AN EXPERIMENTAL EXAMINATION OF THE EVI-DENCE FOR THE PRESENCE OF PHOSPHATIDES

IN THE LIMITING SURFACE OF THE LIVINGPROTOPLAST

BY FREDERICK CAMPION STEWARD.

(From the Botany Department, University of Leeds.)

(Received 24th January 1928.)

AN outstanding problem of plant physiology is the nature of the mechanism whichcontrols the movement of water-soluble substances into, and out of, the living cell.Recent investigations confirm the long established impression that in the vacuolarsap of living cells substances are present, which, although soluble in water, arealmost unable to diffuse out when the cell is surrounded by water. It is clear thatthe living protoplast is responsible for this, since complete permeability is associatedwith death. Moreover, it is well established that the degree of permeability ofliving cells to solutes is dependent upon the composition of the medium with whichthey are in contact, and its consequent effect upon the plasma surface. Leaving onone side the controversy concerning the existence and constitution of a specificplasma membrane, which may be.responsible for these attributes of living cells,it is clear that the phenomena associated with boundary phases give adequategrounds for anticipating that the external surface of the protoplast will have adifferent composition from the main protoplasmic mass. It is natural that thecomposition of this surface layer should be stressed in theories of cell permeability,but it is clearly undesirable that our knowledge of its nature should rest largely onspeculation.

Thus lipoids, or substances soluble in fat solvents and themselves exerting thesolvent properties characteristic of fats, are often assumed to be an essential featureof the protoplast surface. Considerable impetus has been given to this idea by thefact that lipoids are conspicuous by their ability to reduce the surface tension ofwater and might thus predominate in the interphase cell wall/protoplast. Manyauthors have postulated the existence of specific lipoidal constituents in the proto-plasmic surface layer. Outstanding among these are Overton (1900), who emphasisedthe importance of sterols and phosphatides, Czapek (1910, 1915) who regards thesurface layer as a concentrated emulsion of neutral fats in water, and Lepeschkin(1910-1923), who regards it as a protein-lipoid (lecithin) colloidal complex in whichthe continuous phase is not water. Until recently, however, there has been verylittle direct experimental evidence for the presence of such lipoids in the living

Phosphatides in the Surface of Living Protoplast 33

protoplasmic surface, their hypothetical presence only offering a convenient inter-pretation of certain phenomena of cell permeability. Particular importance attaches,therefore, to a series of papers by Hansteen Cranner (1910-1926), and to the ex-tension of this work by V. Grafe and his co-workers (1925-1927).

The outstanding contribution made by Hansteen Cranner in this series of papersis that fatty materials are to be regarded as constant constituents of the cell walls ofall normal, physiologically active cells, and may be obtained from seedling cultures,or leaching extracts of slices of storage tissue, under such conditions that the semi-permeability of the cell is unimpaired, as indicated by the retention of the organicsolutes such as water-soluble pigments and sugars. Any substances lost by thetissue to the surrounding medium, therefore, are assumed to come from the surfaceof the protoplast or the cell walls. Examining cell wall preparations, obtained asfree from protoplasmic constituents as possible by a process of grinding and wash-ing, Hansteen Cranner again concluded that similar lipoids could be readily demon-strated. The lipoids obtained, it was concluded, were not neutral fats, but well-defined, water-insoluble and soluble phosphatides of the lecithin type, togetherwith some phytosterol-like substance in small amount. The water-insoluble andsoluble phosphatides were stated to be present in reversible equilibrium determinedby the temperature. Hence Hansteen Cranner regarded the limiting protoplasmicsurfaces of the cell as colloidal systems whose dispersion media are water-insolublephosphatides capable of swelling, and whose liquid disperse phases are water-soluble phosphatides. Further, the peripheral protoplasmic surface is to be re-garded as closely adjoining the cell wall, which, in the case of all living plant cells,he regarded as a colloidal network of cellulose and hemicellulose whose meshes arefilled with the phosphatides from the peripheral protoplasmic surface. On the basisof this conception, Hansteen Cranner offers an explanation of the injurious effectsof culture solutions deficient in calcium, and the so-called antagonism betweencalcium and sodium, potassium and magnesium.

V. Grafe and his collaborators have published data supporting and extendingthe results of Hansteen Cranner. Before any of their papers had reached thiscountry, Hansteen Cranner's earlier data had been under experimental re-examina-tion with quite contrary results. On perusal of Grafe's earlier papers, the wholeground was re-traversed experimentally but the result, as set out below, is to con-firm the earlier conclusion that in leaching experiments of this type, when carefullycarried out with healthy, parenchymatous tissues, negligible quantities of phos-phatide and only small quantities of fatty substances are released.

