Fat metabolism in higher plants

Fat metabolism in higher plants

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 126, 932-941 Fat Metabolism XXXV. Partial Primary Structure S. MATSUMURA Department (1968) in Hig...

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ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

126,

932-941

Fat Metabolism XXXV.

Partial

Primary

Structure

S. MATSUMURA Department

(1968)

in Higher

Plants

of Spinach

Acyl Carrier

AND

P. IX. STUMPFl

of Biochemistry and Biophysics, University Davis, California 96616

Received

December

Protein

18, 1967; accepted

January

of California, 17, 1968

Escherichia coli, Brthrobacter, and spinach acyl carrier proteins were treated with iodoacetamide-1°C. Peptic hydrolysis of the resulting derivatized proteins has led to the formation of several radioactive peptides, which contain S-carbamoylmethyl[I%]-4’-phosphopantetheine. The prosthetic group, 4’-phosphopantetheine, of spinach acyl carrier protein is linked through a phospho-diester to the serine residue. The amino-terminal residue of Arthrobacter and spinach acyl carrier proteins are serine and alanine, respectively. The amino acid core of 9 residue, containing the substratebinding site and t,he prosthetic group, appears to be identical in E. coli, Arthrobacter, and spinach acyl carrier proteins. Possibly two closely related species of acyl carrier proteins occur in spinach leaves.

Recently, it has been established that acyl carrier proteins isolated from various bacteria and plantsplay an essential role in fatty acid biosynthesis as well as in a number of other biosynthetic reactions (1-14). Acyl carrier protein from Escherichia coli has been purified to homogeneity and extensively characterized by both Vagelos and Wakil and their groups (3, 15). Very recently, these groups (26, 27) independently established the partial amino acid sequence near the prosthetic group, 4’-phosphopantetheine. Furthermore, Simoni et al. (14) reported on the purification and characterization of ACP2 from spinach, avocado, and Arthrobatter. Plant ACPs have a molecular weight of approximately 10,000 and contain 4’phosphopantetheine. Although the total amino acid composition of E. coli and spinach ACPs are quite similar, the major difference is that the spinach ACP has five less glutamic but five more lysine 1 This work was supported in part by grant GB 5879 from the National Science Foundation and grant 12-14-lOO-7990(74) from the Unit,ed States Department of Agriculture. 2 Abbreviation used: ACP, acyl carrier protein. 932

residues. The similarity in properties between E. coli and spinach ACP and their partial interchangeability in fatty acid biosynthesis suggest that a more detailed investigation of the molecular architecture of plant ACP was merited. Such comparative studies could, for example, suggest the relative importance of different amino acids in the acyl carrier activity of the protein, that is, the complete absence of arginine in spinach ACP and its presence in E. coli would suggest that this amino acid is not critical in the biochemical activity of ACP. Because less than 4 pmoles of spinach ACP were available, investigations were confined to the isolation and the partial determination of the iodoacetamide-14C peptic peptides. EXPERIMENTAL

PROCEDURE

ACP preparation. Escherichia coli ACP was purified essentially according to Sauer et al. (15) with the exception of addition of another DEAEcellulose column fractionation. Spinach ACP was purified according to Simoni et al. (14). Arthrobatter ACP was kindly prepared by Dr. R. Simoni. Pepsin digestion of ACP. S-Carbamoylmethyl[WY] ACPs (0.5 pC/pmole) were chemically prepared according to Simoni et al. (14). These sub-

