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Effect of nucleotides on the incorporation of myo-inositol into phosphatidylinositol in rat liver microsomes
Int. J. Biochem. Vol. 16, No. 12, pp. 1367-1371, 1984 Printed in Great Britain. All rights reserved
0020-711X/84 $3.00 + 0.00 Copyright (‘1 1984 Pergamon Press Ltd
EFFECT OF NUCLEOTIDES ON THE INCORPORATION OF mp-INOSITOL INTO PHOSPHATIDYLINOSITOL IN RAT LIVER MICROSOMES J~ZEF ZBOROWSKI
Department
of Cellular
Biochemistry,
and
GRAZYNA
SZYMA~~SKA
Nencki Institute of Experimental Warsaw, Poland
Biology,
3 Pasteur
Street, 02-093
(Received 1 December 1983) Abstract-l. In rat liver microsomes the incorporation of inositol in the presence of Mn’+ was stimulated by cytidine nucleotides, whereas it was inhibited by other nucleotides. 2. At low concentrations of CMP, AMP and other nucleotides stimulated inositol incorporation. No such effect was observed when the concentration of CMP was 2mM or higher. 3. It was found that an appreciable hydrolysis of CMP to cytidine and inorganic phosphate occurred during incubation with microsomes in the presence of Mg’+ or Mn2+. AMP was hydrolysed at a
comparable rate. 4. The activatory effect of AMP and other nucleotides on the CMP-dependent incorporation of inositol could be ascribed to protecting CMP against hydrolysis. INTRODUCTION
(standard laboratory diet) and water before the experiments. Livers, removed immediately after killing the animals, were homogenised in cold 225 mM mannitol75 mM sucrose-O.1 mM EGTA-2 mM Tris-I-ICI (pH 7.4). After sedimenting the mitochondrja, the supernatant was centrifuged at 20,OOOg for 15 min and the pellet discarded. The microsomal fraction was sedimented at 105,OOOg for 60 min and suspended in 300 mM sucrose (pH 7.4) containing 1 mM dithiothreitol. The suspended microsomes were divided into small portions and stored at -20°C. Within 1-3 months no essential loss of activity of the enzymic reactions studied was observed.
It is now well established that the incorporation of myo-inositol into phosphatidylinositol of animal tissues proceeds in two different reactions. One of them is an exchange of the m_vo-inositoi moiety with of membrane-bound phosphatidylinositol exogenous myo-inositol: the other one is de nouo synthesis of phosphatidylinositol from CDP-
diacylglycerol and phatidyltransferase
myo-inositol catalysed by phos(EC 2.7.X. I 1). Both enzymes are
present in the microsomal fraction of liver (Paulus and Kennedy, 1960; Strunecka and Zborowski, 1975; Takenawa and Egawa, 1917; Takenawa et ul., 1977). The enzyme catalysing the first reaction requires specificaily Mn2+, whereas the Iatter reaction can be stimulated by either Mg2+ or Mn2* (Paulus and Kennedy, 1960; Takenawa et al., 1977; Zborowski and Brindley, 1983). Hokin-Neaverson et al. (1977) using microsomal preparation of mouse pancreas and Bleasdale e? al. (1979) for rabbit lung microsomes provided evidence that phosphatidyltransferase can also operate in the reverse direction resulting in formation of CDPdiaphos~JJO -inositol from and cylglycerol phatidylinositol and CMP. Therefore, in the presence of CMP this enzyme can catalyse the inco~orat~on of [3H]inositoI into phosphatidylinositol even in the absence of added CDPdiacylglycerol. In this case the two exchange systems can be differentiated by their dependence on divalent metal cations, Mg2+ and Mn’+. in the present investigation these two exchange reactions were followed in rat liver microsomes and the effect of various nucleotides thereupon was studied. MATERIALS
Incubation This was carried out under constant shaking at 37’ C in 0.25 ml samnles. The standard medium contained: 100 mM Tris-HCl (pH 7.4). I mM dithiothreitol, 0.1 mM EGTA, 2 mM ~~y~-[2-~H~inositol, fatty acid-poor bovine serum albumin 6 mg/ml and microsomai fraction 2-3 mg protein/ml. Mn?+, Mg’+, CMP, AMP and other nucleotides were added where indicated. The incubations were stopped by adding 1.88 ml of CHCl,/CH,OH (1:2, v/v). Lipid exrraction and ident#ication Lipids were extracted by the method of Hajra et al. (1968) as described by Brindley and Bowley (1975). The top phase was separated from the CHCI, layer, which was then washed three times with synthetic top phase (Brindley and Bowley, 1975). Aliquots of the washed bottom phase were taken for determination of radioactivity. Phosphatidylinositol was identified by thin layer chromatography using silica gel 60 plates (Merck A.G., Darmstadt, F.R.G.) in two-step, one dimensional system according to Neskovic and Kostic (1968) or in two-dimensional system described by Rouser et ai. (1970). Radioactive phospholipid was visualised by autoradiography, whereas the unlabelled lipids by iodine vapour. Hydrolysis of monophosphate nucleosides
AND METHODS
Animals and the preparation of liwr microsomes Rats of both sexes of the Wistar strain (150-200 g) were used. The animals were allowed free access to food
This was determined by liberation of inorganic phosphate from CMP and AMP. The incubation medium was as described abcve for inositol incorporation experiments with the exception that it contained unlabelled inositol. Controls without the microsomal fraction were run.
