[CANCER RESEARCH 35, 1536-1541, June 1975]

Substantiation of biochemical reactions on the outersurface of an intact cell is a delicate problem. It is necessaryto maintain the cells intact during the preparation as well asthe assay procedure in order to avoid any leakage of solubleintracellular components into the incubation medium.

In the present investigation, we have determined theactivities of several enzymes, including those that catalyzethe ATP formation on the cell surface of the intact Ehrlichcell as well as the corresponding activity of the cellhomogenate representing the total activity. The cells havebeen handled in physiological buffers at 0°during preparation as well as during the assay procedure at 25°,and it isassumed that their functional characteristics are the same asthose pertaining under in vivo conditions as far as integrityis concerned. Known cell membrane “stabilizers―likealbumin and dextran (cf Refs. 9, 14, and 16) were includedin the incubation media. Finally, a plasma membranefraction from Ehrlich cells was prepared and analyzed forGAPDH and PGK.

The results presented strongly support the view that partof the GAPDH and PGK of the Ehrlich cell is bound to theouter surface, reacting with external substrates and cofactors. Finally, the unequivocal role of albumin as a cellmembrane stabilizer, in the sense of preventing the appearance of any intracellular constituent in the extracellularmedium, is emphasized.

MATERIAL AND METHODS

All enzymes and substrates for the assays except ADP,ATP, and NADH were purchased from Boehringer andSoehne, Mannheim, Germany. The ingredients of the KRBand phosphate buffer were products from E. Merck AG,Darmstadt, Germany. ADP, ATP, NADH, and bovinealbumin (crystalized and lyophilized; No. A 4378, Lot No.430-8120) were obtained from Sigma Chemical Company,St. Louis, Mo. The fatty acid content of the albumin rangedbetween 0. 15 and 0.50% (Dr. F. Cohn, personal communication). The lipid content was less than 0.5% as determined bythe Soxhlet procedure. Dextran Tl 10 and T40 were purchased from Pharmacia Fine Chemicals AB, Uppsala,Sweden. All chemicals were of analytical grade.

Preparation of Tumor Cells. The Ehrlich ascites tumorcells were grown for 7 to 8 days in 5-week-old male Swissalbino mice obtained from the Anticimex Breeding Farm,Norrviken, Stockholm. The tumor cells were separated bycentrifugation of the ascitic fluid, which had been diluted

SUMMARY

Glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase are, together with some other enzymes,present on the surface of intact Ehrlich tumor cells.Aldolase, on the contrary, represents cytoplasmic enzymesnot present at all on the external surface, provided 2.5% ofbovine albumin is included in the isotonic assay medium. Aflux of aldolase from the cell interior to the cell exteriorcould be demonstrated in the absence of albumin. Therefore, any enzymatic activity monitored when keeping theEhrlich tumor cells in the isotonic assay medium containing2.5% albumin was considered to be primarily related to theoutside of the plasma membrane.

Of thetotalglyceraldehyde3-phosphatedehydrogenase,0.7% was located on the outer surface of the tumor cell,while the corresponding figure for 3-phosphoglycerate kinase was 2.7%. Eighty % of this surface-located 3-phosphoglycerate kinase was released into the assay medium duringincubation, while the release of glyceraldehyde 3-phosphatedehydrogenase, at the same time, was minimal.

A plasma membrane preparation of Ehrlich cells, mainlyconsisting of vesicles, showed the presence of 3-phosphoglycerate kinase but the absence of glyceraldehyde 3-phosphate dehydrogenase. Because of the vesicular nature of themembrane preparation, it was assumed that only one side ofthe membrane was exposed during assay.

The specific binding properties of the two enzymes to theplasma membrane, as well as possible differences in theirintramembranous location, are discussed.

INTRODUCTION

Ehrlich ascites tumor cells are able to form extracellularATP involving enzymes on the external cell surface, provided that the necessary substrates and cofactors are presentin the isotonic incubation medium (2). The membranebound enzymes catalyzing this reaction are GAPD2 [Dglyceraldehyde 3-phosphate:NAD@ oxidoreductase (phosphorylating), EC I.2. 1. 12] and PGK (ATP:D-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3).

1 This investigation was supported by grants from the Swedish Medical

ResearchCouncil (Project B74-l3X-228-IOC).2 The abbreviations used are: GAPDH, glyceraldehyde 3-phosphate

dehydrogenase;PGK, 3-phosphoglyceratekinase; KRB, Krebs-Ringerbicarbonate buffer.

