The hydrolysis of phosphatidylcholine by phospholipase A2 in hamster heart

1984 ◽  
Vol 62 (12) ◽  
pp. 1269-1274 ◽  
Author(s):  
Stanley W. Tam ◽  
Ricky Y. K. Man ◽  
Patrick C. Choy
Keyword(s):  
Microsomal Fraction ◽  
High Specificity ◽  
Subcellular Fractions ◽  
Phospholipase A ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Absolute Requirement ◽  
Rate Of Hydrolysis ◽  
Hamster Heart ◽  
Hydrolysis Of

The hydrolysis of acyl esters in phosphatidylcholine and phosphatidylethanolamine by phospholipase A in hamster heart subcellular fractions was investigated. Phosphatidylcholine was found to be a much poorer substrate than phosphatidylethanolamine for the cardiac phospholipase A. The rate of hydrolysis of phosphatidylcholine by microsomal phospholipase A was 10-fold less than with phosphatidylethanolamine as substrate. When 1-[1-14C]palmitoyl-2-acyl phosphatidyl-[Me-3H]choline was used as substrate, both phospholipase A1 and A2 activities were detected in all subcellular fractions, but the highest specific activities for both enzymes were located in the microsomal fraction. However, phospholipase A2 activity in all hamster heart particulate fractions was three to six times higher than phospholipase A1 activity. The hydrolysis of phosphatidylcholine by microsomal phospholipase A2 displayed an alkaline pH optimum and an absolute requirement for Ca2+ or Mg2+. The enzyme also depicted high specificity towards polyunsaturated acyl groups at the C-2 position of phosphatidylcholine.

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.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

1956 ◽  
Vol 34 (5) ◽  
pp. 967-980 ◽  
Author(s):  
Morris Kates
Keyword(s):  
Ethyl Ether ◽  
Phosphatidyl Choline ◽  
Phosphatidyl Ethanolamine ◽  
Enzymatic Reaction ◽  
Phosphatidyl Serine ◽  
Ph Optimum ◽  
Soybean Phosphatide ◽  
Optimum Ph ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

1956 ◽  
Vol 34 (1) ◽  
pp. 967-980 ◽  
Author(s):  
Morris Kates
Keyword(s):  
Ethyl Ether ◽  
Phosphatidyl Choline ◽  
Phosphatidyl Ethanolamine ◽  
Enzymatic Reaction ◽  
Phosphatidyl Serine ◽  
Ph Optimum ◽  
Soybean Phosphatide ◽  
Optimum Ph ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.

scholarly journals The catabolism of plasmenylcholine in the guinea pig heart

1986 ◽  
Vol 236 (2) ◽  
pp. 475-480 ◽  
Author(s):  
G Arthur ◽  
L Page ◽  
T Mock ◽  
P C Choy
Keyword(s):  
Guinea Pig ◽  
Phospholipase A2 ◽  
High Specificity ◽  
Experimental Conditions ◽  
Ph Optimum ◽  
Major Pathway ◽  
Guinea Pig Heart ◽  
Mitochondrial Fractions ◽  
Wide Range ◽  
Hydrolysis Of

The hydrolysis of the alkenyl bonds of plasmenylcholine and plasmenylethanolamine by plasmalogenase, followed by hydrolysis of the resultant lysophospholipid by lysophospholipase, has been postulated as the major pathway for the catabolism of these plasmalogens. However, the postulation was based solely on the presence of plasmalogenase activity towards plasmenylethanolamine and plasmenylcholine in the brain. In this study we have demonstrated the absence of plasmalogenase activity for plasmenylcholine in the guinea pig heart under a wide range of experimental conditions. Plasmenylcholine was hydrolysed by phospolipase A2 activities in cardiac microsomal, mitochondrial and cytosolic fractions. Phospholipase A2 activities in these fractions had an alkaline pH optimum and were enhanced by Ca2+. The enzymes also displayed high specificity for plasmenylcholine with linoleoyl or oleoyl at the C-2 position. Lysoplasmalogenase activity for lysoplasmenycholine was also detected and characterized in the microsomal and mitochondrial fractions. Since the cardiac plasmalogenase is only active towards plasmenylethanolamine but not plasmenylcholine, the catabolism of these two plasmalogens must be different from each other. We postulate that the major pathway for the catabolism of plasmenycholine involves the hydrolysis of the C-2 fatty acid by phospholipase A2, and hydrolysis of the vinyl ether group of the resultant lysoplasmenylcholine by lysoplasmalogenase.

