Toxlron, 1976, Vol . 14, pp. 319-327. Peraamon Prou. Printed In Ore~t Brkain.

EFFECT OF PHO5PHOLIPASES A, AND C ON STRUCTURE AND PHOSPHOLIPIDS OF THE ELECTROPLAX PHILIP ROSENBERG

Section of Pharmacology and Toxicology of the School of Pharmacy, University of Connecticut, Storys, Corm ., 06268, U.SA (Acceptedforprrbllcation 291anuory 1976) P. RasElvs>:Ita. Effect of phospholipases A, and C on structure and phospholipids of the electroplax. Toxicon 14, 319-327, 1976.- Phospholipid hydrolysis after 30 min exposure to phospholipase A, or phospholipase C was determined in intact electrophtx cells and in the separated conducting and non~onducting membranes. These enzymes, in concentrations of 0"2 and 2"0 mg per ml, caused approximately the same percentage hydrolysis of phosphatidylcholine phosphatidylethanolamine and phosphatidylserine (40-80n ; in addition phospholipaso C hydrolyzed sphingomyelin. Phospholipase A2 (0~2 and 2"0 mg per ml) caused mitochondrial swelling, and a pinching off of the membrane inpocketings into clusters of small rounded vesicles external to the membrane. Lysophosphatidylcholine (2 mg per ml) caused some vesicular formation, although not nearly as numerous as with phospholipase A2 ; and no mitochondrial alterations. Phospholipase C (2 mg per ml) caused some mitochondrial swelling, but no vesicle formation. Disruption by phospholipase C of hydrophilic or electrostatic interactions between phospholipids and proteins has less effect on the ultrastructural organization of the membrane than does disruption of hydrophobic interactions by phospholipase A, . The results are discussed in relationship to the 9uid mosaic model of membrane organization. INTRODUCTION

SNAxE venoms and bacterial toxins are the richest sources of PhA2 and PhC* respectively, enzymes which have been extensively used as enzymatic probes to study the structural and functional organization of biological membranes. PhAz (phosphatide acyl hydrolase; EC 3.1 .1 .4) hydrolyzes the fatty acid ester at the 2 position of the phospholipid and would be primarily expected to disrupt hydrophobic bonding between phospholipids and proteins . PhC (phosphatidylcholine : choline phosphohydrolase ; EC 3.1.4.3) hydrolyzes the bond between the phosphorylated base (polar head group) of the phospholipid and the diglyceride portion of the molecule. This should primarily decrease hydrophilic forces of interaction between phospholipids and proteins, since it is known that subsequent to PhC action, the polar head groups of the phospholipids leave the membrane (LENARD and SINGER, 1968 ; ROSENBERG, 1970 ; McILWAIx and RAPPORT, 1971) . The relative potencies of PhA2 and PhC in altering membrane structure might therefore aid us in evaluating the appropriateness of the fluid-mosaic model of membrane structure (GLASER et al., 1970 ; SINGER and NICOLSON, 1972), in which it is proposed that the membranal protein extends

*Abbreviations used : PhA2, phospholipase A2 ; PhC, phospholipase C; PC, phosphatidylcholine ; PE, phosphatidylethanolamine ; PS, phosphatidylserine ; PI, phosphatidylinosotol ; LPC, lysophosphatidylcholine (lysolecithin) SM, sphingomyelin. 319 TOXICON 1976 Vol. l~

