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1.
The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase,
EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found
to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents
caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization
step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium
deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified
preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid
complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity
of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold
enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase
(CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched
with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor
of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable
inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization
of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate,
diacylglycerol. 相似文献
2.
In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate
fraction of rat brain. PLD activity was assayed by measuring the [3H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[3H]choline (dipalmitoyl [3H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to
markedly enhance PLD activity, whereas other detergents including sodium doexycholate, sodium cholate, CHAPS and Lubrol-PX
caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1–0.2% (w/v) and decreased
at higher concentrations. The optimal pH was 7.1–7.3. Both Ca2+ and Mg2+ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized
PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by
0.5–1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine)
were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain,
followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit
brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are
the preferred sources. 相似文献
3.
In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase
(PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate
from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may
affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate
than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence
and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate
substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100 The stimulatory
effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on32P-labeled substrate are rapid and sensitive. It is shown here that33P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus
posing less potential health hazards for the user. 相似文献
4.
Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape 总被引:1,自引:1,他引:0
Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase
(DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO4 and MgCl2 stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme
2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg2+ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease
when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small
organic compound(s) that stimulated microsomal DGAT activity. 相似文献
5.
Acyl-CoA elongases are important in producing high-erucic acid rapeseed. The effects of Triton X-100,N-octyl-β-D-glucopyranoside and deoxycholate on the C18:1-CoA and C20:1-CoA elongase(s) have been studied by using a 15,000
×g pellet from developing rapeseed. The synthesis of very long chain monounsaturated fatty acids (VLCMFA) and, in particular,
that of erucic acid were stimulated by Triton X-100, whatever the substrate used. In the presence ofN-octyl-β-D-glucopyranoside, the elongase activity was practically unchanged, whereas deoxycholate strongly inhibited VLCMFA
synthesis. Triton X-100 was chosen for the solubilization, at an optimal Triton X-100/protein (w/w) ratio of 2.5. Acyl-CoAs
were the major products synthesized by the solubilized acyl-CoA elongase(s). The analysis of the reaction intermediates showed
that the entire elongation complex has been solubilized and was still functional. 相似文献
6.
Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration,
and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported.
A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities
showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and
18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation
is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence
of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the
purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this
enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the
fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis
of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex. 相似文献
7.
Denis J. Murphy 《Lipids》1988,23(3):157-163
The subcellular distribution of the enzymes of triacylglycerol biosynthesis has been studied in developing oilseed rape. All
in vitro enzymatic activities from oleoyl-CoA to triacylglycerol were sufficient to account for the known rate of oleate deposition
in triacylglycerol in vivo. The enzymatic activities from oleoyl-CoA to diacylglycerol preferentially were localized in a
150,000 g supernatant fraction, while the diacylglycerol acyl-transferase mostly was associated with the microsomal (20,000
g pellet and 150,000 g pellet) and oil-body fractions. The soluble (150,000 g supernantant) fraction rapidly incorporated
oleate from [1-14C]oleoyl-CoA into diacylglycerol with rates of 40 nm min−1 g−1 FW at 20 μM oleoyl-CoA. The pH optimum was 7.5–9.0, and normal saturation kinetics were seen with oleoyl-CoA; the S0.5 was about 32 μM. Exogenous acyl acceptors, such as glycerol 3-phosphate, lysophosphatidic acid and lysophosphatidylcholine
stimulated oleate incorporation into diacylglycerol. The detergents Triton X-100 and sodium cholate inhibited diacylglycerol
formation at concentrations in the region of their critical micellar concentration, while n-octyl-β,D-glyco-pyranoside had
no effect, even at high concentration. The significance of these findings for the mechanism of oil-body formation in developing
oilseeds is discussed. 相似文献
8.
Rebecca W. Wilcox Tom Thuren Patricia Sisson Gregory L. Kucera Moseley Waite 《Lipids》1991,26(4):283-288
Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral
lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory
for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Vmax values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The Vmax values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg
for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface
dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When
the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual
combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only
for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in
similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid
substrates. 相似文献
9.
DAG derived from phosphatidylcholine (PtdCho) acts as a lipid second messenger. It can be generated by the activation of phospholipase
D (PLD) and the phosphatidic acid phosphohydrolase type 2 (PAP2) pathway or by a PtdCho-specific phospholipase C (PtdCho-PLC).
