scholarly journals Studies on the phospholipases of rat intestinal mucosa

1970 ◽  
Vol 118 (2) ◽  
pp. 233-239 ◽  
Author(s):  
P. V. Subbaiah ◽  
J. Ganguly
Keyword(s):  
Fatty Acid ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Sodium Deoxycholate ◽  
Phospholipase A ◽  
Phospholipase B ◽  
Ester Linkage ◽  
Hydrolysis Of ◽  
Soluble Fractions

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (β[1-14C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the β-ester linkage of the lecithin molecule.

scholarly journals Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine

1979 ◽  
Vol 184 (2) ◽  
pp. 283-290 ◽  
Author(s):  
V A Ziboh ◽  
J T Lord
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Specific Activity ◽  
Feedback Regulation ◽  
Phospholipase A ◽  
Particulate Fraction ◽  
Prostaglandin F2 Alpha ◽  
Low Concentrations ◽  
Freeze Dried ◽  
Hydrolysis Of

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.

scholarly journals A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine

1983 ◽  
Vol 214 (2) ◽  
pp. 377-386 ◽  
Author(s):  
N S Gee ◽  
R Matsas ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Cell Types ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Hybridoma methodology has been used to produce a monoclonal antibody, GK 7C2, that binds specifically to microvillar endopeptidase-24.11 (EC 3.4.24.11). The antibody (an immunoglobulin G) was generated by fusion of mouse plasmacytoma cells with splenocytes from a Balb/c mouse immunized with pig kidney microvillar membranes. The identity of the antigen recognized by GK 7C2 was established by immuno-precipitation from detergent-solubilized pig kidney microvilli. The protein had an apparent Mr of 90 000 and contained endopeptidase activity sensitive to phosphoramidon. The identity was confirmed by immunoadsorbent purification of endopeptidase-24.11 by a column to which GK 7C2 had been attached. The endopeptidase, purified in a yield of 40%, was electrophoretically homogeneous and of specific activity comparable with that purified by other means. Fluorescence microscopy established that GK 7C2 bound specifically to the luminal membranes of kidney tubules and the intestinal mucosa. Thus endopeptidase-24.11 is located in the brush-border membranes of both cell types.

STUDIES ON THE ACYLATION OF LYSOLECITHINS

1963 ◽  
Vol 41 (9) ◽  
pp. 1983-1990 ◽  
Author(s):  
N. H. Tattrie ◽  
J. R. Bennett
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Sodium Methoxide ◽  
Cadmium Chloride ◽  
Phospholipase A ◽  
Chloride Complexes ◽  
Egg Lecithin ◽  
Hydrolysis Of

"Semi-synthetic" lecithins were prepared by the acylation of lysolecithin cadmium chloride complexes (the lysolecithins were produced from egg lecithin by hydrolysis with phospholipase A) in the presence of pyridine with 1-C14 myristoyl chloride. The labelled lecithins were hydrolyzed with phospholipase A to yield free fatty acids (94% of the label) and lysolecithins (6% of the label). However, when lysolecithins were enzymically acylated with 1-C14 linoleic acid and the resulting labelled lecithins cleaved with phospholipase A, no labelled fatty acid was detected in the lysolecithin fraction.Labelled lecithins were also enzymically synthesized with 1-C14 linoleic acid and lysolecithins prepared from egg lecithin by reaction with sodium methoxide. The labelled lecithins were hydrolyzed with phospholipase A and the labelled lysolecithins were isolated and reacylated enzymically with oleic acid. The hydrolysis of these lecithins with phospholipase A showed that the lysolecithins retained all the label.

