TIIE JOURNAL OF B~LOGIC.~L CHE~STRY Vol. 218, No. 6, Issue of March 25, pp. 2086-2071, 1973

Printed in U.S.A.

Isolation, Purification, and Characterization of Pipecolic Acid-

containing Actinomycins, Pip 2, Pip la, and Pip lp*

(Received for publication, October 5, 1972)

JOSEPH V. FORMICAI AND EDWARD KATZ

From the Departme?lt of Miwobiology, Georgetown Unkersity Xchools of Medicine and Dentistry, Washington, D.C. 20007

SUMMARY

Streptomyces antibioticus synthesizes several new actino- mycins when DL-pipecolic acid is provided in the culture medium. Three of the compounds (Pip 2, Pip lar, and Pip Ifi) were isolated, purified, and characterized. Amino acid hydrolysates revealed that these antibiotics contain threo- nine, valine, sarcosine, and N-methylvaline but differ from actinomycins normally synthesized in the imino acid site of the molecule. The data reveal that: actinomycin Pip 2 contains 2 residues of pipecolic acid, actinomycin Pip lp possesses 1 residue each of pipecolic acid and proline, and actinomycin Pip la! contains pipecolic acid and 4-oxopipecolic acid per mole of actinomycin. 4-Oxopipecolic acid is a normal constituent of a number of peptide antibiotics, e.g. ostreogrycin, vernamycin Ba, and staphylomycin.

Schmidt-Kastner (1) first reported that new actinomycins were synthesized by Sfrepfomyces ch.rysomallus during antibiotic formation in the presence of sarcosine. Quantitative analyses revealed that the new actinomycins contained additional sarco- sine but less proline than the parent compounds elaborated (2, 3). On the basis of these observations it was suggested that sarcosinc substituted for proline in the actinomycin peptides during antibiotic biosynthesis. These results were confirmed (4, 5), and it was further demonstrated that several new actino- mycins were produced by Xtreptomyces antibioticus in the pres-

cnce of pipecolic acid (4), the higher analogue of proline. i2s pipecolic acid was found in hydrolysatcs of actinomycin mixtures, it was proposed that pipecolic acid also replaced proline in the actinomycin molecule.

11-c wish to describe the isolation, purification, and charac- terization of three pipecolic acidcontaining actinomycirls syn- thcrized by S. antibioticus in t,he presence of uL-pipecolic acid. Sonic of the biological properties of these compounds have been dttiicaribed in an earlier publication (6).

* This investigation was supported by Research Grant CA 06926 from the National Cancer Institute and by Training Grant TI-AT 298 from the National Institutes of Health.

2 Present address, Department of Microbiology, Medical Col- lege of Virginia, Health Science Division, Virginia Commonwealth University, Richmond, Virginia.

EXPERIMEKTAL PROCEDURE

Chemicals--DL Pipecolic acid :HCl was purchased from Schwarz-Mann Research Laboratories, Orangeburg, N.Y. All other amino acids and chemicals were obtained from commercial sources with the exception of N-methyl-m-valine which was synthesized by the method of Cook and Cox (7). Sephadex G-25 was purchased from Pharmacia, Uppsala, Sweden.

Organism and Conditions of Cultivation--S. antibioticus strain 3720 (ATCC 14888) was used for production of actinomycin mixtures. The organism was first grown in NZ-amine medium for 48 hours at which time the mycelium was washed and inocu- lated into glutamic acid-galactose medium as previously de- scribed (4, 8, 9). For the production of the pipecolic acid-con- taining actinomycins, m-pipecolic acid:HCl (250 pg per ml, final concentration) was added to the medium after 24 hours, and the culture was reincubated for an additional 96.hour period.

Isolation and Purijcafion of Pipecolic Acid-containing Acfino- mycins-The mycrlium was separated from the medium by suction filtration on a Huchner funnel, and the actinomycin was recovered from the medium by ext,raction with ethyl acetate (9). The extract was evaporated to dryness under reduced pressure at 50”. Subsequent evaporation of solvents was per- formed in the same manner. The crude pipecolic acid-contain- ing actinomycin mixture was resolved by Sephadex G-25 chro- matography (10) as described under “Chromatography and High Voltage Electrophoresis.”