ReceivedOctober 22, 1974;acceptedFebruary 7, 1975.

CANCER RESEARCH VOL. 351536

Enzyme Activities at the Surface of Intact Ehrlich Tumor Cells withAlbumin in the Isotonic Assay Medium'

Christer 0. Wernstedt, Gunnar K. Agren, and Gunnar Ronquist

Institute of Medical and Physiological Chemistry, University of Uppsala, Biomedical Center, Box 575, S-751 23 Uppsala 1, Sweden

SupernatantafterIntact

cellsincubationHomogenateGAPDH1,2340180,000PGK4,0973,252150,000

Enzymes in the Surface Membrane of Ehrlich Cells

without delay 10-fold with ice-cold Krebs-Ringer bicarbonate medium in order to diminish the tendency of cellagglutination. If not otherwise stated, 2.5% (w/v) of crystalline bovine albumin was also included in the KRB. In theexperiments recorded in Table 4 the cells were washed in thesame medium as the corresponding experimental medium.The buffer content of albumin was replaced by Dextran T40or Tl 10 in the experiments using dextran as a cell membrane stabilizer. The cells were washed 3 times in the bufferused. All preparatory steps were performed at 0°and alltogether never exceeded 15 mm. After the 3rd washing thecells were adjusted to a cytocrit value of 40% (±l %)(Clay-Adams, Inc., New York, N. Y.; Serial No.AC20870). The cells were maintained in an atmosphere of93.5% oxygen and 6.5% carbon dioxide at 0° and usedwithin 45 mm after the preparation started.

Preparation of Plasma Membrane Fraction. The procedure described by Ronquist and Christensen (27) wasfollowed exactly with the exception of some experiments inwhich 1 mM glutathione (reduced form) was present duringpreparation. The final membrane fraction was suspended inthe KRB in a concentration corresponding to 22 mgmembrane protein per ml.

Preparation of Cell Homogenate. The prepared cellsuspension with a cytocrit value of 40% was diluted 4-foldwith distilled water containing glutathione and deoxycholicacid, giving a final concentration of 0.5 mM and 0.05%(w/v), respectively. This suspension was frozen and thawedonce. The cells were disrupted in the Dounce homogenizerusing the tight pestle and 5 strokes immediately afterthawing. Ten to 50 zl of the homogenate were used forenzyme assays.

Assay of Enzymes. All enzymatic analyses were performed in an assay medium (final volume, 3 ml) bufferedwith the Krebs-Ringer bicarbonate medium now containingonly one-tenth its Ca2@concentration and freshly bubbledwith carbon dioxide (6.5%) and oxygen (93.5%), pH 7.4. Insome experiments the assay medium did not contain anyalbumin at all, in others it contained varying amounts ofalbumin or dextran.

The enzymes were determined in accordance with themethods given in the Biochemica Information, Boehringer(6), except that the triethanolamine-buffer used in theBoehringer Information Sheets was replaced by the KRBcontaining 1/ 10 of Ca2@ with or without albumin ordextran. Furthermore, EDTA was always excluded in theassay medium and 1 mr@iglutathione was included instead.

All enzymes were assayed by means of enzymatic reactions terminating in the stoichiometric oxidation or reduction of pyridine nucleotide with conditions adjusted so thatthe enzyme to be measured was rate limiting. Reducedpyridine nucleotide was measured at 340 nm in a Zeiss PMQII spectrophotometer. For these analyses the originalstabilized transformer was replaced by an Oltronix Regulated Power Supply C-7-20R. The photometric measurement was followed by a synchronously coupled recording(Servogor RE-Sl 1). The assay medium was in a 1-cm quartzcuvet at 25°.A molar extinction coefficient of 6.22 xl03/cm for both NADH and NADPH was assumed (10).

Enzyme activities are expressed in units or milliunits. OneUnit is defined as the amount of enzyme that converts 1smole of pyridine nucleotide per mm at 25°.

Intact Ehrlich cells to be analyzed for enzyme activitieson the cell surface were transferred (5 to 50 @zlof the 40%cytocrit-cell suspension) into the quartz cuvet containing theisotonic buffered medium. The cells were immediatelyevenly distributed in the cuvet and the oxidation or reduction was followed on the recorder with the cells in the lightpath. The reaction was usually allowed to proceed for 20mm at the most, keeping the cells evenly distributed in theassay medium during recording.

