Proc. Nati. Acad. Sci. USAVol. 73, No. 8, pp. 2803-2807, August 1976Cell Biology

Vitamin K-dependent synthesis and modification of precursorprothrombin in cultured H-35 hepatoma cells

(preprothrombin/radioimmunoassay/cell culture)

THEODORE W. MUNNS, MARILYN F. M. JOHNSTON, MARY K. LISZEWSKI, AND ROBERT E. OLSONDepartment of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104

Communicated by Van R. Potter, May 14, 1976

ABSTRACT The ability of confluent monolayers of H-35cells, originally obtained from a rat hepatoma, to synthesizeprothrombin in response to vitamin K1 (phylloquinone) wasstudied. As demonstrated by radioimmunoassay, selectivebarium salt adsorption, and two coagulation assays which dis-criminate between precursor- and mature-prothrombin, thesecells retained their ability to synthesize precursor prothrombin(preprothrombin) in the absence of exogenous phylloquinone(vitamin K). When phylloquinone was added to the medium (100ng/ml), the existing intracellular concentration of prepro-thrombin was reduced to 50% within 1 hr after exposure to thevitamin and slowly declined thereafter to approximately 30%of control levels by 36 hr. Concomitant with the rapid loss ofintracellular pre rothrombin was the appearance of matureprothrombin in the medium. The appearance of prothrombinwas biphasic: occurring during the initial 0-6 hr interval, andagain at an increased rate during the next 18-24 hr interval. Theamount of prothrombin appearing in the medium exceeded byseveralfold the amount of precursor mobilized. These datademonstrate that monolayer cultures of H-35 hepatoma cellsretain their ability to synthesize preprothrombin and other en-zymes, responsible for post-translational modification of pro-thrombin and its subsequent secretion, under the influence ofvitamin K.

Numerous hypotheses have been advanced regarding themechanism by which vitamin K (phylloquinone) catalyzesprothrombin biosynthesis (1, 2). These hypotheses includeregulation at the levels of transcription (3), translation (4-6),and, more recently, at the post-translational level (7-10). Duringthe past several years, much attention has been focused on thepossibility that the inactive "abnormal" prothrombins (11-13)present in animals deficient in vitamin K or receiving coumarinanticoagulants may represent an unmodified species of matureprothrombin, i.e., precursor prothrombin. Evidence to supportthis view includes the presence and absence of y-carboxyglu-tamate residues in prothrombin and preprothrombin, respec-tively (14-16), the ability of inactive prothrombin to be con-verted to thrombin by Echis carinatus venom (8, 17), the invitro conversion of preprothrombin to prothrombin by rat livermicrosomes (18), and the vitamin K-dependent incorporationof 14CO2 into prothrombin from liver microsomes of vitaminK-deficient rats (10).

In the present communication, we describe a hepatoma cellculture system which is suitable to investigate the vitamin K-dependent synthesis and/or modification of prothrombin. Wepresent data which support the hypothesis that the conversionof preprothrombin to prothrombin in H-35 hepatoma cells isvitamin K-dependent. Further, these data indicate that afterthis conversion an acceleration in the overall rate of synthesisof prothrombin occurs which suggests regulatory controls forboth translation and transcription of preprothrombinmRNA.

2803

MATERIALS AND METHODSCells and Cell Culture Systems. The cell line H4-11-E-C3,

employed in the present investigation, was obtained from VanR. Potter, McArdle Laboratory, University of WisconsinMedical School, Madison, Wisc. Intially established by Pitot etal. (19) from a Reuber H-35 hepatoma (20), these cells (com-monly referred to as H-35 cells) are epithelial in shape andreminiscent of the liver parenchymal cell. In our laboratory,H-35 cells were grown in monolayer culture in an atmosphereof 95% air and 5% C02, in the presence of 15 ml of Swim's S-77media (21), and supplemented with 20% horse serum and 5%fetal calf serum. Under these conditions the cells have a dou-bling time of approximately 24 hr. In certain instances (seebelow), a serum-free medium fortified with 1 jug/ml insulin(Iletin, Eli Lilly) was employed. Media, serum supplements,and trypsin were purchased from International Scientific In-dustries, Inc. Phylloquinone (10 mg/ml), as the water-solublepreparation AquaMephyton, was obtained from Merck, Sharpand Dohme and diluted with Swim's 77 medium. Another cellline tested which did not show significant prothrombin bio-synthesis was the Morris minimum-deviation hepatoma number7795 (22).Radioimmunoassay of Prothrombin. The radioimmu-