The peak fractions were combined and the organic solvents were removed by evaporation. Contaminating sodium o- cresotinate in the fractions was removed by dissolving the dried actinomycin residue in a 0.570 solution of sodium carbonate (10 to 25 ml) and extracting three to four times with an equal volume of chloroform (11, 12). The chloroform extract was washed three times with an equal volume of water and, finally, the chloroform was removed by evaporation. The actinomycin was dissolved in acetone to a final concentration of 50 mg per ml. Two-tenths milliliter (10 mg) was applied as a streak to Whatman No. 3hIJI chromatographic paper (9 X 20 inches) previously impregnated with the aqueous phase of the chromato- graphic system: 10% aqueous sodium o-cresotinate-n-butyl alco- hol-dibutyl ether (5:2 :3) and developed with the organic phase of the same solvent system by the descending technique for 20 hours. The papers were subsequently air-dried at room tcm- perature in subdued light. The colored zones were cut out and the actinomycin eluted from the paper with methanol-water (9 : 1). ,1fter evaporation, sodium o-cresotinate was removed

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from an actinomycin preparation as detailed above. Some of the actinomycin fractions were rrchromatographed to achieve homogeneity.

Further purification was carried out by silicic acid chroma- tography (9, 11, 12). An actinomycin preparation was dis- solved in a small volume of benzene and applied to a column (1 X 5 cm) of silicic acid (Xallinckrodt No 2847). Colored impurities were removed by washing the column successively with benzene, benzene-methanol (99 : 1)) and benzene-methanol (98 : 2). Actinomycin was eluted with a benzene-methanol mix- t,ure (97:3), and the eluate was evaporated to dryness under reduced pressure.

Crystallization of Actinomycin-Actinomycins Pip lcr and Pip 2 were suspended in 10 ml of benzene and rendered soluble by the addition of approximately one-tenth volume of ethanol. Actinomycin Pip lp was dissolved in 0.5 ml of chloroform to which were added 10 volumes of ethanol. Each solution was evaporated to approximately one-half volume under a stream of nitrogen in a water bath at 60”. Crystallization took place at room temperature during a 24.hour period.

Crystals were harvested by centrifugation at 4”, washed twice with ligroin, suspended in a small volume of ligroin, and trans- ferred to a tared vial. The ligroin was allowed to evaporate and the residual solvent removed by heating in an Abderhalden pistol-vacuum apparatus for 1 hour at 80”.

HJJ~TO~YS~.S of Actinomycin-Hydrolysis of an actinomycin preparation in 6 N HCl was carried out in a Teflon-lined screw- capped test tube as described in an earlier publication (9).

Chromatography and I1igh Voltage Electrophoresis-Partition chromatography on Sephades G-25 was used to achieve a partial separation of compounds in an actinomycin mixture (10). Ei&y-five grams of Sephadex G-25 were suspended in 800 ml of the aqueous phase of the solvent system 10% aqueous sodium o-cresotinate-butyl acetate-dibutyl ether (4 :3 : 1) and equili- brated overnight at room temperature. The slurry was placed in a column (3.5 cm in diameter) which gave a settled height of 40 cm. After equilibration ov-ernight the aqueous phase was displaced by the addition of the organic phase to the column. The sample was prepared by suspending approximately 350 mg of the crude actinomycin mixture in 10 ml of the organic phase. The suspension was placed on the column and developed with the orgaiiic phase of the solvent system.

The method of preparation of silicic acid columns has been described elsewhere (9, 11, 12).

Circular paper chromatography was used to establish the homogeneity of actinomycin components as well as the qualita- tive and quantitative composition of an actinomycin mixture. The solveut systems used were as follows. Solvent System A: 10% aqueous sodium o-cresot,inate-butyl acetatedibutyl ether (4: 3 : 1) ; Solvent System B : 10 5% aqueous sodium o-cresot’inate- n-but’yl alcohol-dibutyl ether (5 : 2 : 3) ; Solvent System C : lo’% aqueous sodium o-crcsotinate-n-amyl acetate-dibutpl ether (4:3: 1). Details of the procedure have been published (4, 5, 11).

