Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

440

Biochimica ef Biophysics Acta 877 (1986) 440-446 Elsevier

BBA 52351

Synthesis of polyphosphoinositides

in vertebrate photoreceptor membranes

N.M. Giusto and M.G. Ihncheta

de Boschero

Institute de Investigaciones Bioquimicas (INIBIBB), Universidad National del SW and Consejo National Investigaciones Cientificas y Tknicas (CONICET), Gorriti 43, 8000 Bahia Blanca (Argentina) (Received

Key words:

Photoreceptor;

de

April 21st. 1986)

Polyphosphoinositide; Phosphatidylinositol Rod outer segment membrane

kinase;

Diacylglycerol

kinase;

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [y-32PjATP in a Mg*+ -containing medium.’ Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5bisphosphate labelling in rod outer segments (S- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.

Introduction A close relationship between augmented polyphosphoinositide turnover and increased levels of intracellular calcium ions has been proposed for a wide variety of tissues [l-3]. Inositol 1,4,5trisphosphate, a product of phosphatidylinositol 4,Sbisphosphate hydrolysis, has been found to stimulate the release of calcium ions from intracellular stores in photoreceptor cells from

Abbreviations: Ptdlns, phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns4,5Pz, phosphatidylinosito1 4,5-bisphosphate; Ins1,4,5P,, inositol 1,4,5_trisphosphate. 0005-2760/86/$03.50

0 1986 Elsevier Science Publishers

Limufus [4] and to elicit electrical responses that mimic several aspects of light-induced excitation [5,6]. Intracellular Ins1,4,5P, in vertebrate rods might also be involved in the modulation of membrane voltage during vertebrate phototransduction

171. Recent evidence has shown that cGMP, and not calcium, is the agent acting on the surface of photoreceptor membrane, modulating the membrane current [8,9]. However, calcium/phospholipid-dependent protein kinase C activity [lo], a rapid phosphorylation of diacylglycerols [ll] and a light-activated phosphatidylinositol 4,5-bisphosphate phospholipase C [12,13] have been reported in isolated rod outer segments from bovine retinas. This could imply that other biochemical

B.V. (Biomedical

Division)

441

systems may also be involved in the regulation of the phototransduction mechanism in vertebrate rods. Recent work suggests that, rather than rising, as in the case of the calcium hypothesis, the internal calcium concentration in rods drops when light is applied [14]. A lower level of calcium is known to diminish the light sensitivity of the cGMP-phosphodiesterase [15] and to stimulate the guanylate cyclase, converting GTP to cGMP [16]. Thus, light-induced calcium decreases may well contribute to light adaptation. The turnover of photoreceptor membrane lipids is different from that of proteins [17,18]; however, the enzyme activities involved in the lipid metabolism of these membranes have still not been clearly identified. For instance, it is not yet known how polyphosphoinositides are synthesized in these membranes. Evidence is presented here of the presence of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase as well as an active diacylglycerol kinase in isolated rod outer segments from bovine retina. Specific locations are proposed for these enzymes. Materials and Methods Materials. [y- 32P]ATP (specific activity 10.6 Ci/mmol) was purchased from New England Nuclear Co., Boston, MA. Dioleoylglycerol was purchased from Sigma Chemical Co., St. Louis, MO. Phosphatidylinositol was obtained from cattle retina and purified by two-dimensional TLC [19]. All other chemicals used were analytical grade., Fresh bovine eyes were obtained from a local abattoir and stored in crushed ice within 10 min of decapitation and were dark-adapted for l-2 hours. All subsequent procedures were conducted under dim red light and carried out at 2-4°C. Isolation of rod outer segments and discs. Retinas were removed and rod outer segments were detached by shaking the retinas twice in a 40% sucrose solution as described by Papermaster and Dreyer [20]. The remainder of the retina was sedimented at 4000 r-pm for 4 min and the supernatants were centrifuged for 15 min at 12 000 ‘pm. The crude rod outer segment preparations were then purified by discontinuous density gradient