1367
JOSEF:ZB~R~WSKI and GRA~YNA SZYMA~SKA
I368
Qualitative identification of cytidine and adenine as hydrolvsis products was perfomled using TLC aluminium sheets (2i x 20cm) covered with cellulose FzGii(0.1 mm of layer thickness) (E. Merck. Darmstadt, F.R.G.). The solvent system was isopropanol-25”,;, NH, aq.-H,O (4: I :4, V/V).
Radioactivity was measured by liquid scintillation counting. Protein content was estimated by the method of Lowry et ui. (1951). Inorganic phosphate was determined according to Fiske and Subbarow (1925). Solutions of sucrose and nucleotides were adjusted to pH 7.4 with solid KHCOl.
~~~-~~-~H]lnositol (3.9- II.65 Ci:mmol) was obtained from the Radiochemical Centre (Amersham. U.K.). nr.~oinositol and the nucleotides were from Sigma (St Louis. Missouri, U.S.A.) except of ATP, CTP and UTP that were purchased from Pharma-Waldhoff (Dusseldorf, F.R.G.). Boehringer (Mannheim GmbH, F.R.G.) and Calbiochem (Los Angeles, California, U.S.A.) respectively. All other reagents were of analytical grade. RESULTS
The Mn*+-dependent incorporation of ‘ftyoinositoi into phosphatidylinositol of rat liver microsomes showed a linearity upon protein up to 6.6 mg protein/ml and upon time up to 30 min in the standard medium in the presence of 20 mM MnCl,. This inco~o~~t~on was inhjbited by adenine nucleotides and other nucleoside triphosphates except CTP (Table 1). The inhibitory effect, however, could not be fully ascribed to the binding of Mn” by nucleotides, since a low concentration of ATP inhibited inositol incorporation by about 50% whereas the decreasing concentration of Mn” from 20mM to 2 mM gave only 2Oyj; inhibition (Table 1, Expt 1). A slight inhibition was exerted by inorganic phosphate and ribose 5-phosphate whereas D-ribose was without effect (not shown).
Figure 1 shows the effect of AMP concentration on inositol incorporation in the presence and absence of CMP. The reaction was measured in the presence of either Mn’+ or ME’+. In the absence of CMP the incorporation proceeded only with Mn’+ thus confirming an earlier finding (Paulus and Kennedy, Table t. Effect of nucleotides on inositoi incorporation ni~~et>ce of MI?+
1960; Takenawa et al. (1977); Takenawa and Egawa. 1980; Zborowski and Brindley, 1983) that this cation is required for the inositol exchange reaction. In full accordance with the data of Table I, the addition of AMP inhibited the reaction, showing a more pronounced effect with increasing concentration of the nucleotide (Fig. 1A). Addition of CMP stimulated both MI?‘and Mg”-dependent incorporation. With Mg’ ! , AMP had a strong stimulatory el‘fect (Fig. IB), whereas a slight inhibitory effect was observed in the presence of Mn’+ (Fig. 1A). This latter effect could be ascribed to the inhibition of inositol incorpor~ition catalysed bv the inositol exchange enzyme (see lower curve in F”ig. IA). As shown in Fig. I B, a threefold stimulation of the CMP-dependent incorporation of inositol in the presence of Mg’+ was obtained by addition 01 5510mM AMP. However, even then the amount of inositol incorporated was lower than in the presence of Mn”. This can be explained taking into consideration that in the presence of Mn” both the phosphatidyltransferase reaction and the inositol exchange take place, whereas in the presence of Mg’ ’ only the former reaction can proceed. It may also be worthwhile to stress that the values found for inositol incorporation in the presence of Mg’+ at each AMP concentration corresponded well to those with Mn’ ’ after subtraction of the (IMP-independent incorporation. Therefore, the incorporation of inositol in the presence of Mg.” could be taken as a measure of phosphatidyltransferase activity. The optimum concentration of Mg’+ and Mn” for the CMP-dependent inositol incorporation was lo-20mM (Fig. 2). The optimum CMP concentration in the presence of 10 mM Mg’+ was 1 mM {Fig. 3). The stimulatory effect of AMP on CMPdependent inositol incorporation in the presence of Mg” was not observed at the concentrations of CMP of 2mM and higher with AMP in the range of 0.55IOmM (data not shown). AMP was not the only nucleotide able to stimu~~~te the inositol incorporation at low concentration of CMP in the presence of Mg’+. Other mono-, diand triphosphate nucleosides were effective as well (Table 2). The nucleotides stimulated the inositol incorporation only in the presence of CMP but could not substitute for CMP in the presence of either Mg’ ’ (Table 2) or Mn2+ (not shown). The stimuiatory effect of CMP disappeared if microsomes were pre-
in the
Inositol incorporation (nmoliml: protein) Nucleotide added
Expt
None None (Ma’+, 2mM) AMP, 2mM
2.94 2.37 1.76
ADP. ATP, ATP,
1.71
2mM 2mM 6mM
GTP. 6mM ITP, UTP,
6mM 6mM
(TP
6mM
Incorporation
I .8?
I
Expt 2 2.99
1.54 1.36 I .25 0.59 0.84 7.76
of inositol was measured in the presence of 20 mM Mn”. Other conditions as in “Materials and Methods”. In both experiments microsomes amounted to 2.18 mg protein:ml.