Preparative descending paper chromatography using What- man 30. 3MM paper was employed to isolate pure actinomycin components with Solvent System B listed above.

The qualitative amino acid composit,ion of actinomycins Pip 2, Pip lcr, and Pip I/3 was determined by ascending paper chro- matography using the upper phase of an n-but+ alcohol-acetic acid-phenol-water (3 : 1: 1: 5) solvent system. Amino acid hydrolysates were also examined by two-dimensional chroma- tography using t-butyl alcohol-methyl ethyl ketone-formic acid-

water (40:30:15: 15) in the first dimension and phenol-water (I : 1) in the second dimension. Separation of amino acids in an act,inomycin hydrolysate was also achieved by high voltage elect’rophoresis with 4O/, formate buffer at 4800 volts for 3 hours (9).

Amino acids were visualized with 0.3% ninhydrin in acetone containing 1% collidine. Imino acids also were detected wit,h 0.2y, isatin in acetone. Ehrlich’s reagent was employed after the isatin reaction (13).

Chemical and Physical Properties-Optical rotation of the pipecolic acid-contaiuing actinomycins was measured in methanol at a concentration of 3 mg per ml in a. Rudolph polarimeter. Jnfrared spectral analysis was carried out in KBr with a Perkin- Elmer model 421 spectrometer. Ultraviolet and visible ab- sorption spectra of actinomycin samples were determined in ethanol in a recording Bausch and Lomb spectrophotometer.

Melting point of the crystalline actinomycins was determined on a Koflrr hot plate. Specific extinction coefficients were ob- tained in ethanol in a Gilford model 2000 spectrophotometer.

Elemental analyses of carbon, hydrogen, and nitrogen were determined gravimetrically after incineration.

Analytical Methods-Proline in hydrolysates was quantitated by the method of Troll and Lindsley (14) ; pipecolic acid assays were carried out by a modification of the method of Silberstrin et al. (15). Actinomycin concentration was determined spec- trophoton~etrically at 442 nm in methanol or ethanol (9).

Actinomycin mixtures were examined by circular paper chro- matography to determine the relative quantities of the various actinomycin components produced (4, 8). Solvent System A was used for resolving actinomycins IV, V, Pip 1 ((Y and p), and Pip 2. Slow moving components did not separate in this system. Consequently, the area was cut from the paper and the actino- mycins eluted with methanol-water (9: 1). After removal of the sodium o-cresotinate, the slow moving components were re- chromatographed using Solvent System B. The latter system separated actinomycins I, II, III, and Pip s which then could be estimated. After air-drying chromatograms, the respective colored areas wvcre cut from the chromatogram and the actino- mycin cluted with methanol-water (9: 1). The concentration of an actinomycin was determined by measuring the optical density at 442 nm in a spectrophotomcter (9).

RESULTS

Effect OJ” Pipecolic Acid on Synthesis of Actinomycin Mixtures by X. antibioticus-Initially, the effect of various concentrations of m-pipecolic acid on t’he production of actinomycin mixtures was examined. Generally, synthesis of actinomycin began 24 hours after inoculation of the organism and continued for a 5- day period. Pipecolic acid was somewhat inhibitory to the formation of actinomycin mixtures (Table I) ; however, even at the highest level of DL-pipeCOliC acid employed the inhibit,ion was only 40 to 505;.

At the corrclusion of the iucubation, the actinomycin mixture synt,hesized in each case was isolated and examined by circular paper chromatography. It can bc seen (Fig. 1) that the relative percentage of the actinornycins in the region designated as ac- tinomycins I, II, III, IV, and V in a given mixture was greatly diminished. On the other hand, the relative amount of three new components (designated as Pip x, Pip 1, and Pip 2) in- creased with increasirig concentrations of pipecolic acid in t,he medium. It should be stressed that’ hydrolysates of material obtained from the various actinomycin regions contained sig- nificant amounts of pipecolic acid.