The assay medium, after the removal of the intact cells bycentrifugation, was again subject to analysis regardingactivity to determine the degree of elution of every singleenzyme concerned on the Ehrlich cell surface (supernatantafter incubation; Tables 1 and 3). Likewise, samples fromthe cell-free supernatant of the 40% cytocrit-cell suspensionwere also subjected to enzymatic analysis to determine thedegree of elution by the buffer only (Table 4).

Determination of Total Enzyme Activity of Ehrlich Cells(Cell Homogenate) and of the Membrane Fraction. Ten to50 zl of a KRB suspension containing the cell homogenate(containing 14.6 mg protein per ml) or the membranefraction (containing 22 mg membrane protein per ml) weretransferred to the cuvet containing the assay medium andthe reaction was followed as described above.

RESULTS

The results given in Table 1 clearly show that GAPDHand PGK are present on the surface of intact Ehrlich cellswith 2.5% of albumin in the assay medium. Of the totalGAPDH of the Ehrlich cell, 0.7% is located on the outersurface of the plasma membrane, while the correspondingfigure for PGK is 2.7%. All the surface-bound activity of theformer enzyme is closely associated with the membrane. Itmeans that when the cells are removed from the assaymedium, no activity is left in the medium (supernatant afterincubation). About 80% of the PGK, on the other hand, iseluted by I or several effector substances (29) and remainsin the cell-free medium. Albumin per se did not influence atall any of the enzyme activities studied. This was tested withthe homogenate and also with as small amounts of the

Table I

GAPDH and PGK activities ofintact Ehrlich cells with albumin in theisotonic assay medium

Intact tumor cells incubated in the Krebs-Ringer bicarbonate assaymedium containing 2.5% albumin. Cytocrit of the cell suspension was 40%(±1%).Enzymeactivities areexpressedin milliunits deriving from 5 x 10'cells ( I ml of packedcells).

JUNE 1975 1537

MembranefractionIntactcellsGAPDH01234PGK3624097

SupernatantIntact

cellsafterincubationHomogenateHexokinase001,565Phosphoglucose

isomerase15716044,600Aldolase008,4003-phosphoglucomutase60124,820Enolase14512121,700Pyruvate

kinase0040,500G-6-PDH'60841,687Glutathione

reductase004,650Lacticacid dehydrogenase0059,200

1 5 10 20

C. 0. Wernsiedt et a!.

separate enzymes as were found to be present on the cellsurface.

A purified plasma membrane fraction was also analyzedfor the presence of these 2 enzymes. As is seen in Table 2,362 milliunits of PGK were recovered in this membranousmaterial from 5 x 108 Ehrlich cells (corresponding to 22 mgmembrane protein), amounting to 0.24% of the total activityof the cell. On the contrary, no GAPDH could be detectedin the membrane fraction.

The membrane-bound part of this enzyme can be mactivated by the oxidation of its sulfhydryl groups ( 17, 20).Glutathione ( 1 mM, reduced form) was therefore included inall solutions during preparation and in the assay medium toprevent any spontaneous oxidation. Yet, it was not possibleto demonstrate the presence of GAPDH in the membranefraction. We should, however, keep in mind that thismembrane fraction consists mainly of vesicles (27), andsince we do not know with certainty the “sideness―of thevesicles (cf Ref. 12), the actual enzyme might be located onthe “wrong―side ofthe membrane, i.e., the side not exposedto the assay medium.

The possible presence of several other enzymes on theEhrlich cell surface was also tested, as is shown in Table 3.Of the total glucose 6-phosphate dehydrogenase, 3.5% issurface located, all ofwhich is eluted into the assay medium.3-Phosphoglucomutase, enolase, and phosphoglucose isomerase are some other enzymes that are present, to someextent, on the cell surface. Other enzymes showed nosurface location at all, namely, hexokinase, aldolase, pyru

Table 2GAPDH and PGK activities ofaplasma membranepreparation of Ehrlich

cells

A particulate membrane preparation from Ehrlich cells incubated in theKrebs-Ringer bicarbonate assaymedium containing 2.5% albumin. Enzyme activities are expressedasgiven in Table 1.

vate kinase, glutathione reductase, and lactic acid dehydrogenase.