FAT

METABOLISJI

IN

strates were incubated with pepsin (a twicecrystallized preparation from Worthington) at 37” for 24 hours according to Majerus et al. (16). Paper chromatography. Two solvent systems were employed for the chromatography of peptides: Sobenl 1, butanol-acetic acid-water, 3:l:l (17) ; Solvent 8, bL~taI~ol-p~r~di~le-a.ceti~ acidwater, 15:10:3:12 (18). The papers were sprayed wit.h a ninhydrin reagent containing O.lc10 collidine in 95oj, ethanol. Development was accomplished by heating at 80-85” for 20-30 minutes. The radioactive regions of the paper st,rips were located on a Nuclear Chicago strip counter. Paper electrapharesis. Electrophoresis on an analytical or preparative scale was carried out on a General Medical Electronics apparatus under the following conditions: (a) pyridine-acetate buffer at pH 3.5 (pyridine-acet,ic acid-water, 1:10:89, Ref. 19) at approximately 2500 V for 5 hours; (h) pyridine-acetate buffer at pH 6.5 (pyridineacetic acid-water, 25:1:225, Ref. 19) at 3000 V for 1 hour. 4rnino acid analyses. The amino acid compositions of the protein or of the various peptides were determined with the Phoenix amino acid analyzer model, K 8090 VG, aft,er hydrolysis with 6 N HCl, according to the procedure of Moore and Stein,

(20). Curboxypeptiduse A. digestions. Carboxypeptidase A digestions were carried out in 0.2 M (NH4)JZ01 buffer at pEI 8.5 at 37”. Three-times crystallized carboxypeptidase A (Worthington) was treat,ed with diisopropylfluorophosphate and dialyzed against water before use. The approximate substrate and enzyme concentrations were 0.01 pmole/O.l ml and 0.01 mg/O.l ml, respectively. Glacial acetic acid was added to stop digestion. Amino terminus determination. The aminoterminal amino acid residue of ACPs was determined by preparing t,he ~\~-dinitrophe~~~rl derivative. The DNP amino acids were identified by t,hin-layer chromatography in two dimensious toluene-pyriclinewith the solvent systems, ethylene chlorohydrin-0.8 N ammonia solution (100:30:60:60), and chloroform-methanol-acetic acid 95:5:1 {21). I)ansyl amino acid derivatives were obtained as follows: 2 mpmoles of the peptide was dissolved in 15 ~1 of 1% NaHCOz , and 15 ~1 of 0.1% solutiou of dansyl chloride (California Corporation for Biochemical Research, Los Angeles) in acetone, was added, forming a one-phase system. After 8 hours at room temperature in t,he dark room, the solvent was evaporated in aac~o, and 60 pl of 6 N HCl was added. The tube was then sealed aud heated at 105’ for 12 hours. Aft,er acid was removed in VOCZLO, the residlle was dissolved in ace-

IlT(:fIII:II.

1’LhlXTS.

XSSV.

9x3

tone-acetic acid-water. Aliquots of this solution were used for the identification of DNS amino acids on thin-layer plates of silica gel in one dimension under the following systems: (a) benzenepyridine-acetic acid, 80:20:2; (b) chloroform-tamyl alcoh(~l-formic acid, 70:30: 1; (c) chloroformt-amyl alcohol-acetic acid,70:30:1/2; and (tl) ethyl acetate-isopropyl alcohol-eoncd. ammonia, 8:20:6

(2‘4 Hydruzinolysis. The COOH-terminal amino acid residue of the peptides was determined by combining the method of Akabori ef al. (23) with the I)NS teehnique of Nedkov and Genov (24). Phasphute ~eternz~nut~on. Analysis for organic phosphate in peptides was carried out according to Ames and Dubin (25). Tryptic digestion. One-tenth micromole of a peptic peptide was dissolved in 0.5 ml of water, and the solution was adjusted to pH 7.5 wit,h sodium bicarbonate solution. Twice-crystallized trypsin was first treat,ed in 0.063 i\‘ HCl at 30” for 24 hours to remove chymotrypsin activity. The approximate substrate and the acid-treated trypsin concentrations were 0.1 /*mole/ml and 0.1 mg/ ml, respectively. The resulting radioactive peptides were purified by paper ~~lromat,ograph~ and electrophores~s on paper as described above. Sepwntial degradations. Sequential degradation of peptides from the amino terminus was performed by the Edman procedure. Two-tenths milliliter of the peptide, equivalent to 0.05 pmole, was placed in a stoppered tube and dissolved in 0.5 ml of 5y0 sol&on of phenyl isothio~~rarlate in pyridine. The mixture was left for a short time in a stream of nitrogen, and the test tube was then stoppered and incubated at 40” for 3 hours. Excess amounts of phenyl isothiocyanate and pyridirle were removed by extracting four times with 2 ml of benzene. The aqueous solution, containing the PTC-peptide, was evaporated on a rot~ary evaporator and t,hen taken to dryness in a high varuum over PROS. The test tube was flushed with a stream of nitrogen. One hundred microliters of trifluoroacetic acid was added and the reaction mixture was allowed to stand at room temperat,urc for 2 hours. The trifl~~ort~a~etic acid was removed in astream of nitrogen. A drop of benzene was then added and evaporated in the same way to remove completely the acid. Two-millimicromole aliquots of the peptide were then coupled with dansyl chloride before the first Edman degradation and after the first and second Edman degradation, and then hydrolyzed as described above for DNS derivatives. The I)NS amillo acids were 1 hen identified using t,binlayer chromat,ography systems.