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Xolar Ratio of Pipecolic Acid to Proline in Actinomycin, Xix-

tures-The ratio of proline to pipecolic acid in the actinomycin

mixtures synthesized in the presence of uL-pipecolic acid is presented in Table I. Incorporation of pipecolic acid into the

TABLE I

Injluence of pipecolic acid concentration on synthesis of acti,komycin mixtures and of peptide-bound pipecolic acid and p/.oline

Streptomyces antibioticus was grown in glutamic acid-galactose- mineral salts medium; after 24 hours, oI,-pipecolic acid was added and the incubation was resumed for an additional 4 days. At the conclusion of the experiment the concentration of actino- mycin per ml of culture medium was determined spectrophoto- metrically (9). Actinomycin mixtures were harvested and hy- drolyzed, and the proline and pipecolic acid in hydrolysates were assayed as described under “Experimental Procedure.”

m-Pz;Tlic

concentration in medium

M/ml !&ml

0 80 10 82 50 78

100 64 250 48 500 41

1000 49

Actinomycin synthesized Proline

Pipecolic Pi;3ilic acid/praline

, + pipecolic ! acid X 100

1.79 1.75 1.47 1.25 0.63 0.48 0.48

0 0 0.37 ~ 2: 0.64 3-l

actinomycin molecule was observed at concentrations of 50 to

100 pg of m-pipecolic acid per ml of medium; as much as 20 to 307, of the proline residues were replaced by pipecolic acid.

At higher concentrations of pipecolic acid (500 to 1000 pg per ml), approximately 757, of the proline was substituted by pipe-

colic acid in the antibiotic mixture.

Production, Isolation, and Purification of Pipecolic Acid-con- taining Actinomycins-Fifty liters of culture filtrate yielded 3 g

of crude actinomycin material. Initial fractionation was

achieved by Sephadex G-25 column chromatography; the rlu- tion profile shown in Fig. 2 represents a typical separation of

the antibiotic mixture.

The first fraction (l), amounting to less than 1% of the ma- t’erial, constituted a degradation product which was devoid of

biological activity. Fractions 2 and 3 represented 15 and 25’$&,

respectively, of the material placed on the column, whereas the shoulder region (Fraction 4) constituted 15%. The material

remaining (Fraction 5) was eluted with water and represented 40% of the starting material.

Examination of the various fractions by circular paper chroma-

tography (Solvent System A) revcalrd that Fraction 2 was greatly enriched in actinomycin Pip 2 and that Fraction 3 con-

tained actinomycin l’ip 1. The shoulder region (Fraction 4) consisted largely of a component with the chromatogrnphic

properties of actinomycin V, whereas Fraction 5 was enriched

with a new actinomycin component which xl-as tentatively desig- nated actinomycin l’ip x. Further examination of Fraction 3

in two other chromatographic systems (B and C) revealed that

3

2

FIG. 1. The relative amounts of the actinomycin components in FIG. 2. Fractionation of a crude actinomycin mixture by Seph- the antibiotic mixture produced by Streptomyces antibioticus in adex G-25 chromatography with the solvent system 1070 aque- the presence of various concentrations of DL-pipecolic acid. In- ous sodium o-cresotinate-butyl acetate-dibutyl ether (4:3:1). cubation was for a 5-day period. Actinomycin mixtures were The elution profile is plotted as a function of optical density harvested, separated by circular paper chromatography, and the (O.D.) at 442 nm. percentage of the components in the mixture was determined as described under “Experimental Procedure.”

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actinomycin Pip 1 material separated into two components designated as Pip la and Pip lfl.