The purification and characterization of a phospholipase A2 activity from the 106 000 × g pellet (microsomal fraction) of bovine brain acting on phosphatidylinositol

1982 ◽  
Vol 60 (2) ◽  
pp. 108-117 ◽  
Author(s):  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Fatty Acid Distribution ◽  
Phospholipase A ◽  
Strong Inhibition ◽  
Ph Optimum ◽  
Triton X

A phospholipase A2 acting on phosphatidylinositol (PI) has been purified from the 106 000 × g pellet (microsomal fraction) of bovine grey matter. The purification steps included extraction with Triton X-100 (0.05%), ammonium sulfate fractionation (20–50% fraction), consecutive column chromatographic runs on Sephadex G-200 and DEAE-Sephacel, and preparative gel electrophoresis (on 10.5% polyacrylamide gel). These steps achieved a purification of 1614 times. The purified enzyme ran as a single band on sodium dodecyl sulfate (SDS) gel electrophoresis. Molecular weight estimations gave values of 18 300 by SDS gel electrophoresis and 18 521 based on amino acid analysis. Amino acid analysis showed the presence of 173 residues with aspartic acid (46), glutamic acid (26) and glycine (21) being the most abundant. Single residues of cysteine, tyrosine, and arginine were measured. The remaining 11 amino acids were present in amounts ranging from 3 to 11 residues.The purified enzyme had a pH optimum of 7.4, was heat stable (to 70 °C), and was activated by Ca2+ (5 mM). Other divalent cations were either slightly inhibitory (Mg2+ and Mn2+) or strongly inhibitory (Zn2+). The nonionic detergents, Triton X-100 (0.02 to 0.03%) and octyl glucoside (30 mM) showed 70 and 25% stimulations, respectively. Other detergents showed no effect (Cutscum), slight inhibition (G3634A), or strong inhibition (cetyltrimethylammonium bromide). Determination of the apparent Km and Vmax by an Eisthenal–Cornish-Bowden plot gave values of 0.52 mM and 1440 nmol [1-14C]oleic acid min −1∙mg protein −1, respectively, for 1-acyl-2-[1-,14C]oleoyl-sn glycerol-3-phosphoinositol as substrate. The above plot confirmed the presence of a strong inhibition by substrate (i.e., PI) beyond 0.4 mM. The properties of this enzyme and its location (microsomal) make it uniquely different from other phospholipase A2 activities reported for brain. The microsomal location and preference for PI shown by this enzyme lend support to the view that it may function to form lyso-PI in a deacylation–reacylation cycle for altering the fatty acid distribution in PI.

scholarly journals sn-Glycero(3)phosphoinositol glycerophosphohydrolase. A new phosphodiesterase in rat tissues

1979 ◽  
Vol 182 (1) ◽  
pp. 39-45 ◽  
Author(s):  
R M C Dawson ◽  
N Hemington ◽  
D E Richards ◽  
R F Irvine
Keyword(s):  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Temperature Stability ◽  
Rat Tissues ◽  
Alkaline Ph ◽  
Stability Studies ◽  
Ph Optimum ◽  
Excess Substrate ◽  
Membrane Bound ◽  
Cyclic Phosphate