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PHILIP ROSENBERG

into the interior of the membrane, in close approximation to the fatty acid chains of the phospholipids, while the polar head groups of the phospholipids are at the surface of the membrane. Hydrophobic (non-polar) interactions would be expected to be of major importance in such an arrangement of lipid and protein . By using PhAz and PhC on the squid axon it was concluded that PhC caused a marked change in the phospholipid portion ofthe membrane, but much less of a change in the protein portion, whereas PhA z caused drastic changes in both the phospholipid and protein portions, due to disruption of lipid-protein binding (RosExsExG and ICxAiRALLAx, 1974). In the squid axon, however, there is no known specific functional role for phospholipids. It has in fact been shown that extensive phospholipid hydrolysis by PhC has no effect on the action potential or permeability of the squid axon, even though, subsequent to enzymatic hydrolysis, most of the hydrolyzed polar head groups leave the membrane and appear in the external bathing solution (R06ENBERG, 1970 ; ROSENBERG and CONDREA, 1968). All apparent effects of PhA on the action potential and permeability of the squid axon were in fact due to production by PhA oflysophosphatides which have strong detergent properties (ROSSNBER(i and COIVD1tEA, 1968; ROSBNBERG and No, 1963 ; ROSSNBERG and PODLESKI, 1962). It was of interest to investigate the effects of PhA z and PhC on the structural organization of a biological membrane in which phospholipids do appear to have specific functional roles to subserve . It has been suggested that phospholipids are involved in synaptic transmission . Increased metabolism of PI accompanies synaptic activation of neurons apparently due to the action of the synaptic transmitter on the post-synaptic cell (LA1tRABEE et al., 1963). Theoretical proposals have also been made regarding relationships between synaptiotransmitter functions and phospholipids (WATxiNS, 1965 ; DulutELL et al., 1967) . Both PhAz and PhC depolarize and block the action potential in the isolated single electroplax (BA1tTELS and ROSENBERG, 1972), a synapse containing preparation from the electric eel (Fig. 1). Electrical excitability was maintained when 75 and 89 ~ of PE and PS were hydrolyzed by PhAz but was blocked when more than about a third of the PC was hydrolyzed (BARTELS and ROSExsL~tG, 1972) . Further evidence for an essential phospholipid function in the electroplax was obtained by the observation that both electrical stimulation and the application of acetylcholine increased the rate of incorporation of "P and "C into PI and PC of the isolated electroplax (ROSENBERG, 1973). The fine structure of normal electric tissue has been extensively studied (LuFr, 1956 ; 1958 ; MATHEWSON et al., 1961 ; WACHTEL et al., 1961 ; SHERIDAN, 1965); however, no analyses have been made in this preparation of the ultrastructural effects of PhAz and PhC. This study was therefore initiated in an effort to determine the relative importance of hydrophobic and hydrophilic interactions between phospholipid and protein in a synapse containing preparation where phospholipids may be essential for functioning . MATERIALS AND METHODS Materials PhC, a salt free, lyophilized and partially purified preparation from Ciostridirun perfringens, was obtained from Schw~r~-Mann (Orangeburg, New York). The preparation (lot no. X 3452) has an activity of 13 units per mg. One unit is the amount of enzyme which liberates 1 umole of water soluble organic phosphorus from lecithin per min at pH 7~2 and 37~ (MwcFutr+xs and Kruax~r, 1941). This preparation had no proteolytic activity towards bovine serum albumin asjudged by protein determinations (LowßY et a1.,1931) . Other investigators have also found commercially obtained PhC to be devoid of proteoly.ic and peptidase activity (Gonnox et a/.,1969 ; McIr-w.+nv and RnrPOxT,1971 ; RwY et a1.,1972). TOXICONl976 Vol. It