Actinomycins Pip lcu, Pip 10, and Pip 2 were obtained as homogeneous substances by preparative descending paper chromatography. Crystallization of actinomycin Pip lcr from benzene-ethanol yielded red needles; actinomycin Pip 2 formed red plates from the same solvent mixture. By contrast, actino- mycin Pip lb was crystallized as red prisms from chloroform- ethanol. Good recoveries (80 to 90%) were obtained at all steps except during the crystallization of Pip I/? (53%). The final yield in milligrams was: Pip 2, 272; Pip LX, 102; Pip lb, 136.

Examination of the crystalline actinomycins by circular paper chromatography in Solvent Systems A, B, and C revealed that the components were homogeneous substances. Fig. 3 reveals the homogeneity of the purified components as well as the reso- lution of actinomycin Pip 1 into the components Pip la! and Pip lp.

Properties of Actinomycins Pip 2, Pip ICY, and Pip 1&Ex- amination of acid hydrolysates of the actinomycins by paper chromatography as well as by high voltage electrophoresis (Fig. 4) revealed the presence of threonine, valine, sarcosine, and N-methylvaline in actinomycins Pip loo, Pip 10, and Pip 2. In addition, actinomycin Pip 2 was found to contain pipecolic acid but no proline, whereas actinomycin Pip l/3 contained both proline and pipecolic acid. Actinomycin Pip lor hydrolysates contained pipecolic acid and a ninhydrin-positive compound which gave a yellow color with ninhydrin characteristic of imino acids. The compound proved to be isatin-negative but gave a

reddish pink color with Ehrlich’s reagent. Comparison of hydrolysates of the peptide antibiotics, vernamycin Bar (16) and ostreogrycin (17) nith actinomycin Pip la revealed that a

FIG. 3. Circular paper chromatograms showing the actinomycin mixture produced in the presence of nn-pipecolic acid and the crystalline preparations of actinomycins Pip lor, Pip lp, and Pip 2. Solvent system A consisted of 10% aqueous sodium o-creso- tinate-butyl acetate-dibutyl ether (4:3:1); Solvent System B consisted of 10% aqueous sodium o-cresotinate-1-butanol-dibutyl ether (5:2:3). 1, the actinomycin mixture produced in the pres- ence of nn-pipecolic acid (250 pg per ml) ; 2, actinomycin Pip 2; 3, act.inomycin Pip lay; 4, actinomycin Pip lp.

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similar compound was present in these antibiotics which possessed identical electrophoretic and chromatographic properties with an authentic sample of 4-oxopipecolic acid. Moreover, reduc- tion of the oxoimino acid in hydrolysates with sodium boro- hydride yielded a compound with the properties of cisl-hy- droxypipecolic acid. RF data and the electrophoretic mobility of various amino acids relative to sarcosine are presented in Table II.

Quantitative data have corroborated the findings obtained by paper chromatography (Table III). It was established that actinomycin Pip 2 contains 2 residues of pipecolic acid and no proline, whereas actinomycin Pip l/3 possesses 1 residue each of proline and pipecolic acid per mole of actinomycin. One mole of pipecolic acid is also present per mole of actinomycin Pip 1~. It is assumed that there is 1 mole of oxopipecolic acid per mole of actinomycin Pip lcr.

The absorption spectra of actinomycins Pip 2, Pip lcr, and Pip 10 are similar to each other and to actinomycin IV in both the ultraviolet and visible regions and are quite characteristic of actinomycins previously described. The infrared absorption spectra are also quite similar, showing only minor differences in the 800 to 1400 cm+ region. The minor differences in the “fingerprint” region may reflect the differences in the amino acid composition of the peptide chains. Some physical data and elemental analysis of these actinomycins are presented in Tables III and IV.

DISCUSSION

The actinomycins synthesized by X. antibioticus differ solely in the imino acid site of the molecule (Fig. 5), whereas those

1. 4-OXOPIPECOLIC ACID

2. 3. HyPROLINE 0 NMeVALlNE 4. PROLINE 5. THREONINE 6. PIPECOLIC ACID 7. VALINE 8. SARCOS INE

oo@ooo

8 : 00 c3

A B C Cl E F

FIG. 4. Separation of amino acids in antibiotic hydrolysates by high voltage electrophoresis (4% formate, 3 hours, 4300 volts). A, actinomycin Pip 2; B, actinomycin Pip la, C, standard amino acids; D, actinomycin Pip la; E, vernamycin Bru; F, actinomycin IV.

