cmp-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve-endings

Vol. 112, No. 3, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 16, 1983 Pages 817-821

CMP-DIEPENDENT PHOSPHATIDYLINOSITOL:MYO-INOSITOL EXCHANGE ACTIVITY

IN ISOLATED NERVE-ENDINGS

Gerard Berry, John R. Yandrasitz and Stanton Segal

Division of Biochemical Development and Molecular Diseases,

The Children’s Hospital of Philadelphia and the Departments

of I?ediatrics and Medicine, the University of Pennsylvania School of Medicine,

Philadelphia, PA. 19104

Received Plarch 7, 1983

Greatly enhanced manganese-dependent phosphatidylinositol:myo-inositol exchange activity was observed when isolated, intact nerve-endings were incubated with the nucleotide, CMP, suggesting that the enzyme, CDP-diglyceride:inositol phosphatidyl transferase, catalyzes this exchange. CMP, at 10 u M, produced as much myo-[ Z-3H] inositol incorporation into phosphatidylinositol as did 1 mM. This CMP-stimulated exchange activity may reside on the plasma membrane.

The incorporation of myo-inositol into phosphatidylinositol (Phi) by exchange

of the free and lipid-bound moieties has been demonstrated in various mammalian

tissues (l-13). This exchange activity shows a strong preference for Mn2+ as a

cofactor and results in incorporation of labeled myo-inositol into lipid in the

absentee of de novo synthesis of Phi. Two Mn 2+ -- -dependent enzymes may account

for this activity. The work of Takenawa, Egawa and coworkers (9,141 suggests the

existence of a nucleotide-independent enzyme which catalyzes myo-inositol

specific base exchange with Phi in a manner analgous to that seen for

phosp:hatidylethanolamine and ethanolamine or serine. The enzyme responsible for

Phi synthesis, CDP-diglyceride:inositol phosphatidyl transferase (EC 2.7.8.111, also

catalyzes exchange of free myo-inositol into Phi by its reversible reaction:

CDP-diglyceride + myo-inositol _ Phosphatidylinositol + CMP

The h,allmark of exchange activity catalyzed by the transferase enzyme is it’s

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dependence on CMP (2,4,15,16,13) and it results in both the exchange of free and

lipid myo-inositol and the exchange of phosphatidyl moieties between Phi and CDP-

diglyceride (16).

Since this laboratory reported a Mn 2+ -dependent exchange of free myo-

inositol into Phi in preparations of isolated nerve-endings or synaptosomes (lo), it

was suggested that this manganese stimulation of exchange must be cytidine

nucleotide independent since radioactive phosphate incorporation into Phi was

essentially unaffected (1’7). We have further investigated this exchange

phenomenon and its dependence on cytidine nucleotide, and report here a striking

increase in synaptosomal Mn2+-dependent phosphatidylinositol:myo-inositol

exchange activity following incubation with CMP.

Materials and Methods

Synaptosomes were prepared from the cerebral hemispheres of four rats using differential centrifugation and a S-step Ficoll gradient as previously described (18). Synaptosomal membranes were prepared by hypotonic lysis of synaptosomes followed by centrifugation at 40,OOOg for 30 min. After a 10 min preincubation at 370, intact synaptosomes or synaptosomal membranes were incubated at 370 in a shaker bath for 10 min with 0.1 mM myo- [2-3Hlinositol (45 mCi/mmole) in a modified Krebs-HEPES buffer, pH 7.4 (lo), containing 0.5 mM MnC12. For time dependence studies incubations were continued up to 40 min. Incubations were terminated by dilution with a cold trichloroacetic acid (TCA) solution containing 20 mM myo-inositol; the TCA precipitate was centrifuged for 30 min at 40,000 g and washed with cold 20 mM myo-inositol. Phi was extracted as previously described (lo), except that the lipid extract was partitioned with 20 mM myo- inositol in 100 mM HCl.

The lipid extract was evaporated in 20 ml glass scintillation vials and counted in 10 ml of 0CS:ethanol (70:28) in a Packard Tri-Carb liquid scintillation counter. Counting efficiency was determined with [3H] toluene. Identical amounts of myo-[ 2-3Hi inositol were added to synaptosomal suspensions while in an ice bath after the addition of TCA and 20 mM myo-inositol. These samples were analyzed in parallel with the experimental samples and served as controls for the non-specific carry-over of free myo-[ 2-38 linositol into the lipid extract. When selected samples were analyzed by high-pressure liquid chromatography (191, the radioactivity in the Phi peak accounted for the tritium in the extract following correction for free myo- [2-3H linositol carry-over. The protein content of the synaptosomal suspensions was determined at the end of the incubation by the method of Lowry et al (20) using bovine serum albumin as the standard.

Sprague-Dawley rats were obtained from the Charles River Breeding Labs (Wilmington, Mass.). Myo-[ 2-3I-Il inositol and OCS were obtained from the Amersham, Arlington Heights, IL; solvents were from Burdick & Jackson Labs. (Muskegan, Mich.) or J.T. Baker (Philipsburg, N.J.); sulfuric acid used in HPLC solvents was Ultrex grade from Baker; and HEPES was obtained from Calbiochem (LaJolla, CA). All other chemicals were of reagent grade.

