamide gels in the presence of Ca++, contains 1 mol of trimethyllysine/17 kdaltons, and has the amino acid composition characteristic of calmodulins. Calmodulin is a major component of purified, extruded trichocysts, of which it represents between 1 and 10% by mass. The other proteins of the trichocyst also resemble calmodulin in several properties. Possible roles for calmodulin in the Ca++-activated extrusion of trichocysts is discussed.

scholarly journals Properties of phosphatidate phosphohydrolase in rat adipose tissue

1994 ◽  
Vol 301 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S C Jamdar ◽  
W F Cao
Keyword(s):  
Adipose Tissue ◽  
Plasma Membranes ◽  
Lipid A ◽  
Mesangial Cell ◽  
Intraperitoneal Administration ◽  
Subcellular Fractions ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound ◽  
Rat Adipose Tissue ◽  
Triton X

Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5′-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.

scholarly journals An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins

1999 ◽  
Vol 341 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Jože PUNGERĆAR ◽  
Igor KRIAJ ◽  
Ning-Sheng LIANG ◽  
Franc GUBENŠEK
Keyword(s):  
Bovine Brain ◽  
Enzymic Activity ◽  
Aliphatic Chain ◽  
Wild Type ◽  
Basic Residue ◽  
Enzyme Active Site ◽  
Single Mutant ◽  
Adsorption Surface ◽  
Interfacial Adsorption ◽  
Triton X

Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124 → I/K128 → E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128 → E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108 → N/K111 → N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128 → E) is equivalent to the AtxC(I124 → F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.

scholarly journals Glycosylation of ribonuclease A catalysed by rabbit liver extracts

1975 ◽  
Vol 146 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Z Khalkhall ◽  
R D Marshall
Keyword(s):  
Metal Ion ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Rabbit Liver ◽  
Absolute Requirement ◽  
Pancreatic Ribonuclease A ◽  
Triton X ◽  
Increased Activity ◽  
Asparagine Residue

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.

scholarly journals The nucleoid as a scaffold for the assembly of bacterial signaling complexes

2017 ◽  
Author(s):  
Audrey Moine ◽  
Leon Espinosa ◽  
Eugenie Martineau ◽  
Mutum Yaikhomba ◽  
P J Jazleena ◽  
...  
Keyword(s):  
Dna Binding ◽  
Independent Manner ◽  
Signaling Systems ◽  
Bacterial Signaling ◽  
Aminoacid Sequence ◽  
Membrane Bound ◽  
Gliding Bacterium ◽  
Positively Charged

ABSTRACTThe FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.AUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

scholarly journals Cross-regulation between RpfG and HtsHs to regulate antibiotic biosynthesis in Lysobacter

2020 ◽  
Author(s):  
Kaihuai Li ◽  
Gaoge Xu ◽  
Bo Wang ◽  
Guichun Wu ◽  
Fengquan Liu
Keyword(s):  
Gene Expression ◽  
Environmental Changes ◽  
Antibiotic Biosynthesis ◽  
Two Component System ◽  
Independent Manner ◽  
Heat Stable ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Sense And Respond

AbstractBacterial two-component systems (TCSs) sense and respond to environmental changes and modulate downstream gene expression. However, the mechanism of cross-talk between multiple TCSs is unclear. In this study, we report a previously uncharacterized mechanism by which the TCS protein RpfG interacts with hybrid two-component system (HyTCS) proteins HtsH1, HtsH2 and HtsH3 to regulate antibiotic biosynthesis in Lysobacter. RpfG, a phosphodiesterase (PDE), can degrade c-di-GMP to 5’-pGpG and can regulate antibiotic heat-stable antifungal factor (HSAF) biosynthesis in a PDE- independent manner. Thus, we wondered whether RpfG regulate HSAF biosynthesis through interactions with other factors. Subsequently, we demonstrated that RpfG interacts with three HyTCS proteins (HtsH1, HtsH2 and HtsH3), that can inhibit the PDE enzymatic activity of RpfG. Importantly, deletion of htsH1, htsH2 and htsH3 resulted in significantly decreased HSAF production, and we showed that HtsH1, HtsH2 and HtsH3 depend on their phosphorylation activity to directly regulate HSAF biosynthesis gene expression. Our results reveal that RpfG does not depend on PDE activity to regulate HSAF biosynthesis, rather it interacts with HtsH1, HtsH2 and HtsH3 to do so, a regulatory mechanism that may be a conserved paradigm in Lysobacter and Xanthomonas.

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