AMP
tmMi
AMP
imM)
Fig. I. Effect of AMP on inositol incorporation in the absence and presence of CMP. Incubations were performed in the presence of either 2OmM Mn’ ’ (A) or Mg” (B). Microsomes were added in amount of 2.4 mg proteinml. Other conditions as in Materials and Methods. CMP absent (0). 0.5 mM CMP present (a).
mj&nositol
I
,
IO
5
Cotton
I
1
15
20
(mM)
Fig. 2. The requirement of CMP-dependent inositol incorand Mg’+. Microsomes (2.4 mg poration for Mn’+ protein/ml) were incubated in the presence of Mg’+ (0) or Mn* + (0). CMP concentration was 0.5 mM.
incubated with this nucleotide for 30min at 37°C prior to the addition of [3H]inositol. The addition of 2 mM AMP or other nucleotides to the preincubation medium partly protected the stimulatory effect of CMP (not shown). This protective effect of AMP on the CMPdependent incorporation of inositol was also evident in the time dependence. In the presence of AMP the
o-
IO CMP (mM)
Fig. 3. Effect of CMP on inositol incorporation in the presence of Mg’+. Microsomes (2.58 mg protein/ml) were incubated in the presence of IOmM MgCI,.
Table 2. Effect of nucleotides on inositol incorporation oresence of Me” Nucleotide added None CMP, CMP, GMP, IMP, ADP, AT?, ITP. UTP, CMP, CMP, CMP, CMP, CMP, CMP, CMP,
0.5 mM 5.0mM 5.0 mM S.OmM 5.0mM 5.0mM S.OmM 5.0 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM
i AMP, 5.0 mM + GMP, 5.0 mM + IMP, 5.0 mM + ADP, 5.0 mM + ATP, 5.0 mM + ITP, 5.0 mM + UTP, 5.0 mM
incorporation
1369
Fig. 4. Effect of AMP on the time-course of CMPdependent inositol incorporation. Microsomes (2.66 mg protein/ml) were incubated with 0.5 mM CMP without (0) or with 2.0 mM AMP (e). 10 mM Mg’+ was present in the incubation medium.
reaction was proceeding linearly for 30 min, whereas in the absence of AMP it levelled off after 15min (Fig. 4). The linearity of CUP-dependent incorporation was also protected when AMP was substituted by equimolar concentration of GMP or IMP which is in accordance with the data of Table 2. The levelling off of CMP-dependent inositol incorporation observed in the absence of other nucleotides suggested that CMP hydroiysis may be a possible explanation. Indeed, when microsomes were incubated with CMP in the presence of Mg”+ or Mn2+ cytidine could be detected chromatographically after 30min at 37°C. A quantitative estimation of CMP hydrolysis was done by measuring the liberation of inorganic phosphate. As shown in Fig. 5, the hydrolysis rate was maximal at l-2 mM Mn2+ and declined at higher concentrations, whereas for Mg*’ a plateau was observed for concentrations 5-20 mM. With the optimum concentration of either cation more than 45% of CMP was hydrolysed within 10 min of incubation. In order to ascertain whether the effect of AMP on inositol incorporation in the presence of CMP was due to the protective action against hydrolysis of the
in the
Inasitol incorooration (nmoi/mg protein) 0.56 2.44 2.24 0.97 0.87 0.22 0.38 0.18 0.25 3.69 5.79 6.44 5.25 5.48 6.84 7.13
Incubation was carried out in the presence of 10 mM Mg”. Microsomes in amount 2.58 mg protein/ml were added.
Cotlon
I mM i
Fig. 5. Dependence of CMP hydrolysis on varying concentrations of Mn*+ and Mg2+. Microsomes (2.18 mg protein/ml) were incubated in the presence of Mg*+ (0) and Mn2+ (a) in the medium containing unlabelled myoinoitol. The concentration of CMP was 5.0mM. Incubation was carried out for 10 min at 37°C. The reaction was terminated by addition of ice-cold trichloroacetic acid. The samples were centrifuged and an aliquot of each supernatant was taken for determination of liberated phosphate.
I370
JOSEF ZROROWSKI and GKA~YNA SZVM,&KA
Time ImJni Fig. 6. Hydrolysis of CMP and AMP in the presence of Mg’+. The concentration of each nucleotide was 5.0 mM and Mg” was IOmM. Other details as in the legend to
Fig. 5. (0) CMP, (0) AMP.
an experiment was performed in which the rate of hydrolysis of both nucleotides was measured. It is evident from Fig. 6 that the rate of hydrolysis of each of these nucleotides was the same. 96gi of the nucleotide was hydrolysed after 30min under conditions used.