Our purpose was to study PtdCho-PLC activity in rat cerebral cortex synaptosomes (CC Syn). DAG production was highly stimulated
by detergents such as Triton X-100 and sodium deoxycholate. Ethanol and tricyclodecan-9-yl-xanthate potassium salt decreased
DAG generation by 42 and 61%, respectively, at 20 min of incubation. These data demonstrate that both the PLD/PAP2 pathway
and PtdCho-PLC contribute to DAG generation in CC Syn. PtdCho-PLC activity remained located mainly in the synaptosomal plasma
membrane fraction. Kinetic studies showed K
m and V
max values of 350 μM and 3.7 nmol DAG × (mg protein × h)−1, respectively. Western blot analysis with anti-PtdCho-PLC antibody showed a band of 66 KDa in CC Syn. Our results indicate
the presence of a novel DAG-generating pathway in CC Syn in addition to the known PLD/PAP2 pathway. 相似文献
10.
Acylated glycoglycerolipids were identified in the total lipid extract from cyanobacerium Synechocystis sp. PCC 6803. These compounds have a palmitoyl group esterified to the hydroxyl group at the C-6 position of the terminal
glycosyl moiety of digalactosyl monoacylglycerol and digalactosyl diacylglycerol. Their structural elucidation was accomplished
by tandem mass spectrometry coupled with fast atom bombardment ionization. Acylated digalactosyl monoacylglycerol has a structure
of 1-hydroxy-2-palmitoyl-3-O-[(6-O-palmitoyl)-α-d-galactopyranosyl-(1→6)-β-d-galactopyranosyl]-sn-glycerol. This compound has not been reported previously. 相似文献
11.
Aida Ibrik Henri Chahinian Nathalie Rugani Louis Sarda Louis-Claude Comeau 《Lipids》1998,33(4):377-383
An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes
emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against
partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35°C for 60 min and has maximal activity
in a pH range of 8–10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of
P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol. 相似文献
12.
Effect of acylation stimulating protein on the triacylglycerol synthetic pathway of human adipose tissue 总被引:3,自引:0,他引:3
Zivart Yasruel Katherine Cianflone Allan D. Sniderman Mark Rosenbloom Mark Walsh Miguel A. Rodriguez 《Lipids》1991,26(7):495-499
Acylation stimulating protein (ASP) is a 14 kDa plasma protein which causesin vitro triacylglycerol synthesis in human adipocytes and fibroblasts to increase substantially. ASP was found to stimulate human
adipose tissue microsomal glycerophosphate acyltransferase and diacylglycerol acyltransferase activities by 23% and 90%, respectively.
However, phosphatidate phosphohydrolase activity showed no increase in activity, nor did microsomal acyl-CoA synthetase activity.
Moreover, ASP did not decrease the apparent Km of diacylglycerol acyltransferase (DGAT), but rather increased its apparent Vmax suggesting direct interaction of ASP with DGAT. 相似文献
13.
Previous experiments showed that during the early stages (20–30 seconds) of aggregation induced by adenosine diphosphate (ADP,
2 μM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA
[ethylene glycol-bis-(β-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release
of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and
found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated
cytoskeletons. In both species and with either ADP or thrombin as aggregating agent, 70–85% of the label in both intact platelets
and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was
similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in
the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol
was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets.
The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the
intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and
palmitic acid into the cytoskeleton. Although intact rabbit platelets had more label in ceramide (6.7±2.9%) than intact human
platelets (1.5±0.9%), platelets of both species exhibited a three- to four-fold enrichment of labeled ceramide in the cytoskeletons.
Thus phospholipids and ceramide that are readily labeled with palmitic acid are selectively incorporated into the cytoskeleton
during the initial stages of platelet aggregation. 相似文献
14.
15.
Yan Feng Yong-Goe Joh Kazuhiko Ishikawa Hiroyasu Ishida Susumu Ando Tohru Yamagaki Hiroshi Nakanishi Shugui Cao Ikuo Matsui Yoshitsugu Kosugi 《Journal of the American Oil Chemists' Society》2000,77(11):1147-1152
Hyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A2 in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn-2 fatty acids in the following order: linoleoyl> oleoyl>arachidoyl phosphatidylcholine. Phospholipase A2 had similar activities toward l-α-1-palmitoyl-2-arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium.
The activity was enhanced more than 5–18-fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by
the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544
disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A2 was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using
10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). No activity of phospholipase A2 activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS-PAGE.
Portions of this article were presented at the Biocatalysis Symposium at the 91st Annual Meeting and Expo of the American
Oil Chemists’ Society in San Diego, CA, April, 2000. 相似文献
16.
Tim Stöveken Rainer Kalscheuer Alexander Steinbüchel 《European Journal of Lipid Science and Technology》2009,111(2):112-119
Bacterial acyltransferases of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) family possess a highly conserved HHXXXDG motif. In this study, we describe the first experimental evidence that this motif is part of the active site of WS/DGAT from the Acinetobacter baylyi strain ADP1 and that it is crucial for enzymatic activity. The second histidine residue of this motif (H133) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His132) also led to explicitly decreased activity. A complete loss of activity was only observed upon substitution of both histidine residues by leucine, revealing that both are necessary for maximal activity. In contrast, the replacement of Asp137 and Gly138 against alanine had only little effect on enzyme activity, thus demonstrating that they are not essential for WS/DGAT catalysis although belonging to the highly conserved motif. One peculiarity of WS/DGAT enzymes is their little substrate specificity regarding hydrophobic compounds. In this study, we demonstrated the inability of WS/DGAT to accept polar compounds as substrates. 相似文献
17.
