Effects of the antioxidants dithiothreitol and vitamin E on phospholipid metabolism in isolated rod outer segments

Exp. Eye Res. (1991) 52, 607-612

Effects of the Antioxidants Phospholipid Metabolism WILLIAM Department (Received

F.ZIMMERMAN*

of Biology,

20 June

Dithiothreitol and Vitamin E on in Isolated Rod Outer Segments

1990

Amherst

AND

College,

and accepted

SUSAN

Amherst,

in revised

KEYS

MA

form

07002,

U.S.A.

13 September

7990)

Phospholipidperoxidation and the activities of phospholipaseA, acyl CoenzymeA :lysophospholipid acyltransferaseand lysophospholipase were measuredin isolatedbovine rod outer segments(ROS)that wereincubatedin the presenceor absenceof the addedantioxidants,vitamin Eand dithiothreitol (DTT), and additionally in light or dark. DTT and vitamin E significantly inhibit both lipid peroxidationand the enzyme activities. Theseresultssuggestthat one function of the enzymesfor molecularreplacementof fatty acids in ROS, is removal of peroxidized fatty acids and thus protection of the membrane phospholipidsand proteinsfrom further oxidative damage. Key words: rod outer segments;lipid peroxidation; phospholipaseA: antioxidants; vitamin E: dithiothreitol.

1. Introduction

2. Materials and Methods

Bovine rod outer segments (ROS) contain enzymes that remove, activate and replace the fatty acids of their membrane phospholipids (Giusto et al., 1986 ; Zimmerman and Keys, 1986, 1988, 1989; Jelsema, 1987) ; these enzyme activities are probably the basis of the phospholipid ‘molecular replacement’ that has been inferred from autoradiographic and biochemical studies(Bibb and Young, 19 74 ; Fliesler and Anderson, 1983) to occur within the organelle. ROS phospholipids are also renewed by ’ membrane replacement ’ (Young, 1976), in which rhodopsin as well as phospholipids are synthesized de novo in the inner segment and assembledinto new membrane discs at the base of the outer segment. Extending the original hypothesis of Young and Droz (1968) that membrane replacement in ROS functions to replace oxidatively damaged molecules, we suggested that one function of the fatty acid replacement reactions in ROS is to replace oxidized fatty acids (Zimmerman and Keys, 1988) and thus prevent the further propagation of such fatty acid peroxidation. We adduced indirect evidence for this hypothesis by showing that the presence of an antioxidant, dithiothreitol (DTT), inhibits both the endogenous phospholipase A activity toward added radiolabeled phospholipids and the acylation of endogenous phospholipids with added radiolabeled fatty acids. Here we present evidence that in isolated bovine ROS, both DTT and a differently acting antioxidant, vitamin E,inhibit ROSphospholipaseA activity toward endogenous phospholipids and the removal of lysophospholipids by lysophospholipase and acyl-CoA : lysophospholipid acyltransferase activities.

Chemicalsand Substrates

* For correspondence.

00144835/91/050607+06

$03.00/O

Organic chemicals were purchased from Sigma Chemical Co. (St Louis, MO) or Fisher Scientific (Pittsburgh, PA). [Choline-3H]lysophosphatidylcholine (1ysoPC) at a specific activity of 30 Ci mmol-* was prepared from dipalmitoyl-[3H-methyl choline]phosphatidylcholine (New England Nuclear, Boston, MA) by methods described in Zimmerman and Keys (1989). High performance, silica gel thin layer chromatography plates, (10 x 10 cm, 200 pm) were purchased from Whatman Chemical Separation, Inc. (Clifton, NJ). Optima grade HPLC solvents were purchased from Fisher Scientific. Isolation of ROS Bovine ROSwere purified from retinal homogenates by methods that have been described and discussed with regard to the purity and protein content of the final fractions (Godchaux and Zimmerman, 1979 ; Zimmerman and Keys, 1988). Enzyme Assays The preparation and use of [choline-3H]lysoPC in the assay of ROS lysophospholipase and acyl-Coenzyme A (CoA) : lysophospholipid acyltransferase activities have been described (Zimmerman and Keys, 1989). In this assay, removal of the remaining fatty acid of a lysophospholipid, the most common type of lysophospholipase activity (Waite, 1985), results in the formation of [3H]glycerophosphocholine (GPC), a water soluble product. Removal of the phosphocholine or of the choline group, before or after removal of the fatty acid from the glycerol backbone, would also 0 1997 AcademicPressLimited