latter,
DlSCLlSSlON
In the present study a different effect of nucleotides on the inositol-exchange reaction and CMPdependent incorporation of inositol into phosphatidylinositol is established. AMP, ADP, ATP and other mono-, di- and triphosphate nucleotides inhibit the Mn’+-dependent exchange reaction (Table I, Fig. 1A). Similar observation for triphosphate nucleotides including CTP has also been made by Takenawa and Egawa (1980) with the partially purified enzyme. According to these authors also other phosphorus containing acidic substances, among them phospholipids, phosphatidylserine and phosphatidic acid, act as inhibitors. In our experiments with rat liver microsomal membranes CTP stimulated inositol incorporation in the presence of Mn’+. A stimulatory effect of CTP has also been reported by Holub (1975). This effect may be, however, ascribed to the reaction catalysed by phosphatidyltransferas~, since CTP can be hydrolysed in the microsomal preparation to CMP. Such a mechanism of stimulation of inositol incorporation has also been proposed for rabbit lung microsomes (Bleasdale and Wallis, 1981). In fact. the presence of enzymes that hydrolyse tri- and diphosphate nucleosides in liver microsomes was demonstrated (Eppler and Morre, 1982). As shown in this work (Table 2) AMP and other nucleotides stimulate the CMP-dependent inositol incorporation. Upon addition of AMP the linearity of the inositol incorporation was maintained, whereas in its absence it levelled off after 15 min of incubation (Fig. 4). Such results strongly suggested that during incubation a hydrolysis of CMP occurred and the addition of AMP protected CMP by supplying another equivalent substrate. Indeed, it was found that both these nucleotides were hydrolysed with the same rate (Fig. 6). S-Nucleotidase (EC ~_.._ 3.1.3.5) and alkaline phosphatase (EC 3.1 .J.l) that
may be responsible for this hydrolysis most probably originated from plasma membrane fragments. However, 5’nucleotidase has also been reported as a constituent of the rough endoplasmic reticulum (Widnell, 1972). On the other hand, the possibility that the inhibition of CDPdiacylglycerol hydrolysis by AMP and other nucleotides (Raetz et ul., 1976) could contribute to the level of incorpoi-ation of inositol in the presence of CMP cannot be ruled out. Recently, Bleasdale and Wallis (1981) have provided an evidence that CMP-dependent incorporation of inositol into phosphatidylinositol of rabbit is not catalysed by phoslung microsomes phatidyltransferase but by separate enzymes less susceptible to heat exposure and distinct on the basis of inhibition by either Ca’+ or Hg’+. Whether this is also the case in liver tissue remains to be established. A~~no~~l~~~~~~~nt~~e would like to thank Professor Lech Wojtczak for his contin~l interest during this study and for critically reading the manuscript. REFERENCES BJeasdaJe J. E. and Wallis P. (1981) Phosph~tidy~inositolinositol exchange in rabbit lung. Bjffclzit~. hiuphy.~. Acra 664, 4288440. Bleasdale J. E., Wallis P., MacDonald P. C. and Johnston J. M. (1979) Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol: inositol transferase in rabbit lung tissue. Biochim. hiophys. Ada 575, 135147. Brindley D. N. and Bowiey M. (1975) Drug afhecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amcinchocaine. phetamine, chlorpromazine. demethylimipramine. mepyramine and some of their derivatives ~i~~~~i~~~.J. 148, 461-469. Eppler C. M. and Morre D. J. (1982) Flow kinetics of a nucleoside phosphatase common to endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat liver. Eur. .I. C?ll &/I. 29, 13-23. Fiske C. H. and Subbarow Y. (1925) The calorimetric determination of phosphorus. d. hi&. Chcnz. 66, 375400. Hajra A. K.. Seguin E. B. and Agranoff B. W. (1968) Rapid labeiling of mitochondrial lipids by labeled orthophosphate and adenosine triphosphate. J. hiol. Chem. 243, 1609-1616. Holub B. J. (1975) Role of cytidine triphosphate and cytidine diphosphate choline in promoting inositol entry into microsomai phosphatidylinositol. &iris 10,483-490. Hokin-Neaverson M., Sadeghian K.. Harris D. W. and Merrin J. S. (1977) Synthesis of CDP-diglyceride from phosphatidylinositol and CMP. Biochem. hiophl~s. Res. ?-o&run. is, 364-37 I t Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein m~dsurement with the Folin phenol reagent. J. hid. Chem. 193, 265-275. Neskovic N. M. and Kostic D. M. (1968) Quantitative _ analysis of rat liver phospholipids by a two-step thin-layer chromatographic procedure. J. Chrumutogr. 35,297-300. Paulus H. and Kennedy E. P. (1960) The enzymatic synthesis of inositol monophosphatide. J. hid. Chem. 235, 1303~13il. Raetr C. R. H., Dowhan W. and Kennedy E. P. (1976) Partial purification and characterization of Cytidine 5’-diphosphate-diglyceride hydrolase from membranes of Escherichiu co& J. LZacterioi. 12.5, 855-863. Rouser G.. Fleischer S. and Yamamoto A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analvsis of spots. Lipids 5, 494496. _
myo-Inositol Strunecka A. and Zborowski J. (1975) Microsomal synthesis of phosphatidylinositol and its exchange between subcellular structures of rat liver. Comp. Biochem. Physiol. 50B, 55-60. Takenawa T. and Egawa K. (1977) CDP-diglyceride: inositol transferase from rat liver. J. biol. Chem. 252, 5419-5423. Takenawa T. and Egawa K. (1980) Phosphatidyl inositol: mqjo-inositol exchange enzyme from rat liver: Partial purification and characterization. Archs Biochrm. Biophys. 202, 601-607. Takenawa T., Saito M., Nagai Y. and Egawa K. (1977) Solubilization of the enzyme catalyzing CDP-diglyceride-
incorporation
1371
independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride: inositol transferase. Archs Biochem. Biophys. 182, 244-250. Widnell C. C. (1972) Cytochemical localization of 5’-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5’nucleotidase activity in microsomes. J. Cell Biol. 52, 542-558. Zborowski J. and Brindley D. N. (1983) The metabolism of CDP-diacylglycerol and phosphatidylinositol in the microsomal fraction of rat liver. Effects of chlorpromazine, magnesium and manganese. Biochim. biophys. Acra 751, 81-89.