MATSUMUHA

933 RESULTS

Since the con~ent,ration of ACP in plant tissuesis about GO-fold lessthan that, found in bacterial cells (14), it is difficult to obtain sufficient quantities of pure plant acyl carrier protein for complete chemical analyses. In addition, it has recently been observed in this laboratory that spinach ACP is associated with another protein (henceforth called Protein I) whose amino acid composition is similar to that of spinach ACP, as indicated in Table I. Since this protein migrates more slowly than spinach ACP in a starch gel electrophoresis system, the preparative starch gel electrophoresis has been employed3 for the further purification of spinach ACP and Protein I. However, for the structural work of the peptic peptides to be reported in this paper, protein containing 50 % spinach ACP and 50% Protein I was employed. After treatment, with dithiot~eitol, 50 % pure spinach ACP {containing an equal amount of Protein I) leas nlkylated with iodoacetamideJ4C. DEAE-Cellulose column chromatography of alkylated 50 % pure spinach ACP yielded an elution pattern with a sy~etri~al protein peak with constant specific radioactivity throughout the peak. The material from this peak was used for further studies on the partial amino acid sequence and amino acid composition surrounding the prosthetic group, 4’-phosphopantetheine, of spinach ACP. Amino ermines of ACPs. Majerus et al. (26) and Pugh et ~2. (27) independently determined t’hat E. coli ACP has serine as its amino-terminal residue. The aminoterminal residues of t’he pure spinach ACP and ~~~~r~~~~r ACP, isolated as described above, were found to be alanine and serine, respectively. In case of spinach ACP, traces of DNP-serine, DNP-glutamic, and DNPmethionine were observed besides t,he major spot of DNP-alanine. Control experiments with E. co& ACP confirmed the reports of Majerus and Pugh that serine is the aminoterminal residue. Peptic hydmlysis of ACPs. As noted in Pig. 1, several radioactive peptic peaks were obtained by paper electrophoresis of the pepsin hydrolysates of 14C-alkylated spin8 Dr. R. Simoni, ~~npublished observation.

ANII

STUMPF

TABLE I ACID COMPOSITION OF SPINACH ACP ARD PROTEIK I Amino acid analyses were performed as described under EXPERIMENTAL PROCEDURE. No corrections for losses of serine and threonine due to hydrolysis have been made. Cysteic acid was determined after performic acid oxidation (2Q}. No tryptophan estimat~io~ were carried out. Burn-o

Spinach Amino

Cy&eic acic Taurine &Alanine Aspartic Threonine Serine Glutamic Protine Glycine Alanine 1.aline Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine llist,idine Arginine NII,-Terminal amino acid

ACP (14)