Phaspholipases on Electrophu

ROSTRAL

SPINAL CORD 31rIY . bLAppER

321

CAUQ4L

Fro. 1. Ttssurs oa Electrophorus dectrkus. Upper left : position of Main (A) Sechs (B) and Hunter (C) electric organs. Middle left : fragment of electric organ showing relationships between rows of electroplax and other organs of electric eel. Lower left : changes in frequency of electroplax per cm of Main organ. Numbers indicate distance in cxn from tho anterior end of the organ. Upper right : method of dissection of single electroplax. Cutting at 1 and 3 isolates a single electroplax. Noa-innervated, nonconducting membrane is indicated by deep invaginations. The conducting synaptic-containing membraao is represented by straight line to the left of 3. Lower right : arrangement for mounting a single electrophu for electrical recording. Shown are one chamber for a pool of fluid (other chamber not shown), tho shcet of r>,ylon containing a window, tl~ single electroplax and the grid used for pressing the cell against the window [figure modified from N~c~f+xsoRx (1959)]. Highly puriSed PhA, (L~rsz and RosBxssxa, 1974) was used as the enzyme sours in some experiments and gave results identical to those with acid boiled venom (described below) . Cottonmouth mocassin (.lgkistrodon piscivoruspiscivorus) venom was obtained as a lyophilized preparation (lot no . 10206 from Ross Allen Reptile Institute (Silver Springs, Florida) . A solution of this venom, heated for 15 min at 100°C and pH 5, was used as the source of PhA,, after centrifugation and adjustment of the supernatant fluid to pH 7~0 beforo use. PhA, is the only enzyme prosent in snake venoms known to be resistant to boiling at acid pH : it is destroyed by boiling at alkaline pH (HUC3HES, 1935 ; HRAC3ANCA and Quesret, 1952 ; Meow and THOMP90N, 1960). Using bovine serum albumin as substrate, we confirmed the observation (Sr~xnvs et aL, 1971) that acid-boiled venom has no proteolytic activity . LPC, which is also known as lysolecithin, was obtained from Pierce Chem . Co. (Rockford, Ill.) . It is a white crystallino powder prepared from purißed egg lecithin, by PhA, with further purification. TLC revealed a single spot without contamination . 7Yssue studied Eels (Electrophorus electrtcus), 1~2-1~5 m long purchased from Paramount Aquarium (Elmsford, New York) wero maintained for several days prior to use at 25° in aerated, filtered, aged tap water and wero fed daily. Single cells (approx. 10 x 4 x 0~2 mm), wero isolated from the Sechs electric organ by cutting at points 1 and 3 (Fig.1). It was also posarblo to separate the innervated membrane of tho cell, which receives neural inputs and which is electrically excitable, from the non-innervated, non~xcitable membrane. All dissections and incubations wero carried out in eel Ringer's solution of the following rnmposition (mM) : NaC1,160 ; KCI, S; CaCI,, 2 ; MgCI,, 2 ; Tris buffer, pH 7, 5 and glucose, l0. Phosphoh'pid analysis Foreach analysis on isolated Sechs cells,l0-15 cells (400-600 mg wet weight) were pooled after a 30 min TOXICON 1976

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PHILIP ROSENBERG

exposure of the isolated individual cells to either normal eel Ringer's or a solution of PhAz (0~2 and 2 mg per ml), PhC (0~2 and 2 mg per ml) or LPC (0~2 and 2 mg per ml) in eel Ringers. Innervated (50-150 mg) and aon-innervated (400-800 mg) membranes were dissected from the Sachs cell after exposure of rows of cells to either normal eel Ringers or to PhA, PhC or LPC in eel Ringer's solution. Lipids were extracted by the methods of Mnßnvez-n et al. (1959) and Form et al . (1957) as previously described (Bnx~rs~s and Rosexs~ea, 1972). Phospholipids were separated by two-dimensional thin layer chromatography (Cormx~ et al., 1967) and the phosphorus content of the total lipid extract and of the phospholipid spots (identified by iodine staining and scraped from thin layer chromatograms) was assayed by the method of BnxTtsrr (1959) . The percentage of each phospholipid split by exposure to PhAz or PhC was calculated as previously described (Roserresxa and Cormxax, 1968 ; Corroxsx and Roser>esaa, 1968) . Electron-microscopy Small slices of the Sachs organ consisting of a few electroplaques wero removed and either fixed immediately or placed in eel Ringer's solution for further dissection to isolate individual eloctroplaques. The slices which were fixed directly consisted of a single column of up to six undamaged electroplaques surrounded by columns of cells that had been cut . Primary fixation was in a mixture of 3 ~ formaldehyde (freshly prepared from paraformaldehyde) and 0~5 ~ glutaraldehyde in 005 M phosphate buffer, pH 7~2, containing 0~ 1 magnesium chloride for 20-24 hr. The tissue was washed in the same buffer solution for 2 hr. and then postfixed in 2 ~ OsO, for 1 hr . After washing in 005 M maleate buffer at pH 5~2 for 15 min, the tissue was stained in a solution of 0~5 ~ uranyl acetate in maleate buffer for 2 hr, then washed again in buffer and distilled water, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. All fixation and dehydration steps through 70~ ethanol were performed at 0~°C ; the remainder at room temperature . Individual electroplaques isolated from the slices of tissue that had been plead in Ringer's solution were fixed after a total of 2~} hr in this solution, or after 2 hr in Ringer's solution and } hr in Ringer's solution containing either 0~2 or 2~0 mg of PhAz, PhC or LPC per ml . The fixation and embedding schedule was the same as that used for columns of cells. Thin sections were cut on Porter-Blum or LKB ultn~microtomes mounted on bare copper grids, stained with lead citrate, and photographed in a Philips EM 300 electron microscopy or a Hitachi HU-11A .

