Macromolecular synthesis: Effects of vitamin K and warfarin on macromolecular synthesis in escherichia coli

Pergamon Press

Life Sciences Vol . 10, Part II, pp . ß1-89, 1971 . Printed in Great Britain

MACROMOLECULAH SYNTHESIS :

EFFECTS OF VITAMIN ~ AND WAHFAR,IN

ON MACROMOLECULAH SYNTHESIS IN SSCHERICHIA COLI 1 H. Bruce Bosmann2 Department of Pharmacology and Toxicology IIniversity of Hochester Medical School Hochester, New York 14620

(Received 21 August 1970; in final form 2 December 1970) SDlAlAHY : At 2 : 10-3M, vitamin ~1 increased E . coli protein synthesis more than two fold but decreased the incorporationof glucoeamine into acid insoluble material (74X lipopolysaccharide) to li the control rate ; DANA and RNA rates of synthesis were unaffected . Warfarin at 2 .x 10 -3M inhibited RNA synthesis to 25X the control rate, DNA synthesis to 87X, protein synthesis to 24X and the incorporation of glucoeamine into acid insoluble material to 40X the control rate . In the presence of 250 ug/~ of chlorampheaicol vitamin R1 at 2 z 10 -3M increased the rate of protein synthesis is E, côli three fold over the rate with chloramphenicol alone. In a very recent report from this laboratory (1) vitamin

Rl

and, to a

lesser eztent, warfarin have been shown to substantially stimulate protein and glycoproteia synthesis, but not RNA or DNA synthesis is isolated rat liver mitochondria in vitro .

Also, in contradistinction to these results,

vitamin R.l and warfarin at elevated levels were found to severely inhibit protein, glycoprotein, DNA and RNA synthesis is L5178Y cells (mouse leukemia cell line) in vitro (1) .

Mitochondria are capable of the autonomous syn-

thesis of nucleic acids (2-4),

lipids (5,6), glycolipids (7,8), proteins

(8-12) sad glycoproteins (8,13) .

Because of this autonomy, mitochondria are

thought to be similar in some respects to bacteria .

Furthermore, mammalian

mitochondria and certain bacteria show similar responses to challenge by certain antibiotics (14,15) .

Indeed, mitochondria are thought, by some, to

1Supported in part bq Grant Nos. 1-Pll~i-15190 from the National Institute of General Medical Sciences and P529 from ~he American Cancer Society. 2Hesearch Career Development Awardee of the National Institute of General Medical Sciences .

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Macromolecular SyN:heeie

have arisen (from an evolutionary standpoint) from a bacterial invasion of mammalian cells and the subsequent formation of a symbiotic relationship between these cells and the "bacteria."

In light of these similarities between

mitochondria and bacteria and because of the aforementioned stimulation of protein and glycoprotein synthesis in mitochondria by vitamin R1 and varfarin and the inhibition of macromoleculer synthesis in a mammalian cell line by vitamin B.l and varfarin, it seemed of interest to determine whether E. cola responded to vitamin cells .

&1

and warfaria like the mitochondria or the leukemic

The present com~nication describes the effects of warfaria and vita-

min Rl on macromoleculer synthesis in E. cola . MATERIALS AND METHODS Cell culture .

E. coli B was grove in nutrient broth (Baltimore Biologi-

cal Laboratory) supplemented with 5 g/1 of NaCl and vas used is mid-log phase. Materials .

Vitamin H was used in as aqueous colloidal solution as l

Aquamephyton (Merck, Sharp b Dohme) . (Endo Laboratories) .

Sodium warfaria vas used ae Coumadin

Thymidine-methyl-~ (sp. ac . 15 Ci/mmole), 1-leucine-

4,5- 3H (sp . ac . 30 C1/mmole), uridine-5-~ (sp . ac . 20 Ci/mmole), and glucosamine-1-14 C HC1 (sp . ac . 40 mCi/mmole) were purchased from Nev England Nuclear .

Chloramphenicol and cyclohezimide in crystalline form were purchased

from Sigma Chemicals . Determination of macromoleculer synthesis .