noassay for prothrombin was performed as described byJohnston et al. (23). Antigenic determinants in both theNH2-terminal and COOH-terminal regions of prothrombinwere recognized by this antibody, and by the conditions of theassay, both preprothrombin and prothrombin were mea-sured.

Coagulation Assays of Prothrombin. Two coagulation assayswere used to discriminate between prothrombin and prepro-thrombin. These included the Echis carinatus assay (7, 13), andthe physiological two-stage assay method of Shapiro and Waugh(24) employing thromboplastin. Since the Echis carinatus assaywas capable of generating clotting activity from both prepro-thrombin and prothrombin preparations while the two-stageassay generated a clotting activity only from prothrombin (13,25), employment of both assays provided a means of assessingthe preprothrombin and prothrombin content of various celland media fractions. Clotting times for both assays were mea-sured with the aid of a Fibrometer Coagulation Timer usingpurified rat prothrombin as a standard. For quantitativemeasurement of clotting activity, aliquots of extracts from cellsand media were used to yield clotting times in the range 20-100sec. Beyond 100 sec, the method was considered semi-quanti-tative (see Table 1).Determination of Intra- and Extra-Cellular Levels of

Prothrombin Following Vitamin K Administration. Cellcultures were initially grown to a confluent state in the presence

Dow

nloa

ded

by g

uest

on

Apr

il 25

, 202

1

Proc. Natl. Acad. Sci. USA 73 (1976)

-serum-i nsulin(o)) |'Xiii0.6B: ~~~~~~~-K_~~~~~~ -f

0 2 6 12 18 24 36

Time (hr) After Phylloquinone Administration**u * .u. * FIG. 2. Time course of the extracellular appearance and intra-

24 48 72 96 120 144 cellular loss of prothrombin antigenic equivalents from monolayersHours of H-35 cells after phylloquinone addition (100 ng/ml of medium).

FIG. 1. The effect of supplements to the medium on the growthof H-35 cells. Cells were subcultured in plastic tissue culture dishes(15 X 60 mm) and grown in Swim's 77 medium in the presence ofserum supplements (20% horse serum, 5% fetal calf serum) until ap-

proximately 30% confluent. At this time, the cultures were dividedinto three groups and the media changed at 24 hr intervals to provideserum supplements (0), insulin (o), or Swim's 77 medium withoutsupplements (0). Insulin was added to the last group at 120 hr.

of serum supplements and then maintained at confluency for24 hr with fresh serum-free medium containing 1 Ag/ml ofinsulin. Medium was then replaced with Swim's S-77 +1 ag/mlof insulin containing 100 ng/ml of phylloquinone and at se-

lected intervals thereafter media and cells were processed forthe determination of prothrombin and preprothrombin. Me-dium was collected in 1.8 ml aliquots to which 0.2 ml of 10%bovine serum albumin (BSA) was added prior to storage at-80°. Aliquots of thawed media were used for clotting assays

and radioimmunoassay. One antigenic equivalent was definedas the antibody binding capacity for 1 ug of rat plasma pro-thrombin.

