Turnover time prothrombin and of prothrombin messenger RNA and evidence for a ribosomal site of action of vitamin K in prothrombin synthesis

Vol . 5, pp . 385-392, 1966 . Life Sciences Printed in Great Britain .

Pergamon Press Ltd .

TURNOVER TIME OF PROTHRCd~IN AND OF PROTFIROMBIA MESSENGER RNA Al® EVIDENCE FOR A RIBOSOMAL SITE OF ACTION . OF VITAMIN K IN PROTHROMRIN SYNTHESIS H . Connor Johnson,

Roberta Bleíler Hill,

Rosemary Alden sad G. S . Ranhotra

Division of Nutritional Biochemistry, Department of Animal Science, University of Illinois, Urbann, Illinois (Received 3 December 1965)

A mbar of roles of vitamin K have been proposed ; however, the only observed symptom of vitemln K deficiency m the aalmal appears to be the decreased level of blood prothrambm, factor IX, end possibly other clotting factors .

Attempts

to find a role of vitamin K m general protein synthesis have not been successful (Hill et e1 ., 1963), sad it is natural to turn to a possible role of vitamin K at the level of specific prothrombin synthesis .

Olsan (1964, 1965),

using the chick and actmamycin D, has recently reported experiments indicating that vitamin K functions at the genetic level .

We would like to present evid-

ence es to the site of action of vitamin K and data on the turnroer of prothrambiu in the body . METHODS Male Sprague-Dawley rata were used in these experiments .

All animals were

fed the vitamin K-deficient diet (Johnson et el ., 1960), but the controls were treated orally with the diphosphosodium ester of menadione .

The vitamin K

deficient rata were housed in tubular cages (Metta et al ., 1961) to avoid coprophegy .

Prothrombm times were measured according to Quick (1957) .

Pro-

thrombin concentrations were expressed ss percent of normal prothrombia time on the basis of rat blood dilution curves run against prothro®bin times . protein synthesis, actinomycin D or cycloheximide were used .

To block

Actinomycin D has

been given to normal, vitamin K-deficient sad warfarin-treated rate at the rate of 2000 g and 500 g/100 g body weight by mtraperitoneal injection. 385

Cyclohexi-

386

vol . 5,

PROTHßOYBIR 8Y1~THEBIB

wide was given at the rates of 750 pg, 25o Ng and 50

No .

5

100 g body weight .

Following administration of the blocking agent to normal rats, prothrombin times were followed at intervals up to 30 hr or until the animals had died .

At

0, 1, 3 or 5 hr after administration of the blocking agent to vitamin K-defident animals or warfarin-treated animals, a 40 4+g dose of vitamin K1 was given per rat.

Control deficient rats were given the same dose of vitamin K1 without

a blocking agent . RESULTS The results for actinomycin D treated rats are given in figure 1 and for cycloheximide treated rats in figures 2 and 3,

(See Fig.l, Fig.2, Fig .3) .

From

the data it is evident that both blocking agents have blocked synthesis of pro thrombin in normal rats, prothrambm concentrations decreasing rapidly following cycloheximide and somewhat more slowly following actinomycin D . From the dotted line curves of figures 1 and 2, it can be seen that when no blocking agent was administered the prothrombin level of the vitamin K-defident rats returnéd to normal within 1 br after administration of vitamin Kl . The data in figure 1 show that following the blocking of prothrombin synthesis by actinomycin D the rats still responded to the administration of vitamin K1 . However, from the data given in figure 2, it can be séen that vitamin K-defident rata did not respond norme.lly to vitamin Kl , whether given at 0, 1, 3, or 5 hr after administration of 750

100 g body weight of cycloheximide,

although at zero time some response to vitamin K1 was obtained .

Since at this

level of cycloheximide (which was used initially because it inhibited C14 -amino acid incorporation into liver protein over 80~) all rats dieti within 10 hr, the effect of lcnrer levels of cycloheximide were examined .

It was found that 250

4+g and even 50 4+g of cycloheximide~100 g body weight caused approximately the same rate of disappeaaance of protein from the blood as did the 750 I~ level, but that all rats recovered within 30 hr aí'ter the 50 4+g dose . This latter level was thus given to vitamin K-deficiént rats, some of

vol . 5, No . y

PROTHROYBIN siTHEBIS

FIG. 1 Effect of vitamin Kl treatme:rt on rats treated with 2000 Ng actinamycin D per ]AO g body weight . Antagonists in 0 .1 ml 0 and vitamin Kl in 0 .1 mls of 6~ Tween 80, 94~0 .85~ NsC1, were administered by intraperitoneal injection. Curve A - Four normal rats given actinamycin D át zero time . Curve B - Four vitamin K-deficient rata, not treated with actinoaQycin D, given ~+0 4.ßg of vitamin S1 . Curves C, D and E - Vitamin K-deficient rats given actinamycin D at zero time and vitamin K1 at 1 hr (5 rats 40 Ng), 3 hr (2 rats, 40 4+g) and 5 hr (3 rats, 500 ugi respective],y . which were then treated with vitamin K at 1 hr . i figure 3 .

These data are plotted in

387

388

Qol . 5, No . 5

PROTSROI®I1~ sYNTHF,8I8

C

FIG. 2 Effect of vitamin Ky treatment on rats treated with cycloheximide per 100 g body Weight . Chart A - Curve A: Curve B:

750

Lig

As curve B, figure 1 . Four normal rats given cycloheximide at zero time .

