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1.
Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain 总被引：12，自引：0，他引：12
Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC. 相似文献
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George N. De Martino 《Archives of biochemistry and biophysics》1981,211(1):253-257
Soluble extracts from rat liver cytoplasm contained substantial endoprotease activity which was totally dependent on calcium ions. This activity was accounted for by two distinct proteases which were separated by anion-exchange chromatography. One protease was half maximally activated by 7 μm calcium while the other protease required 150 μm calcium for half-maximal activation. 相似文献
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Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69,000 and 63,000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57,000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lower than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that are distinct from at least one cytosolic form (PLC-II) of the enzyme. 相似文献
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Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex. The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase. The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex. 相似文献
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Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM
carboxymethyl
- FPLC
fast protein liquid chromatography
- NEPHGE
non-equilibrium pH gradient gel electrophoresis
- PAP
pokeweed antiviral protein
- RIP
ribosome-inactivating protein
A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK. 相似文献
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Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes. 相似文献
10.
Lin FG Cheng HF Lee IF Kao HJ Loh SH Lee WH 《Biochemical and biophysical research communications》2001,286(2):274-280
Four different isoforms of mammalian phospholipase C delta (PLCdelta) have been described. PLCdelta1, the best-understood isoform, is activated by an atypical GTP-binding protein. It has been suggested that it is a calcium signal amplifier. However, very less is known about other subtypes, including PLCdelta3. Therefore, in the present study, we examined the expression of PLCdelta3 in different human tissues. Moreover, the cellular underlying regulation for PLCdelta3 was studied in different cell lines. Our study showed that the mRNA and protein levels differed significantly among human tissues. The human PLCdelta3 gene was composed of 15 exons and 1 putative cAMP response element in the 5'-end promoter region. PLCdelta3 mRNA expression was downregulated by cAMP and calcium in both the human normal embryonic lung tissue diploid WI38 cell line and the glioblastoma/astrocytoma U373 cell line. However, mRNA expression showed no impact by PKC activators or inhibitors. This study shows the human PLCdelta3 expression pattern and is the first report that PLCdelta3 gene expression is downregulation by cAMP and calcium. 相似文献
11.
Electrophoretic characterization of hepatic alkaline phosphatase released by phosphatidylinositol-specific phospholipase C. A comparison with liver membrane and serum-soluble forms. 总被引：3，自引：1，他引：3 
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下载免费PDF全文 Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane. 相似文献
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N A Babenko 《Biokhimii?a (Moscow, Russia)》1991,56(2):346-353
The changes in the functional activities of sphingomyelinase and phospholipase C from rat liver cell plasma membranes were studied in postnatal ontogenesis in the presence of thyroxin and mercasolyl. It was found that endogenous phospholipases of plasma membranes control of phospholipid content in rat liver cells. The sphingomyelinase activity is under control of thyroid hormones, whereas that of phospholipase C which is phosphatidyl choline-specific, is unaffected by them. The data obtained testify to the possible involvement of diacylglycerols formed via enzymatic hydrolysis of phosphatidylcholine, in the regulation of the sphingomyelinase activity. 相似文献
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Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements. 总被引：5，自引：3，他引：5 下载免费PDF全文
下载免费PDF全文 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. 相似文献
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Presence of different phospholipase C isoforms in the nucleus and their activation during compensatory liver growth 总被引：1，自引：0，他引：1
Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the total PLC-activity corresponded to beta1b isoform, 30% to PLC-gamma1 and less than 10% to PLC-delta1. PLC-beta1b and -gamma1 were found in the nuclear matrix, while PLC-delta1 was detected in the chromatin. Two peaks of an increase in the total PLC-activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC-beta1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC-gamma1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC-delta1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy. 相似文献
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两种肚倍型蚜虫之两种同功酶及可溶性蛋白的比较 总被引：2，自引：0，他引：2
应用电泳分析方法,对青麸杨上幼锤形信子与长枣形倍子内蚜虫进行了酯酶同功酶、过氧化物同功酶及可溶蛋白质的比较研究。结果表明：经形态学鉴定同为肚倍蚜可致瘿形成纺锤形与长枣形倍子。2种倍形内蚜虫在可溶性蛋白及酯酶同功酶上有较大差异。相异系数分别为02571,0067。说明肚倍蚜种内已出现分化,不同变形体倍子内蚜虫在分子水平上已具有向不同生物型分化的趋势。 相似文献
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Properties of a novel GTP-binding protein which is associated with soluble phosphoinositides-specific phospholipase C 总被引：1，自引：0，他引：1
Recently we demonstrated the presence in calf thymocytes of a GTP-binding protein (G-protein) composed of three polypeptides, 54, 41, and 27 kDa, which was physically and functionally associated with a soluble phosphoinositides-specific phospholipase C (PI-phospholipase C). The properties of this G protein were further investigated with the following results. 1) In addition to the ability to bind [35S]guanosine-5'-[gamma-thio]triphosphate (GTP gamma S), the G-protein exhibited GTPase activity, which was enhanced by Mg2+, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but inhibited by sodium cholate, GTP gamma S and F-.2) The 54-kDa polypeptide was ADP-ribosylated by pertussis toxin and also by endogenous membrane-bound ADP-ribosyltransferase, but none of these three polypeptides was ADP-ribosylated by cholera toxin. 3) The G-protein did not cross-react with either anti-rat brain alpha 1 (alpha-subunit of inhibitory G-protein, G1), alpha 0 (alpha-subunit of other G1-like G-protein, G0) or beta gamma antibodies. 4) Incubation of this G Protein with GTP gamma S caused dissociation of the three polypeptides. 5) The 27 kDa polypeptide showed GTP-binding activity and enhanced the phosphatidylinositol 4,5-bisphosphate hydrolysis by purified PI-phospholipase C. These results suggest that the PI-phospholipase C-associated G-protein in calf thymocytes may be a novel one and that it is involved in the regulation of PI-phospholipase C activity. 相似文献
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Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076). 相似文献
