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 THE USE OF FORMALDEHYDE-TREATED 131I-ALBUMIN IN THE STUDY OF DIGESTIVE VACUOLES AND SOME PROPERTIES OF THESE PARTICLES FROM MOUSE LIVER

1967 ◽  
Vol 32 (3) ◽  
pp. 699-707 ◽  
Author(s):  
John L. Mego ◽  
Francisco Bertini ◽  
J. Donald McQueen
Keyword(s):  
Trichloroacetic Acid ◽  
Mouse Liver ◽  
Inhibitory Effect ◽  
Protein Hydrolysis ◽  
Cathepsin Activity ◽  
High Concentrations ◽  
Triton X ◽  
Hydrolysis Of ◽  
High Sucrose ◽  
Stimulation Of

The trichloroacetic acid-soluble radioactivity released during incubation of mouse liver particles containing intravenously injected formaldehyde-treated 131I-albumin consisted almost entirely of 131I-iodotyrosine. The material was shown to be excreted into the medium and was not due to disruption of the particles by acid. Triton X-100 or the absence of sucrose in the medium inhibited hydrolysis of the particle-associated labeled protein. This inhibition was due to disruption of the digestive vacuoles and dilution of the protein and cathepsins in the suspending medium. These results and other experimental evidence strongly suggest that the 131I-albumin-containing liver particles are digestive vacuoles. The results also establish that 131I-albumin may be used to study these vacuoles. High concentrations of sucrose (1 M) inhibited degradation of intraparticulate protein. However, 1 M salts inhibited only the rate of the digestion. Sucrose had an inhibitory effect on a crude cathepsin preparation, and salts stimulated the activity when 131I-albumin was used as substrate. The effect of high sucrose concentrations as an inhibitor of protein hydrolysis within digestive vacuoles was, therefore, most likely due principally to an inhibition of cathepsin activity within the vacuoles. The effect of salt was probably caused by a stimulation of both intra- and extra-particulate cathepsin activities, although 0.5–1.0 M KCl appeared to protect the particles.

scholarly journals The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1979 ◽  
Vol 180 (3) ◽  
pp. 515-522 ◽  
Author(s):  
James Wilson ◽  
Arnaldo Corti ◽  
Margaret Hawkins ◽  
H. Guy Williams-Ashman ◽  
Anthony E. Pegg
Keyword(s):  
Rat Liver ◽  
Homoserine Lactone ◽  
Adenosylmethionine Decarboxylase ◽  
Constant Rate ◽  
14Co2 Production ◽  
Membrane Bound ◽  
Lag Period ◽  
High Concentrations ◽  
Triton X

1. The production of 14CO2 from S-adenosyl[carboxyl-14C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of 14CO2 could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56–69]. 2. The formation of 14CO2 by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5′-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce 14CO2 when incubated with [U-14C]-homoserine, and 14CO2 production was greater when S-adenosyl[carboxyl-14C]methionine that had been degraded by heating at pH6 at 100°C for 30min (a procedure known to produce mainly 5′-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-14C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that 14CO2 is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-14C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5′-methylthioadenosine and homoserine lactone.

scholarly journals Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys

1983 ◽  
Vol 211 (3) ◽  
pp. 743-753 ◽  
Author(s):  
I S Fulcher ◽  
A J Kenny
Keyword(s):  
Chelating Agents ◽  
Gel Filtration ◽  
Small Region ◽  
The Other ◽  
Kidney Cortex ◽  
Dimeric Molecule ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Hydrolysis Of ◽  
Positively Charged

The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.

Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes

1993 ◽  
Vol 264 (2) ◽  
pp. L146-L152
Author(s):  
R. Goke ◽  
F. Kolligs ◽  
G. Richter ◽  
B. Lankat-Buttgereit ◽  
B. Goke
Keyword(s):  
Divalent Cations ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Rat Lung ◽  
Single Class ◽  
Binding Data ◽  
Membrane Bound ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Triton X

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7–36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7–36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)- dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7–36)amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7–36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 microM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments 125I-labeled GLP-1(7-36)amide was covalently attached to GLP-1(7–36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTP gamma S or GDP beta S) were unable to reduce GLP-1(7–36)amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1974 ◽  
Vol 143 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Hans Vilhardt ◽  
Derek B. Hope
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Fractions ◽  
Neural Lobe ◽  
Bivalent Cations ◽  
Pituitary Glands ◽  
Membrane Bound ◽  
Triton X ◽  
Hydrolysis Of

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+or Ca2+and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.

scholarly journals Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion

1986 ◽  
Vol 234 (1) ◽  
pp. 13-20 ◽  
Author(s):  
H M'Zali ◽  
F Giraud
Keyword(s):  
Phosphatidic Acid ◽  
Membrane Vesicles ◽  
Acid Synthesis ◽  
Cholesterol Depletion ◽  
Membrane Domains ◽  
Human Erythrocyte Membrane ◽  
Phosphatidylinositol 4 ◽  
Triton X ◽  
Hydrolysis Of ◽  
Synthesis And Degradation

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].

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