Protein

~-

acid

I

-

Residues/ molecule __.-

Nearest integer

Residues/ molecule -~

0.15 0.89 0.93 12.0 5.7 1.3 16.3 1.8 4.2 9.0 7.0 0.92 5.0 7.0 0.08 2.1

0

0.82

1 1 12 6 4 16 2 4 9 7 1 5 7 0 2

8.8 1.0 0.08

9 1 0

0.0 0.0

10.8 4.6 5.2 12.4 4.0 6.0 8.1 7.2

~-

Nearest integer 1

0 0 11 5 5 12 1 F 8 7

3.6 (5.2 0.3 2.0 7.2 2.1 0.3

Ala

-

-

ach ACP at pH 6.5. Several radioactive patterns were observed at both the cathode and anode sides. These peptides were eluted separately and further purified by both paper chromatography as described above and by paper electrophoresis at pH 3.5. Since the S-3,4,5 and 6 peptic peptides contained essentially only the cystea~ne residue and no cyst,eine, they were obviously derived from the spinach ACP and were examined in considerable detail. Since the S-l, 2,3 and 7 peptides contained cysteine and not cysteamine, they were clearly derived from Protein I and were not further charac~rized,

FAT

hlETABOI,ISM

IN

IIIGI~EX

PLANTS.

NSS\‘.

ORIGIN

I ORIGIN

E-l

I ORIGIN

A-l FIG. 1. Paper (at pH 6.5).

electrophoresis

of peptic

peptides

of carbamoylmethyl-W

derivatives

of various

ACPs

The clear-cut results obtained by using the in good agreement with that of E. coli carbamoyl-14C methyl derivatives of cys- peptic peptide E-l-B.4 In addition, the teine and cysteamine containing peptides amino acid composition of spinach peptic obviously greatly simplified the use of 50% peptide S-6-Ca is similar to that of E. coli pure ACP since we could readily separate peptide E-I-Ca and Adhrobacter A-l-Ca. Two radioactive peptic peptides, S-5 and the peptic peptides derived either from ACP or from Protein I. S-4-B, isolated from spinach ACP, have in Peptic peptides from 14C-alkylated E. coli common t,he amino acid residues of the ACP and Arthrobucter ACP show similar S-6-B fragment, which is the site of the prosthetic group, 4’-phosphopantetheine. electrophoretic patterns. Table II indicates the RF values of some They differ from each other in that, as inof t,he important radioactive peptic peptides dicated in Table IV, peptic peptide S-5 has from spinach, E. coli, and Arthrobacter. These one more aspartic, one more glutamic, and data showed that the Group II and Group one more alanine than peptic peptide S-4-B. III peptic peptides from the three organ- The S-5 peptide also has an isoleucine residue isms have the same Rp values and the same which is absent in S-4-B, but no lysine, and electrophoretic behaviors at pH 3.5 and 6.5 S-4-B has one lysine residue. As would be as well as the sameninhydrin colors, respec- expected, on treatment with trypsin, no change in amino acid composition was obtively. Amino acid composition of radioactive served with S-5, but with S-4-B a decrease peptic peptides. Data are reported as the of one residue with respect to glutamic acid number of residues of each amino acid per and serine and a loss of phenylalanine and mole of radioactive peptic peptides. As in- lysine were observed. The radioactive peptic dicated in Table III, the amino acid com- peptides S-l-A and S-4-A, which mere iso4 S, Spinach; E, E. coli; A, .lrthrobacter. position of spinach peptic peptide S-6-B is

936

MATSUMURA

AND TABLE

PROPERTIES

OF

SIMILAR

GROUPS

OF

E. coli, Group

I

Peptide

RF

Py:But:HAc:HaO (10:15:3:12)

STUMPF II

RADIOACTIVE

Arthrobacter

AND

RF But:HAc:HzO (3:1:1)

PEPTIC

NHz-Terminal

S-6-A E-l-A A-l-A

0.27 0.266 0.266

S-6-B E-l-B A-l-B

0.38 0.38 0.38

0.18 0.19 0.19

Gly, Gly, Gly,

III

S-6-C. E-l-C, A-l-C,

0.51 0.51 0.51

0.22 0.22 0.22

GUY Gly GUY

IV

S-6-& E-l-&

0..51 0.51

0.35 0.38

II

TABLE AMINO

ACID

COMPOSITION

OF

PEPTIC Group

Amino

Taurine” Aspartic Threonine Serine Glutamic Glycine Alanine Valine Isoleucine Leucine p-Alanine amino

