The role of ribonucleic acid in the formation of prothrombin activity by rat-liver microsomes

41o

PRELIMINARY NOTES

1 G. E. 1DALADE,in ROBERTS, Microsomal Particles and Protein Synthesis, P e r g a m o n P re s s Inc., N e w Y o r k , 1958 . 2 H. T. SHIGEURA AND E. CHARGAFF, Biochim. Biophys. Acta, 24 (1957) 45 o. 3 I~. T. SHIGEURA AND E. CHARGAFF, .]. Biol. Chem., 233 (1958) 197. 4 H. SACHS, J. Biol. Chem., 233 (1958) 643. 5 M. TAKANAMI, J. Histochem. and Cytochem., 7 (1959) 126. 6 y . MOULE, C. ROtlILLER AND J. CHAUVEAU, J. Biophys. Biochem. Cytol., 7 (196o) 547. 7 ]E. REID, Biochim. Biophys. Acta, 49 (1961) 218. 8 M. B. I-IOAGLAND, M. L. STEPHENSON, J. F. SCOTT, L. I. HECHT AND P, C. ZAMECNIK, J. Biol. Chem., 231 (1958 ) 241. * R. LIPSCHITZ AND E. CHARGAFF, Biochim. Biophys. Acta, 42 (196o) 544. 10 D. 13. DUNN, Biochim. Biophys. Acta, 34 (1959) 286. 11 V. G. ALLFREY AND &. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 45 (1959) 132512 T. WILCZOK AND l~. CHORAZY, Nature, 188 (196o) 516. 13 j . N. DAVlDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 594. 14 R. 13ARER, S. JOSEPit AND G. A. MEEK, Proc. Roy. Soc. London, t3 152 (196o) 353. 15 U. STENRAM, Acta Pathol. Microbiol. Scand., 44 (1958) 239. 1~ D. W. FAWCETT, J. Natl. Cancer Inst., 15 (1955) 147.5.

Received December 29th , 1961 Biochim. Biophys. Acta, 55 (1962) 4 o 8 - 4 1 o

The role of ribonucleic ocid in the formation of prothrombin activity by rot-liver microsomes LASCH AND ROKA1 have claimed that, in the presence of serum, rat-liver mitochondria can form coagulation factor V r I and convert it to prothrombin. However, POOL ANn ROBINSON2 failed to obtain evidence of synthesis unless intact liver cells were incubated. We have now observed that the prothrombin content of rat-liver microHomes increases during incubation and that this effect is dependent on the RNA associated with the microsomal membrane. Rat livers were homogenised in io vol. of 0.25 M sucrose and fracfionated by centrifugation 3 into mitochondria (65oo×g for io rain after removal of nuclei), h e a v y microsomes (18 o o o × g for I h), light microsomes (lO 5 o o o × g for I h) and cell sap. The particulate fractions were resuspended in the original volume of 0.25 M sucrose used for homogenisation, and 2 ml of each fraction were incubated at p H 6. 9 with 2 ml of Krebs-Ringer bicarbonate medium modified by omission of CaC12, which would interfere with prothrombin assays. The gas phase was 95 % 02-5 % C02. Samples taken at intervals up to 3 h incubation were estimated for prothrombin content by the ALLINGTON4 modification of the one-stage procedure, which also measures the factor V I I content of the system. The results have been expressed in units giving the prothrombin activity as a percentage of the prothrombin contained in a standard sample of rat plasma. Protein was estimated b y the LOWRY5 procedure, and RNA 6 b y removal of free nucleotides with cold 0. 4 N HC104, followed b y two 2o-min extractions with 0.4 N HC104 at 7 o°, the RNA in the extract being measured b y ultraviolet absorption. Table I shows that the prothrombin activity of the microsomes increased during incubation, particularly in the case of the heavy-microsome fraction. Experiments not detailed here showed that the increase of activity in this fraction was not influenced b y substitution of nitrogen for oxygen in the gas phase, b y adding dinitroBiochim. Biophys. Acta, 55 (1962) 4 1 ° - 4 1 2

PRELIMINARY

411

NOTES

TABLE

I

INCREASE IN PROTHROMBIN ACTIVITY OF ISOLATED LIVER CELL FRACTIONS, D U R I N G 3-H INCUBATION IN MODIFIED KREBS--RINGER BICARBONATE SOLUTION

The

data

are the

mean

results

obtained

in 3 experiments.

Prothrombin content (units[rag protein) Cell /faction

Initial content

Di//erence

A/tev incubation

Mitochondria

3.4

3.7

+ o.3

Heavy

3.7

8.9

+5.2

2.8

4.5

+ 1.7

i.o

0. 7

--0. 3

Light

microsomes microsomes

Cell s a p

phenol, ATP, DPN or TPN to the system, or by inclusion of a complete mixture of amino acids in the medium. Unlike the particulate system of LASCH AND ROKA x, activity was obtained without addition of serum to the incubation mixture. Since prothrombin activity in the heavy microsomes was not increased when they were disintegrated with ballotini beads, a procedure which has been found to liberate bound amylase from pancreatic microsomes 3, it was concluded that the increase in prothrombin activity during incubation was not due to release of pre-existing prothrombin. The ribosomes on the surface of the microsomal particles can be removed by treatment with pyrophosphate 7, leaving intact the microsomal membrane. Part of the RNA of the microsomes is not released by pyrophosphate treatment and appears to be an integral part of the membrane 8. When heavy microsomes were treated 8 with pyrophosphate and the resulting membrane preparations were incubated in KrebsRinger bicarbonate medium, the increase in prothrombin activity was even more rapid than in the case of whole microsomes (Table II). When the same membrane preparations were incubated in a medium containing ribonuclease (0.25 mg/ml), the TABLE