Chart I illustrates the time dependency of the surfacebound GAPDH and PGK. The catalytic activities of bethenzymes were constant for up to 20 mm, although notillustrated for PGK in the chart. Surface-bound enzymeactivity was also proportional up to at least 20 sl of packedcells (1 x l0@ cells) in the assay medium regarding PGK(Chart 2). The GAPDH consistently deviated from linearityalready at 4 zl of packed Ehrlich cells (2 x 106 cells) in themedium. The explanation for this behavior is not known. Itmight indicate that the ideal ratio between the number ofadded cells and the volume of the assay medium is reachedalready at 4@ of cells under the conditions used. Controlexperiments have been performed and excluded the possibility that any other factor, e.g., substrate, cosubstrate orauxilliary enzyme, is rate limiting in the over-all assayreaction.

Chart 3 illustrates the effectiveness of albumin as a cellmembrane stabilizer. As has already been mentioned (Table2), aldolase is not present on the Ehrlich cell surface when2.5% of albumin is present in the medium. In the absence ofalbumin an aldolase activity is recorded of intact Ehrlichcells in the isotonic assay medium. It is clear from the chartthat albumin is quite effective already at a concentration of0.5% in the assay medium. Dextran T40 has been claimed tohave similar effects (I, 4, 9). It is evident from the chartthat, when using aldolase as a test enzyme, the effectivity ofDextran T40 is less pronounced and higher concentrationsare needed. Dextran Tl 10, with a molecular weight veryclose to that of albumin, has about the same effect asDextran T40 in the concentration range of 2.5 to 10%.

Table 4 shows the release of surface-associated GAPDHand PGK into the supernatant of isotonic Ehrlich cellsuspensions of different composition. The KRB containing2.5% albumin causes a very slow release of GAPDH, incontrast to the assay medium, which did not cause any

40

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TIME. MIN

Chart 1. Time courseof oxidation of NADH by intact Ehrlich cells inthe presenceof 2.5%albumin in the assaymedium.GAPDH (A) and PGK(0) weredeterminedon the surfaceof intact Ehrlich cells by meansof anenzymatic systemterminating in the stoichiometric oxidation of NADH.Enzymeactivities areexpressedas zmolesof NADH oxidizedper 5 x 10'cells. Points, meanvaluesof triplicate samples.

Table 3Activity ofother Ehrlich cell enzymes with albumin in the isotonic assay

mediumIntact Ehrlich cells incubated under the sameconditions as given in

Table I.

a G-6-PDH, glucose 6-phosphate dehydrogenase.

CANCER RESEARCH VOL. 351538

RatioExperimentCompositionof

suspendingmediumGAPDHPGKPGK/GAPDH1NaC1,

KCI, 2.5%albumin(pH7.4)73512821.72NaCl,

KCI, 2.5%albumin(pH6.2)6679571.43NaC1,

KCI, TrisHAc°, 2.5%albumin (pH7.4)49615043.04NaCI,

KCl,Ca2@, 2.5%albumin (pH7.4)86718222.15NaCI,

KCI, Mg2@,2.5%albumin (pH7.4)80918802.36KRB,

2.5%albumin (pH7.4)188189710.07KRP,2.5% albumin (pH6.2)5359831.88NaCI,KC1(albumin omitted)

(pH7.0)384553831.49KRB(albumin omitted)

(pH 7.4)538391431.6

Enzymes in the Surface Membrane of Ehr!ich Cells

release at all of this enzyme (Table 1). The release of PGK ismarkedly enhanced by the albumin-containing buffer.Therefore, the ratio of released amounts of the latterenzyme to those of the former enzyme is high, about 10.

The Krebs-Ringer bicarbonate medium without albumindisplays the highest ability of releasing both enzymes amongthe media investigated. The ratio of the 2 activities is low,

Chart 2. Proportionality betweensurface-boundenzymeactivities andamount of cells in the assay medium. Enzymes were determined andactivities were expressed in nmoles of NADH oxidized per mm and peramount of cellsgiven(ab.fcissa).Points, meanvaluesof triplicate samples.A, GAPDH; 0, PGK.

around 1 6. The release of PGK was 10 times slower andwas lowest in a medium ofNaCl, KCI, and albumin, pH 6.2.The ratio of the 2 enzymes was I.4. When the pH of themedium was increased to 7.4 by the addition of NaOH, therelease of PGK was enhanced by 34% compared with the pH6.2 medium, while the corresponding figure ofGAPDH wasonly 10%. Thus, the elution pattern of the 2 enzymes ishighly dependent upon the composition of the suspendingmedium and, furthermore, what is effective as an eluant forone enzyme is not necessarily effective for the other enzyme.

