scholarly journals The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain

1978 ◽  
Vol 176 (2) ◽  
pp. 475-484 ◽  
Author(s):  
R F Irvine ◽  
N Hemington ◽  
R M C Dawson
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Gel Filtration ◽  
Phosphatidyl Inositol ◽  
Temperature Stability ◽  
Enzyme Systems ◽  
Phosphatidic Acid Phosphatase ◽  
Bivalent Metals ◽  
Hydrolysis Of ◽  
Marked Specificity

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.

scholarly journals The action of phosphatidylinositol-specific phospholipases C on membranes

1976 ◽  
Vol 154 (1) ◽  
pp. 203-208 ◽  
Author(s):  
M G Low ◽  
J B Finean
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Phosphatidyl Inositol ◽  
Detectable Effect ◽  
Erythrocyte Ghosts ◽  
Membrane Phospholipids ◽  
Liver Microsomal Fraction ◽  
Hydrolysis Of ◽  
Low Activity

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte “ghosts” and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte “ghosts” together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.

scholarly journals Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies

1988 ◽  
Vol 256 (2) ◽  
pp. 453-459 ◽  
Author(s):  
O Nakanishi ◽  
Y Homma ◽  
H Kawasaki ◽  
Y Emori ◽  
K Suzuki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Homogenate ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

scholarly journals Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

1979 ◽  
Vol 182 (2) ◽  
pp. 599-606 ◽  
Author(s):  
Donald E. Richards ◽  
Robin F. Irvine ◽  
Rex M. C. Dawson
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Lysosomal Enzymes ◽  
Microsomal Fraction ◽  
Water Soluble ◽  
Membrane Phospholipids ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Hydrolysis Of

(1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.

scholarly journals Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA

1993 ◽  
Vol 295 (1) ◽  
pp. 81-86 ◽  
Author(s):  
J J Mukherjee ◽  
F T Jay ◽  
P C Choy
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Neutral Lipids ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Rat Liver Microsomes ◽  
Terminal Sequence ◽  
Physiological Control ◽  
Apparent Homogeneity ◽  
Hydrolysis Of

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

scholarly journals Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements

1987 ◽  
Vol 248 (3) ◽  
pp. 791-799 ◽  
Author(s):  
S J Taylor ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Maximal Effect ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Membrane Bound ◽  
Hydrolysis Of

The effect of the GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5′-[beta gamma-imido]triphosphate, but not with adenosine 5′-[gamma-thio]triphosphate, and was inhibited by guanosine 5′-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.

scholarly journals Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-l-carnitine hydrolase from rat liver

1979 ◽  
Vol 181 (1) ◽  
pp. 119-125 ◽  
Author(s):  
R K Berge ◽  
B Døssland
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Temperature Stability ◽  
Fatty Acyl ◽  
Mitochondrial Matrix ◽  
Microsomal Enzymes ◽  
Coa Hydrolase ◽  
Hydrolysis Of ◽  
Temperature Optima ◽  
Matrix Enzyme

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.

Biosynthesis of Mitochondrial Phospholipids Using Endogenously Generated Diglycerides

1975 ◽  
Vol 53 (7) ◽  
pp. 784-795 ◽  
Author(s):  
W. C. McMurray
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
De Novo ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Mitochondria ◽  
Hydrolysis Of ◽  
Stimulation Of

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [γ32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.

Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase

1970 ◽  
Vol 48 (7) ◽  
pp. 799-804 ◽  
Author(s):  
C. Hétu ◽  
R. Gianetto
Keyword(s):  
Rat Liver ◽  
Molecular Sieve ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Filtration Step ◽  
Hydrolysis Of ◽  
Menten Constant

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.

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