Cells were detached from T-flasks with trypsin, harvested,and washed by low-speed centrifugation in the presence ofphosphate-buffered saline and stored as pellets at -80°. Cellpellets from one to four T-flasks were thawed at 40 in 1.0 mlof Swim's S-77 containing 1% BSA and 0.5% Nonidet P-40 (ShellOil Co.), sonicated, and differentially centrifuged (2000 X gfor 10 min; 30,000 X g for 20 min; 105,000 X g for 60 min at40) for isolation of 105,000 X g supernatant fractions. Thesefractions were used directly in the Echis carinatus assay andfor the radioimmunoassay. Each T-flask (75 cm2) containedapproximately 2 X 107 cells with a total cell protein, RNA, andDNA content of 8.3 + 0.4, 0.89 ± .07, and 0.34 + .03 mg perT-flask, respectively, as determined by conventional assaymethods (26-28).Barium Adsorption Studies. Because preliminary studies

with Nonidet P-40 indicated that this detergent inhibited theclotting activity associated with the assay of Shapiro and Waugh(24) (but not that of the Echis carinatus assay), the prothrombincontained in 105,000 X g supernatant fractions was initiallyadsorbed to barium sulfate (7, 25) prior to assessing clottingactivity. Adsorption was conducted as previously described (7,25) and preliminary studies with purified rat prothrombin in-dicated recoveries in the order of 70-90%. In some instances,Echis carinatus assays were performed on supernatants after

Eacn point represents the mean or three to live experimental valueswith standard deviations as shown.

barium treatment (see Tables 1 and 2). Thus, the ability todiscriminate between preprothrombin and prothrombin invarious cell and medium fractions was based not only on twodistinct clotting assays, but also on the well-established abilityof prothrombin to be selectively adsorbed to insoluble bariumsalts. This latter feature further implies that the preprothrombindescribed herein is devoid of 'y-carboxyglutamate residues(25).

RESULTSEffects of Insulin on Cell Growth. Because 5% fetal calf

serum and 20% horse serum concentrations exhibited relativelyhigh activities as determined by both coagulation assays, a

means was sought to maintain H-35 cells in a confluent statein the absence of serum supplements. Based on the success ofGerschenson et al. (29) in maintaining rat liver cells in culturewith insulin, the feasibility of this approach in the H-35 cellculture system was investigated. As illustrated by the datapresented in Fig. 1, cell growth and maintenance in the pres-ence of insulin was indistinguishable from that observed withserum supplements. However, medium alone appearedsomewhat detrimental as illustrated by both an abrupt halt incell growth as well as some cell detachment (5-10% of the cellpopulation per 24 hr).Vitamin K Induction of Prothrombin Antigenic Equiva-

lents in Cultured H-35 Cells. The responsiveness of H-35 cellsto vitamin K was initially investigated by assessing the intra-and extracellular levels of prothrombin antigenic equivalentsby means of radioimmunoassay. As illustrated in Fig. 2, anti-genic equivalents to rat prothrombin were detected in bothintra- and extracellular fractions of cell cultures incubated for36 hr in the absence of vitamin K. While the intracellularconcentration of equivalents remained relatively constantthroughout this incubation period (approximately 0.6 ,ug/107cells), the extracellular concentration increased to values ap-proximating 1.6 ,ug/107 cells. These data demonstrated that inthe absence of vitamin K, H-35 cells continue to synthesizeantigenic equivalents to rat prothrombin as reflected by (a) thesteady-state levels of equivalents within the cell and (b) theirslow extracellular accumulation.

Fig. 2 also depicts the effect of vitamin K upon the levels of

(0

0bx

C0C.

U

2804 Cell Biology: Munns et al.

Dow

nloa

ded

by g

uest

on

Apr

il 25

, 202

1

Proc. Natl. Acad. Sci. USA 73 (1976) 2805

Table 1. Identification of intracellularprothrombin antigenic equivalents as preprothrombin.