z2

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x0

4

UI

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4

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“

100 @@AIR@UMIN

1 2DEXTRAN

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DEXTRAN

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10CPERCENTAGE

ALBUMN AND DEXTRAN (W/vLMILLIONSOF CELlS @4THEASSAYMEDIUM

Chart 3. Effectivenessof albumin and dextran in preventing theoccurrenceof aldolase on the intact Ehrlich cell surface. Aldolase wasmeasured on the surface of Ehrlich cells suspended in the isotonic assaymedium containing different concentrationsof albumin and Dextran T40or T I 10.Abscissa,percentagesof albumin or dextran (w/v). Points, meanvalues of triplicate samples.

Table 4

Effect ofdifferen: media on binding of GAPDH and PGK to the Ehrlich cell surface

The concentrationsof NaCI and KCI of the suspensionsin ExperimentsI , 2, 3, 4, 5, and 8 were130and 25 mM, respectively.In Experiments6 and 9, isotonicity wasmaintainedby KRB and, inExperiment 7, by a Krebs-Ringer phosphatebuffer instead of the NaCl-KCI medium. Ca2@concentration in Experiment 4 and Mg2@concentration in Experiment 5 were 2.5 and 1.2 mM,respectively. Adjustment of pH to 7.4 in Experiments 1, 4, and 5 was performed with I M NaOH. InExperiment3, a Tris-acetic acid buffer wasincludedto a final concentrationof 10msi. The tumorcellswerewashed3 timesin the respectivemedium,andthecytocrit valueof the final suspensionwas40%(±1%).The cellswerekept suspendedand aeratedin their respectivemedia for 30mm at 20°before centrifugation and enzymatic analysisof the supernatants.The cell-free supernatantswereanalyzedwith regardto the enzymesgiven in the table. The enzymeactivity is expressedasgiveninTable 1.

a TrisHAc, Tris-acetic acid buffer; KRP, Krebs-Ringer phosphate buffer.

JUNE 1975 1539

C. 0. Wernstedt et a!.

DISCUSSION

It was reported in a previous paper (2) that 32P-labeledATP could be formed at the Ehrlich cell surface, providedthat the necessary substrates and cofactors, together with[32P]PI, were present in the external medium. A prerequisitefor such a reaction to occur is the presence of both GAPDHand PGK on the Ehrlich cell surface. The present investigation clearly shows that both enzymes are present on the cellsurface, PGK being the most abundant one. A relativelylarge release of the latter enzyme could be achieved by theassay medium, while the GAPDH was not disengaged underthis condition.

A plasma membrane preparation mainly consisting ofvesicles (27) was also tested with respect to the presence ofthese 2 enzymes. Only PGK could be demonstrated in thepreparation, while GAPDH could not be detected. Thereason for this is not clear. The possibility of an inactivationof the enzyme due to oxidation of sulfhydryl groups (17, 20)was excluded, since experiments were carried out with I mMglutathione present. Since the membrane material mainlyconsists of vesicles (27), it is possible to explain the negativeresult by a one-side position of the enzyme on the membrane. It means that only I side of the vesicles is exposed tothe necessary components of the assay medium, and theenzyme is not present on this side. If this is true, the vesiclesshould be inside out since the enzyme in question was clearlydemonstrated on the outside of the intact Ehrlich cells. Thesideness of the vesicles has not been established so far. Themembrane preparation was carried out at a weak alkalinepH as well as at a low ionic strength. Furthermore,ethylene glycol bis(@-aminoethyl ether)-N,N'-tetraaceticacid was added as a divalent cation chelator duringpreparation. Such conditions have been shown, for thehuman erythrocyte, to favor the formation of inside outvesicles (12). This might also reflect a kind of crypticity ofthe membrane-bound enzyme, as has been discussed for theerythrocyte membrane (7, 15).

These 2 enzymes have also been shown to occur at thesurface of intact human erythrocytes (21, 22) as well as in ared cell membrane fraction (17, 20, 23). Again, the PGKwas more abundant on the intact erythrocyte surface (22),but instead the GAPDH of the erythrocyte membranefraction occurred in a 10-fold higher amount than the PGK(20). Hence, the 2 plasma membrane-associated enzymesdisplay differences between each other as regards bindingproperties and probably also localization within the plasmamembrane.