Results

Incorporation of 0.1 mM myo- [3H] inositol into lipid soluble material was

readily apparent without added nucleotide after a 10 min incubation of

818


TABLE 1

Effect of CMP and Cytidine on Phosphatidylinositol: myo-inositol exchange activity in intact synaptosomes and synaptosomal membranes.

dmp myo-12-3~~ inositol/mg pi-0t

SYNAPTOSOMES SYNAPTOSOMAL MEMBRANES

No nucleotide 484.5 t 77.2 (4) 281.9 + 64.2 (3)

1mM Cytidine 539.4 t 87.4 (4) 240.1 2 13.7 (4)

lti CM? 14448 2 684 (4) 14945 ? 2378 (4)

(n) = number of determinations

Synaptosomes and synaptosomal membranes were incubated for 10 min in an isotonic buffer containing 0.5 mM MnC12 and 0.1 mM my0-[2-3~]

inositol as described in the text. Results are expressed as mean + SEM.

synaptosomes with 0.5 mM MnC12, while no incorporation was detected in

incubations with the buffer containing only 1 UM Mn2+. (Table 1). These results are

consonant with the effect of Mn2+ on the exchange incorporation of free myo-

inosil:ol into synaptosomal Phi seen with longer incubations (10). Table I shows the

effect of cytidine nucleotides on this Mn 2+-dependent exchange activity during

similar 10 minute incubations. While 1 mM cytidine had little effect on myo-

inositol incorporation, 1 mM CMP markedly increased myo-inositol exchange in

intact synaptosomes as well as synaptosomal membranes. CMP, at lmM, was far in

excess of the amount required to observe maximum stimuation: 10 p M CMP

produced comparable results. Figure 1 shows the time course of myo-[ 2-

3H linositol incorporation into Phi in the presence of 10 UM CMP and 0.5mM

MnC1.2. Incorporation was linear for the first 10 minutes but leveled off at longer

times, perhaps due to consumption of unlabelled endogenous Phi.

Discussion ~-

These experiments show that the Mn2+-stimulated incorporation of myo-

inositol into synaptosomal Phi by an exchange activity is dependent upon CMP.

While Mn2+ stimulates the basal incorporation of myo- [22Hl inositol into Phi

several fold (lo), this activity is less than 5% of that measured in the presence of

Mn2’. and CMP. Preliminary data indicate that the apparent Km for CMP in this

reaction is about 1 u M, suggesting that the Mn2’stimulated exchange seen without

added nucleotide could be supported by nanomolar concentrations of CMP which

might be available from endogenous sources (21). Since a dependence on CMP is

819


Figure 1. Ttme (mans)

Time dependence of CMP-stimulated phosphatidylinositol:myo-inositol exchange activity.

The incorporation of myo- 12-3Hl inositol into Phi is plotted against the time of incubation. Synaptosomes were incubated in an isotonic buffer containing 0.5 mM MnC12, 10 v M CMP and 0.1 mM myo- 1 2-58 1 inositol as described in the text. Results are given as mean + S.E.M. for four determinations. -

the hallmark of phosphatidylinositol: myo-inositol exchange catalyzed by the

reversible CDP-diglyceride:inositol phosphatidyl transferase, it is possible that this

enzyme is solely responsible for exchange activity in synaptosomes. Cytidine

nucleotide dependence of exchange activity has also been described in lung (13)

and thyroid microsomal preparations (6). The effect of CMP was not tested in the

studies which described a nucleotide independent enzyme in liver (14).

Our data suggest that the enzyme catalyzing exchange in our synaptosomal

preparation is directly accessible to CMP outside the synaptosome. The

synaptosomal plasma membrane should exclude CMP and other nucleotides, and the

action of CMP cannot be explained by external hydrolysis and uptake of its

nucleoside moiety by the active uptake system (22) since cytidine itself showed

little activity. Even though previous uptake studies (23) have shown that our

preparation is comprised of metabolically active synaptosomes, it is possible that

the Mn2+-stimulated exchange activity resides in a contaminating membrane

component of our preparation. If this were the case, however, one would expect an

increase in activity in the membrane preparation due to lytic exposure of all of the

synaptosomal membranes. The failure of hypotonic lysis to reveal additional

820


activity suggests that the synaptosomal enzyme was measured in the intact

preparation and the effect of CMP suggests that this enzyme is accessible to the

outside.

Hypotonic lysis results in a loss of cytosolic protein equal to about 30% of the

origin.al protein content. This membrane enrichment should result in an increase in

specific activities of enzymes such as CDP-diglyceride: inositol phosphatidyl

transferase which are localized to particulate fractions (9). In fact, the specific

activity of the membrane preparation was lower than the intact synaptosomes

suggesting that optimal CMP-stimulated phosphatidylinositol:myo-inositol

exchange activity depends on intact synaptosomal structures either because of

inherent organizational features or unknown cytosolic factors.

Acknowledgments

This work was supported by grant no. HD 08536 from the National Institutes

of Health, Bethesda, MD.

Referlences ~-

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

13. 14. 15. 16.

17. 18. 19. 20.

21. 22. 23.

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cmp-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve-endings

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