FROM

SPINACH,

Val Val Val

amino

(t) (t) (t),

Ala

acid

Ninhydrin

color

Yellow Yellow Yellow

(t)

Yellow Yellow Yellow

III S-6-B,

E-l-B,

S-6-Ca,

II

A-l-Ca, Group

AND

E-l-C&

IIId

acid S-6-B

Total

PEPTIDES,

PEPTIDES

ACPs

0.8 (l)c 1.5 (2) 0.8 (1) 0.9 (1) 1.2 (1) 1.7 (2) 1.0 (1) 0.8 (1) 0.5 (1) 0.8 (1) 1.0 (1) acide

11

E-1-B

0.9 1.4 0.6 0.8 1.2 1.8 1.0 1.2 <.l 1.3 1.0

S-&G3

(1) (2) (1) (1) (1) (2) (1) (1) (0) (1) (1) 10

0.5 1.5 0.3 0.9 0.4 1.3 1.0 0 0.4 0.6 1.0

A-l-&

(1) (2) (0) (1) (0) (1) (1) (0) (1)

6

a Calculations are based on the assumption of 1 residue of ol-alanine b The decomposition of S-carbamoylmethylcysteamine is particularly tubes are not evacuated, oxidation lead to the formation of appreciable c Nearest integer. d Two or three analyses. Average figures are presented. e Excluding taurine and B-alanine.

lated from 50% pure spinach ACP, corltained one residue of cysteine instead of cysteamine. Both @-alanine and phosphate were not found in these peptides. These peptides were obviously derived from Protein I and were not further characterized. Amino acid terminus of radioactive peptic peptides. Since t’he amounts of all peptic

0.8 2.2 + 0.8 + 1.1 1.0 0 0.4 1.3 1.0

E-l-&

(1) (2) (0) (1) (0) (1) (1) (0) (1)

0.5 1.5 0.3 0.9 0.4 1.3 1.0 0 0 1.2 1.0

6 in each peptide. sensitive to oxygen. amounts of taurine.

(1) (2) (0) (1) (0) (1) (1)

(1)

6

If

the

peptides were less than 0.05 pmole, the dansyl chloride procedure has been applied for NHz-terminal analysis and sequence determination on the peptic peptides. Although this method is rapid and reproducible, two-dimensional separation resulted in an appreciable decrease in sensitivity. Therefore, the samples have been analyzed

FAT

METABOLISM

TABLE EVIDENCE

FOR

AMINO

Two

acid residues

HIGHER

PL.4NTS.

IV

SPINACH

BASED

S-4-B b

S-5’”

ON

@ELIMINATION

PEPTIC Amino

0.5

(1)

0.5

(1)

0.6

(1)

Aspartic

3.0 1.3

(3) (1)

1.6 0.9

(2) (1)

1.8 0.9

(2) (1)

2.3 3.3

(2) (3)

2.0

Alanine T’aline

1.6

(2)

1.0

(1)

(2)

1.8 2.1

(2) (2)

1.3 1.5

(1) (2)

1.7 1.7

(2) (2)

1.3 1.0

(1) (1)

1.2 1.0

(1) (1)

Isoleucine Leucine

1.1 1.3

(1) (1)

0 1.2

(1)

0 1.3

Phenylalanine

0.7 0

(1)

0.8

(1)

0

1.3 1.0

(1)

0 0.8

Glutamic Glycine

Lysine &Alanine

1.0

NH?-Terminus Total

amino

n One

acids”

preparation,

b Three preparations, c Excluding taurine

Aspartic

Serine

18

14

two

acid

S-4B-T S-4-BT

(0.03

FROM

~MOLE) After

alkali

treatment

S4B-T”

Taurine

Serine

\-

OF 4’.PHOSPHOPANTETHEINE PEPTIDE

Aspartic

Threonine

937

XXX\..