0020-711X/84 $3.00 + 0.00 Copyright (‘1 1984 Pergamon Press Ltd
EFFECT OF NUCLEOTIDES ON THE INCORPORATION OF mp-INOSITOL INTO PHOSPHATIDYLINOSITOL IN RAT LIVER MICROSOMES J~ZEF ZBOROWSKI
Department
of Cellular
Biochemistry,
and
GRAZYNA
SZYMA~~SKA
Nencki Institute of Experimental Warsaw, Poland
Biology,
3 Pasteur
Street, 02-093
(Received 1 December 1983) Abstract-l. In rat liver microsomes the incorporation of inositol in the presence of Mn’+ was stimulated by cytidine nucleotides, whereas it was inhibited by other nucleotides. 2. At low concentrations of CMP, AMP and other nucleotides stimulated inositol incorporation. No such effect was observed when the concentration of CMP was 2mM or higher. 3. It was found that an appreciable hydrolysis of CMP to cytidine and inorganic phosphate occurred during incubation with microsomes in the presence of Mg’+ or Mn2+. AMP was hydrolysed at a
comparable rate. 4. The activatory effect of AMP and other nucleotides on the CMP-dependent incorporation of inositol could be ascribed to protecting CMP against hydrolysis. INTRODUCTION
(standard laboratory diet) and water before the experiments. Livers, removed immediately after killing the animals, were homogenised in cold 225 mM mannitol75 mM sucrose-O.1 mM EGTA-2 mM Tris-I-ICI (pH 7.4). After sedimenting the mitochondrja, the supernatant was centrifuged at 20,OOOg for 15 min and the pellet discarded. The microsomal fraction was sedimented at 105,OOOg for 60 min and suspended in 300 mM sucrose (pH 7.4) containing 1 mM dithiothreitol. The suspended microsomes were divided into small portions and stored at -20°C. Within 1-3 months no essential loss of activity of the enzymic reactions studied was observed.
It is now well established that the incorporation of myo-inositol into phosphatidylinositol of animal tissues proceeds in two different reactions. One of them is an exchange of the m_vo-inositoi moiety with of membrane-bound phosphatidylinositol exogenous myo-inositol: the other one is de nouo synthesis of phosphatidylinositol from CDP-
diacylglycerol and phatidyltransferase
myo-inositol catalysed by phos(EC 2.7.X. I 1). Both enzymes are
present in the microsomal fraction of liver (Paulus and Kennedy, 1960; Strunecka and Zborowski, 1975; Takenawa and Egawa, 1917; Takenawa et ul., 1977). The enzyme catalysing the first reaction requires specificaily Mn2+, whereas the Iatter reaction can be stimulated by either Mg2+ or Mn2* (Paulus and Kennedy, 1960; Takenawa et al., 1977; Zborowski and Brindley, 1983). Hokin-Neaverson et al. (1977) using microsomal preparation of mouse pancreas and Bleasdale e? al. (1979) for rabbit lung microsomes provided evidence that phosphatidyltransferase can also operate in the reverse direction resulting in formation of CDPdiaphos~JJO -inositol from and cylglycerol phatidylinositol and CMP. Therefore, in the presence of CMP this enzyme can catalyse the inco~orat~on of [3H]inositoI into phosphatidylinositol even in the absence of added CDPdiacylglycerol. In this case the two exchange systems can be differentiated by their dependence on divalent metal cations, Mg2+ and Mn’+. in the present investigation these two exchange reactions were followed in rat liver microsomes and the effect of various nucleotides thereupon was studied. MATERIALS
Incubation This was carried out under constant shaking at 37’ C in 0.25 ml samnles. The standard medium contained: 100 mM Tris-HCl (pH 7.4). I mM dithiothreitol, 0.1 mM EGTA, 2 mM ~~y~-[2-~H~inositol, fatty acid-poor bovine serum albumin 6 mg/ml and microsomai fraction 2-3 mg protein/ml. Mn?+, Mg’+, CMP, AMP and other nucleotides were added where indicated. The incubations were stopped by adding 1.88 ml of CHCl,/CH,OH (1:2, v/v). Lipid exrraction and ident#ication Lipids were extracted by the method of Hajra et al. (1968) as described by Brindley and Bowley (1975). The top phase was separated from the CHCI, layer, which was then washed three times with synthetic top phase (Brindley and Bowley, 1975). Aliquots of the washed bottom phase were taken for determination of radioactivity. Phosphatidylinositol was identified by thin layer chromatography using silica gel 60 plates (Merck A.G., Darmstadt, F.R.G.) in two-step, one dimensional system according to Neskovic and Kostic (1968) or in two-dimensional system described by Rouser et ai. (1970). Radioactive phospholipid was visualised by autoradiography, whereas the unlabelled lipids by iodine vapour. Hydrolysis of monophosphate nucleosides
AND METHODS
Animals and the preparation of liwr microsomes Rats of both sexes of the Wistar strain (150-200 g) were used. The animals were allowed free access to food
This was determined by liberation of inorganic phosphate from CMP and AMP. The incubation medium was as described abcve for inositol incorporation experiments with the exception that it contained unlabelled inositol. Controls without the microsomal fraction were run.