W. F.ZIMMERMAN

608

yield these [3H]choline-containing compounds in the water phase of the ROS extract. However, we have not differentiated between these possibilities thus far, and we assume here that the appearance of 3H-containing compounds in the water phase is due to ‘lysophospholipase A’ activity, i.e. deacylation of the lysophospholipid. Acyl CoA : lysophospholipid acyltransferase results in the formation of [3H]PC. Thus, the use of [3Hcholine]lysoPC allows simultaneous assay of both enzyme activities by measuring the disappearance of their substrate (1ysoPC) and the appearance of their products (GPC, PC). In the assay of ROS phospholipase A activity toward endogenous phospholipids, ROS (300-500 ,ug protein) were incubated for 1 hr at 37°C in the presence of 1.0 mM ATP, 30 mM MgCl, 8.8 mM CaCl,, 0.25 mM CoA, in a total volume of 350 ~1, in the presence or absence of incandescent light (130 fc) and in the presence or absence of 1.2 mM vitamin E or 0.5 mM DTT. Vitamin E (D a-tocopherol, derived from vegetable oil: Sigma) was introduced into the incubation medium by dissolving in a small volume of ethanol in the incubation tube, evaporating the ethanol with a stream of nitrogen and sonicating the incubation mixture, including ROS, in the tube for 24 sec. Experiments on the effects of vitamin E included sonication of the incubation mixtures from which vitamin E was absent. The vitamin E was checked for and found to be free of significant contamination by fatty acids. ROS phospholipids and free fatty acids were extracted by methods that have been described (Zimmer-

man

and Keys, 1988).

redissolved

in

AND

S. KEYS

early steps in peroxidation of polyenoic fatty acids was obtained by redissolving a sample of the CHCl:, (phospholipid) extract of the incubated ROS in absolute ethanol and measuring the absorbance at 232 and 210 nm (Kagan, 1988). Absorbance at 232 nm is due to both non-conjugated and conjugated double bonds and therefore measures unsaturated fatty acids as well as hydroperoxides of unsaturated fatty acids. respectively. Absorbance at 2 7 0 nm is due entirely to isolated double bonds, and it decreases as hydroperoxides are formed. Therefore, the ratio A,,,/A,,,, is a measure of the concentration of fatty acid hydroperoxides relative to that of intact, unsaturated fatty acids (Kagan, 1988). This measurement was not carried out on incubation mixtures containing vitamin E as that compound absorbs in the UV. All of the enzyme and conjugated diene assays were repeated on three to eight separate ROS preparations. 3. Results The relative amounts of conjugated diene fatty acids in the ROS mixtures incubated for 1 hr at 37°C are given here as percentage increase in the value of the ratio, A,,,/A,,,. for the incubation mixture that contained DTT and was kept in the dark: dark/-DTT: + 5.7+_0.5% light/ + DTT : + 11.4 F 1.2 % light/-DTT: +20.6+1.7x.

50 ,A of

hexane/isopropanol/H,O (6 : 8 : 1, by volume) and separated by HPLC (Waters 600 Binary Gradient System), using a 4 x 250 mm, 5 pm Lichrospher Si 100 column (EM Laboratories) and an isocratic solvent hexane/isopropanol/CH,OH/2 5 IIIM system of KH,PO, (K,HPO,, buffer at pH 7.0)/CH,COOH (367:490:100:62:0.4, by volume) pumped at a rate of 1 ml min-l (Patton, Fasulo and Robins, 1982). The fatty acids were converted to their methyl esters by procedures that have been described (Zimmerman and Keys, 1988) ; they were separated and quantified by gas chromatography (Waters Dimension 1 GC System) using a 30 mm x 0.5 L&m (megabore), 50% cyanopropyl column (DB-23, J&W Scientific), 140-210°C 4°C min-‘, 8 psi. A standard amount of heptadecanoic acid (17: 0) was added to each incubation mixture before extraction in order to correct for absolute and differential loss of fatty acids during extraction, chemical esterification and chromatographic separation.

Measurement of Fatty Acid Oxidation An approximate estimate of the hydroperoxides formed in the ROS during incubation as a result of the

0

16: 0

18:O

18: I Fatty

20:4

22.4

22:6

acid

FIG. 1. Effects of light/dark and presence/absence of DTT on phospholipase A activity toward endogenous phospholipids in isolated bovine ROS. Histograms give mmol free fatty acids (FFA) mol-1 total phospholipid present after 1 hr incubation of ROS at 37°C. (m) Dark. + DTT: ( 0) light, +DTT; (B) dark, -DTT: (m) light. -DTT. Mean of experiments of four separate ROS preparations : no standard errors exceeded 22% of the mean.