In a later communication the basic assumption that leaching experiments offera means of studying the substances present in the cell wall or peripheral protoplasmicsurface will also be critically examined. In the present paper, the experimentalprocedure and results of Hansteen Cranner and Grafe are briefly stated, followedby a summarised account of the results and conclusions of similar experimentscarried out in the Botany Department, University of Leeds.

BjEB'Vli

34 FREDERICK CAMPION STEWARD

THE CHEMICAL NATURE OF THE LIPOIDS OBTAINED BYHANSTEEN CRANNER AND V. GRAFE.

During extensive culture experiments with various seedlings in physiologicallyunbalanced solutions, toxic effects were noticed by Hansteen Cranner and ascribedto the removal from the cell walls of substances yielding soluble sodium, potassiumand magnesium salts but insoluble compounds with calcium. The excessive removalof such substances, which appeared in the culture solutions as turbidities and weredescribed as resembling the fats and pectins, was regarded as the cause of the toxicityof cultures deficient in calcium (1910, 1914). It was stated, as the result of micro-scopical examination only, that the cultures were sterile and that the turbidity wasnot due to organisms. From the turbid solution was obtained an alcohol-insolublefraction, yielding pectin reactions, and a fatty, ether-soluble fraction which wasseparated into saponifiable and unsaponifiable materials. The former, after re-crystallisation, melted at about 50-530 C , and the latter gave the Liebermanncolour reaction for cholesterol. Glycerol, phosphoric acid and proteins were notdetected. In the same paper is described how cell-wall preparations were prepared,free from cell contents, by a process of trituration and washing, involving theminimum of chemical disturbance. These (o-24-o-86 gm. in weight) gave by treat-ment with alcoholic hydrochloric acid a substance, soluble in ether and 96 per cent,alcohol, but not in absolute alcohol, which proved to be fatty, and partly saponifiableand formed approximately 10 per cent, of the pure wall preparation. The produc-tion of a fatty acid-pectin compound and the analogous behaviour of living cellsand artificial membranes, prepared from cellulose or the pure wall preparationsimpregnated with pectin and colloidal soaps, were also described. On this basisHansteen Cranner emphasised the importance of fat-pectin complexes and soapsin cell walls.

These results were extended to storage tissues (1919). It was noted that whenred beetroot slices were immersed in water at 300 C , a similar turbidity arose,which, again on the basis of microscopical examination, was stated to be independentof bacterial contamination. The anthocyanin pigment did not diffuse appreciablyand the tissue was regarded as still living. At room temperatures clear, or slightlyturbid, colourless extracts were obtained in which lipoid materials could be de-tected. The effect of salt solutions on the appearance of colour and turbidity (whichusually arose simultaneously) during the leaching of red beetroot was noted and theanalogy between this and the precipitation reactions of lecithin cited by Koch (1903)was emphasised. Similar results were obtained with other tissues, notably theepidermal cells of the bulb scales of onion (Allium cepa). It was indicated thatthe lipoids obtained were phosphatides (water-soluble and insoluble) which couldbe fractionated by precipitation with lead acetate and alcohol into closely alliedsub-fractions.

In 1922 the evidence that the lipoidal constituents were phosphatides of varyingsolubilities was considerably augmented. The experimental tissue was again thebeetroot and the essential results may be summarised as follows:

1. Leaching of well-washed slices in single layers for periods of 24 hours at

Phosphatides in the Surface of Living Protoplast 35

Qo Q Q̂gg n o t impair the semi-permeability of the tissue and neither proteins norconstituents of the cell sap (anthocyanin) occur in the extracts.

2. Leaching extracts contain either water-soluble phosphatides alone, or theseare accompanied by water-insoluble phosphatides according to the temperature.At room temperature extracts were clear, or only slightly turbid, but at 300 C. werevery turbid.

•2. The phosphatides were of the lecithin type containing glycerol, fatty acids(both solid and liquid, the latter autoxydisable), phosphorus and a nitrogen base.By lead acetate precipitation was obtained a precipitate divisible into three fractionsby means of alcohol and acetone. The three fractions yielded similar decompositionproducts with betaine as the nitrogen base. Fatty acids and phosphorus werequalitatively identified, but glyceroi was only definitely detected in one fraction.The lead-free fraction from lead acetate precipitation yielded similar decompositionproducts with choline as nitrogen base. Sugar and mineral constituents were alsopresent.