1. A phosphodiesterase that cleaves glycerophosphoinositol into glycerophosphate and inositol has been detected in rat tissues. 2. The enzyme requires Mg2+ (Mn2+) and has a pH optimum of 7.7. 3. The richest sources of the enzyme are kidney and intestinal mucosa. In pancreas subcellular fractions it occurs largely in the microsomal fraction. 4. The enzyme is inhibited by excess substrate and by the reaction product glycerophosphate. 5. Temperature-stability studies and other observations distinguish the enzyme from other membrane-bound phosphodiesterases active at an alkaline pH e.g. glycerophosphoinositol inositophosphohydrolase, glycerophosphocholine diesterase, inositol cyclic phosphate phosphodiesterase and phosphodiesterase I.

scholarly journals Glyceride lipases in nerve endings of guinea-pig brain and their stimulation by noradrenaline, 5-hydroxytryptamine and adrenaline

1973 ◽  
Vol 132 (2) ◽  
pp. 233-248 ◽  
Author(s):  
O. S. Vyvoda ◽  
C. E. Rowe
Keyword(s):  
Guinea Pig ◽  
Synaptic Vesicles ◽  
Protein Concentration ◽  
Synaptic Membranes ◽  
Ph Optimum ◽  
Triglyceride Lipase ◽  
Monoglyceride Lipase ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Guinea Pig Brain

1. Combined guinea-pig cortex and cerebellum was shown to contain triglyceride lipase, diglyceride lipase and monoglyceride lipase, which were assayed by the release of [1-14C]palmitate from [1-14C]palmitoylglycerol esters. Triglyceride lipase and diglyceride lipase were found in all particulate fractions. 2. With osmotically ruptured synaptosomes the rates of release of palmitate from glyceryl tripalmitate and glyceryl dipalmitate were 7–25μmol/h per g of protein and 0.18–0.69mmol/h per g of protein respectively. The logarithm of the rate of hydrolysis of glyceryl monopalmitate increased linearly with the logarithm of protein concentration. The pH optima of triglyceride lipase and diglyceride lipase were between 7 and 8. The pH optimum for monoglyceride lipase was approx. 8. 3. Triglyceride lipase and diglyceride lipase of osmotically ruptured synaptosomes were stimulated by noradrenaline, 5-hydroxytryptamine and adrenaline. Triglyceride lipase of isolated synaptic membranes was stimulated by 0.01–1mm-noradrenaline. Aging of membranes at 0°C decreased activity, which could still be stimulated by noradrenaline. Diglyceride lipase of isolated membranes was stimulated by 1μm–1mm-noradrenaline. The activity of triglyceride lipase in isolated synaptic vesicles was diminished by 1mm-5-hydroxytryptamine.

scholarly journals The pur7 Gene from the Puromycin Biosynthetic pur Cluster of Streptomyces alboniger Encodes a Nudix Hydrolase

1999 ◽  
Vol 181 (16) ◽  
pp. 4914-4918 ◽  
Author(s):  
J. C. Espinosa ◽  
J. A. Tercero ◽  
M. A. Rubio ◽  
A. Jiménez
Keyword(s):  
Biosynthetic Pathway ◽  
High Specificity ◽  
Reaction Rates ◽  
Amino Group ◽  
Nudix Hydrolase ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Strong Inhibitor ◽  
His Tag ◽  
Nudix Hydrolases

ABSTRACT Pur7 is the product of a gene from the puromycin biosyntheticpur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni2+ column. It showed a 3′-amino-3′-dATP pyrophosphohydrolase (nudix) activity which produced 3′-amino-3′-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg2+. Among a large variety of other nucleotides tested, only 3′-amino-3′-dTTP was a Pur7 substrate, although at lower reaction rates than 3′-amino-3′-dATP. These findings suggest that Pur7 has a high specificity for the 3′ amino group at the ribofuranoside moiety of these two substrates. The Km andV max values for these dATP and dTTP derivatives were 120 μM and 17 μM/min and 3.45 mM and 12.5 μM/min, respectively. Since it is well known that 3′-amino-3′-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3′-amino-3′-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.

Sign in / Sign up

Export Citation Format

Share Document