1367
JOSEF:ZB~R~WSKI and GRA~YNA SZYMA~SKA
I368
Qualitative identification of cytidine and adenine as hydrolvsis products was perfomled using TLC aluminium sheets (2i x 20cm) covered with cellulose FzGii(0.1 mm of layer thickness) (E. Merck. Darmstadt, F.R.G.). The solvent system was isopropanol-25”,;, NH, aq.-H,O (4: I :4, V/V).
Radioactivity was measured by liquid scintillation counting. Protein content was estimated by the method of Lowry et ui. (1951). Inorganic phosphate was determined according to Fiske and Subbarow (1925). Solutions of sucrose and nucleotides were adjusted to pH 7.4 with solid KHCOl.
~~~-~~-~H]lnositol (3.9- II.65 Ci:mmol) was obtained from the Radiochemical Centre (Amersham. U.K.). nr.~oinositol and the nucleotides were from Sigma (St Louis. Missouri, U.S.A.) except of ATP, CTP and UTP that were purchased from Pharma-Waldhoff (Dusseldorf, F.R.G.). Boehringer (Mannheim GmbH, F.R.G.) and Calbiochem (Los Angeles, California, U.S.A.) respectively. All other reagents were of analytical grade. RESULTS
The Mn*+-dependent incorporation of ‘ftyoinositoi into phosphatidylinositol of rat liver microsomes showed a linearity upon protein up to 6.6 mg protein/ml and upon time up to 30 min in the standard medium in the presence of 20 mM MnCl,. This inco~o~~t~on was inhjbited by adenine nucleotides and other nucleoside triphosphates except CTP (Table 1). The inhibitory effect, however, could not be fully ascribed to the binding of Mn” by nucleotides, since a low concentration of ATP inhibited inositol incorporation by about 50% whereas the decreasing concentration of Mn” from 20mM to 2 mM gave only 2Oyj; inhibition (Table 1, Expt 1). A slight inhibition was exerted by inorganic phosphate and ribose 5-phosphate whereas D-ribose was without effect (not shown).
Figure 1 shows the effect of AMP concentration on inositol incorporation in the presence and absence of CMP. The reaction was measured in the presence of either Mn’+ or ME’+. In the absence of CMP the incorporation proceeded only with Mn’+ thus confirming an earlier finding (Paulus and Kennedy, Table t. Effect of nucleotides on inositoi incorporation ni~~et>ce of MI?+
1960; Takenawa et al. (1977); Takenawa and Egawa. 1980; Zborowski and Brindley, 1983) that this cation is required for the inositol exchange reaction. In full accordance with the data of Table I, the addition of AMP inhibited the reaction, showing a more pronounced effect with increasing concentration of the nucleotide (Fig. 1A). Addition of CMP stimulated both MI?‘and Mg”-dependent incorporation. With Mg’ ! , AMP had a strong stimulatory el‘fect (Fig. IB), whereas a slight inhibitory effect was observed in the presence of Mn’+ (Fig. 1A). This latter effect could be ascribed to the inhibition of inositol incorpor~ition catalysed bv the inositol exchange enzyme (see lower curve in F”ig. IA). As shown in Fig. I B, a threefold stimulation of the CMP-dependent incorporation of inositol in the presence of Mg’+ was obtained by addition 01 5510mM AMP. However, even then the amount of inositol incorporated was lower than in the presence of Mn”. This can be explained taking into consideration that in the presence of Mn” both the phosphatidyltransferase reaction and the inositol exchange take place, whereas in the presence of Mg’ ’ only the former reaction can proceed. It may also be worthwhile to stress that the values found for inositol incorporation in the presence of Mg’+ at each AMP concentration corresponded well to those with Mn’ ’ after subtraction of the (IMP-independent incorporation. Therefore, the incorporation of inositol in the presence of Mg.” could be taken as a measure of phosphatidyltransferase activity. The optimum concentration of Mg’+ and Mn” for the CMP-dependent inositol incorporation was lo-20mM (Fig. 2). The optimum CMP concentration in the presence of 10 mM Mg’+ was 1 mM {Fig. 3). The stimulatory effect of AMP on CMPdependent inositol incorporation in the presence of Mg” was not observed at the concentrations of CMP of 2mM and higher with AMP in the range of 0.55IOmM (data not shown). AMP was not the only nucleotide able to stimu~~~te the inositol incorporation at low concentration of CMP in the presence of Mg’+. Other mono-, diand triphosphate nucleosides were effective as well (Table 2). The nucleotides stimulated the inositol incorporation only in the presence of CMP but could not substitute for CMP in the presence of either Mg’ ’ (Table 2) or Mn2+ (not shown). The stimuiatory effect of CMP disappeared if microsomes were pre-
in the
Inositol incorporation (nmoliml: protein) Nucleotide added
Expt
None None (Ma’+, 2mM) AMP, 2mM
2.94 2.37 1.76
ADP. ATP, ATP,
1.71
2mM 2mM 6mM
GTP. 6mM ITP, UTP,
6mM 6mM
(TP
6mM
Incorporation
I .8?
I
Expt 2 2.99
1.54 1.36 I .25 0.59 0.84 7.76
of inositol was measured in the presence of 20 mM Mn”. Other conditions as in “Materials and Methods”. In both experiments microsomes amounted to 2.18 mg protein:ml.