In more recent (posthumous) papers, Hansteen Cranner (1925-1926) hasextended the evidence that the water-insoluble turbidity is due to phosphatides. Thehydrolysis of the water-soluble and insoluble fractions from various species ofPisum has been followed by an estimation of the fatty acid content, and the phos-phorus-nitrogen ratio in the small amounts of material obtained, by microchemicalmethods. For example:

Sol. part*3-98%

Pisum sativum

Pisum arvense unicolor.

fNN

[pfNPNP

Insol. part.n %

3*3%i

0*15

6-6%

z

I*I6

i-35%4-85%

i

It is noteworthy that in the leaching extracts from peas during 24 hours at300 C , protein reactions were obtained. The vitamin properties of these extractswere extensively examined.

In 1926 were published the results of the examination of "pure cell-wallpreparations" from carrot (Daucus carotd), with microchemical estimations of theirnitrogen and phosphorus content, and also the fatty acid liberated by acid hydro-lysis.

Finally, the evidence is presented which, it is claimed, confirmed the earliersuggestion that there is a definite equilibrium, determined by temperature, betweenthe water-insoluble and soluble phosphatides. This consists of the followingobservations:

i. The turbid, leaching extract from potato, if filtered through a Berkefeldfilter, yields a water-clear solution. The residual turbid suspension, if incubatedat 280 C. for 48 hours in presence of 1 c.c. of ether to 250 c.c. of suspension, againyielded a water-clear solution on filtration, which contained fatty acid, phosphorus

3-3

36 FREDERICK CAMPION STEWARD

and a nitrogen base detected qualitatively after treatment with 5 per cent, sulphuricacid. The base was detected by phosphotungstic acid. Hence Hansteen Crannerpostulates the conversion in recognisable amount of the insoluble part to thesoluble on standing at 280 C. for 48 hours.

2. From a clear, water-soluble extract from potato, two portions of 750 c.c.each were incubated in stoppered bottles, with the addition of 4-5 c.c. of ether, indark thermostats, one at 6-100 C , the other at 300 C. After 30 hours, that at 300 C.was very turbid; the other was only slightly opalescent. Hence he concluded thatat 300 C. the water-soluble fraction had yielded an insoluble phosphatide, and thatthe production of soluble from insoluble phosphatides or vice versa is a reversibleequilibrium governed by the temperature.

During the years 1925-1927, seven papers have appeared by V. Grafe and colla-borators which claim to substantiate the earlier conclusions of Hansteen Cranner.

Grafe and Horvat (1925) supplied the complete chemical analysis of the phos-phatide complex leaching from sugar beet slices, thus supporting Hansteen Cran-ner 's qualitative results. The leaching technique was that of Hansteen Cranner. Theclear extract free from proteins (i.e. negative results given with Molisch, Biuret,Xanthoproteic and Adamkiewicz reactions) was concentrated at 280 C. by means ofa vigorous air current. The somewhat concentrated solution was precipitated witha strong solution of normal lead acetate, and from the constancy of composition ofthis material (in P and N content), with varying conditions of precipitation, it wasregarded as a pure chemical compound. The elementary analysis of the lead acetateprecipitate was determined, and its saponification products identified and suitablederivatives prepared. It was not possible to purify the original product as treatmentof the lead acetate precipitate with sulphuric acid or hydrogen sulphide yielded asubstance, regarded as a decomposition product, which did not again precipitatewith lead acetate. The phosphatide content was entirely in the lead acetate precipi-tate, and this, instead of being fractionated, was hydrolysed directly. From theidentification of the hydrolytic products of 50 gm. of the crude washed, dried leadacetate precipitate obtained directly from the somewhat concentrated aqueous,leaching extract, and from the elementary analysis of the same, Grafe and Horvatconcluded that it contained a lecithin-like compound and could be represented veryexactly by the following formulae:

CH2. OOC. CigH31 C17H33COO. CH2

CH. OOC. C17H33 C15H31COO. CH +4 Pb

CH 2 .0 P . O. CH2. CH2. N O P OCH2

I III IOH (CH3)3 OH

% found % calculated

H 678 6717N 0-624 o-6ioP 2-78 2705Pb 36-10 36-10O — 13-55

Phosphatides in the Surface of Living Protoplast 37

All the appropriate saponification products (oleic, palmitic and glycerophos-phoric acids and choline) were qualitatively obtained, and appropriate derivativesquantitatively analysed. It is noteworthy that Grafe and Horvat, contrary to HansteenCranner, state that the nitrogen base contained in the lead acetate precipitate wascholine and not betaine. It would appear then, that in all essential respects, Grafeand Horvat have effected the most complete confirmation of Hansteen Cranner.