From the results in Table 4 it seems likely that the Ca2'and also Mg2@of the KRB may serve as negative effectorsin the sense of being inhibitors of the enzymatic release. TheCa2@effect might be accomplished by a so-called membranecoarctation (I 1). It means that Ca2@ easily binds to proteinstructures of the membrane, thereby establishing crosslinked protein conjugates leading, for example, to morecompact membrane structure of the cell (8, 18, 19, 28, 30,31). Also, Ca2@ might well exert such an influence on theplasma membrane by direct action on a special Mg2@- and

Ca2@-stimu1ated ATPase at the tumor cell surface recentlydescribed in our laboratory (26).

The unique albumin effect might be explained by itsability of being adsorbed to the surface of certain cells. Suchan adsorptionhas beenshownto increasethe mechanicalresistance of fresh human erythrocytes to lysis by hydrodynamic shear forces (3 1). Also, a protective effect of albuminon membranes, particularly those of neoplastic cells, is wellknown(16).

The presence of GAPDH and PGK at the tumor cellsurface is necessary for the ATP formation in the surfacemembrane. Important ATP-requiring reactions at thetumor cell surface have recently been described from thislaboratory and by others. One is the phosphorylation ofsurface-located proteins by a surface-located protein kinase(3—5, 13, 24, 25). The other is an ATPase, which isspecifically stimulated by both Mg2@and Ca2@and whichmight be related to cell motility and/or contraction (26).The end products of both reactions, provided there is also aphosphoprotein phosphatase present, are ADP and P@.These metabolites are at the same time the immediatesubstrates for GAPDH and PGK in the de n@vo synthesis ofATP on the cellsurface. Therefore, all these reactions seemto be connected with each other, and the basis for areciprocal influence might be ADP and orthophosphate onthe cell surface.

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2. Agren, G., and Ronquist, G. Formation of Extracellular AdenosineTriphosphate by Tumour Cells. Acta Physiol. Scand., 75: 124-128,1969.

3. Agren, G., and Ronquist,G. Isolation of “P-labeledPhosphorylserinefrom Ehrlich Mouse-AscitesTumor Cells Suspendedin an IsotonicMedium Containing ‘2P-labeledAdenosine Triphosphate. Acta Physiol. Scand.,79: 125-128, 1970.

4. Agren,G., and Ronquist,G. Isolation of “P-labeledPhosphorylserineand Phosphorylthreoninefrom Ehrlich Mouse-AscitesTumor Cells,Suspended in a Medium Containing ‘2P.labeledNucleoside Triphosphate or Inorganic Pyrophosphate. Acta Chem. Scand., 25:2931-2934,1971.

5. Agren, G., and Ronquist, G. (32P)Phosphoryl Transfer by EndogenousProtein Kinaseat the Surfaceof Glia andGlioma Cells in Culture intoExtrinsic Acceptor Proteins. Acta Physiol. Scand., 92: 430—432,1974.

6. Boehringer Biochemica Information Sheets, January, 1961, November, 1961,andJanuary, 1962.Mannheim, Germany:C. F. Boehringerand Soehne, GmbH.

7. Duchon, G., and Collier, H. B. Enzyme Activities of HumanErythrocyte Ghosts: Effects of Various Treatments. J. MembraneBiol., 6: 138-157, 1971.

8. Hempling, H. G., Stewart, C. C., and Gasic, G. The Effect ofExogenous ATP on the Electrolyte Content of TA, Ascites TumorCells. J. Cellular Physiol., 73: 133-140, 1969.

9. Hjelm, M., Ostling, G. S., and Persson,A. E. G. The Lossof CertainCellular Components from Human Erythrocytes during HypotonicHemolysis in the Presenceof Dextran. Acta Physiol. Scand., 67:43-49, 1966.

CANCER RESEARCH VOL. 351540

Enzymes in the Surface Membrane of Ehrlich Cells

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I I . Horvath, C., and Sovak, M. MembraneCoarctation by Calcium asaRegulator for Bound Enzymes. Biochim. Biophys. Acta, 298: 850-860,1973.

12. Kant, J. A., and Steck, T. L. Cation-impermeable Inside-out orRight-side-out Vesicles from Human Erythrocyte Membranes. NatureNew Biol., 240: 26-28, 1972.