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formed by S. chrysomallus each contaiu 2 L-proline residues but

differ at the “n-valine-n-alloisoleucine” site (18). Previous studies have shown that new actinomycins are synthesized in

the presence of sarcosine (l-5) or a number of proline analogues (e.g. pipecolic acid (4), 4-methylproline (19), azetidine-2 carbox-

TABLE II

Electrophoretic mobility and IL’s valuer of amino acids

ylic acid (18, 20), haloprolines (20), and thiazolidine 4-carbox- ylic acid (21)). Presumably the csogenously supplied analoguc

competes with endogeneously formed proline and is incorporated in lieu of proline into the antibiotic molecule. As a consequence,

synthesis of trace actinomycin components is enhanced or new

actinomycins are formed at the cspense of normal actinomycin components. These results suggest that the enzyme system

catalyzing the activation and incorporation of proline into the

antibiotic peptide possesses a low or broad specificity. Such broad specificity for chemically related amino acids and their

analogucs has been frequ(‘ntly observed during antibiotic syn-

thesis (see References 20 and 22 for review). As we had noted previously, several new actinomycins are

produced in the presence of DL-pipecolic acid by X. antibioticus

(4). To account for the number of actinomycin analogues formed it was postulated (4, 18) that actinomycin prptides

would bc found containing pipecolic acid-pipecolic acid, pipe- colic acid-proline, pipecolic acid-osoproline, pipecolic acid-

hydroxyproline, and pipecolic acid-sarcosine in the imino acid

High voltage electro-

phoresis

Paper chromatography”

Amino acid

Sarcosine Valine Threonine N-Methylvaline. Pipecolic acid.. Proline. cis-4-Hydroxyproline trans+Hydroxyproline. trans.4-Hydroxypipecolic

acid. cis-4-Hydroxypipecolic

acid. 4-Oxopipecolic acid..

RF 0.74 0.71 0.49 0.92 0.88 0.85 0.69 0.63

0.72

0.69 0.53

1.00 0.20 0.90 0.45 0.82 0.21 0.71 0.49 0.86 0.38 0.78 0.28 0.75 0.16 0.64 0.15

0.74

0.70 0.63

0.24

0.20 0.21

0.53 0.20 0.72 0.43 0.45 0.16 0.81 0.59 0.70 0.48 0.57 0.34 0.45 0.16 0.41 0.15

0.53

0.48 0.31

0.23

0.18 0.16

I I COpCH(L) (L)CH --CO I I I I

I;CH, tiCHJ tiCHJ I I

SIX SIX I I

L- Pro L- Pro

I I D- Val D- Val

I I t0 CO

,,,c,-Eli -CHJ -CHJ (L)CH- I

NH ..H

Sar I

L- Pro I

a Solvent systems for paper chromatography were as follows: 1, N-butyl alcohol-acetic acid-water (4:1:5, upper phase); 2, phenol-water (4 g:l); 3, t-butyl alcohol-methyl ethyl ketone- formic acid-water (40:30: 15:15) ; 4, n-butyl alcohol-acetic acid- phenol-water (3:l:l g:5).

D- Vol

I co

I -CH(L)