Synthesis of macromolecules

vas determined ae previously described (1,8,13-15) .

DNA, RNA, and protein

synthesis and the incorporation of glucosamine into acid insoluble material were determined by the incorporation of radioactively labelled thymidine, uridine, leucine, and glucosamine, respectively, into trichloroacetic acid, ether-ethanol insoluble material . the folloviag:

The complete incubation system included

10 mM MgC1 2, 10 mM sodium phosphate (pH 7 .6),

5 mM phosphoenol-

pyruvate, 20 yg of pyruvic kinase (rabbit skeletal muscle, 1 mg of protein converts 430 umoles of phoephoenolpyruvate to pyruvate per min), 10 mM ATP,

Macromolecular 8y~hesis

Yol . 10, No. 2

83

2 mM SDTA, 22 .5 mg/ml of a complete amino acid mizture mimic leucine, RCI, 100

ul

of the S. coli

(pH 7 .6) and 50

ul

0.154 M

(between 0.3 and 0.7 mg protein) in 0.1 M Trie

of 0.1 M Tris (pH 7 .6) containing the required concentra-

boas of vitamin Rl , warfarin, or antibiotic . dine methyl- 3H or uridiae-5-3 H,

In addition 10 uCi of thymi-

5 uCi of 1-leucine-4,5- 3H, or 50 pCi of

glucosamine-1- 14C were added; in all instances the final volume was 280

ul .

After incubation at 37 ° for the time stated (usually 20 min) the protein or glycoprotein bound radioactivity was precipitated with 1X phosphotungetic acid in 0 .5 N HC1, washed three times with lOX trichloroacetic acid and once with a mutate of ethanol and diethyl ether (2 :1 by volume) .

The resulting

pellet was dissolved in 1 .0 N NaOH, plated on a glass fiber filter, and the radioactivity was determined by counting in a liquid scintillation counter. Protein. et al . (16) .

Protein determinations were made by the procedure of Lowry Samples were prepared by precipitation with 1X phoephotungstic

acid is 0 .5 N HC1, washing 3 times with lOX trichloroacetic acid and washing once with diethyl-ether ethanol (1 :2, v/v) .

The resulting residue was dis-

solved in 1 ml of 1.0 N NaOH and analyzed .

Crystalline bovine serum albumin

was used ae standard . Identification of glucoeamine-1-14C labelled macromolecules .

Thirty

control incubations were prepared with glucosamiae-1- 14 C in the usual ma+Lr and the precipitates pooled .

Twenty ml of water were added to the pooled

precipitates, and these pooled precipitates were extracted with phenol by the procedure of Weatphal et al . (17) except that the suspension was heated at 66 ° for 15 min.

Lipopolyeaccharide (LPS) was finallyr precipitated succes-

sively from 70Z ethanol, absolute ethanol, acetone, and diethyl ether. sample was dried is vacuo over CaC12 . protein and vas termed crude LPS.

The

The sample did not test positive for

Crude LPS was hydrolyzed in 4 N HC1 at

100° for 6 hours and the neutralized hydrolyzate chromatographed for identification of hesosamine as described previously (18) .

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Macromolecular Synthesie

Vol . 10, No. 2

RESULTS AND DISCOSSION The data in Table 1 indicate that at 2 z

10_M,

warfarin inhibited

E. cola RNA synthesis to 25X the control rate, DNA synthesis to 87X, protein synthesis to 24X and the incorporation of glucosamine into acid insoluble material to 40X the control rate .

Lower concentrations of warfarin caused

laver inhibition of macromolecular synthesis ; at 2 z

10~ warfarin

and lower

concentrations there was essentially no change in rates of macromolecular synthesis from the control rates . At a concentration of 2 a

10 ~,

vitamin Rl had little effect on DNA or

RNA synthesis in the E. cola , accelerated protein synthesis to greater than twice the control rate, and severely inhibited the incorporation of glucosa

nino into acid insoluble material to 1X of the control rate .

