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Fig. 1. From a section through a ganglion of the myenteric plexus of rat stomach. This figure was obtained by preparing a montage with several electron micrographs (microscope enlargement: 10 000 x ; printed at a final enlargement of 40 000 x) ; profiles of glial and nervous structures were picked out on tracing paper, and a heliografic reproduction obtained (approximately 43 x 210 cm). Glial cell bodies and processes of glial cells have been shaded in; nuclei of both glial and nerve cells are grey. The section is perpendicular to the stomach wall; to the left (not reproduced) is the inner muscle coat, to the right the outer muscle coat; small dots around the ganglion represent collagen fibres. 229 x. This is the enlargement of the original print I sent you; I hope you did

not reduce it.

Fig. 2. Myenteric plexus of the rat stomach. A laminar glial process with gliofilaments longitudinally sectioned and glyco-

gen granules. 90 000 x.

Fig. 3. Myenteric plexus of the rat stomach. In a large glial process microtubules and gliofilaments, mainly transversally

sectioned, are apparent. 62 000 x.

PHOSPHOLIPASES 247

used, was kept as low as possible. When studying phos-pholipase A from Crotalus adamanteus the incubation (in Tris-HCl buffer) contained 2 x 1 0 - 3 M CaCl2 8.

For phospholipase C, the particles were incubated as above but in Tris-HCl-buffer with 2 x 1 0 - 3 M CaCl2.

For phospholipase D, the particles were incubated as above but in acetate buffer with 2 x 1 0 - 3 M CaCl2 .

The conditions for the assays of the different elec-tron transfer enzymes were those described in detail in a preceding paper1 and elsewhere9-11. For assay of succinate-cytochrome c-reductase the enzyme was incubated in the reaction mixture for 120 sec before the reaction was initiated with substrate. Activity was calculated from the time period when a steady reaction velocity was readied (usually 2 —3 min after addition of substate).

The procedure for measuring solubilized protein was that previously described1. Protein was deter-mined by the method of LOWRY et al.12.

Results and Discussion

Although phospholipase A is generally known as a Ca 2 0 requiring enzyme (see e . g . 1 3 ) , it has been shown by SALACH et al . 1 4 that phospholipase A from the venom of Naja Naja does not have Ca2® necessary for its activity. This property is confirmed here. It was found that the effects of this lipolytic activity on the electron transport containing mem-brane fraction of R. rubrum are given as well in the absence of Ca2®. By inactivating phospholipase A with a combined heat and alkali treatment it was secured that the effects which will be presented below were in fact produced by the enzyme.

There are reports in the literature that phospho-lipase A prepared from Crotalus adamanteus venom is unable to release N A D H dehydrogenase 8 ' 1 5 and other enzymes 16 from membrane structures. It was found here that this venom when used as the source of phospholipase A, with or without addition of C a 2 0 (see "Materials and Methods"), is completely ineffective with the membrane system of R. rubrum. As a reason for the inability of the rattlesnake venom its inert nature towards cardiolipin (diphos-

8 Y . C . AWASTHI, F . J. RUZICKA, and F . L . CRANE, Biochim. biophysica Acta [Amsterdam] 203, 233 [1970].

9 M . BOLL, Arch. Mikrobio l . 6 2 , 9 4 [ 1 9 6 8 ] . 1 0 M . BOLL, Arch. Mikrobio l . 6 4 , 8 5 [ 1 9 6 8 ] . 1 1 M . BOLL, Arch. Mikrobiol . 69 , 3 0 1 [ 1 9 6 9 ] . 1 2 O . H . LOWRY, M . J. ROSEBROUGH, A . L . FARR, and R . J.

RANDALL, J. biol . Chemistry 1 9 3 , 2 6 5 [ 1 9 5 1 ] . 1 3 T . E . KING, R . L . HOWARD, J. KETTMAN, JR., B . M . HEDGE-

KAR, M . KUBOYAMA. K . S. NICKEL, and E . A . POSSEHL, i n : Flavins and Flavoproteins, p. 441, E. C. SLATER, ed., Else-vier, Amsterdam 1966.

phatidyl-glycerol) is discussed8. This could apply also to the membrane system of R. rubrum as the organism is known to contain this phospholipid 17.

While studying the effects of a number of deter-gents on the membrane fraction containing the elec-tron transport system 7 ' 1 8 ' 1 9 , it was observed that two of its activities, NADH- and succinate-cyto-chrome c-reductase become stimulated. This be-haviour is different with the detergents tested. Only in the presence of deoxycholate both activities are increased while with Triton X -100 and dodecyl-sulfate only one of the two reveals this stimulation. The other activity is at the same time decomposed. Based on these findings different modes of action of the detergents on the individual NADH—> ubiqui-none and succinate —> ubiquinone sections of the electron transfer chain are to be assumed.