Charts B, C~ D and E - Vitamin K-deficient rats given cycloheximide at zero time and vitamin K; at 0 hr (5 rats, 40 Lig), 1 hr (6 rats, 40 4~g), 3 hr (2 rats, 4o Ng) and 5 hr (2 rats, 40 Ng ; 2 rats,~500 N8 ), respectively.

Vol .

5, Ro . 5

PBOTHROIiBïF SYFTfIESIB

389

FIG. 3 Effect of vitamin 1~ treatme~ an rats treated with he~d mide per 100 g body weight .

50

Ng cyclo-

Curve

5 -

Five normal rats given cyclohezimide et zero time .

Curve

6 -

91x Warfarm-treated rats (1 Ng 24 hr before zero time) given cyclohe~dmide et zero time .

Curve 8 - Eight vitamin K-deficient rats given cyclohe~dmide at zero time . Curve A - Four vitamin á-defid.ent rats given cycloheaimide at zero time, vitamin Sl at 1 hr . Curve B - ftine Warfarm-treated rats given cyclohe~d.mide at zero time and vitamin K1 at 1 hr . Macussion By use of the 2 antibiotics actinoaprcin D and. cyclohe~d.mide~, vre hoped to be able to dist inguish between

3

general sites of fraction of vitamin S in

paoTgxo~iN snQTxESis

39o

voi . 5, No . 5

prothrombin synthesis . These are : (1) at the level of .tranacription from DNA

to messenger RNA, (2) at same level between RNA and formation of the ca~mpleted polypeptide precursor of prothrombin on the ribosome, and (3) at some step between peptide chain precursor and final prothrombin. As can be seen from figure l, blood prothro~mbin decreases only gradually for the first

6

hr following actinomycin D administration and then falls very

rapidly, indicating that the turnover time of prothrambin-messenger RNA is about

6

hr . On the other hand, prothrambin levels start falling rapidly follow-

ing cycloheximide administration and the data in figures turnover time for prothrambin of less than reported by Josso et el.,

6

2

and

hr in contrast to

3

indicate a

3 - 5

days

(1962) .

It was assumed that vitamin K deficiency blocks one step only and that up of prothrombin proceeds normally in the

to that step the pathway of synthE

vitamin K-deficient or warfarin-treated animals . This appears probable, since recovery of deficient (or warfarin-treated) rats following vitamin K1 therapy was very rapid (figures 1 and 2). Thus with the precursors present up to the critical vitamin K requiring step, vitamin K should complete prothrombin synthesis, unless another block intervenes . Since Bernhardt et al .

(1962)

using fluorescent. antibody showed prothrom-

bin formation in the parenchyma) cells of the liver within 1 vitamin K treatment, and since Iüll et al .

(1963)

2

hr after

showed prothrombin in the

liver microsames of the normal bu not of the vitamin K-deficient animal, it

seems clear that the site of vitamin K íluictian is in the liver, probably in the microsa~mea and not in the blood.

Actina~rcin D acts to prevent RNA synthesis (Traketellis et al.,

1964),

thus blocking mRNA formation. It has been stated that cycloheadmide in rat

liver preve~s the break-down of ribosomal aggregates which is associated with

the growth of nascent polypeptide chains (Wettstein et al .,

1964),

while in

intact cells (rabbit reticulocytes) it appears that it acts at the level of some polyribosomal structure (Colombo et al .,

1965) .

In yeast it appears to

Yol . 5, No . . 5

PROTHROllBIF SYNTHESIS

inhi bit specifically formation of ribosomal RNA (de Klcet, The data of figures 1 and

3

391 1965) .

indicate clearly that the site of action of

vitamin K is beyond the site of action of actinamycin D in blocking the transcription of DNA to RNA end also beyond the site of cycloheximide block in the formation of the peptide chain an the ribosome . How then can the data of figure 2 be explained?

A final explanation must

await the establishment of the mechanism of cycloheximide inhibition of protein synthesis .

However, it seems possible that at the lethal levels of cyclohex

amide used in figure 2 polyribosa~mal structure was so damaged that existing attached peptide chains were lost .

One can then postulate that vitamin K as

s quinone functions in the stripping of the prothra~mbin precursor peptide frame the ribosam~e . bond formation .

This mqy be due to a requirement for a specific pattern for 3SFieser (1941) has shown these quinonea to be active SH-

reagents . Summaty The data show that the site of vitamin K in prothrambin synthesis is later than the site of blocking of protein synthesis by cycloheximide and that it is involved at the ribosa®al level .

The hypothesis is proposed that vitamin K is

involved in the removal of the properly folded structure fra~m the precursor peptide on the ribosome .

Açknowled~ents Actinomycin D was generously supplied by Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey, through the courtesy of Dr . David F. Green . Actidione (cycloheximide) was generously supplied by Upjohn, Co ., Kalamazoo, Michigan, through the courtesy of Dr . George Savage . at the

1965

This work was presented

meeting of the Federation of American Societies for Experimental

Biology (Johnson et al .,

1965),

and was supported in part by e great from the

United States Public Health Service

(AM-06005) .

PROTHROYHIN BYI~TSEBIB

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5, ao .

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