centrifugation [20]. The discs from retinal rod outer segments were prepared by osmotic shock followed by flotation on 5% Ficoll [21]. The purity of the membrane preparation was controlled by the ratio of absorbances at 278 and 500 nm after solubilization in phosphate buffer (pH 7) containing 1% Emulphogene. Values of 2.3 5 0.2 were typically obtained for this ratio. In addition, sodium dodecyl sulphate-polyacrylamide gel electrophoresis [22] was used to check the purity of the membranes. 85-90% of the photoreceptor membrane protein was rhodopsin, even in heavily overloaded gels. Lipid membrane phosphorylation. Rod outer segments or disc membranes (0.3 mg of protein) were incubated with 0.2 mM [ y- 32P]ATP (specific activity 0.1 pCi/mmol), 5 mM MgCl, and 50 mM sodium acetate-10 mM magnesium acetate (pH 6.5) in a final volume of 0.5 ml [23]. Prior to the incubations in a shaking water bath at 37°C rod outer segments were disrupted by sonication for 30 s to facilitate ATP penetration. Blanks were prepared identically, except that membrane suspensions were boiled for 5 min before incubation. Radioactivity values in lipid spots from these blanks were subtracted from those of experimental values. The incubations were stopped by adding 4.5 ml of ice-cold 5 mM ATP followed by 5 ml of ice cold 10% trichloroacetic acid, thoroughly mixed, chilled and centrifuged. The supernatants were removed and the pellets extracted with chloroform/ methanol/ concentrated HCl (400 : 200 : 3, v/v). Isolation of lipids. After 1 h extraction at 37°C in a shaker bath [24] the pellets were centrifuged and washed with chloroform/methanol (1 : 1, v/v). The extracts were combined and chloroform was added to give a final chloroform/methanol ratio of 2 : 1 (v/v). The extract was subsequently shaken with 0.2 vol. of 1 M HCl. The lower phase was successively washed with two-thirds its volume of chloroform/methanol/O.1 M HCl (3 : 48 : 47, v/v) and chloroform/ methanol/O.01 M HCl (3 : 48 : 47, v/v). The washed extract was made to one phase with methanol, and NH,OH was added to adjust the pH to 7-8. PtdIns4P and PtdIns4,5P, were separated by one-dimensional TLC [25] and other phospholipids were isolated by two-dimensional TLC [19].

442

Lipids were visualized by exposure to iodine vapours and scraped into vials containing 0.4 ml of water, and 10 ml of 0.4% Omnifluor/Triton X-100 (800 : 200, v/v) were added [26]. Radioactivity was quantitated by liquid scintillation counting. Lipid phosphorus was determined according to Rouser et al. [19]. Protein concentration was measured using the method of Lowry et al. [27]. Results -“P incorporation

in rod outer segments

Incubation of rod outer segments with [y32P]ATP in the presence of magnesium promoted the net labelling of phosphatidic acid, PtdIns4P and PtdIns4,5P,, in this order. As early as 20 s after the addition of the precursor, both phosphoinositides were phosphorylated (Fig. la). At this point the label in PtdIns4,5P, represented 25% of that present in PtdIns4P. No other phospholipids were labelled. The labelling of PtdIns4P and PtdIns4,5P, in these membranes was time dependent (Fig. la). The rate of ‘*P incorporations into PtdIns4P was constant for about 10 min (about 4 pmol/min per mg protein), steadily decreasing thereafter. The loss of radiolabelled polyphosphoinositides after 30 min indicates that the turnover of these lipids is very active and that ATPases are probably operative in this preparation. The rate of phosphatidylinositol 4,5-bisphosphate labelling was much lower, reaching a maximum of 0.35 pmol/min per mg protein. The activity of both kinases was protein dependent from 100 to 950 pug (Fig. lb), the production of PtdIns4P being higher than that of PtdIns4,5P,. The time-course of ‘*P incorporation into phosphatidic acid is presented in Fig. 2. The reaction was linear for 10 min when the phosphatidate synthesis reached a maximum. The inset shows the direct dependence of phosphatidic acid production on the amount of protein in the range of 150-950 pg. The assay was performed in the presence of 0.1 mM exogenous diacylglycerol in order to avoid substrate dependence. The level of 32P incorporation into phosphatidic acid was similar to that observed without the addition of this neutral lipid. This could imply that the exogenous diacylglycerol was not employed as substrate by the enzyme.