13. Kinzel, V., and Mueller, G. Phosphorylation of Surface Proteins ofHeLa Cells Using an ExogenousProtein Kinase and (‘y-32P)ATP.Biochim. Biophys. Acta, 322: 337-351, 1972.

14. Kromphardt, H. Chlorid Transport and Kationenpumpf in EhrlichAscites Zellen. European J. Biochem., 3: 377—384, 1968.

15. McDaniel, C. F., and Kirtley, M. E. The Interactionof Glyceraldehyde 3-PhosphateDehydrogenasewith Human Erythrocyte Membranes.J. Biol. Chem., 249: 6478-6485, 1974.

16. Mehard, C. W., Packer,L., andAbraham, S. Activity and Ultrastructure of Mitochondria from Mouse Mammary Gland and MammaryAdenocarcinoma. Cancer Res., 31: 2148—2160,1971.

17. Nilsson, 0., and Ronquist, G. Enzyme Activities and Ultrastructure ofa Membrane Fraction from Human Erythrocytes. Biochim. Biophys.Acta, 183: 1-9, 1969.

18. Palek, J., Curby, W. A., and Lionetti, F. J. Effects of Calcium andadenosine Triphosphate on the Volume of Human Red Cell Ghosts.Am. J. Physiol., 220: 19-26, 1971.

19. Palek, J., Curby, W. A., and Lionetti, F. J. Relation of Ca@@Activated ATPase to Ca@-Linked Shrinkage of Human Cell Ghosts.Am. J.Physiol.,220: 1028-1032,1971.

20. Ronquist, G. Formation of AdenosineTriphosphateby a MembraneFraction from Human Erythrocytes. Acta Chem. Scand., 21:1484-1494, 1967.

21. Ronquist, G. Formation of Extracellular Adenosine Triphosphate by

Human Erythrocytes.Acta Physiol. Scand., 74: 594-605, 1968.22. Ronquist, G. Enzyme Activities at the Surface of Intact Human

Erythrocytes. Acta Physiol. Scand., 76: 312-320, 1969.23. Ronquist, G., and Agren, G. Formation of Adenosine Triphosphate by

Human Erythrocyte Ghosts.Nature, 209: 1090-1091,1966.24. Ronquist, G., and Agren, G. Isolation of “P-labeledPhosphorylthreo

nine from Ehrlich Mouse-Ascites Tumor Cells Suspended in anIsotonic Medium Containing 32P-labeledAdenosine Triphosphate.Acta Chem. Scand., 24: 728-729, 1970.

25. Ronquist, G., and Agren, G. (‘2P)PhosphorylTransfer by EndogenousProtein Kinase at the Ehrlich Cell Surface into Extrinsic AcceptorProteins. Upsala J. Med. Sci., 79: 138-142, 1974.

26. Ronquist, G., andAgren, G. A Mg2@- and Ca2@-stimulated AdenosineTriphosphatase at the Outer Surface of Erlich Ascites Tumor Cells.Cancer Res.,35: 1402-1406,1975.

27. Ronquist, 0., and Christensen, H. Amino Acid Stimulation ofAlkali-metal-independent ATP Cleavage by an Ehrlich Cell Membrane Preparation. Biochem.Biophys.Acta, 323: 337-341, 1973.

28. Rorive, G., and Kleinzeller, A. The Effect of ATP and Ca2@on theCell Volume in IsolatedKidneyTubules.Biochim. Biophys.Acta, 274:226-239, 1972.

29. Shin, B. C., and Carraway, K. L. Association of Glyceraldehyde3-PhosphateDehydrogenasewith the Human Erythrocyte Membrane.J.Biol.Chem., 248: 1436-1444,1973.

30. Stewart, C. C., Gasic, G., and Hempling, H. G. Effect of ExogenousAlP on the Volume ofTA3 AscitesTumor Cells.J. Cellular Physiol.,73:125—132,1969.

31. Weed, R. L., La Celle, P. L., and Merritt, E. W. MetabolicDependence of Red Cell Deformability. J. Clin. Invest., 48:1794-1809,1969.

32. Williams, A. R. The Effect of Bovine and Human Serum Albumins onthe Mechanical Properties of Human Erythrocyte Membranes.Biochim. Biophys. Acta, 307: 58-64, 1973.

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