The action of phospholipase A on the R. rubrum membranal system was found to be similar to that of deoxycholate. As can be seen from Table 1, the activities of NADH- and succinate-cytochrome c-re-ductase are both increased by an incubation with the

percent activity

jug phospholipase A/mg membrane

3.5 7.5 protein

30 75 120 250

NADH oxidase 100 90 80 63 48 38 NADH-cytc-

reductase 130 160 170 170 160 170 succinate-cytc-

reductase 120 155 165 165 160 165 NADH

dehydrogenase 100 — — — 95 90 succinate

dehydrogenase 100 — — — 95 90

Table 1. Action of phospholipase A on the different electron transfer activities. Electron transport particles (5.2 mg pro-tein/ml) were incubated in phosphate buffer for 30 min with the desired amounts of phospholipase A. Then the activities were determined. Control incubations without phospholipase A were taken to be 100 percent activity. For experimental de-

tails see "Materials and Methods".

1 4 J. SALACH, P . TURINI, J. HAUBER, H . TISDALE, and T . P . SINGER, Biochem. biophysic. Res. Commun. 33, 936 [1968].

15 G. A. THOMPSON, in: Comprehensive Biochemistry, Vol. 1 8 . p. 1 5 7 , M . FLORKIN and E . H . STOTZ, eds. , Elsevier, Amsterdam 1970.

1 6 G . RENDINA and T . P . SINGER, J. biol . C h e m . 2 3 4 , 1 6 0 5 [1959],

1 7 B . J. B . W O O D , B . W . NICHOLS, and A . T . JAMES, Biochim. biophysica Acta [Amsterdam] 106, 261 [1965].

18 M. BOLL, Arch. Mikrobiol. 71,1 [1970]. 19 M. BOLL, Experientia [Basel] 26, 956 [1970],

2 4 8 M. BOLL

lipolytic enzyme. The increase usually is of the order of 50 — 80 percent, depending on the preparation. It should be noted that very low concentrations of phospholipase A can stimulate the enzymes. In ad-dition to the values of Table 1, a ratio of 1.2 / /g /mg will already lead to a partial stimulation. The deter-mination of succinate-cytochrome c-reductase is somewhat complicated by the fact that phosphate activates the enzyme 10, as shown by the increasing reaction rate during assay. However, by a great number of experiments it was secured that the phos-pholipase A produced increase in activity is equal to that of NADH-cytochrome c-reductase.

The mechanism underlying the stimulation by phospholipase A appears to be the same as that found with deoxycholate. Table 2 shows that the ac-tivity of NADH- and succinate-cytochrome c-re-ductase of particles incubated with phospholipase A

percent activity

NADH-cytc-reductase succinate-cytc-reductase

A 165 155 B 160 160 C 170 160

Table 2. The effect of deoxycholate on the phospholipase A stimulated NADH- and succinate-cytochrome c-reductase. Conditions as in Table 1. A = 25 ag phospholipase A/mg membrane protein, 30 min at 20 °C. B = same as A, after 30 min at 20 °C, 250 [xg deoxycholate/mg membrane protein, 15 min at 0 °C. C = 250 fxg deoxycholate/mg membrane pro-

tein, 15 min at 0 °C.

is not further increased by a subsequent treatment with deoxycholate. It would thus become possible to identify one effect of deoxycholate on the membrane structures as being the same as it is given by phos-pholipase A, i. e. a specific attack on the 2-position of the glycerol moiety of phospholipids where either saturated or unsaturated fatty acids may be present. This could then also apply to Triton X - 1 0 0 and suc-cinate-cytochrome c-reductase and to dodecylsulfate and NADH-cytochrome c-reductase, both activities being stimulated in the presence of the detergents.

Unlike the situation with deoxycholate and with the other detergents where stimulation is observed only within a very limited concentration range and higher amounts of the detergent will cause rapid destruction of the activities7 '18, no such decay is found with high phospholipase A concentrations (see Table 1) . This means that the detergent, what is to be expected, exerts multiple effects on the mem-

brane system and that higher concentrations of it will not only stimulate the enzymes but at the same time also decompose other active structural elements thus leading to inactivation.

Once stimulated by phospholipase A, the activities in this state are stable and they still remain com-pletely bound to the membrane fraction. No change in the Km values for both substrate and electron ac-ceptor is detectable as a result of the stimulation with phospholipase A. Thus molecular modifications of the enzymes with altered catalytic properties can apparently be excluded.