PHOSPHOLIPID

METABOLISM

IN

ROS

609

Figure 1 shows the effects of the four combinations of conditions, i.e. light/dark and presence/absence of DTT, on ROS phospholipase A activity toward endogenous phospholipids : ROS samples (300-500 ,ug) were incubated for 1 hr at 37°C under the four condition combinations, the samples were then assayed for the amounts of free fatty acids present. Light did not significantly affect the ROS phospholipase A activity, but the presence of DTT inhibited phospholipase A activity toward all esterified fatty acids and, in particular, toward the polyunsaturated species, 22 : 6. Table I compares the total amounts of free fatty acids in ROS that were incubated for 1 hr at 37°C in light/dark and presence/absence of DTT or added vitamin E. The data from experiments with DTT are thus a summary of the data for individual fatty acids shown in Fig. 1. In the experiments with DTT, calcium was added to the incubation medium, and in those with vitamin E it was not. Light did not significantly affect the hydrolysis of fatty acids, but the presence of DTT and of added vitamin E significantly decreased it. Unlike DTT, vitamin E did not cause a differential decrease in phospholipase A activity toward 22 :6 (data not shown). In fresh. untreated ROS, the free fatty acids (FFA) comprise approximately 1.1% of the total fatty acids (Zimmerman and Keys, 1988), so a small increase in the percent of phospholipid-esterified fatty acids that are hydrolysed during the 1-hr incubation (column 3) results in a large percentage increase in the total FFA pool (column 2).

I

TABLE

Inhibitory effects of DTl’and vitamin E on phospholipase A activity toward endogenous phospholipids in isolated bovine ROS Conditions compared light, dark, light, dark,

+ DTT +DTT +vit E f vit E

% change in FFA

Change in % of total FA hydrolysed

+46.4+ 13.6 + 54.1 rfr15.2

+ 1.750.3 +1.9+0.2

+48.4+15-7 + 56.6 + 17.2

+4.8+ 1.4 + 3.9kO.4

Column1. Incubationconditionsbeingcompared:ROSwere incubatedfor 1 hr at 37°Cin light or darkandin the presence or absence of @5 mM DTT or 1.2 mM added vitamin E. Column 2. Percent change in total free fatty acids in ROS samples incubated without antioxidants. relative to ROS samples incubated with these compounds. Column 3, Net change in percent of total phospholipid-esterified fatty acids that were hydrolysed in ROS samples incubated for 1 hr without added antioxidants. relative to ROS samples incubated with these compounds. Values are mean+s.~. of experiments on four to eight ROS preparations.

TABLE

II

Effects of DTT and light/dark on lysophospholipaseand acyl-CoA: Zysophospholipidacyl transferase activities in isolatedbovine ROS y0 total [3H]dpm Incubation condition light, + DTT light. - DTT dark, +DTT dark, -DTT

I 40 t

0’

,,:1__

y 0

73.5 + 6.6 24.7k3.6

PC

water phase

17.9 +_ 6.2

5.1 kO.3 10.4 & 1.3 59 * 0.4

61.5k3.3

81.4+0.9 10.6+0.7 45.15 6.8 38.7f 7.4

ll.Of1.5

Column 1. Conditions under which ROS were incubated for 1 hr at 37°C in the presence of [3H-choline]lysoPC: light or dark and presence or absence of @5 mM DTT. Columns 2. 3 and 4, Percentages of total [“H]dpm recovered from organic and water phases of ROS extract that co-chromatographed on thin layer plates with IysoPC or PC, or that were present in water phase. Values are mean+s.~. of experiments on four separate ROS preparations.

60

2o/

1ysoPC

I IO

20

30 Time

40

50

60

(mm)

FIG. 2. Effects of added vitamin E on lysophospholipase and acyl-CoA : lysophospholipid acyltransferase activities in isolated bovine ROS, using [3H-choline]lysoPC as substrate. Presented are percentages of total [3H]dpm (in water and organic phases of ROS extracts) that co-chromatographed on thin layer plates with 1ysoPC (0. n ) and PC (0, a), or that were present in the water phase of the ROS extract (a, A). Data from incubation mixtures with added vitamin E are shown as 0-0, a-a. A--A; data from experiments without added vitamin E are shown as O---O, rJ-----lJ, n---n. Values are mean)-S.E. of six experiments (duplicate experiments on three separate ROS preparations).