Fig. 1. Phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities in photoreceptor membranes. a, Time-course of phosphatidylinositol kinase (0) and phosphatidylinositol 4-phosphate kinase (0) -catalyzed reactions; b, effect of protein concentration on phosphatidylinositol kinase (0) and phosphatidylinositol4-phosphate kinase (0) reactions. Rod outer segments, obtained according to Papermaster and Dreyer [20], were incubated with 0.2 mM [Y-~~P]ATP (spec. act. 0.1 pCi/mmol), 5 mM CI,Mg and 50 mM sodium acetate/l0 mM magnesium acetate (pH 6.5) in a final volume of 0.5 ml. In (a) the time was varied and in (b) 2 min of incubation were used. Membranes were previously sonicated for 30 s. The incubations were stopped by addition of 4.5 ml of ice-cold 5 mM ATP followed by 5 ml of ice-cold 10% trichloroacetic acid, thoroughly mixed, and centrifuged. Lipids were extracted from the pellets with acidified solvents [24]. Phosphoinositides were separated by one-dimensional TLC [25] and phosphatidate by two-dimensional TLC [19]. The incorporation values represent the average of two individual samples.

The endogenous content of phosphatidic acid, PtdIns4P and PtdIns4,5P, was also measured. These lipids amount to 14.86,0.19 and 0.24 nmol/ mg protein, respectively, representing approximately 0.9, 0.01 and 0.02% of the rod outer segment phospholipids. Time course of j2P incorporation

in disc membranes

Phosphatidic acid, PtdIns4P and PtdIns4,5P, were the only phospholipids labelled with 32P in discs obtained from rod outer segments. The labelling of polyphosphoinositides from [ y- 32P]ATP was linear for 5 min and reached a plateau thereafter (Fig. 3). The time course of phosphorylation of endogenous phosphatidylinositol in disc membranes also showed a peak of maximal incorporation at 5 min, slowly decreasing thereafter. The synthesis of PtdIns4P proceeded at a higher rate in disc membranes than in rod outer segments (6.12 pmol/min per mg protein and 3.22 pmol/ min per mg protein, respectively). Phosphati-

443

dylinositol 4,5_bisphosphate labelling was also higher in discs (1.24 pmol/min per mg protein) than in rod outer segments (0.78 pmol/min per mg protein). Although the pattern of diacylglycerol phosphorylation kinetics (Fig. 3, inset) was similar to that of rod outer segments (Fig. 2) the level of 32P incorporated was 10% or less of that present in the latter. A TP-dependence

for

polyphosphoinositides

“P

incorporation

and phosphatidic

into

acid

Since the enzymatic conversion of PtdIns and PtdIns4P into PtdIns4P and PtdIns4,5P, requires the appropriate phosphoinositides and ATP as the dependence of PtdIns4P and substrates, PtdIns4,5P, labelling on ATP concentration in rod outer segments and discs was investigated. At 5 mM MgCl,, increased concentrations of ATP induced a stimulation in the phosphorylation of these lipids and phosphatidic acid in both preparations (Fig. 4). The response was linear in the time range tested. As mentioned above, PtdIns and PtdIns4P phosphorylation in discs was higher at all ATP concentrations than that in rod outer segments. In contrast, phosphorylation of diacylglycerol was lower in discs than in rod outer segments. Fig. 4

shows the percentual change of PtdIns4P, PtdIns4,5P, and phosphatidic acid labelling at 200 PM ATP in rod outer segments with respect to discs. Dependence

of PtdIns,

PtdIns4P

and phosphatidic

acid phosphorylation on exogenous dylinositol. Effect of Trixon X-100

phosphati-

The extent to which endogenous phosphatidylinositol is phosphorylated was also analyzed. After 20 min incubation and at maximal levels of incorporation, only 1% of the PtdIns was labelled in rod outer segments. However, in the presence of 0.4% Triton X-100, the reaction rate was stimulated l-fold (Table I). A concomitant increase was observed in the labelling of PtdIns4,5P,, and diacylglycerol phosphorylation was drastically reduced (98% inhibition). In order to investigate whether exogenously added phosphatidylinositol would also act as a substrate for the kinase, incubation of rod outer segments was carried out in the presence of various concentrations of PtdIns with Triton X-100. The addition of phosphatidylinositol (obtained

s

E CL

N

‘9

10

20 time

30

(mtn)

10

time Fig. 2 Profile of 32P incorporation into phosphatidic acid of bovine rod outer segments. Conditions as in Fig. 1. The 32P incorporation as a function of protein concentration is depicted in the inset. Membranes were incubated in the assay medium containing a suspension of 0.1 mM dioleoylglycerol for 2 min. The reaction was stopped and the lipids were extracted and analyzed as described in Fig. 1. Values represent the average of two individual samples.