In a recent paper 1 it was described that proteases will inactivate the electron transport enzymes located on the membranal structures. When phospholipase A treated membranes are subsequently incubated with pronase or other proteases, the kinetics of the inactivation of NADH- and succinate-cytochrome c-reductase are different from that of particles not treated with phosppholipase A. This is shown in Fig. 1. It can be seen that the phospholipase A sti-mulated NADH-cytochrome c-reductase becomes in-activated by pronase at a significantly slower rate than the untreated enzyme. Results identical to those described for the N A D H enzyme are found with the succinate-oxidizing activity.

c 2 0 o

"rt >

c

CL

80

Fig. 1. Effect of digestion with pronase on the NADH-cyto-chrome c-reductase activity stimulated with phospholipase A. 1 = electron transport particles (4.7 mg protein/ml) were treated with 20 /ug phospholipase A/mg membrane protein at 20 °C for 30 min. After determination of activity, pronase was added (time zero) to the incubation mixture at a ratio of 20 /ug/mg and inactivation was determined at the stated time intervals. 2 = The particles were incubated with pronase as

above and inactivation was determined.

Time (min)

PHOSPHOLIPASES 249

The increase in activity of the two enzymes men-tioned as a result of the action of phospholipase A and, as the mechanisms are obviously identical, also of deoxycholate is thought to be new activity arising upon modification of structural elements in the mem-brane system by the agents. The differential inacti-vation by proteases observed here might well be a reflection of a greater stability against proteolytic digestion of that part of activity which arises after treatment with phospholipase A. An alternative explanation would be, as the phospholipase A is still present in the incubation mixture during the action of the protease, that by the proteolysis structures containing electron transfer activity are modified and exposed in such a way that they become sus-ceptible for the action of phospholipase A. Thus new active sites of NADH- and succinate-cytochrome c-re-ductase would be laid open, resulting in an increase in activity and, therefore, in the slower overall in-activation by the protease which is measured.

As can also be seen from Table 1, the N A D H oxidase system is increasingly inactivated by higher concentrations of phospholipase A. On the other hand, NADH- and succinate-dehydrogenase are only very slightly affected. These results are in complete accordance with those made with the detergents. Digestion of membrane structures with phospho-lipase A produces fatty acids and lysophosphatides, substances which could inhibit activities of the elec-tron transfer20. However, this obviously does not account for the decrease of N A D H oxidase as no alteration in the extent of inactivation was observed with a sample which, after phospholipase A treat-ment, was washed with bovine serume albumine to remove inhibitory compounds 20 .

The activity of N A D H dehydrogenase, under none of the experimental conditions employed, is ren-dered soluble by phospholipase A. Up to 3 5 0 jug/mg membrane protein together with prolonged periods of incubation were found to be ineffective in solubi-lizing any protein in either enzymatically active or

2 0 S. FLEISCHER and B. FLEISCHER, i n : Methods in Enzymo-logy, V o l . X , p. 4 0 6 , R . W . ESTABROOK and M . E . PULL-MAN, eds., Academic Press, New York 1967.

in inactive form. In contrast, approximately 50 per-cent of the bound N A D H dehydrogenase could be solubilized by a treatment with deoxycholate7 'n . Apparently the linkage of N A D H dehydrogenase to the electron transport system is susceptible to the detergent but not to phospholipase A. As, however, only high concentrations of deoxycholate will solu-bilize the activity (about 8 — 1 0 times the concen-tration that will activate NADH- and succinate-cyto-chrome c-reductase) it is reasonable to assume that by the strong and diverse action of the detergent membrane structures are opened up so that the linkage becomes accessible for a detachment, a situation which cannot be achieved by phospho-lipase A, acting in a more specific manner on the membrane system.

Phospholipase C and D, the other two lipolytic activities which were studied here, are completely without any effect on the electron transport parti-culate system. Similar observations have been made with the membranes of Mycoplasma laidlawii21, where only phospholipase A effectively decreased turbidity of the membrane suspension (taken as a measure of hydrolytic breakdown) while phospho-lipase C and D showed no effect. In contrast, re-spiratory chain-linked enzymes from the mitochon-drial membrane and a number of microsomal enzy-mes are well affected by phospholipase C 8 and D 22 .

In this connection it is noteworthy that the pre-paration of phospholipase C used in the present studies revealed strong inactivating effects on all electron transfer activities. These, however, were evidently not resulting from an action of phospho-lipase C since after removal of over 95 percent of the protein from the phospholipase preparation by heat and by acid precipitation, approximately the same degrees of inactivation were still observed.

This work has been supported by the Deutsche For-schungsgemeinschaft and by the Stiftung Volkswagen-werk. The author wishes to thank Prof. Dr. G. DREWS for his interest in the work.

2 1 P . F . SMITH, W . L . KOOSTRA, and W . R . MAYBERRY, J. Bacteriol. 100 ,1166 [1969].

2 2 L . LUMPER, Z . ZUBRZYCKI, and H . STAUDINGER, Hoppe-Sey-ler's Z. physiol. Chem. 350 ,163 [1969].