Table II shows the effects of light/dark

presence/absence

of DTT on lysophospholipase

and

and

acyl-CoA : lysophospholipid acyltransferase activities. ROS were incubated with [3H-choline]lysoPC under the four condition combinations. Lysophospholipase activity, measured as the amount of [3H]cholinecontaining compounds in the water phase of the ROS extract, was inhibited approximately 50% by the presenceof DTT, but there were no effects induced by light/dark. Acyl-CoA : lysophosphatidylcholine acyltransferase activity was : (a) inhibited 39 % by dark (average percent decrement of [3H]dpm in PC for [ + DTT, light vs. dark] and [ - DTT, light vs. dark]) ; (b)

610

inhibited 72 % by the presence of DTT (average percent decrement of r3H]dpm in PC for [dark, +DTT vs. - DTT] and [light, + DTT vs. - DTT) ; and (c) inhibited 8 3 % by the combination of dark and the presence of DTT (percent decrement of [3H]dpm in PC for [light, -DTT] vs. [dark, +DTT]). Figure 2 shows the results of experiments with vitamin E analogous to those shown in Table I with DTT, except that the vitamin E was introduced into the ROS by brief sonication, no calcium was added to the incubation medium and samples were taken from the incubation mixtures at timed intervals. Because the differences between light and dark-incubated samples were small and inconsistent in direction, the data from incubation mixtures differing only in lighting conditions were combined, thus yielding only a comparison of the percent of total C3H]dpm in lysoPC, GPC and PC in the presence vs. absence of exogenously added vitamin E. In the ROS incubation mixtures to which vitamin E was added, the rates of decrease in 1ysoPC and increase in PC were slightly but significantly lower than in incubation mixtures from which vitamin E was absent. There were no such effects on the accumulation of [3H]choline-containing compounds in the soluble phase of the ROS extract. These results indicate an inhibitory effect of added vitamin E on acyl-CoA : 1ysoPC acyl transferase activity and no effect on lysophospholipase activity.

4. Discussion Our previous investigations of phospholipid metabolism in isolated bovine ROS have demonstrated the presence in this organelle of enzyme activities required for the synthesis and degradation of fatty acid CoA compounds and for the acylation and deacylation of phospholipids (Zimmerman and Keys, 198 6, 1988, 1989). Most of the latter two reactions take place at the sn-2 position of the phospholipids (Zimmerman and Keys, 1988). As in any study of intersecting and concerted enzyme catalysed reactions in an isolated cellular organelle, it should be emphasized that estimates of these enzyme activities in vitro are net or apparent ones. The enzyme activities might: (a) affect and be affected by each other’s substrate concentrations ; (b) be differently affected by ROS isolation and incubation conditions; (c) be affected by competition for substrates with other reactions [e.g. the acylation of rhodopsin with palmitic acid (O’Brien and Zatz, 1984)]; and (d) catalyse reactions that reverse each other’s effects (acyl transferase and phospholipase A). Since most of these factors would contribute to underestimations of enzyme activities, it is noteworthy that our in vitro measurements of acylation and deacylation rates suggest a capacity for in situ exchange or ‘molecular replacement ’ of the fatty acids of ROS phospholipids that is comparable (and additional) to the complete replacement, in the course of