30

20 (min

)

Fig. 3. Phosphorylating activities as a function of time in discs obtained from rod outer segments. Phosphatidylinositol 4phosphate (0) and phosphatidylinositol 4,5_bisphosphate (0) were labelled by incubation of disc membranes as described in Fig. I. Disc membranes were obtained according to Smith et al. [21]. In the inset the synthesis of phosphatidic acid (A) by phosphorylation of endogenous diacylglycerol is shown. Values are the average of two individual samples.

444

A

-

800 .i

2 P

L -400

‘i a R z E a

0.1

0

0.1

0.2 03 ATP CmM)

02 0.3 ATP (mM)

04

from bovine retina), at a concentration of 0.1 mM, stimulated 7-fold PtdIns4P labelling. There was no further increase in the labelling of this phospholipid when the concentration of Ptdlns was raised from 0.5 to 1 mM. On the other hand, 32P incorporation into PtdIns4,5P, was linear up to 0.5 mM PtdIns, reaching a plateau thereafter. This pattern is indicative of a precursor-product relationship between PtdIns4P and PtdIns4,5P,. The inhibitory effect of Triton X-100 on diacylglycerol phosphorylation was partially reversed by exogenously added PtdIns. Phosphatidate labelling increased from 6.80 + 1.48 to 100.61

TABLE

0.4

Fig. 4. ATP dependence of phosphatidylinositol kinase (o), phosphatidylinositol 4-phosphate kinase (0) and diacylglycerol kinase (A) activities in rod outer segments and in discs obtained from them. Disc membranes were obtained according to Smith et al. [21]. Membranes were incubated for 5 min as described in Fig. 1. Values represent the average of two individual samples or the mean f S.D. of four individual samples at 200 PM ATP. Percentual changes are the ratio of dpm in rod outer segments to dpm in disc membranes at 200 pM ATP. W, PtdIns4P; 0, PtdIns4,5Ps; q phosphatidic acid.

+ 6.07 pmol/mg Ptdlns.

protein

in the presence

of 1 mM

Discussion Evidence is presented here that the incubation of rod outer segments with [Y-~~P]ATP in a magnesium-containing medium promotes 32P labelling of polyphosphoinositides, mainly phosphatidylinositol 4-phosphate and phosphatidic acid. Furthermore, it is shown that discs obtained from rod outer segments also exhibit this phosphorylating activity. These results indicate that bovine

I

EFFECT OF Ptdlns AND TRITON IN ROD OUTER SEGMENTS

X-100 ON THE PHOSPHORYLATION

OF PtdIns,

PtdIndP

AND

DIACYLGLYCEROL

Membranes (0.3 mg protein) were incubated in the standard assay medium supplemented with PtdIns (prepared from bovine retina) and 0.4% Ttiton X-100. The 32P incorporation was initiated by addition of 0.2.mM ATP (4.5 pCi), 5 mM MgCl, and sonication for 30 s. The reaction was stopped after 2 min of incubation at 37 OC as described in Fig. 1. Values are expressed as pmol/mg protein and are the mean f S.D. of three individual samples. Additions