Diana Linke Manfred Nimtz Ralf G. Berger Holger Zorn 《Journal of the American Oil Chemists' Society》2009,86(5):437-444
Two extracellular esterases were produced in submerged cultures of the basidiomycete Pleurotus sapidus. A foam fractionation device was designed and employed for the isolation of the esterolytic enzymes. The recovery of enzyme
activity in the liquefied foam strongly depended on the superficial gas velocity. High purification and enrichment factors
(E
a = 62.0, P = 15.5) were achieved using nitrogen at 1.87 cm min−1 within 100 min. Increasing the superficial gas velocity to 2.49 cm min−1 improved the recovery of total esterase activity in the foam to >95% at the expense of reduced enrichment and purification
factors. Differences in their physicochemical characteristics resulted in differing foaming properties of the two esterases
secreted by P. sapidus. By variation of the pH value of the culture medium and addition of Triton X-100, both esterases were successively and quantitatively
transferred into the foam in a two-step fractionation process. 相似文献
18.
Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 μM, strongly
increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 μM) or the combination of ferric ion
(10 μM) and ascorbic acid (500 μM), in the presence of reduced glutathione (GSH, 800 μM). This effect was not achieved using
heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic
acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent),
but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal
lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 μM). ATG similarly reversed an inhibition
of phosphatidyl-choline hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide
glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids.
ATG inhibited, in addition to the classifical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal
and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 μM
ATG. ATG, at concentrations up to 250 μM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence
of Triton X-100 (0.1%). These data demonstrate the potential of ATG to increase toxicity of lipid peroxidative stimuli by
inhibition of microsomal and cytosolic defense mechanisms. Although ATG did not inhibit Triton-enhanced PHGPX activity, overall
evidence points toward inhibition of this enzyme as the mechanism for ATG-augmented lipid peroxidation and supports the conclusion
that PHGPX plays a major role in the cellular defense mechanism. 相似文献
19.
Studies on the model plant Arabidopsis thaliana have uncovered the identities of most enzymatic components involved in seed storage lipid biosynthesis. However, much remains to be learned on how pathway interactions operate in the seed metabolic network. In this study, we dissected seed glycerolipid molecular compositional changes in the Arabidopsis mutant deficient in diacylglycerol acyltransferase 1 (DGAT1). Our results indicate that metabolic adjustments occurred in both phosphatidylcholine synthesis and deacylation in developing seeds. Ultrastructural changes of perturbed oil and protein bodies were also evident in cotyledon parenchyma cells. To unmask the physiological and developmental role associated with DGAT1-mediated neutral lipid biosynthesis, we attempted to combine dgat1 mutation with lpcat2 that harbors a defect in lysophosphatidylcholine acyltransferase 2 (LPCAT2). Disruption in both DGAT1 and LPCAT2 led to an apparent defect in pollen development that manifested as pollen sterility. Collectively, our results highlight a role of DGAT1 in both storage lipid synthesis and plant development. 相似文献
20.
The light mitochondrial fraction from rabbit liver was found to catalyze the hydrolysis of platelet-activating factor (PAF,
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the phospholipase C reaction to form 1-O-alkyl-2-acetyl-glycerol and phosphocholine. The highest
specific phospholipase C activity occurred in the liver and kidney. A subcellular survey showed that the enzyme was of lysosomal
origin. The enzyme was solubilized with 2% Triton X-100 from rabbit liver light mitochondria and purified ca. 600- to 700-fold
with a 17% yield using procedures that included hydroxyapatite, Sepharose 4B and isoelectric focusing column chromatography
followed by fast protein liquid chromatography. The enzyme consists of two forms having a pl of 4.7 and 5.8. Each form was
purified to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme migrated
to positions corresponding to apparent molecular weights of 33,000 and 75,000, respectively. The purified enzymes of pl 4.7
and 5.8 had pH optima of 8.2 and 8.5 and apparent Km values of 55.6 and 45.5 μM for PAF, respectively. Furthermore, their
phospholipase C activity was significantly inhibited by the addition of 1 mM EDTA. EDTA-inactivated enzyme, however, recovered
completely upon addition of Ca2+ to the original level. p-Chloromercuribenzoate markedly inhibited enzyme activity, suggesting that phospholipase C is a—SH
enzyme. The physiological role of the enzyme should be evaluated, considering its specificity for a highly potent, biologically
active ether-phospholipid. 相似文献