W. F.ZIMMERMAN

AND

S. KEYS

11 days, of those fatty acids by ‘membrane replacement’ of the ROS phospholipids (Zimmerman and Keys, 1988). On the basis of the findings that (a) exogenously added snake venom phospholipase A, is stimulated by and preferentially hydrolyses peroxidized fatty acids from membranes (Sevanian and Kim, 19 8 5) and (b) that phospholipase A, is required for the reductive detoxification of fatty acid hydroperoxides (van Kuijk, Handleman and Dratz, 1985) Van Kuijk et al. (1987) proposed that an important function of phospholipase A, in membranes is to protect them from oxidative injury. This hypothesis was also suggested earlier by Sevanian, Muakkassah-Kelly and Montestruque (198 3). If this is one function of the high endogenous capacity for fatty acid exchange in the ROS, we would expect some sort of functional link between either oxidizing conditions per se or the amounts of oxidized fatty acids esterified to ROS phospholipids and the activities of the enzymes required for removal and replacement of the oxidized fatty acids. Therefore, we compared these enzyme activities and the extent of fatty acid oxidation in ROS that had been incubated in the presence or absence of the antioxidants DTT and vitamin E. As we stated in Results, the addition of DTT significantly decreased the amount of lipid peroxidation during incubation of the ROS in vitro. Figure 1 and Table I show that if either DTT or vitamin E is added to the incubating ROS, there is also significant inhibition of the endogenous phospholipase A activity : more fatty acids are released in the absence of the added antioxidants. Similarly, Douglas, Chan and Choy (1986) found that the activity of platelet phospholipase A toward added, radiolabeled phospholipids is inhibited by added vitamin E. Vitamin E. an efficient free radical scavenger that breaks chain autoxidations (Witting, 1980). is relatively highly concentrated in ROS membranes (Farnsworth and Dratz, 1976; Stephens et al.. 1988). DTT, a water soluble, thiol compound that protects against oxidation of other thiol groups, may also directly react with free radicals and participate in the reductive recovery of vitamin E radicals (Cadenas, 1985). Exogenously added vitamin E inhibits ROS phospholipase A activity more than DTT (Table I). This may be due to its greater proximity to the membrane phospholipids than DTT and to its more direct action in scavenging of free radicals. However. whereas the vitamin E-containing incubation mixtures were sonicated in order to introduce that hydrophobic compound into the ROS membranes, the DTT-containing incubation mixtures were not, and as sonication may be expected to cause additional oxidation of phospholipids the two kinds of experiments cannot be directly compared. We and other workers have reported that light stimulates ROS phospholipase A activity toward radiolabeled phospholipids that had been either introduced into isolated bovine ROS by sonication (Zimmer-

PHOSPHOLIPID

METABOLISM

IN

ROS

man and Keys, 1986, 1988) or mixed with ROS as liposomes (Jelsema, 1986, 1987). The latter worker adduced evidence that the G-protein, transducin, is involved in mediating such light effects. However, we find no effects of light on the enzyme’s activity toward endogenous phospholipids (Fig. 1, Table I). This difference could be due to the fact that use of exogenously added, fatty acid-radiolabeled phospholipids allows measurement of phospholipase A activity alone. The released, radiolabeled fatty acids would be diluted by endogenous non-radioactive ones, and therefore few of them would be re-incorporated, by acyl transferase activity, into phospholipids. However, measurement of the ROS phospholipase A activity toward endogenous phospholipids by the release of free fatty acids is a ‘ net ’ measurement of activity. The fatty acids released might be re-incorporated into phospholipids by the acyl transferase activity, which is, in fact, stimulated by light (Table II, see discussion below). This difference between the two assays of ROS phospholipase A also suggests the need for caution in interpreting general light effects on ROS enzyme activities in vitro: they might be due to unnaturally high concentrations of retinoids caused by extensive bleaching of rhodopsin in the absence of the pigment epithelium and/or sonication-induced disruption and oxidation of the membranes. We are therefore reluctant to attribute any functional significance to the finding that light significantly stimulates the acyl transferase and lysophospholipase activities in isolated ROS (Table II), especially considering that there are no such light effects in the presence vs. absence of added vitamin E (Fig. 2). The results in Table II and Fig. 2 show that in the incubation mixtures without added DTT or vitamin E, lysophospholipase and acyl-CoA : lysophospholipid acyltransferase activities are both stimulated. Both of these enzyme activities remove membrane-disruptive lysophospholipids : deacylation (lysophospholipase activity) results in the formation of water soluble products and reacylation (acyltransferase activity) results in an intact phospholipid (PC). The extent to which these two enzyme activities are stimulated is actually considerably more than that suggested by the results. This is because the added [3H]lysoPC comprises only 0.002 ‘X0of the initial 1ysoPC pool, and lysoPC, in turn, initially comprises less than 0.2 % of the total PC (Anderson, Feldman and Feldman, 19 70). Although we have not measured the amounts of lysophospholipids present during the incubation of ROS. it can be inferred from the increase in free fatty acids (Fig. 1) and from the increase in acylation of radiolabeled free fatty acids (Zimmerman and Keys, 1988) that the size of the non-radioactive lysophospholipid pool must have been at least five times higher in stimulatory than inhibitory conditions. So the much greater dilution of the J3H]lysoPC by nonradioactive lysophospholipids under stimulatory conditions makes the indirect measure of these enzyme

611

activities (by the proportions of radiolabel in the substrate and products) an underestimation of the true differences. The experiments presented here demonstrate a correlation between the peroxidation of ROS fatty acids and the activity of the enzymes within the ROS that remove and replace the fatty acids of their phospholipids. In their study of the effects of lipid peroxidation on the membrane structure of liposomes and on the activity of snake venom phospholipase A,, Sevanian et al. (1988) found that lipid peroxidation increases membrane viscosity, which is associated with vesicle instability and enhanced phospholipase A, attack. They inferred that snake venom phospholipase A, recognizes and is activated by structural perturbations in the membrane. Whether the association we find between lipid peroxidation and the activities of the endogenous enzymes of phospholipid metabolism in ROS is due to such a direct mechanism, or whether there are indirect signaling mechanisms between peroxidized fatty acids or oxidizing conditions and the enzyme activities, are questions that remain to be investigated.