Effects of phylloquinone administrations

Assays (jMg of prothrombin/107 cells)b

Shapiroand

Echis carinatusc Waugh(24)

Time of Before After (BaSO4incubation (hr) RIAd BaSO4 BaSO4 adsorbed)

Vitamin absent:0 0.67 0.47 0.45 o.Oe2 0.65 0.47 0.49 NDf6 0.59 ND ND ND

24 0.61 0.45 0.41 0.0Vitamin present:

0 0.67 0.47 0.45 0.01 0.34 0.26 0.16 +92 0.30 ND ND ND6 0.29 0.20 0.12 +

24 0.23 ND ND ND

a Confluent H-35 cells were maintained in Swim's medium con-

taining 1 Mzg/ml of insulin for 24 hr prior to addition of new mediawith or without vitamin K (100 ng/ml). At the designated times,cells were collected and processed for determination of intracellu-lar prothrombin via the cited assays.

b Assays based on purified rat prothrombin as standard.c Echis carinatus assay: before BaSO4 represents clotting activitybefore barium treatment and is a measure of both preprothrombinand prothrombin; after BaSO4 represents clotting activity dueonly to preprothrbmbin since prothrombin is quantitatively ad-sorbed on BaSO4.

d RIA: radioimmunoassay. Standard deviations are shown in Fig. 2.e Aliquots possessing clotting times greater than 150 sec in eitherassay were considered to be 0.

f ND = not determined.g Values presented as + reflect the presence of traces of clottingactivity in the semi-quantitative range of the method with clottingtimes of 100-150 sec. Each value represents the average of two tothree independent determinations.

prothrombin antigenic equivalents. Most pronounced duringthe initial hour after vitamin addition was an abrupt decreasein intracellular equivalents (from 0.7 to 0.3 ,ug/107 cells) witha concomitant increase in extracellular levels (1.2 ,ug/107 cells).Progressively smaller decreases and increases in the levels ofequivalents were further observed in cells and medium, re-

spectively, during the next 16 hr of incubation, i.e., 2 through18 hr. At this stage of the response, a second marked increasein prothrombin antigenic equivalents became apparent in themedium (from 3.5 ,ug at 18 hr to 6.3 ,g/107 cells at 24 hr) whichwas not accompanied by significant changes in the level ofintracellular equivalents.Determination of Antigenic Equivalents as Prothrombin

and/or Preprothrombin. To determine the nature of the aboveantigenic equivalents, various cell and medium preparationswere assayed for Echis carinatus and normal two-stage clottingactivities. The results of these assays are presented in Tables 1

and 2 and revealed the following information. First, antigenicequivalents detected intracellularly in the absence of vitaminK represented preprothrombin since only the Echis carinatusassay demonstrated clotting activity. In the presence of vitamin,however, not only did the level of preprothrombin declineduring the first several hours, but the level of intracellularprothrombin appeared to increase slightly as demonstrated bya barely detectable increase in normal two-stage activity and

Table 2. Identification of extracellularprothrombin antigenic equivalents as prothrombin.

Effects of phylloquinone administration

Assays (mig ofprothrombin/ 107 cells)

Echis carinatus Shapiroand

Time of Before After Waughincubation (hr) RIA BaSO4 BaSO4 (24)

Vitamin K absent:0 0.00 0.0 0.0 0.02 0.69 0.0 ND 0.06 1.05 0.0 ND 0.0

24 1.32 0.0 ND 0.0Vitamin K present:

0 0.00 0.0 0.0 0.01 1.21 0.0 0.0 0.02 1.67 + 0.0 +

6 2.26 + 0.0 +

24 6.30 3.73 0.0 3.8736 6.55 4.04 0.0 3.93

For details, see Table 1.

by a decrease in Echts catrnatus activity after barium treatment(Table 1). These data indicate that vitamin K is required for theconversion of preprothrombin to prothrombin in this system.

Table 2 shows that cells exposed to vitamin K secrete into themedium significantly higher amounts of prothrombin antigenicequivalents than deficient cells. Evidence that this material wasprothrombin is based on the finding that media obtained fromcells exposed to vitamin K for 24 and 36 hr contained equivalentamounts of prothrombin as assessed by both clotting assays.Further identification was obtained by assessing clotting ac-tivities before and after barium adsorption. As expected (Tables1 and 2), vitamin K-deficient cells showed no adsorption ofintracellular Echis carinatus thrombin equivalents to bariumsalts, while the prothrombin secreted into the medium in re-sponse to vitamin K was fully adsorbable.