AMP
tmMi
AMP
imM)
Fig. I. Effect of AMP on inositol incorporation in the absence and presence of CMP. Incubations were performed in the presence of either 2OmM Mn’ ’ (A) or Mg” (B). Microsomes were added in amount of 2.4 mg proteinml. Other conditions as in Materials and Methods. CMP absent (0). 0.5 mM CMP present (a).
mj&nositol
I
,
IO
5
Cotton
I
1
15
20
(mM)
Fig. 2. The requirement of CMP-dependent inositol incorand Mg’+. Microsomes (2.4 mg poration for Mn’+ protein/ml) were incubated in the presence of Mg’+ (0) or Mn* + (0). CMP concentration was 0.5 mM.
incubated with this nucleotide for 30min at 37°C prior to the addition of [3H]inositol. The addition of 2 mM AMP or other nucleotides to the preincubation medium partly protected the stimulatory effect of CMP (not shown). This protective effect of AMP on the CMPdependent incorporation of inositol was also evident in the time dependence. In the presence of AMP the
o-
IO CMP (mM)
Fig. 3. Effect of CMP on inositol incorporation in the presence of Mg’+. Microsomes (2.58 mg protein/ml) were incubated in the presence of IOmM MgCI,.
Table 2. Effect of nucleotides on inositol incorporation oresence of Me” Nucleotide added None CMP, CMP, GMP, IMP, ADP, AT?, ITP. UTP, CMP, CMP, CMP, CMP, CMP, CMP, CMP,
0.5 mM 5.0mM 5.0 mM S.OmM 5.0mM 5.0mM S.OmM 5.0 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM
i AMP, 5.0 mM + GMP, 5.0 mM + IMP, 5.0 mM + ADP, 5.0 mM + ATP, 5.0 mM + ITP, 5.0 mM + UTP, 5.0 mM
incorporation
1369
Fig. 4. Effect of AMP on the time-course of CMPdependent inositol incorporation. Microsomes (2.66 mg protein/ml) were incubated with 0.5 mM CMP without (0) or with 2.0 mM AMP (e). 10 mM Mg’+ was present in the incubation medium.
reaction was proceeding linearly for 30 min, whereas in the absence of AMP it levelled off after 15min (Fig. 4). The linearity of CUP-dependent incorporation was also protected when AMP was substituted by equimolar concentration of GMP or IMP which is in accordance with the data of Table 2. The levelling off of CMP-dependent inositol incorporation observed in the absence of other nucleotides suggested that CMP hydroiysis may be a possible explanation. Indeed, when microsomes were incubated with CMP in the presence of Mg”+ or Mn2+ cytidine could be detected chromatographically after 30min at 37°C. A quantitative estimation of CMP hydrolysis was done by measuring the liberation of inorganic phosphate. As shown in Fig. 5, the hydrolysis rate was maximal at l-2 mM Mn2+ and declined at higher concentrations, whereas for Mg*’ a plateau was observed for concentrations 5-20 mM. With the optimum concentration of either cation more than 45% of CMP was hydrolysed within 10 min of incubation. In order to ascertain whether the effect of AMP on inositol incorporation in the presence of CMP was due to the protective action against hydrolysis of the
in the
Inasitol incorooration (nmoi/mg protein) 0.56 2.44 2.24 0.97 0.87 0.22 0.38 0.18 0.25 3.69 5.79 6.44 5.25 5.48 6.84 7.13
Incubation was carried out in the presence of 10 mM Mg”. Microsomes in amount 2.58 mg protein/ml were added.
Cotlon
I mM i
Fig. 5. Dependence of CMP hydrolysis on varying concentrations of Mn*+ and Mg2+. Microsomes (2.18 mg protein/ml) were incubated in the presence of Mg*+ (0) and Mn2+ (a) in the medium containing unlabelled myoinoitol. The concentration of CMP was 5.0mM. Incubation was carried out for 10 min at 37°C. The reaction was terminated by addition of ice-cold trichloroacetic acid. The samples were centrifuged and an aliquot of each supernatant was taken for determination of liberated phosphate.
I370
JOSEF ZROROWSKI and GKA~YNA SZVM,&KA
Time ImJni Fig. 6. Hydrolysis of CMP and AMP in the presence of Mg’+. The concentration of each nucleotide was 5.0 mM and Mg” was IOmM. Other details as in the legend to
Fig. 5. (0) CMP, (0) AMP.
an experiment was performed in which the rate of hydrolysis of both nucleotides was measured. It is evident from Fig. 6 that the rate of hydrolysis of each of these nucleotides was the same. 96gi of the nucleotide was hydrolysed after 30min under conditions used.