Acknowledgements This research was supported by a grant from the National Eye Institute, NIH, EY00675-17. We thank Dr Robert E. Anderson and his co-workers at the Baylor College of Medicine for discussions of this work, and we thank the anonymous reviewers for their helpful suggestions.

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Jelsema. C. (1986). Light activation of phospholipase A, and C in rod outer segments of bovine retina: roie of transducin. Fed. Proc. 45, 1560. Jelsema, C. (1987). Light activation of phospholipase A, in rod outer segments of bovine retina and its modulation by GTP-binding proteins. J. Biol. Chem. 282. 163-168. Kagan. V. E. (1988). Lipid Peroxidation in Biomembranes. CRC Press: Boca Raton, FL. O’Brien, P. J. and Zatz, M. (1984). Acylation of bovine rhodopsin by [3H]palmitic acid. 1. Biol. Chem. 259, 5054-7.

Patton, G. M., Fasulo, J. M. and Robins, S. J. (1982). Separation of phospholipids and individual molecular species of phospholipids by high performance liquid chromatography. J. Lipid Res. 23. 190-6. Sevanian, A. and Kim, E. (1985). Phospholipase A,dependent release of fatty acids from peroxidized membranes. 1. Free Rad. Biol. Med. 1, 26 3-71. Sevanian, A., Muakkassah-Kelly, S. F. and Montestruque, S. (1983). The influence of phospholipase A, and the glutathione peroxidase on the elimination of membrane lipid peroxides. Arch. Biochem. Biophys. 223, 441-52. Sevanian, A.. Wratten, M. L., McLeod. L. L. and Kim, E. (1988). Lipid peroxidation and phospholipase A, activity in liposomes composed of unsaturated phospholipids : a structural basis for enzyme activation. Biochim. Biophys. Acta. 961, 316-27. Stephens, R. J.. Negi, D. S.. Short, S., Dratz. E. A. and Thomas, D. W. (1988). Vitamin E distribution in ocular tissues resulting from long-term depletion and supplementation as determined by gas chromatographymass spectrometry. Exp. Eye Res. 47, 237-46.

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van Kuijk, F. G. M., Handelman, G. J. and Dratz. E. A. (1985). Consecutive action of phospholipase A, and glutathione peroxiase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes. I. Free Rad. Biol. Med. 1, 421-7. van Kuijk, F. G. M., Sevanian, A., Handelman, G. J. and Dratz, E. A. (1987). A new role for phospholipase A,: protection of membranes from lipid peroxidation damage. TIBS. 12, 314. Waite, M. (1985). Phospholipases. In Biochemistry of Lipids and Membranes. (Eds Vance, D. E. and Vance, J. E.) Pp. 299-324. Benjamin, Cummings: Menlo Park, CA. Witting, L. A. (1980). Vitamin E and lipid antioxidants in free radical-initiated reactions. In Free Radicals in Biology. Vol. 4. (Ed. Pryor, W. E.) Pp. 295-320. Academic Press : N.Y. Young, R. W. (1976). Visual cells and the concept of renewal. Invest. Ophthalmol. 15. 700-2 5. Young, R. W. and Droz, B. (1968). The renewal of protein in retinal rods and cones. I. Cell Biol. 39. 169-84. Zimmerman, W. F. and Keys. S. (1986). Acyl transferase and fatty acid coenzyme A synthetase activities within bovine rod outer segments. Biochim. Biophys. Acta 138, 988-94.

Zimmerman, W. F. and Keys, S. (1988). Acylation and deacylation of phospholipids in isolated bovine rod outer segments. Exp. Eye Res. 47, 24760. Zimmerman. W. F. and Keys, S. (1989). Lysophospholipase and the metabolism of lysophosphatidylcholine in isolated bovine rod outer segments. Exp. Eye Res. 48. 69-76.