io

Phospholipid hydrolysis

RESULTS

The total lipid phosphorus value (measure of phospholipid content) of single Sachs cells was 25 f 6 ~g per g wet weight (Mean ~ S.E., 5 expts) . The per cent distribution of individual phospholipids in the Sachs cell is as follows (Mean ~ S.E., each based on 5 expts) : PC, 50 ~ 5 ; PE, 29 ~ 3 ; PS, 7 ~ 2; SM, 5 ~ 1 ; PI, 4 ~ 0~5; LPC, 3 ~ 1 . The above results are in excellent agreement with previous data (BARrec.s and R1Ä&NBERG, 1972). The per cent hydrolysis of phospholipids by PhAz and PhC in isolated Sachs cells and its innervated and non-innervated membrane are shown in Table 1. The extents of hydrolysis of PC, PE and PS by PhC are only slightly less than that by equal concentrations of PhAz, the differences not being significant. PhC hydrolyzes SM, whereas PhAz has no effect on SM. The per cent hydrolysis of PE and PS by the higher concentration of the enzymes is not much greater than that by the lower, whereas the difference is quite significant in the case of PC. Electron microscopy Control tissue (Fig. 2) .

The innervated face is indented by frequent relatively broad inpocketings of the surface membrane. The inpocketings usually are simple, but may be branched, and are relatively short. More complex and longer inpocketings cover the non innervated face . They are usually branched and form an interconnected cannalicular reticulum like that found in electroplaques of other strongly electric fish. Near both surfaces of the cells there are many mitochondria and nuclei and a sparse endoplasmic reticulum. The interior of the cell is filled with fine filaments and contains few membranous organelles . Fixation artefacts in the form of swelling of the perinuclear space of the endoplasmic reticulum are sometimes seen in control material . This may be related to the large size of TOXICON 1976 VoJ. l~

FIG . 2. CONTROL ISOLATED SINGLE ELECTROPLAX FROM SACHE ORGAN OF THE ELECTRIC EEL. Left : innervated surface ( ~ 7200 . Right: non-innervated surface ( -: 7200). Note deep

invaginations of mcmbrane and large nucleus.

FIG. 3.

PhA y

(2

mg per ml) TREATED ISOLATED SINGLE

ELECTROPLAX FROM SACHS ORGAN OF THE ELECTRIC EEL, Ringers for 30 min. Left : innervated surface ( x 7200).

Cells were eXposed to PhA, in eel Note formation of clusters of vesicles and swelling of some of the mitochondria . A large nerve is near the lower left-hand corner . Right : non-innervated surface ( :c 13,500), Note massive vesicle formation and swelling of mitochondria.

TOXICON 1976 Val, ld

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FIG. 4. INNERVATED SURFACE OF PI1C AND LPC TREATED ISOLATED SINGLE FLE(TROPLAX FROM SACHS ORGAN OF THE ELECTRIC EEL.