TABLE ACPs

ACID COMPOSITIONS OF SOME PEPTIDES OF SPINACH ACP

Amino

IN

(1)

acid

1.8

Threo”ine

0.86

1.8 0.8

Serine

1.0

0.4

1.3

1.4

1.5 1.2

1.0 1.2

1.0 1.3

1.0

Leucine Taurine @-Alanine

0.6 0.8

Glutamic Glycine Alanine Valine

acid

1.2 0.4 0.7

(1)

Glytine 10

analyses.

three analyses. and p-alanine.

by one-dimensional separation using the two solvent systems as described in EXPERIMENTAL PROCEDURE. As indicated in Table II, glycine was the major amino terminal residue in Group II peptic peptides. In addition, however, a trace amount of alanine and valine was always found. The only amino terminus found in Group III peptic peptides was glycine. The peptic peptide S-4-B was Oreat’ed with fluorodinitrobenzene to determine the amino-terminal residue. After acid hydrolysis of t,he DNP-peptide, DNP-serine was identified by thin-layer chromatography. The dansyl chloride procedure on the tryptic peptide S-4-BT showed dansyl-glycine as the amino t,erminal residue (see Table IV). Incubation of these peptides with leucine amino-peptidase and carboxypeptidase A did not result in the release of any amino acid. Similar difficulties were observed by other workers in this field (26, 27). Linkage of 4’-phosphopantetheine to radioactive spinach peptic peptide. Vagelos and Wakil have concluded that t’he prosthetic group, 4’-phosphopantetheine, is linked as

a phosphodiester to a serine residue of E:. coli ACP (26, 27). Similar studies were carried out with spinach ACP. The peptic peptide, S-4-BT, was treated with 0.2 N sodium hydroxide in a stream of nitrogen at 37” for 1 hour. After neutralization and lyophilization, the peptide was hydrolyzed with 6 N HCI. A loss of half of the serine residue from the initial peptide was observed as indicated in Table V. These are the expected results since treatment with base allows the phosphopantetheine group to be eliminated with the resultant formation of a dehydroalanine residue from serine. This compound on hydrolysis rearranges to pyruvie acid with a concomitant decrease in serine. Thus, the prosthetic group, 4’-phosphopantetheine, of spinach ACP is presumably linked to a serine residue. The appearance of pyruvic acid after hydrolysis was not determined. Control experiments with synthetic Gly-Ala-Asp-Ser-Leu at 40” for 2 hours in 1 K NaOH showed no loss of serine. &u&we of Group III peptic peptides. The Group III radioactive peptic peptides were hydrolyzed with HCl. The amino acid compositions of these peptides are indicated in Table III. The amino acid terminus of Group III peptides is glycine as determined by t,he dansyl chloride t’echnique. The Group III peptic peptides were then degraded by the Edman procedure, and the subtractive Gray and Hartley procedure was employed.

938

RIATSUMURA

Two-millimicromole aliquots of the peptide were removed before the first Edman degradation and after the first Edman degradation and were coupled w&h dansyl chloride, hydrolyzed, and chromotographed on silica gel thin-layer plates. As was expected, the spot obtained before t,he first degradatio~l corresponded to DNS-glycine. After the first degradation the major fluorescent spot was DNS-alanine with a trace of DNS-glycine. After the second Edman degradation, DNSaspartic was detected with smaller amounts of DNS-glycine and DNS-alanine. The persistent traces of fluorescent spots in addit,ion to those of the amino-terminai residue can be explained by incomplete reaction by the Edman degradation cycle of bhe original peptide. This observation is not surp~sing since Majerus et al. and Wakil et al. have already observed incomplete reactions of these peptides with Edman reagent (26, 27), Since, as indicated in Table II, onIy one amino terminal residue is detected in the Group III peptides, namely, glycine, and since with each cycle of degradation one new DNS amino acid is revealed plus the preceding DNS amino acid residue, it can be argued that the peptides under consideration are pure and that the additional DNS amino acid derivative is derived from an incomplet,ely reacted peptide. The same di~culties were observed in Edman degradation of S-4-BT under the same conditions, namely, the use of pyridine as the solvent. However, t,his difficultly was avoided in the degradation of S-4-BT by using a solution of phenyl isothiocyanate in pyridine containing trimethylamine rather than pUyridine alone according to the method of Margoliash (30). These results would argue that spinach and ~rt~ro~~~r Group III peptides have the amino terminus and have the sequence Gly-Ala-Asp. Majerus et al. (26) have already established that the pentapeptide containing the prosthetic group isolated from E. co& ACP has the sequence GIy-Ala-AspSer-Leu-. Since the amino acid compositions, the R, values in two solvent systems, the amino terminus, and the ninhydrin color are identical (see Table II), Group III peptides appear to overlap the pentapeptide of Majerus et al. (26). Therefore, these results