Thus, two significant and distinctive responses were notedwhen H-35 cells were exposed to vitamin K. The first whichoccurred during the first several hours after vitamin treatment(early phase), resulted in a significant decrease in the intra-cellular content of preprothrombin of 0.37 ug/107 cells witha concomitant increase in extracellular prothrombin of 2.26,og/107 cells. The second occurring at approximately 18 hr (latephase) resulted in a 2-fold increase in extracellular prothrombinwithout an apparent change in intracellular preprothrombinlevels. Levels of cellular DNA, RNA, and protein remainedunaltered throughout the entire course of the response.

Although the immunoassay detected somewhat more pro-thrombin or preprothrombin than did either clotting assay, thisdiscrepancy was attributed to the presence of immunoreactiveincomplete peptides which did not possess biological activityand/or deterioration of biologically active molecules duringextraction. In the case of extracellular prothrombin, the pref-erential loss of clotting activity may also be due to denaturationof secreted prothrombin during the incubation period. In theaging of pure rat prothrombin at 40, biological activity disap-pears before antibody binding activity.Dose-Response Relationship. Depicted in Fig. 3 is a dose-

response relationship which presents the concentration ofphylloquinone required to maximally depress the intracellularpreprothrombin content of H-35 cells 1 hr after exposure to the

Cell Biology: Munns et al.

Dow

nloa

ded

by g

uest

on

Apr

il 25

, 202

1

2806 Cell Biology: Munns et al.

CD'0.4

0.3

0.20. 0.1 1.0 10.0 100.0 500.0

Phylloquinone (ng/ml medium)

FIG. 3. Dose of phylloquinone versus intracellular concentrationof preprothrombin in H-35 cells 1 hr after exposure to the vitamin.Concentration of preprothrombin based on Echis carinatus assay.

For experimental details, see section on Materials and Methods. Allpoints represent the average of three to five independent determi-nations with standard deviations as shown.

vitamin. From these data it was concluded that these cells re-

spond maximally to phylloquinone at doses approximating 20ng/ml of medium. This value is comparable to those derivedfrom liver perfusion studies (30). A similar dose-response curve

for H-35 cells was obtained when the concentration of extra-cellular prothrombin was measured 24 hr after various vitamindoses.

DISCUSSION

Two rat cell lines, a Morris minimum-deviation hepatoma(number 7795) (22) and the H-35 hepatoma cell (19), both ofwhich synthesize rat serum albumin and respond to hydro-cortisone with increases in tyrosine aminotransferase (19,81-34), were selected for investigation because of their capacityto synthesize prothrombin under the influence of vitamin K.We found that the administration of phylloquinone to these twocell lines at confluency resulted in a minimal output of im-munochemically detectable prothrombin with hepatomanumber 7795 and a 5-fold greater amount with the H-35 cells.Hence, we continued our studies with the H-35 cells. That mostof the features of vitamin K-dependent prothrombin synthesisare preserved in the H-35 line of hepatoma cells maintainedin a chemically-defined serum-free medium is indicated bytheir: (a) synthesis of preprothrombin in the absence of exog-

enous vitamin K, (b) response to physiological amounts of vi-tamin by rapidly decreasing the existing intracellular pool ofpreprothrombin while increasing extracellular prothrombin,and (c) latent response at 18 hr to administered vitamin K whichresulted in a further expansion of the extracellular prothrombinpool.Two particularly intriguing features associated with this

response were (a) the disproportionately greater increase inextracellular preprothrombin during the early phase of theresponse and (b) the second "latent" increase in extracellularprothrombin that occurred approximately 18 hr after vitaminadministration. These results raised new questions about manyof the proposed mechanisms for vitamin K action (i.e., post-translational, translational, and transcriptional controls). Evi-dence for post-translational regulation rests on the vitamin-dependent decrease in intracellular preprothrombin with a