(~el Is were exposed to PhC or LPC in eel R fingers for 30 min. Left : PhC (2 mg per ml) ; - 7200). There is slight mitochondrial swelling, but no vesicle formation . Right: LPC (2 mg per ml ; ~. 7200). Mitochondria were not markedly affected . Some vesicles are seen but these are not nearly as numerous as was observed with PhA (Fig . 3) .

TOXlCON 1976 Vol. l4

323

Phospholipases on Electroplax TABLE

l.

HYDROLYSIS OF PHOBPHOLIPIIa3, IN INNSRVAT~ AND NON-INDIERVAT'® ME!®RANBS AND IN SACFD crass, BY PhA, AND PhC. R(~iipasa 1~eParation" mi 1â~2

0.2

Intact Inn. äoa-inn.

2.0

Ph0

0.2

Pc 50 t 4 58t4

ra

re

stc

76 t 3

64 t 7

0 t0 0t0

60 t 17

0 *0

58t5

69t6

58 ± 3

60 * 3

Intact

85 t 3

80 t 7

80t5

65±4

73 t 8

0 t0

Im .

80t2

0*0

8oo-inn.

88 t 2

70 t z

T9 t 3

o t

Intact

42 t 4

49 t 3

46 * 5

4T t 3

59 t 2 60 t t

49 t 2

55 t 2

Intact

78 t 3

77 * 2

Imm.

75t5

68t3

62t3

70t2

66 t ç

Inn. xoa-inn. 2.0

f $Idro~tis

IIoa-inn.

Bo t 4

INrACr

o

55 t T

47 ± 4

57 t 6

40 * 3

59 t 5

75 t 4

6o t 3

78 t 5

"Intact, isolated singlo cells from the Sachs organ were exposed for 30 min to the indicated concentrations of enzymes prior to lipid extraction ; Inn. and Non-inn . innervated and non-innervated membranes of Sachs cells, which were isolated after exposure of rows of cells (one~ell thick) to phospholipases followed by extensive rinsing with cel Ringer's solution. Valuesrepresent means ~ S.E.,eachbased upon4experiments. the slices that were fixed in order to insure the presence of intact electroplaques, and perhaps also to retardation of diffusion of the fixative by the dense connective tissue. When individual electroplaques were fixed, an additional challenge to ideal fixation was the duration ofthe exposure to eel Ringer's solution prior to fixation, which was based upon the time required to dissect enough free electroplaques for control and enzymatic treatment . PhA z (Fig. 3). Some effects of PhA z treatment are seen after ~ hr of exposure to 0~2 mg per ml solution. Some mitochondria have areas of swelling in which the electron dense matrix and the cristae are displaced, though the mitochondria as a whole maintain their form. A few small vesicles of uncertain origin appear at the surfaces of the electroplaques . Other structures are normal. After ~ hr in 2~0 mg of this enzyme per ml both of these changes are greatly increased . Many of the mitochondria are rounded and swollen to the point of being recognizable mainly by the remnants of cristae remaining around their peripheries . The membranous inpocketings at both surfaces are completely disrupted and have pinched off into clusters of small rounded vesicles which are now external to the electroplaques . However, the ground substance of the cytoplasm appears to be normal, even to the surface of the cells, and the cell membrane of the electroplaque probably is continuous, having reformed after exclusion of the clusters of vesicles . PhC (Fig. 4). Treatments with this enzyme have less drastic effects upon cellular morphology than those with PhAz . At the lower concentration cellular structures did not routinely differ from those ofthe controls. At the higher concentration some xnitochondrial swelling with displacement ofthe cristae is seen. Vesiculation of the plasma membrane with the formation of extracellular vesicles was not found. LPC (Fig. 4). At the lower concentration, structural changes were not noted, however, TOXICON J976 Vo1. Jf

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PHILIP ROSENBERG

at the higher concentration extracellular clusters of vesicles produced by the degradation of the inpocketings of the plasma membrane were found. These were not so numerous nor were the inpocketings as completely obliterated as was observed with PhAz. Mitochondria were not affected by either concentration of LPC . DISCUSSION