Phosphatidic acid

PtdIns4P

PtdIns4,5P,

None

441.9*41.9 6.8* 1.5 14.9* 3.8 55.3 f 10.2 100.6+ 6.1

9.2+ 1.3 20.0+ 3.4 72.2 k 13.3 5.1 65.4* 80.7 f 19.4

1.4 f 0.3 3.5 f 0.3 6.1 + 1.3 11.5+0.9 10.2*0.7

Triton X-100 + PtdIns 0.1 mM 0.5 mM 1.0 mM

445

photoreceptor membranes have the enzymes for the phosphorylation of diacylglycerol, phosphatidylinositol and phosphatidylinositol 4-phosphate. Endogenous phosphatidylinositol, which represents only 1% of rod outer segment phospholipids (Giusto, N.M. et al., unpublished observations) was employed as the substrate for phosphatidylinositol kinase. The levels of radioactivity incorporated into phosphatidic acid with respect to PtdIns and PtdIns4P differed, suggesting that pre-existing pools of polyphosphoinositide precursors could be depleted. This is consistent with the concomitant increase of PtdIns4P and PtdIns4,5P, syntheses stimulated by exogenous phosphatidylinositol. Polyphosphoinositides are minor phospholipid components of rod outer segment membranes; together, they constitute less than 0.03 mol% of the total. However, these levels may not necessarily be those present in native rod outer segment membranes. The well-known lability of these compounds and the relatively long time between the animal’s death and obtainment of the membranes could certainly modify the polyphosphoinositide pool content. Phosphatidylinositol kinase has been reported to be located predominantly ‘at the plasma membrane [28]. However, recent evidence indicates that this enzyme can also be found in liver Golgi [29] or nuclear envelopes [30]. Similar discrepancies are apparent in the localization of phosphatidylinositol 4-phosphate kinase. This enzyme has been recovered mainly from the cytosolic fraction [28], though a less specific distribution was later reported [31]. Moreover, phosphatidylinositol 4phosphate kinase activity has also been found in membrane preparations [32]. In sealed rod outer segment this activity is probably higher than that found in our preparations, because during their purification they do not maintain an intact plasma membrane which is essential to prevent loss of endogenous proteins [33]. It would therefore be interesting to analyze the activity of the enzyme in intact rod outer segments. The fact that disc membranes exhibit enhanced phosphorylating activity compared with rod outer segments can partly be accounted for by a greater accessibility of the precursor to the membranes during incubation. But more importantly, these findings strongly indicate that phosphatidylinosi-

to1 4-phosphate kinase is predominantly a membrane-bound enzyme in disc membranes, although a contribution from soluble forms cannot be totally excluded. The present experiments do not rule out the presence of enzymes or activating factors responsible for polyphosphoinositide metabolism in the soluble fraction and/or in the plasma membrane of rod outer segments. As in the case of the phosphatidylinositol kinase activity reported for different subcellular fractions [34,35], the rod outer segment enzyme analyzed here exhibits the highest specific activity in the presence of a nonionic detergent and exogenous PtdIns. It seems likely that the detergent stimulation is the consequence of a higher amount of substrate (PtdIns) being made available to the enzyme. Phosphatidylinositol 4-phosphate kinase also exhibits a similar behaviour. The degradation of PtdIns4P and PtdIns4,5P, within 30 min (by which time ATP is likely to be partially depleted) is in agreement with the notion that these molecules have an active turnover. Recent studies have shown that the hydrolysis of PtdIns4,5P, is an early event in the receptor-mediated cascade for a wide variety of stimuli [36] and that the products resulting from the cascade, diacylglycerol and Ins1,4,5P,, have potent intracellular effects. It has also been suggested that the latter metabolite is involved in the phototransduction mechanism in vertebrates [7]. Furthermore, in rod outer segment the fatty acid composition of diacylglycerol resembles that of phosphatidylinositol and polyphosphoinositides [37], which is consistent with the presence of a phospholipase C activity. In agreement with Seyfred et al. [ll] we have also found an active diacylglycerol kinase in rod outer segments. Such an enzyme may play a role in the removal of diacylglycerol, one of the metabolites known to stimulate protein kinase C. This would ensure a steady supply of phosphatidic acid in rod outer segments, which can in turn be converted to PtdIns4P and PtdIns4,5P, (the latter being involved in the response to light [12,13]). The finding that diacylglycerol kinase activity is lower in discs than in rod outer segments merits further comment. It is likely that the enzymes responsible for phosphatidic acid synthesis and/or activating factors are mainly present in the soluble