I NH

TABLE III

Analyses of Actinomycins IV, Pip Lx, Pip 10 and Pip 2 I CO

I CO

Actino- mycin / “\ \ NH2 cm \ \ 10

0

_____ ~__~. Calm- 59.2 6.85 13.4

lated: 1.90 0.00 Found: 59.0 6.98 13.22’ Calcu- 59.2 6.8 12.8

lated: 0.00 0.86 Found: 56.9 6.84' 12.22 Calcu- 59.6 6.95 13.25

lated: 0.95 1.01 Found : 59.21 7.29 13.59 Calcu- 59.9 7.02 13.1

lated: 0.00 2.21 Found : 60.6( 7.61 11.81

IV

Pip lol

Pip lg

Pip 2

C”, CHJ

FIG. 5. Structure of actinomycin IV. Sequence of amino acids is L-threonine, D-valine, L-proline, sarcosine, and N-methyl-L- valine. Actinomycin I: 1 residue of proline is replaced by 1 resi- due of hydroxy-L-proline; actinomycin II: 2 residues of proline are replaced by 2 residues of sarcosine ; actinomycin III : 1 proline residue is replaced by 1 sarcosine residue; and actinomycin V: 1 proline residue is replaced by 1 residue of 4.oxoproline. Q Moles per mole of actinomycin.

TABLE IV

Plopedies of aclinovrycins IV, Pip ICU, Pip lp ad Pip 2

Optical rotation

Absorption spectrum (in ethanol) Extinction

coefficient (e 443)

Melting point (decomp) Actinomycin Crystalline form

-I -

IV Plates 245-247 Pip la Needles 232-235 Pip lp Prisms 230-232 Pip 2 Plates 218-220

I Minimal Maximal

Ial; nm

-261.8 242, 426, 444 -100.4 236, 425, 443 -240.7 240, 426, 442

-56.0 234, 426, 442

230, 334 19.4 233, 334 17.2 230, 333 18.4 230, 332 15.1

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sites. Thus far, this prediction has only been partially realized in the case of actinomycin Pip 2 (pipccolic acid-pipecolic acid) and actinomycin Pip Ip (pipecolic acid-proline). The third actinomycin that we have purified contains pipecolic acid and osopipecolic acid in lieu of oxoproline. Thus, the organism ap- pears to mimic the normal biosynthetic reactions that occur with proline when presented with the analogue, pipecolic acid. Synthesis of oxoproline from proline may be partially or com- pletcly inhibited in the presence of pipecolic acid. Based on the finding that oxopipecolic acid is found in an actinomycin molecule, it appears likely that hydroxypipecolic acid will also be present in actinomycins synthesized in the presence of pipe- colic acid.

I,-Pipecolic acid has been found as a major constituent of the free amino acid pool in the common bean Phaseolus vulgaris (23, 24), and it is also present as a free component in many other plants (25). The observation that pipecolic acid is in- corporated into the actinomycin molecule was the first report that this imino acid can be found in a peptide structure (4). Subsequently, the n-enantiomorph of pipecolic acid has been described as a normal constituent of the peptide antibiotics aspartocin (26) and glumamycin (27). To our knowledge there are no experimental data to show that pipecolic acid can be incorporated into cellular proteins in Go. Moreover, recent studies with highly purified preparations of prolyl-tRNA syn- thetasc (28, 29) have revealed that this imino acid is not a sub- strate for the enzyme. The findings presented here indicate t,hut S. antibioticus can synthesize and incorporate 4-oxopipe- colic acid into the antibiotic peptide actinomycin when provided with the appropriate substrate. 4-Osopipecolic acid has been described as an amino acid component of a number of chemically related antibiotics, including vcrnamycin Bar (16), ostreogrycin (17), mikamycin (30)) and staphylomgcin (31). It is conceivable that the organisms producing these antibiotics can synthesize osoproline and incorporate the oxoimino acid into the antibiotic peptidcs they elaborate when provided with an exogenous source of proline.

Further investigations are in progress to purify and charac- terize additional pipecolic acid-containing actinomycins spnthe- sized by X. antibioticus.

ilcknowledgments-The authors kindly thank Lord Toddr Cambridge University, for a generous sample of ostreogrycin, Dr. M. Ondetti, Squibb Institute for Medical Research, for a sample of vernamycin Bcu, and Dr. H. Vanderhaeghe for a samnle of 4-oxo-n-uinecolic acid.

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Joseph V. Formica and Edward Katzβ, and Pip 1αActinomycins, Pip 2, Pip 1

Isolation, Purification, and Characterization of Pipecolic Acid-containing

1973, 248:2066-2071.J. Biol. Chem. 

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