PhAz causes a much greater disruption of the structural organization of the electroplax cell than PhC, even though the extent of phospholipid hydrolysis produced by these two enzymes is similar. It would thus appear that disruption of hydrophilic or electrostatic interactions between phospholipids and proteins by PhC has less effect on the ultrastructural organization of the membranes than does disruption of hydrophobic interactions by PhA z. Similar electron microscopic changes to what we find produced by PhAz in the electroplax were also found following the application of PhA z to the squid giant axon (MnRTnv and ROSSNBERG, 1968). Following hydrolysis of phospholipids by PhC in the squid axon, almost all of the aqueous soluble phosphorylated bases (polar head groups) of the phospholipids leave the membrane appearing in the external aqueous bathing solution, whereas most of the lipid-soluble diglyceride, produced as a result of PhC action, remains firmly bound to the membrane (Ros$xBIIta, 1970). We would expect similar findings to be observed in the electroplax. Other investigators have in fact observed, following exposure of nerve (TosL4s, 1958), mitochondria or human red-blood cell ghosts (OTTOLSNGHI and 13oWMnN, 1970) or muscle microsomes (Fua$aN and MARTONOSI, 196 to PhC, the appearance of discrete electron-dense particles or droplets which it has been suggested may represent diglyceride produced as a result of PhC action . It is also possible, however, that these droplets may, at least in some preparations, represent unesterified fatty acids produced as a result of the hydrolysis of diglycerides by membrane associated lipase (MICÜßLL et al., 1973). We did not observe the formation of these droplets in our study ; however, no information is available concerning the lipase activity of the isolated electroplax . On the basis of our phospholipid and electron-microscopic analyses, we might conclude that PhC causes marked changes in the phospholipid portion of the membrane, but much less of a change in the protein portion . Using electron spin resonance spin label studies, similar conclusions were reached using erythrocyte ghosts and submitochondrial particles (Sn~xnvs et al., 1971) . Using red blood cell membranes it was found that PhC released about 70 ~ of the phosphorylated bases but no protein, nor was the circular dichroism spectrum of the protein portion of the membrane affected (LSNARD and SnvGt;Hx, 1968 ; GLnsfiR et al., 1970) . It has been reported that hydrolysis of two-thirds ofthe phospholipids of rat liver plasma membranes by PhC causes the release of only about 15 ~ of the membrane protein : whereas 1 M NaCI released 33 ~ of the protein (RnY et al., 1972), although in contrast to our results on intact cells they found marked structural changes when PhC was applied to suspensions of rat liver plasma membranes . In agreement with our findings, others have also noted that membranes treated with PhC do not show any striking morphological changes (LENARD and SINGER, 1968 ; MCILwanv and RAPPORT, 1971 ; Fn~x and MARTONOSI, 1965; TRUE et al., 1970), which may also be related to its lack of effect on the protein component ofthe membrane. In contrast to these findings with PhC, marked alterations in protein structure of membranes have been observed after PhAz . Using a fluorescent probe or optical spectra, it was found that PhA z modifies the architecture of red blood cell membrane proteins (CORDON et al., 1969 ; WIEDExA~ et al., 1971) . Somewhat in contradiction are the findings of SnKPtüNS et al. (1971) who found with both red TOXICON1976 Vol. II