corresponding increased rate of accumulation of extracellular

prothrombin. These results parallel those observed in the ratand support the view that vitamin K is necessary for the mod-ification of preprothrombin (presumably via -y-carboxylation)to prothrombin and its subsequent transport to the medium (8,10, 17). Evidence for translational control of preprothrombinsynthesis during the early phase of the response stems from theobservation that the net decrease in intracellular prepro-thrombin represents only a small percentage of the prothrombinaccumulating in the medium. For example, whereas vitaminK reduces the intracellular pool of preprothrombin to an extentof 0.37 lug/107 cells during the initial phase of the response, theaccumulation of extracellular prothrombin increases to valuesof 1.2 and 1.6 .ug/107 cells at 1 and 2 hr. respectively (Tables1 and 2). It is thus likely that the vitamin K-dependent car-boxylation of preprothrombin induces a secondary increase inoverall biosynthesis of preprothrombin which is modified andsecreted into the medium at an enhanced rate.The time lag between exposure of the H-35 cells to vitamin

K and the onset of the second or "latent" response (beginningat approximately 18 hr) suggests a tertiary control mechanism,possibly at the transcriptional level, and reflects an acceleratedrate of synthesis or availability of preprothrombin mRNA. Thelateness of this response further suggests that vitamin K doesnot participate directly at the level of tertiary control. Ratherits primary action at the post-translational level may eventuallyresult in the accumulation of by-products which conceivablycould act to release bound mRNA for preprothrombin (35) orderepress the preprothrombin gene.The data presented here are in essential accord with those

obtained by other investigators who have demonstrated theexistence of preprothrombin in the livers of vitamin K-deficientand coumarin anticoagulated rats (1, 2, 8, 13, 25, 30) and itssubsequent conversion to prothrombin following vitamin ad-ministration. With regard to three different characteristics ofvitamin K-prothrombin system, i.e., (a) the quantity (gg) ofpreprothrombin per g of liver cell protein in the absence ofvitamin, (b) the concentration of vitamin K required to obtaina maximal response, and (c) the rate of synthesis of prothrombinin the steady state after vitamin administration, the H-35 cellsparallel the intact or isolated perfused rat liver. In deficientH-35 cells, the steady-state preprothrombin levels were 9-13,ug/g fresh weight as compared to 6-8 ug/g in fresh rat liver(13,25). The dose of phylloquinone required to give a maximalresponse in H-35 cells was 10-20 ng/ml as compared to 5-10ng/ml in the isolated perfused rat liver (30). Finally, the rateof prothrombin biosynthesis estimated for both H-35 cells andrat liver (in the final steady state), assuming a turnover of 6 hrhalf-life, was 1-2 ng/min per mg of cell protein (1, 36).

In summary, this report demonstrates a new model systemfor the study of the vitamin K-dependent synthesis of pro-thrombin in vitro. The responses of these H-35 cells to vitaminK are in accord with those of other investigators who havedemonstrated the accumulation of preprothrombin in liver cellsof vitamin K-deficient and coumarin drug-anticoagulatedanimals and its subsequent conversion to prothrombin and se-cretion upon administration of vitamin K. The system alsopossesses certain features (high output of prothrombin and la-tent burst) which may help explain some of the controversieswhich have arisen regarding the action of the vitamin (1, 2).

The authors wish to express their appreciation to WilmaClaxton,John P. Jones, Adelaida Villadiego, and James Campo for excellenttechnical assistance and to Dr. Van R. Potter and Joyce Becker forproviding the initial stocks of Reuber H-35 cells and for helpful advice.This work was supported by U.S. Public Health Service Grants HL-15619 and AM-09992.

Proc. Natl. Acad. Sci. USA 73 (1976)

Dow

nloa

ded

by g

uest

on

Apr

il 25

, 202

1

Proc. Natl. Acad. Sci. USA 73 (1976) 2807

1. Olson, R. E. (1974) Vitam. Horm. (N.Y.) 32, 483-511.2. Suttie, J. W. (1974) Vitam. Horm. (N.Y.) 32,463-481.3. Olson, R. E. (1964) Science 145,926-928.4. Olson, R. E. (1965) Can. J. Biochem. 43,1565-1573.5. Johnson, B. C., Hill, R. B., Alden, R. & Ranhotra, G. S. (1966) Life

Sci. 5,3892.6. Suttie, J. W. (1967) Arch. Biochem. Blophys. 118, 166-171.7. Shah, D. V. & Suttie, J. W. (1971) Proc. Nati. Acad. Sd. USA 68,

1653-1657.8. Suttie, J. W. (1973) Science 179, 192-194.9. Girardot, J. M., Delaney, R. & Johnson, C. B. (1974) Biochem.