Lower concen-

trations of vitamin R1 had lesser effects on the macromolecular synthesis of E . coli ; however even at 2 x 107M vitamin Rl the incorporation of glucosamine into acid insoluble material was inhibited 15X (Table 1) . Table 1. Effect of vitamin R1 and warfarin oa macromolecular synthesis in E . coli . Data are pmoles incorporated per mg E. coli protein . Precursor ;

Uridine

Control

Thymidine

1-Leucine

Glucosamiae

20

53

67

200

Boil control 0° control 3 2 a 10 M warfarin 1 z 10_4 M warfarin 2 z 10 M warfarin 2 a 10 5 M warfarin 6 2 a 10 M warfarin 2 a 10 7 M warfarin

1 1

2 3

2 2

2 1

5 9 17 20 20 20

46 48 53 51 53 53

16 30 52 66 67 67

80 120 182 200 200 200

2 1 2 2 2 2

18 20 22 23 20 20

50 53 51 53 54 53

140 106 99 77 67 67

2 2 4 50 80 170

a x z x x x

10_ 10 10~M 10 5M 10_~I 10 M

vitamin vitamin vitamin vitamin vitamin vitamin

Rl R Ri Rl Rl Rl

Experiments were performed as given in the teat ; means from 6 observaThe precursor in each instance was tions . Incubation was for 20 min at 37° . the radioactively labelled compound mentioned in the tent .

Vol. 10, No . 2

Macromolecular 3y~hesis

85

Of the radioactivity from incubations with labelled glucosamine, fractionated into the LPS fraction . was recovered ae glucosamiae-14C.

74x

Of the total radioactivity in LPS, 81x It was concluded that LPS was the major

macramolecular class being synthesised by the E . cola with the glucosamiae14 C . The results presented in Table 2 demonstrate that cyclohe:imide did not inhibit the increase in protein synthesis found with vitamin Rl , and did not adversely affect any of the other effects of vitamin ICl or warfarin on macromolecular synthesis is E . coli .

Chloramphenicol inhibited protein synthesis

in E . cola and inhibited the increase in protein synthesis caused by vitamin ICl.

However, with 2 z

10 ~

vitamin R1 and 250 ug/ml of chloramphenicol the

rate of protein synthesis was three times that occurring in the presence of 250 yg/ml of chloramphenicol alone (Table 2) .

Similarly, with the 2 a

10

vitamin K1 and 100 or 40 ug/ml of chloraapheaicol the rate of protein synthesis was 2 .5 times tha rate which occurred with the chloramphenicol alone . The data given in Table 3 demonstrate the temporal sequence of the effects of vitamin K.l and warfarin on nacromolecular synthesis is E . cola . The moat striking results were that the vitamin R.l inhibition of the incorporation of D-glucosamine into acid insoluble material by E, coli and acceleration of protein synthesis occurred at the first time interval measured

(5 min) and increased with time .

The warfarin inhibition of pro-

tein and RNA synthesis was evident after 5 min incubation, but the inhibition was approximately to the same degree as that for the satire 20 min incubation period

(Table 3) .

The experiments described herein show that in E . coli vitamin

-1

accelerates protein synthesis (two fold) but not to the same extent as in mammlian mitochondria (five fold) (1) .

Furthermore,

in E. coli vitamin B.l

greatly inhibits the incorporation of D-glucosamine into LPS while in the rat liver mitochondria equimolar vitamin thesis seven fold (1) .

$1

accelerates glycoprotein syn-

The effects of warfarin is E. coli , causing inhibition

88

Macromolecular Synthesis

Table 2 .

vol . 10, No . 2

Effects of chloramphenicol and cyclohezimide on vitamin Rl and warfarin effects in _E . coli . Data are pmolee incorporated per mg E . coli protein

Precursor:

IIridine

Thymidine

1-Leucine

Glucosamine

chloramphenicol chloramphenicol chloramphenicol cycloheximide cycloheaimide cyclohezimide

20 30 26 21 20 19 20

53 50 52 51 50 50 53

67 2 4 8 66 67 65

200 160 174 191 185 190 192

2 z 10 3M warfarin +250 ug/ml chloramphenicol +100 ug/ml chloramphenicol + 40 ug/ml chlorampheaicol +250 ug/ml cyclohezimide +100 pg/ml cyclohezimide + 40 ug/ml cycloheaimide