The extensive data derived by Grafe and his collaborators will not be summarisedfurther. They serve to indicate the great variation in molecular composition of theproducts obtained by the lead acetate method, so much so that Grafe (1927) haspostulated that phosphatides are specific for a given tissue or organ, even closelyrelated plants yielding quite different phosphatides. The results of experimentsupon the products leached from parenchymatous tissues under different conditionswill now be briefly summarised. Details of chemical manipulations will be givenelsewhere.

LEACHING EXPERIMENTS ON STORAGE TISSUES.

Slices of Scotch seed potatoes (Sharpe's Express) were leached in distilled waterin shallow trays in the preliminary experiments. 6 to 8 kg. of fresh slices were thusleached by the same methods as used by Hansteen Cranner, the extracts beingsubsequently precipitated with basic lead acetate. 100 gm. of this precipitate werethen hydrolysed and freed from lead and a turbid filtrate obtained which containedcalcium, magnesium, potassium, chloride, phosphate and carbonate. Only a traceof substance reducing Fehling solution was present even after inversion with mineralacid. Definite traces were found of a globulin, which precipitated upon dialysis.The hydrolysis of the whole 100 gm. of this precipitate only yielded 0-5 gm. of anether soluble fatty acid. No ether soluble unsaponifiable material was present andno choline, betaine or glycerol. Minute traces of choline were found in the filtratefrom the original extracts after precipitation with basic lead acetate, but there wasno evidence of the presence of any phosphatide in the original extract.

Leaching experiments, in which the extracts were kept sterile, showed that inthis manner the turbidities described by Hansteen Cranner could be avoided, Aspecial apparatus was therefore devised in which slices of tissue, after sterilisationwith 0-25 per cent, aqueous formaldehyde, could be leached with boiled distilledwater, the water being changed daily, without bacterial contamination. Red beet-root slices and sugar beets were extracted in this apparatus and the extracts pre-cipitated by lead acetate. This precipitate was hydrolysed and freed from lead andexamined in the usual manner. In Table I the results thus obtained in such sterileleaching experiments are compared with others obtained when the shallow trayswere used for leaching, and also with the results of Grafe and Horvat.

It will be seen that the results obtained in the Leeds experiments by the twomethods are in good general agreement. There is a complete absence of choline,glycero-phosphoric acid or glycerol amongst the hydrolytic products, whilst thequantities of fatty acid are very small when compared with those recorded by Grafe

F R E D E R I C K C A M P I O N S T E W A R D

and Horvat. The analyses of the precipitate supplied in Table II show that thebulk of the precipitate is inorganic in nature.

Table I.

Leachingmethod

"Approx.sterile""Shallowtray"Grafe andHorvat

Wt. ofppt.gm.

55

20

PbSO4gm.

61

21

%Pb

75-3

71-2

36*10

Fatty acid

Wt. ingm.

0-50

O'I2

0/

/o

I

o*6

47-0

Wt. ofphospho-

tung-statesgm.

0-37

I'O

0/

/ocholine

#

#

53

/oglycero-

phos-phoricacid

#

#

15

* None detected.

Table II.Leaching method % N % CO2

"Approx. sterile" 0-115 8*26"Shallow tray" 0-47 9-3

Nitrogen estimated by Kjeldahl method.

% P b75'271-2

%P0*42

Carbon dioxide in a modified Schrotter flask.Phosphorus estimated by the Pemberton-Neumann method.

The filtrates, after precipitation of the extracts with lead acetate were alsoexamined. Betaine was obtained in appreciable quantity, 0-2 gm. of the purecrystalline hydrochloride being prepared. Traces of choline were also probablypresent. Glycerol, however, was not present, nor was there any evidence of thepresence of either phosphatides or fatty acids. The main bulk of the residue onevaporation of the filtrate was a deliquescent syrup, almost free from nitrogen andphosphorus, very soluble in water or moist solvents but insoluble in all other drysolvents. No chemical crystalline derivatives of this substance have so far beenobtained and its chemical nature remains uncertain.

The precipitation of the leaching extracts by lead acetate is not the usual pro-cedure in the isolation of phosphatides. Its use was dictated by the necessity ofstrict comparison of results with those of previous workers in this field. In otherexperiments the leaching extracts were'directly extracted with ether, with resultsthat confirmed the practically complete absence of phosphatides from these extracts.