Blophys. Res. Commun. 59, 1197-1203.10. Esmon, C. T., Sadowski, J. A. & Suttie, J. W. (1975) J. Biol. Chem.

250,4744-4748.11. Stenflo, J. & Ganrot, P. 0. (1972) J. Biol. Chem. 247, 8160-

8166.12. Stenflo, J. (1972) J. Biol. Chem. 247, 8167-8175.13. Esmon, C. T., Grant, G. A. & Suttie, J. W. (1975) Biochemistry

14, 1595-1600.14. Stenflo, J., Ferniund, P., Egan, W. & Roepstorff, P. (1974) Proc.

NatI. Acad. Sci. USA 71, 2730-2733.15. Nelsestuen, G. L. & Zytkovicz, T. H. (1974) J. Biol. Chem. 249,

6347-6350.16. Magnussen, S., Sottrup-Jensen, L., Peterson, T. E., Morris, H. R.

& Dell, A. (1974) FEBS Lett. 44, 189-193.17. Morrissey, J. J., Jones, J. P. & Olson, R. E. (1973) Biochem. Bio-

phys. Res. Commun. 54,1075-1082.18. Shah, D. V. & Suttie, J. W. (1974) Blochem. Biophys. Res.

Commun. 60, 1397-1402.19. Pitot, H. C., Peraino, C., Morse, P. A. & Potter, V. R. (1964) NatI.

Cancer Inst. Monogr. 13, 229-242.

20. Reuber, M. D. (1961) J. Nati. Cancer Inst. 26,891-895.21. Morse, P. A. & Potter, V. R. (1965) Cancer Res. 25,499-508.22. Richardson, U. I., Tashjian, A. H. & Levine, L. (1969) J. Cell Biol.

40,236-243.23. Johnston, M. F. M., Kipfer, R. K. & Olson, R. E. (1972) J. Biol.

Chem. 247, 3987-3993.24. Shapiro, S. S. & Waugh, D. F. (1966) Thromb. Diath. Haemorrh.

16,469-479.25. Carlisle, T. L., Shah, D. V., Schlegel, R. & Suttie, J. W. (1975)

Proc. Soc. Exp. Biol. Med. 148,140-144.26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol. Chem. 193,265-215.27. Burton, K. (1959) Biochem. J. 62, 315-323.28. Ceriotti, G. (1955) J. Biol. Chem. 214,59-70.29. Gerschenson, L. E., Okigaki, T., Andersson, M., Molson, J. &

Davidson, M. B. (1972) Exp. Cell. Res. 71, 49-58.30. Kipfer, R. K. & Olson, R. E. (1970) Biochem. Biophys. Res.

Commun. 38,1041-1048.31. Lee, K. L., Reel, J. R. & Kenny, F. T. (1970) J. Biol. Chem. 245,

5806-5812.32. Barnett, C. A. & Wicks, W. D. (1971) J. Biol. Chem. 246,

7201-7206.33. Butcher, F. R., Bushnell, D. E., Becker, J. E. & Potter, V. R. (1972)

Exp. Cell. Res. 74, 115-123.34. Potter, V. R., Watanabe, M., Becker, J. E. & Pitot, H. C. (1967)

Adv. Enzyme Regul. 5, 303-316.35. Zahringer, J., Baligg, B. S. & Munro, H. N. (1976) Proc. Natl.

Acad. Sci. USA 73,857-861.36. Bell, R. G. & Matschiner, J. T. (1969) Arch. Biochem. Biophys.

135, 152-159.

Cell Biology: Munns et al.

Dow

nloa

ded

by g

uest

on

Apr

il 25

, 202

1