446

fraction or are loosely associated to the disc membrane within rod outer segments. The present work provides a starting point for further studies of the individual steps of polyphosphoinositide formation and breakdown in rod outer segments and for the understanding of their physiological significance in vertebrate photoreceptors. Acknowledgements We thank Drs. MI. Aveldatio and F.J. Barrantes for reading the manuscript. This investigation was supported by Consejo National de Investigaciones Cientificas y Tecnicas (CONICET). References 1 Berridge, M.J. (1983) B&hem. J. 212, 849-858 2 Berridge, M.J., Dawson, R.M.C., Downes, C.P., Heslop, J.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482 3 Creba, J.A.. Downes, C.P., Hawkins, P.T., Brewster, G., Michell, R.H. and Kirk, C.J. (1983) B&hem. J. 212, 733-747 4 Brown, J.E. and Rubin, L.J. (1984) B&hem. Biophys. Res. Commun. 125, 1137-1142 5 Brown, J.E., Rubin, L.J., Ghalayini, A.J., Tarvar, A.P., Irvine, R.F. Berridge, M.J. and Anderson, R.E. (1984) Nature (Lond.) 311, 160-163 6 Fein, A., Payne, R., Corson, D.W., Benidge, M.J. and Irvine, R.F. (1984) Nature (Lond.) 311, 157-160 7 Waloga, G. and Anderson, R.E. (1985) B&hem. Biophys. Res. Commun. 126, 59-62 8 Fesenko, E.E., Kolesnikov, S.S. and Lynbarsky, A.L. (1985) Nature 313, 310-313 9 Nakatami, K. and Yau, K.W. (1985) Biophys. J. 47, 356-360a Biophys. 10 Kapoor, C.L. and Chader, G.J. (1984) B&hem. Res. Commun. 122, 1397-1403 11 Seyfred, M.A.. Kohnken, R.E., Collins, C.A. and McConnell, D.G. (1984) B&hem. Biophys. Res. Commun. 123, 121-127 12 Ghalayini, A.J. and Anderson, R.E. (1984) Biochem. Biophys. Res. Commun. 124, 503-506 T. (1985) Biochem. Biophys. 13 Hayashi, F. and Amakawa, Res. Commun. 128, 954-959

14 Yau, K.W. and Nakatami, K. (1985) Nature 313, 579-582 15 Kawamura, S. and Brownds, M.D. (1981) J. Gen. Physiol. 77, 571-576 16 Troyer, E.W., Hall, L.A. and Ferrendelli, J.A. (1978) J. Neurochem. 31, 825-829 17 Fliesler, S.J. and Anderson, R.E. (1983) Prog. Lipid Res. 22, 79-131 18 Anderson, R.E., Maude, M.B., Kelleher, P.A., Maida, T.M. and Basinger, 212-226

J.F.

(1980)

Biochim.

Biophys.

Acta

620,

19 Rouser, G., Fleischer, S. and Yamamoto, A. (1970) Lipids 5, 494-496 20 Papermaster, D.S. and Dreyer, W.J. (1974) Biochemistry 13, 2438-2444 21 Smith, H.G.J.R., Stubbs, G.W. and Litman, B.J. (1975) Exp. Eye Res. 20, 21 I-21 7 22 Laemmh, U.K. (1970) Nature 227, 680-685 23 Akhtar, R.A., Taft, W.C. and Abdel Latif, A.A. (1983) J. Neurochem. 41, 1460-1468 24 Uma, S. and Ramakrishnan, C.V. (1983) J. Neurochem. 40, 914-916 25 Shaikh, N.A. and Palmer, F.B.S. (1977) J. Neurochem. 28, 395-402 26 Rodriguez de Turco, E.B. and Aveldaho de Caldironi, M.I. (1980) Anal. Biochem. 104, 62-69 27 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 28 Harwood, J.L. and Hawthorne, J.N. (1969) J. Neurochem. 16, 1377-1387 29 Jergil, B. and Sundler, R. (1983) J. Biol. Chem. 13, 7968-7973 30 Smith, CD. and Wells, W.W. (1983) J. Biol. Chem. 258, 9365-9375 31 Cooper, P.H. and Hawthorne, J.N. (1976) Biochem. J. 160, 97-105 32 Jolles, J., Zwiers, H., Van Dongen, C., Schotman, P., Wirtz, K.W.A. and Gispen, W.H. (1980) Nature 286, 623-625 D.B. (1984) Biochemistry 23, 33 Shuster, T. and Farber, 515-521 34 Collins, CA. and Wells, W.W. (1983) J. Biol. Chem. 258, 2130-2134 J.N. (1976) 35 Lefevre, Y.A., White, D.A. and Hawthorne, Can. J. B&hem. 54, 746-753 36 Abdel-Latif, A.A., Akhtar, R.A. and Hawthorne, J.N. (1977) Biochem. J. 162, 61-73 37 Bazan, N.G., Di Fazio de Escalante, MS., Carega, M.M., Bazan, H.E.P. and Giusto, N.M. (1982) Biochim. Biophys. Acta 712, 702-706