Properties of phospholipase A2 isolated from rat serum

1991 ◽  
Vol 69 (5-6) ◽  
pp. 358-365 ◽  
Author(s):  
R. R. Baker ◽  
H.-y. Chang
Keyword(s):  
Fatty Acid ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Purification Step ◽  
Ph Optimum ◽  
Rat Serum ◽  
Absolute Requirement ◽  
Hydrolysis Of

Phospholipase A2 was extensively purified (1300- to 1400-fold) from rat serum using Sephadex G-100 chromatography. It eluted at a position corresponding to a molecular mass of about 15 kDa. This one purification step gave two bands on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The faster component had a molecular mass of 16 kDa and the slower band likely contained an aggregate of the faster component. Activity was associated with protein bands on nondenaturing gels. Enzyme activity was assessed using phosphatidylcholine or phosphatidylethanol-amine labelled at sn position 2 with radioactive arachidonate. Phosphatidylethanolamine gave higher specific activities than phosphatidylcholine. The enzyme has an absolute requirement for Ca2+ and a pH optimum at 7.4. This pH optimum was more prominent for phosphatidylethanolamine. Activity was inhibited by oleate or arachidonate when phosphatidylcholine was used as substrate, but added free fatty acid did not significantly affect the hydrolysis of phosphatidylethanolamine. Addition of bovine serum albumin (fatty acid free) to assays increased the rate of release of arachidonate from phosphatidylcholine, but not from phosphatidylethanolamine. Phospholipase A2 is present in serum likely as a consequence of blood coagulation and may release fatty acids from cellular membranes following hemorrhage.Key words: phospholipase A2, phosphatidylcholine, phosphatidylethanolamine, rat serum.

The interfacial calcium ion concentration as modulator of the latency phase in the hydrolysis of dimyristoylphosphatidylcholine liposomes by phospholipase A2

1991 ◽  
Vol 69 (10-11) ◽  
pp. 722-727 ◽  
Author(s):  
Marta S. Fernández ◽  
Ricardo Mejía ◽  
Eunice Zavala
Keyword(s):  
Fatty Acid ◽  
Electrostatic Potential ◽  
Calcium Ion ◽  
Time Course ◽  
Latency Period ◽  
Phospholipase A ◽  
Ion Concentration ◽  
Time Changes ◽  
Calcium Ion Concentration ◽  
Hydrolysis Of

Analysis of the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes catalyzed by porcine pancreatic phospholipase A2 at 18 °C shows that, in the presence of 10 mM NaCl, the length of the latency period in the presteady-state phase increases from 3 to 10.5 min when the CaCl2 concentration is reduced from 15 to 1 mM. This inverse dependence of the lag period on calcium ion concentration is seen more readily at 1 M NaCl, where the induction time changes from 13.5 to 42 min by decreasing the concentration of CaCl2 from 15 to 1 mM. To interpret these results, we took into account the small amount of fatty acid that is produced during the latency phases. The fatty acid generates a negative surface electrostatic potential and makes the interfacial concentration of calcium ions different from the concentration in the bulk solvent. Variations in the analytical concentrations of NaCl and CaCl2 affect both the interfacial calcium ion concentration and electrostatic potential, as estimated theoretically from Grahame and Boltzmann equations. According to these estimates, the length of the latency period diminishes with the increase of the interfacial calcium concentration, but does not show any logical dependence on the change in surface electrostatic potential.Key words: phospholipase A2, latency phase, interfacial calcium ion concentration, liposomes.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns

1982 ◽  
Vol 60 (4) ◽  
pp. 434-443 ◽  
Author(s):  
A. Berteloot ◽  
J. S. Hugon
Keyword(s):  
Membrane Proteins ◽  
Organ Culture ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Marker

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 ± 0.9) as compared with scrapings of intestinal mucosa (14.8 ± 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase–glucoamylase–isomaltase (band 4), alkaline phosphatase (bands 9–10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.