II

INCREASE IN PROTHROMBIN ACTIVITY OF WHOLE MICROSOMES AND OF PYROPHOSPHATE-TREATED MICROSOMES D U R I N G INCUBATION IN THE PRESENCE OR ABSENCE OF R . ~ A A S E The

Preparation incubated

data

are the

mean

microsomes

obtained

in 2 experiments.

Prothrombin content (unitslrng protein)

RNA ase added

RNA content (t~g/ml flask contents)

Period o/ incubation (It) o

Whole

results

I

2

Period o[ incubation (it) 3

o

r

2

3

o

I.I

2. 3

3.7

4.7

23.5

21.9

21.o

2o.2

Pyrophosphate-treated microsomes

o

1. 7

3.8

5.2

6.2

5.9

5 .8

4.6

3 .1

Pyrophosphate-treated microsomes

+

1.2

1. 3

1.3

1-4

6.0

1.5

I .o

i.o

RNA content of the preparations was rapidly reduced and the capacity to form prothrombin was abolished (Table II). This indicates that the mechanism of the increment in prothrombin activity is not likely to be the auto-activation described" when Biochim.

Biophys.

Acta,

55 (1962)

41o-412

412

PRELIMINARY NOTES

prothrombin, inactivated by heat, is incubated with mitochondria. The function of R N A in the phenomenon remains obscure. The conditions of incubation are such that synthesis of protein de novo seems unlikely, and it is more probable that the increase in activity represents finalisation of prothrombin and/or factor V I I from some precursor molecule. This work was carried out with the aid of a grant from the British Empire Cancer Campaign.

Department of Biochemistry, University o/ Glasgow, Glasgow (Great Britain

P.

GOSWAMI

H. N. MunRo

z H. G. LASCH AND L. I{OKA, Z.

physiol. Chem., Hoppe-Seyler's, 294 (1953) 3 °. 2 j . G. POOL AND J. ROBINSON, Am. J. Physiol., 196 (1959) 423 . s T. A. DOUGLAS AND H. N. MUNRO, Exptl. Cell Research, 16 (1959) 148. 4 M. J. ALLINGTON, J. Clin. Pathol., i i (1958) 62. 6 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND 1~. J. RANDALL, f . Biol. Chem., 193 (1951 ) 265. R. RENDI AND P. N. CAMPBELL, Biochem. J., 72 (1959) 4357 H. SACHS,. J. Biol. Chem., 233 (1958 ) 643. s p. GOSWAMI, G. C. BARR AND H. N. MUNRO, Bioehim. Biophys. Acta, 55 (1962) 4o8. 9 ~NT.ALKJAERSIG AND W. H. SEEGERS, Am. J. Physiol., 183 (1955) I I I .

Received December 29th , 1961 Biochim, Biophys. Acta, 55 (1962) 41o-412

Sequences of nucleotides in deoxyribonucleic .acid containing 5-bromouracil This article reports a study of nucleotide sequences in bromouracil-DNA with the use of the diphenylamine-induced degradation1, ~ which releases the sequences of pyrimidine nucleotides quantitatively as compounds of the general structure (pyrimidine nucleoside)n (phosphate)c~+x~. A thymine auxotroph of Escherichia cull (strain B3 from Dr. S. BRENNER of the Cavendish Laboratory, Cambridge) was grown overnight with aeration at 37 ° in 2.5 1 of a glucose and inorganic salts medium which contained 5 mg/1 t h y m i n e . This culture was added to 141 of sterile growth medium which contained 50 mg/1 5-bromouracil and 4 rag/1 thymine. After aeration for 14 h at 37 °, the bacteria were collected b y centrifuging and lysed b y sodium dodecyl sulphate in E D T A 3. Proteins were denatured b y agitation with chloroform and octan-I-ol in the presence of NaC104 and nucleic acids were precipitated b y ethanol. They were treated with RNAase and then with charcoal 4 to remove RNA. For preparation I, base analysis 5 values were (in moles per IOO bases recovered): adenine, 25.4; guanine, 24.7; cytosine, 27.1; 5-bromouracil, 11.4; thymine, IO.O. For preparation I I the values were 24.1, 23.1, 28.0, io.o and 14.5, respectively. On degradation b y diphenylamine and formic acid for 17 h at 3 o°, 23.3 % of the phosphorus of both bromouracil preparations was released as inorganic orthophosphate; normal DNA of strain B3 gave 23.2 °/o under the same conditions. No more inorganic phosphate was liberated in a further 48 h, indicating that there was no hydrolysis of the glycosidic linkage between bromouracil and deoxyribose. The inorBiochim. Biophys. Acta, 55 (1962) 412-415