latter,
DlSCLlSSlON
In the present study a different effect of nucleotides on the inositol-exchange reaction and CMPdependent incorporation of inositol into phosphatidylinositol is established. AMP, ADP, ATP and other mono-, di- and triphosphate nucleotides inhibit the Mn’+-dependent exchange reaction (Table I, Fig. 1A). Similar observation for triphosphate nucleotides including CTP has also been made by Takenawa and Egawa (1980) with the partially purified enzyme. According to these authors also other phosphorus containing acidic substances, among them phospholipids, phosphatidylserine and phosphatidic acid, act as inhibitors. In our experiments with rat liver microsomal membranes CTP stimulated inositol incorporation in the presence of Mn’+. A stimulatory effect of CTP has also been reported by Holub (1975). This effect may be, however, ascribed to the reaction catalysed by phosphatidyltransferas~, since CTP can be hydrolysed in the microsomal preparation to CMP. Such a mechanism of stimulation of inositol incorporation has also been proposed for rabbit lung microsomes (Bleasdale and Wallis, 1981). In fact. the presence of enzymes that hydrolyse tri- and diphosphate nucleosides in liver microsomes was demonstrated (Eppler and Morre, 1982). As shown in this work (Table 2) AMP and other nucleotides stimulate the CMP-dependent inositol incorporation. Upon addition of AMP the linearity of the inositol incorporation was maintained, whereas in its absence it levelled off after 15 min of incubation (Fig. 4). Such results strongly suggested that during incubation a hydrolysis of CMP occurred and the addition of AMP protected CMP by supplying another equivalent substrate. Indeed, it was found that both these nucleotides were hydrolysed with the same rate (Fig. 6). S-Nucleotidase (EC ~_.._ 3.1.3.5) and alkaline phosphatase (EC 3.1 .J.l) that
may be responsible for this hydrolysis most probably originated from plasma membrane fragments. However, 5’nucleotidase has also been reported as a constituent of the rough endoplasmic reticulum (Widnell, 1972). On the other hand, the possibility that the inhibition of CDPdiacylglycerol hydrolysis by AMP and other nucleotides (Raetz et ul., 1976) could contribute to the level of incorpoi-ation of inositol in the presence of CMP cannot be ruled out. Recently, Bleasdale and Wallis (1981) have provided an evidence that CMP-dependent incorporation of inositol into phosphatidylinositol of rabbit is not catalysed by phoslung microsomes phatidyltransferase but by separate enzymes less susceptible to heat exposure and distinct on the basis of inhibition by either Ca’+ or Hg’+. Whether this is also the case in liver tissue remains to be established. A~~no~~l~~~~~~~nt~~e would like to thank Professor Lech Wojtczak for his contin~l interest during this study and for critically reading the manuscript. REFERENCES BJeasdaJe J. E. and Wallis P. (1981) Phosph~tidy~inositolinositol exchange in rabbit lung. Bjffclzit~. hiuphy.~. Acra 664, 4288440. Bleasdale J. E., Wallis P., MacDonald P. C. and Johnston J. M. (1979) Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol: inositol transferase in rabbit lung tissue. Biochim. hiophys. Ada 575, 135147. Brindley D. N. and Bowiey M. (1975) Drug afhecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amcinchocaine. phetamine, chlorpromazine. demethylimipramine. mepyramine and some of their derivatives ~i~~~~i~~~.J. 148, 461-469. Eppler C. M. and Morre D. J. (1982) Flow kinetics of a nucleoside phosphatase common to endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat liver. Eur. .I. C?ll &/I. 29, 13-23. Fiske C. H. and Subbarow Y. (1925) The calorimetric determination of phosphorus. d. hi&. Chcnz. 66, 375400. Hajra A. K.. Seguin E. B. and Agranoff B. W. (1968) Rapid labeiling of mitochondrial lipids by labeled orthophosphate and adenosine triphosphate. J. hiol. Chem. 243, 1609-1616. Holub B. J. (1975) Role of cytidine triphosphate and cytidine diphosphate choline in promoting inositol entry into microsomai phosphatidylinositol. &iris 10,483-490. Hokin-Neaverson M., Sadeghian K.. Harris D. W. and Merrin J. S. (1977) Synthesis of CDP-diglyceride from phosphatidylinositol and CMP. Biochem. hiophl~s. Res. ?-o&run. is, 364-37 I t Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein m~dsurement with the Folin phenol reagent. J. hid. Chem. 193, 265-275. Neskovic N. M. and Kostic D. M. (1968) Quantitative _ analysis of rat liver phospholipids by a two-step thin-layer chromatographic procedure. J. Chrumutogr. 35,297-300. Paulus H. and Kennedy E. P. (1960) The enzymatic synthesis of inositol monophosphatide. J. hid. Chem. 235, 1303~13il. Raetr C. R. H., Dowhan W. and Kennedy E. P. (1976) Partial purification and characterization of Cytidine 5’-diphosphate-diglyceride hydrolase from membranes of Escherichiu co& J. LZacterioi. 12.5, 855-863. Rouser G.. Fleischer S. and Yamamoto A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analvsis of spots. Lipids 5, 494496. _
myo-Inositol Strunecka A. and Zborowski J. (1975) Microsomal synthesis of phosphatidylinositol and its exchange between subcellular structures of rat liver. Comp. Biochem. Physiol. 50B, 55-60. Takenawa T. and Egawa K. (1977) CDP-diglyceride: inositol transferase from rat liver. J. biol. Chem. 252, 5419-5423. Takenawa T. and Egawa K. (1980) Phosphatidyl inositol: mqjo-inositol exchange enzyme from rat liver: Partial purification and characterization. Archs Biochrm. Biophys. 202, 601-607. Takenawa T., Saito M., Nagai Y. and Egawa K. (1977) Solubilization of the enzyme catalyzing CDP-diglyceride-
incorporation
1371
independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride: inositol transferase. Archs Biochem. Biophys. 182, 244-250. Widnell C. C. (1972) Cytochemical localization of 5’-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5’nucleotidase activity in microsomes. J. Cell Biol. 52, 542-558. Zborowski J. and Brindley D. N. (1983) The metabolism of CDP-diacylglycerol and phosphatidylinositol in the microsomal fraction of rat liver. Effects of chlorpromazine, magnesium and manganese. Biochim. biophys. Acra 751, 81-89.