Effect of chloroquine on intestinal lipid metabolism

1987 ◽  
Vol 253 (5) ◽  
pp. G673-G678
Author(s):  
C. M. Mansbach ◽  
A. Arnold ◽  
M. Garrett
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Free Fatty Acid ◽  
Portal Vein ◽  
Intestinal Mucosa ◽  
Specific Activity ◽  
Acid Lipase ◽  
Storage Pool ◽  
Mesenteric Lymph ◽  
Glyceryl Trioleate

Most studies that have quantitated recovery of infused lipid in the intestinal mucosa and mesenteric lymph have only been able to recapture 50-75%. One possibility is that the missing lipid enters a triacylglycerol (TG) storage pool in the enterocyte and is hydrolyzed by lysosomal lipase, and the free fatty acid released is transported by the portal vein. This postulate was tested by comparing glyceryl trioleate (TO)-infused rats pretreated with the lysosomotropic drug, chloroquine (6.3 mg.kg-1.h-1) with saline controls. Chloroquine increased mucosal TG from 94 +/- 6 to 128 +/- 8 mumol. Additionally, the specific activity of the mucosal TG relative to the infused [3H]TO was reduced in the treated rats. The mucosal TG increase was not due to impaired TG output, which remained the same as controls. We conclude that the TG in the acid lipase-sensitive pool derives most of its glyceride-glycerol from endogenous sources. Furthermore, the increment in mucosal TG caused by chloroquine is not enough to explain the majority of the acyl groups unaccounted for in the mucosa and lymph after a TG infusion. For these a direct passage of acyl groups through the enterocyte is postulated.

STUDIES ON THE ACYLATION OF LYSOLECITHINS

1963 ◽  
Vol 41 (1) ◽  
pp. 1983-1990 ◽  
Author(s):  
N. H. Tattrie ◽  
J. R. Bennett
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Sodium Methoxide ◽  
Cadmium Chloride ◽  
Phospholipase A ◽  
Chloride Complexes ◽  
Egg Lecithin ◽  
Hydrolysis Of

"Semi-synthetic" lecithins were prepared by the acylation of lysolecithin cadmium chloride complexes (the lysolecithins were produced from egg lecithin by hydrolysis with phospholipase A) in the presence of pyridine with 1-C14 myristoyl chloride. The labelled lecithins were hydrolyzed with phospholipase A to yield free fatty acids (94% of the label) and lysolecithins (6% of the label). However, when lysolecithins were enzymically acylated with 1-C14 linoleic acid and the resulting labelled lecithins cleaved with phospholipase A, no labelled fatty acid was detected in the lysolecithin fraction.Labelled lecithins were also enzymically synthesized with 1-C14 linoleic acid and lysolecithins prepared from egg lecithin by reaction with sodium methoxide. The labelled lecithins were hydrolyzed with phospholipase A and the labelled lysolecithins were isolated and reacylated enzymically with oleic acid. The hydrolysis of these lecithins with phospholipase A showed that the lysolecithins retained all the label.

The Hydrolysis of Glycine Oligopeptides by Guinea-Pig Intestinal Mucosa and by Isolated Brush Borders

1973 ◽  
Vol 45 (6) ◽  
pp. 803-816
Author(s):  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Enzyme Reaction ◽  
Intestinal Lumen ◽  
Small Intestinal Mucosa ◽  
Peptidase Activity ◽  
Reaction Products ◽  
Brush Borders ◽  
Hydrolysis Of

1. The relative rates of hydrolysis of a series of glycine homopeptides by guinea-pig small intestinal mucosa and by isolated brush borders have been studied. Oligopeptides up to and including hexaglycine were hydrolysed. No activity was detected against a series of homopolypeptides of molecular weight 10000–100000. 2. Except for activity against diglycine, the peptidase activity was greater in the brush-border fraction than in the original homogenate. The relative activity of the peptidase in the brush borders compared to the homogenate increased with increasing length oligopeptide substrate. 3. Analysis of the enzyme reaction products indicated that the brush border contained an exopeptidase. Studies with peptide derivatives containing N- and C-terminal blocking groups and with tetraglycine synthesized with either the N- or C-terminal residues labelled, demonstrated that the brush borders contained an amino-oligopeptidase. 4. Studies with inhibitors and with purified gastric and pancreatic enzymes indicated that this glycine oligopeptidase activity was not due to enzymes from the intestinal lumen which had been adsorbed to the brush borders.

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