5 8 7 6 6 5 5

46 42 43 44 43 43 45

16 1 2 4 18 17 18

80 74 78 77 78 78 79

3 2 x 10 M vitamin &1 +250 ug/ml chlorampHenicol +100 ug/ml chloramphenicol + 40 ug/ml chloramphenicol +250 ug/ml cyclohesimide +100 ug/ml cyclohezimide + 40 ug/ml cyclohezimide

18 22 22 22 18 17 18

50 50 51 50 50 49 50

140 6 10 20 136 138 140

2 2 2 2 2 1 2

Control +250 ug/ml +100 ug/ml + 40 Ug/ml +250 yg/ml +100 yg/ml + 40 ug/ml

Ezperiments were performed as given in the teat ; means Prom 5 experimeats . Incubation was for 20 min at 37 ° . The precursor was in each instance the radioactively labelled compound mentioned in the text . of RNA and protein synthesis and inhibition of the incorporation of glucosamine into macromolecules, were more similar to the whole L5178Y mammalian cell response to warfarin than the rat liver or brain mitochondrial response to an equimolar concentration of warfarin (1) . The results are of importance to E . cola function since both vitamin %

1

and warfarin are very inhibitory to E . coli LPS synthesis, but the bacterium distinguishes is its response on protein synthesis between vitamin warfarin .

1

and

Thus warfarin at the levels studied in these ezperimente is an

effective inhibitor of E . coli macromoleculer synthesis and in fact might be useful as s bacteriostatic agent .

Since both vitamin

R1

and warfarin

are

lipid soluble agents, the tonic effects of these molecules may be related to a aembrane or cell wall interaction.

Macromolecular Sy~hesis

Vol . 10, No. 2

Table 3 .

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Time course of effects of vitamin &1 and warfarin on macromoleculer

synthesis in 8, cola .

Data are pmolae incorporated per mg of B . coli protein Time

Control

2 z

10~ vitamin

2 a

103Ii

S.l

warfaria

Uridine

Thymidine

1-Leucine

Glucosamine

0 5 10 15 20

2 9 15 18 20

2 16 30 42 53

0 21 40 51 67

0 70 130 170 200

0 5 10 15 20

2 7 14 16 18

2 15 29 41 50

0 40 79 110 140

0 1 1 2 2

0 5 10 15 20

2 3 3 4 5

1 15 26 40 46

0 6 11 14 16

0 26 48 62 80

Experiments were performed as given in the tent ; mesas from 5 observations. The precursor 1a each instance was the radioactively labelled compound mentioned in the teat . Warfarin and especially vitamin S.l accelerate synthesis of glycoprotein and protein is mitochondria ; this acceleration may result in the production of an enzyme, cofactor, or other moiety necessary in the blood clotting aechanism (1) .

The fact that in 8 . cola , protein synthesis but not lipopoly-

saccharida synthesis is increased may indicate that the glycoprotein response of the mitochondrion evolved after the bacterium and cell became eymbiotes. Thus vitamin

-1

and warfaria are not like other molecules such as chlorampheni-

col, cyclohe:imide, and RNAse to which ß. cola and mammalian mitochondria respond similarly is the effect on synthesis of macromolecules . Finally, vitamin

gl

(at 2 :

1041i)

under the present ezperimental condi-

tions is an eacelleat specific inhibitor of LPS synthesis without having any deleterious effect on nucleic acid or protein synthesis .

As such it could

prove to be an effective tool for inhibition of LPS synthesis or cell wall synthesis.

Furthermore warfarin at high concentrations could serve se a tool

for inhibiting all four classes of macromalecular synthesis simultaneously .

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Macromolecular Synthesis

Vol. 10, No . 2

Asknowledgemeata . I thank especially Mra . Gerilya Pike, Mies Meliada Shea, and Mr . Kenneth Caee for valuable technical assistance . REFEBENCES l.

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3.

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4.

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7.

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8.

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9.

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10 .

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11 .

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Macromolecular 3y~heeie

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14 .

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