Phospholipaaea on Elatroplax

32 5

blood cells and walking leg nerves of lobster no release or change in membranal protein after PhAz, as judged by the iodoacetamide spin label or circular dichroism. However, using a N-ethylmaleimide spin label, some proteins deep in the membrane appeared to become accessible to the label after PhAz. Using high angle X-ray diffraction techniques it was shown that PhAz mainly alters the protein component of the red blood cell membrane, an effxt which was not mimicked by LPC, whereas PhC caused a structural change in the lipid region (BSxtvsxao and S~xixs, 1972). Our present results and the above noted reports in the literature can be interpreted in terms of the fluid mosaic model (GLASSR et al., 1970; SINGER and hlICOLSON, 1972) of membrane structure in which the polar head groups of the phospholipids are at the surface of the membrane, with the major protein-lipid binding being of a hydrophobic nature . One can readily visualize how PhC might cause the loss of phosphorylated bases from the membrane without drastically modifying phospholipid-amino acid binding. This would be the case if the electrostatic interactions between proteins and the polar head groups of the phospholipids, at the surface of the membrane, are not of major importance in the maintenance of the phospholipid-amino acid complexes. In contrast, hydrolysis of fatty acids by PhAz would drastically disrupt phospholipid-amino acid complexes. A major hydrophobic bonding force in the membrane, according to the more recent models of membrane structure, is the Van-der-Waals type interactions between the methylene groups of the fatty acids of the phospholipids and the amino acids of the proteins . Hydrolysis of the ß fatty acid esters of the phospholipids by PhAz decreases hydrophobic interactions and thereby interferes with the structural integrity of the membrane because of the loss of phospholipid-protein binding. In our earlier studies on the electroplax (BARTELS and RosexsaRC, 1972) we showed that both PhAz and PhC depolarized and blocked conduction at approximately equal concentrations . This finding, coupled with the observation that acetylcholine and electrical stimulation increased phospholipid turnover (ROSSNBSR(i, 1973) leads one to suggest that phospholipids may have a specific function in the generation of bioelectricity in the electroplax cell . The effects of PhC at least do not appear to be due simply to breakdown of the membranal organization of the cell, since the electron-microscopically visible alterations after PhC treatment are not great. Since LPC had similar effects on the resting and action potential of the electroplax as PhAz (BARTELS and R06ENBERG, 1972), it was important to ascertain in our present study that the effects of PhAz on membrane infrastructure were not simply due to the resultant production of lysophosphatides, which have detergent properties . We found in our present study, however, that LPC had much less of an effect on the structure of the electroplax than PhAz. We can estimate how much lysophosphatides would be produced as a result of the action of PhA on the single electroplax cells used for electronmicroscopy . Knowing that three cells (approx. weight 0~1 g) with a total lipid phosphorus value of 25 ~g per g wet weight were incubated in 10 ml of a solution containing 2 mg PhAz per ml and assuming that the average molecular weight of lysophosphatides is about 18 times that of phosphorus, we can estimate the concentration of lysophosphatides which would be produced, assuming 100 ~ phospholipid hydrolysis . Using these approximations we would estimate that the molar concentration of LPC we applied to the axons when we used 2 mg per ml is about 3~6 x 10'3 ; M, whereas the concentration produced by PhAz action on the electroplax cells, even if 100 ~ of the phospholipids were hydrolyzed would only be about 8 x 10~ M. It would thus appear that the effects of PhAz on the electronmicroscopic appearance of the electroplax is not due to the production of lysophosphatides . TOXICON 1976 Vol. 1~

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PHILIP ROSENBERG

It is of course possible that the effects of lysophosphatides produced by the action of PhAz directly within the membrane would be greater than that of even higher amounts applied externally. In the squid giant axon axoplasmic phospholipids are hydrolyzed by PhAz and PhC (ROSENBERG, 1970 ; ROSENBERG and ICüAIRALLAiI, 1974 ; CONDREA and ROSENBERG, 1968). MCILwAIN and RAPPORT (1971) have even reported that PhC can penetrate the multilayered membranes of myelin fragments. In addition (ROSENBERG, in press), direct assays of PhAz and PhC activities in the axoplasm of squid giant axons exposed to these two enzymes showed that both can penetrate into the axoplasm . Our results on the electroplax, showing that phospholipases can alter the structure of intracellular organelles such as mitochondria, also indicates an ability to penetrate biological membranes. It is also of interest that phospholipids can undergo a `flip-flop' motion from one side of a biological membrane to the other (MCNAI~E and MCCoNNELL, 1973). It is thus possible that `internal' phospholipids could be exposed to phospholipase enzymes even if the enzymes were not able to completely penetrate the membrane. The changes produced in mitochondrial ultrastructure by PhA in the electroplax confirm earlier findings with rat liver mitochondria (AUGUSTYN et al., 1970) in which severe inhibition of electron-transport was caused by PhAz. Other workers also obtained some evidence for morphological and functional changes in mitochondria induced by PhA z (NYGAARD et al., 1954 ; CONDREA et al., 1965 ; AwnsrIII et al., 1970 ; ARAVINDAKSHAN and BRAGANCA, 1961). Acknowledgements-I am greatly indebted to Dr . Ai uuty Wecx~rEL and Mrs. LAMCA xw"m "i r . " w for the preparation and assistance in evaluation of the electron micrographs . This work was supported in part by a grant from the National institute of Health (IRO1N S 09008) .

