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Biomedical subjects

H Chap

Publications and source records attributed to H Chap.

At least 217 records · Page 12Linked to original sources
PubMed1984

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. II. Studies on glycerophospholipids.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various natural and synthetic phospholipids. Natural glycerophospholipids carrying a 1-acyl-bond were degraded in the following order of decreasing activity: phosphatidylcholine = phosphatidylinositol greater than 1-acyl-sn-glycero-3-phosphocholine greater than phosphatidylethanolamine greater than phosphatidylglycerol. Sodium deoxycholate was an activator with all the phospholipids tested, each one requiring its own optimal concentration of detergent. Whereas 1-alkyl-2-acyl-sn-glycero-3-phosphocholine remained fully insensitive to enzyme degradation, 2-acyl-sn-glycero-3-phosphocholine was hydrolysed to some extent. However, additional experiments involving time-course hydrolysis revealed that this was entirely due to the migration of the 2-acyl-chain to the sn-1 position. From studies using racemic or enantiomeric phosphatidylcholines, it was concluded that the enzymes are not stereospecific. Activity against 1-acylpropanediolphosphocholine was much lower than with 1-acyl-sn-glycero-3-phosphocholine, indicating that the 2-hydroxyl group (or the 2-acyl-ester group) participates in the substrate reactivity through a strong inductive effect. Some activity could be detected against 1,3-diacylglycero-2-phosphocholine (beta-phosphatidylcholine) and 1-acylglycol-2-phosphocholine. It is thus concluded that the failure of the lipases to hydrolyse the 2-acyl-bond in a natural phospholipid is due to the steric hindrance brought about by the acyl, alkyl or hydroxyl group present in the sn-1 position. The lipases might also be unable to hydrolyse acyl-ester bonds involving a secondary alcohol.

Animals↗
PubMed1983

Platelet activating factor (PAF-acether) promotes an early degradation of phosphatidylinositol-4,5-biphosphate in rabbit platelets.

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.

Animals↗
PubMed1983

Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.

Humans↗
PubMed1982

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases.

Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [14C]arachidonate/[3H]arachidonoylphosphatidylcholine or free [14C]oleate/[3H]oleoylphosphatidylcholine. Whereas [14C]arachidonate was incorporated at a 10-15-times higher rate than [14C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free 3H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A2 hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [3H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [14C]choline-labelled dipalmitoyl-and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.

Arachidonic Acid↗
PubMed1982

Isolation and characterization of plasma membranes from krebs II ascite cells using Percoll gradient.

1. Plasma membranes were isolated from Krebs II ascite cells grown in the mouse. Cells were disrupted by nitrogen cavitation in an isotonic alkaline buffer containing magnesium and ATP. Isolation was performed in an alkaline-buffered self-generating gradient of Percoll with an angular rotor. At each step of the preparation, the pH appeared as the critical aspect of our procedure. 2. External membrane markers were concanavalin A and 5'-nucleotidase (EC 3.1.3.5). They reached a relative specific activity of 10, whereas this value was only of 0.7 for the endoplasmic reticulum marker, NADH dehydrogenase (EC 1.6.99.3). 3. Plasma membrane from 4 ml packed cells were isolated within 1 h after homogenization with good yield: 50% and 67% of total [3H]concanavalin A and 5'-nucleotidase, respectively, were recovered in the two plasma membrane fractions. 4. Electron microscopy examination showed the presence of vesicles of different sizes devoid of other structural contaminants. 5. Using the specific binding of concanavalin A to the external cell membrane, it was calculated that about 50% of the total cell phospholipid and 10% protein are located in the plasma membrane. Their sphingomyelin content is much higher than in the whole cell, in contrast to phosphatidylinositol, known as a more specific endoplasmic reticulum phospholipid.

Animals↗
PubMed1982

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin.

Phospholipases A2 from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A2-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimics the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.

Animals↗
PubMed1981

Evidence for the lack of classical secretory phospholipase A2 in guinea-pig pancreas.

Several lipolytic enzymes from guinea-pig pancreas have been determined in a soluble extract and in a purified zymogen granule fractions. The positional specificity of phospholipolytic enzymes was detected using phospholipids bearing various radioactive labels. It is shown that guinea-pig pancreatic extracts are able to release both fatty acids from phosphatidylcholine, but with more efficiency towards the fatty acid occupying the 1-position of sn-glycerol. Evidence is given that guinea-pig pancreas lacks the classical secretory phospholipase A2 and that phospholipid digestion is achieved through the sequential action of phospholipase A1 and lysophospholipase.

Animals↗
PubMed1981

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas.

1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100. 2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37,000 and 42,000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3-9.4. 3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1. 4. Using doubly labelled phosphatidylcholine, a specificity, A1, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl2 and MgCl2, whereas sodium deoxycholate is required for maximal activity.

Animals↗
PubMed1981

Studies on topological distribution of arachidonic acid replacement in platelet phospholipids and on enzymes involved in the phospholipid effect accompanying platelet activation.

In this short review recent results obtained on platelet phospholipid metabolism are summarized. The first part reports a topological study of arachidonic acid (AA) replacement in platelet phospholipids. It is shown that incubation of platelets with radioactive free arachidonic acid leads to a labelling of the phospholipids present inside the platelet, whereas the exchange of intact phosphatidylcholine (PC) molecules with the plasma lipoproteins occurs on the platelet outer surface. This should allow a selective labelling of the small external pool of AA in order to follow its behaviour during platelet activation. In the second part, some enzymes involved in the metabolism of phosphatidylinositol (PI) have been further characterized. The first one is a diglyceride-lipase, which is located in the plasma membrane and releases the two fatty acids esterifying the diglycerides formed from PI by the action of the platelet phospholipase C. Such an enzyme is probably responsible for the release of AA from PI occurring upon platelet activation. On the other hand, cytosolic phospholipid exchange proteins able to catalyse the transfer of PI between membranes have been identified. The possible role of the enzymes involved in the acceleration of PI turnover occurring during platelet activation is discussed.

Arachidonic Acid↗