As these results are so contrary to those recorded in the previous section, anattempt was made to extract phosphatides directly from the tissues of the potatotuber after grinding and pulping. The juice was expressed and both juice and solidresidue extracted with ether. The solid residue was also extracted with boilingalcohol, and a protein coagulum from the juice as also the lead acetate precipitate,also extracted with alcohol and ether. The total amount of ether-soluble, acetone-insoluble substance thus obtained from all operations, from 4 kg. of potatoes, waso-i6 gm., of which 0-14 gm. was obtained from the solid residue. Gallagher (1923)obtained from 3 kg. of potato with alcohol extraction i-6 gm. of a substance soluble

Phosphatides in the Surface of Living Protoplast 39

in ether and insoluble in acetone, which contained phosphorus but not choline, andwhich probably consisted in part of phosphatide. This is further evidence that thetotal amount of these substances thus to be extracted from the potato tuber is verysmall.

GENERAL DISCUSSION.

The conclusion to be derived from these extensive leaching experiments is thatphosphatides are not removed from living, parenchymatous tissues (red and sugarbeetroot, and potato) by distilled water. Further, simpler fatty substances couldonly be demonstrated in the extracts in small quantity. Although the isolation anddetection of some of the products of phosphatide hydrolysis may be attended withsome considerable difficulty, yet the extraction of the fatty acids is a relatively simplematter, and the fact that these were only obtained from leaching extracts in verysmall amounts is to be interpreted as strong proof that phosphatides do not diffusefrom the living tissue except in negligible amounts. The knowledge that theseresults are contrary to those previously described by Hansteen Cranner and Grafeand his collaborators has necessitated considerable repetition, and it is to be under-stood that the data cited have been confirmed in many duplicate experiments.Furthermore, the application of the more usual extraction methods for the isolationof fats and phosphatides to potato tissue indicate that lecithin, or similar phospha-tide, forms an almost negligible part of the fresh weight of the tissue, and much ofthis is retained in the dry residue after squeezing out the water-soluble material.Since leaching experiments involve the minimum of extraction of cell contents,it is improbable that these substances would have been obtained under the experi-mental conditions described in this paper.

The disparity between the results of Grafe and those described in this papercannot be adequately accounted for. In view, however, of the general use made byGrafe of the method of lead acetate precipitation and the versatility of this reagentas a general precipitant of plant products, it is submitted that evidence supposed toprove the isolation of pure phosphatide complexes by a single precipitation shouldbe accepted with caution, unless verified by the usual and more trustworthy tech-nique of extraction with fat solvents, etc. The postulation by Grafe (1927) of labilephosphatide complexes, containing pigments, anthocyanin, protein and carbo-hydrate groups, isolated by these methods, would, in the writer's opinion, merelyconfirm the above implication.

Regarding the significance of the turbidity obtained and the possibility of anequilibrium determined by temperature between the water-insoluble and solublephosphatides (see p. 36), the writer believes that the effects of bacterial activityand the possibility of denaturing and coagulation of some small amount of proteinmaterial, which can proceed at the temperature involved, have not been exhaus-tively eliminated by Hansteen Cranner and Grafe. It is interesting in this con-nection that turbidities, which Hansteen Cranner regarded as due to insolublelipoids, have been regarded by other authors as due to varying degrees of bacterialcontamination (Wachter (1905), Merrill (1915), Hibbard (1915)).

40 FREDERICK CAMPION STEWARD

In short, the study of leaching extracts of living tissues does not yield anyevidence which proves that phosphatides are present in the limiting protoplasmicsurface. The extent to which such methods of attack are applicable to the directstudy of the surface layer of the protoplast is a controversial subject which it ishoped to treat more fully in a later communication. Since the data supplied byHansteen Cranner, and their alleged confirmation by Grafe and his co-workers,represent the only direct macrochemical evidence indicating the presence and im-portance of phosphatides in the cell walls and protoplasmic surface, this questionmust be regarded as still unsettled.

SUMMARY.

1. The evidence for the view that phosphatides are present in the surface ofthe protoplast and can thence diffuse from living tissue into distilled water, iscritically reviewed.

2. Further experimental work is briefly summarised, which leads to the con-clusion that phosphatides do not diffuse out into distilled water from healthyparenchymatous tissues.

The author desires to-acknowledge his indebtedness to Prof. J. H. Priestley,in whose laboratory this work was carried out, also to the Department of Scientificand Industrial Research for a grant of maintenance.

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