REFERENCES Awvmwetcs~rr, 1. and HxAaAxcn, B. M. (1961) Studies on phospholipid structures in mitochondria of animals injected with cobra venom or phospholipase A. Btochim . J. 79, 84 . Auous~rrcv, J. N., PAnsA, B, and Er uo~rr, W. B. (1970) Structural and respiratory effects of Agkistrodon ptscivorus phospholipase A on rat liver mitochondria. Btochim. biophys. Acta 197, 185. Awesrm, Y. C., Ruztcx~, F. J. and CR"Na, F. L. (1970) The relation between phospholipase action and release of NADH dehydrogenase from mitochondrial membrane. Btochim. biophys, Acta 203, 233. B.urrazs, E, and RosEiveEao, P. (1972) Correlation between electrical activity and splitting of phospholipids by snake venom in the single electroplax . J. Neurochem. 19, 125, BAx~.e~-r, G. R. (1959) Phosphorus assay in column chromatography . J. btol. Chem. 234, 466. BERNENGO, J. C. and Smtpxnvs, H. (1972) A high angle diffraction study of red cell membranes after treatment with phospholipases AZ and C. Can. J. Btochim. 50, 1260 . Bxwa~vce, B. M, and QUA31'EL, J. H. (1952) Action of snake venom on acetylcholine synthesis in brain. Nature, Lond. i69, 695 . Corroxra, E. and Roser~eaG, P. (1968) Demonstration of phospholipid splitting as the factor responsible for increased permeability and block of axonal conduction induced by snako venom. II . Study on squid axons. Btochim. biophys. Acta 150, 271, CormxEA, E., Rasexs~eG, P. and D~iuv, W. D. (1967) Demonstration of phospholipid splitting as the factor responsible for increasing permeability and block of axonal conduction induced by snake venom. I. Study of lobster axons . Btochim. biophys. Acta 35, 669. CONDREA, E., Avc-Dox, Y. and MAaea, J. (1965) Mitochondria) swelling and phospholipid splitting induced by snake venoms. Btochim. biophys. Acta 110, 337. DvRRF?~r- J G~a,r, .~vn, J. T. and Fxn?oEt., R. O. (1967) Acetykholine action : biochemical aspects. Science 168, 867. FnvEAx, J. B. and MAx~roNOSi, A. (1965) The action of phospholipase C on muscle microsomes : a correlation of electron microscope and biochemical data . Btochim. biophys. Acta 98, 547. Foi.cx, J., LeFS, M. and Sconxe-STAtvcsv, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. l. btol. Chem . 226, 497. G~~e, M., SnKPxuvs, H., Snvosx, S. J., Sm~z, M. and Cxwiv, S. I. (1970) On the interactions of lipids and proteins in the red blood cell membrane . Proc. natn. Aced. Sct., U.S.A . 68, 721. Gosnox, A. S WALLACH, D, F. H. and $TRAUS, J. H. (1969) The optical activity of plasma membranes and its modification by lysolecithin, phospholipase A and phospholipase C. Btochim. biophys. Acta 183, 405. TOXICON 1976 Vol. If

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