AND

STUMPF

suggest t,hat Group III peptic peptides have in common the sequence Gly-Ala-Asp-SerLeu-Asp-. ~t~etu~e of tempts ~~d~o~t~~~peptic peptides. The radioactive peptic peptide S-4-B

was digested with trypsin,

as outlined

in

~XP~RIMENT~LPROCED~~~. Theradioa~tive

trypt’ic peptide S-4-BT was hydrolyzed with HCI. Table IV summarizes the amino acid composition. The electrophoretic and chromato~aphic behaviors of the tryptic peptide S-4-BT are very similar to those of peptic peptide S-6-3 (Group II peptides). The amino terminus of peptide S-4-BT was gfytine. All attempts to determine the carboxyl terminal amino acid failed. Edman degradation of S-4-BT (0.07 pmole) gave the following results. First step (60%): Gly 0.7, Ala 1.1, Asp 1.6, Ser 0.9, Leu 1.2, Thr, 0.52, Val 0.9, Glu 1.2, P-ala, 0.8. Second step (80%): Gly 0.6, Ala, 0.48, Asp 1.5, Ser, 0.85, Leu 1.1, Thr. 0.83, Val, 0.94, Glu, 1.2, @-Ala 0.81. These results suggest the sequence Gly-Ala-, which overlaps Groups II and III peptides. Recently, Vanaman et aL5 showed that a peptide fragment which has the amino acid sequence of Asp-Thr-ValGlu was isolated from a pept,ide core near the prosthetic group in E. co& ACP. Their observation suggests the partial structure of Group II peptides and of S-4-BT would be Gly-Ala,Asp,Ser,Leu,Asp,Thr,VaI,Glu,GIy. Of considerable interest in the observation that S-5 resists tryptic digestion. S-5 has three glutamic residues, and no lysine residue. Since S-4-B has two glutamic residues and a lysine residue, trypsin w-i11attack this peptide. The product of tryptic hydrolysis is S-4-BT, which has 1 Iess serine, phenylalanine, glutamic, and Iysine than has S-4-B. These results would therefore indicate that the amino acids lost in digestion were to the left of the lysine residue and that Iysine was linked through its carboxyl group to the amino group of glycine, the N-terminal amino acid of S-4-BT. Since S-5 has one more glutamic and no lysine, it is tempting to suggest that S-5 has a cluster of two glutamic residues positionally identical to a possible Glu-Lys cluster in S-4-B. This supposition is attractive since the codons for Glu and 5 Private

&omrnL~nicatioll

from Dr. S. Wakil.

* In S-5, lysine

Spinach

E. coli

.I rthrobactcr

VI

Partial

structure

IIO-P-0-Pantetheine

0

site

T

HO-P+0

I

0-Pantetheine

of active

‘; Ser, Leu, Asp, Thr, F

glutamic

residue

is present.

Ser, Phe, Glu, Ly*s-[Gly-Ala, kc

Gly-Ala-Asp, b-E-l-Ca -E-l-B

HO-P-0-Pantetheine I

T

Gly-Ala-Asp, Ser, Leu, Asp, b------A-l-Ca -4 0

-Asp, Ala,

and an additional

Alanine

Scrinc

Serine

TABLE

Asp, Ser, Leu, Asp, Thr, S-6-CaF S-4-BT

7

1 0 t HO--P-0-Pantetheine

Val, Glrr, Gly

Val,

STRUCTURE FOR PEPTIC PE~TIDES ASSOCIATED WITH OF E. coli, Arthrobacter, ANU SPINACH ACP

-Asp-Ala-Ser-Phe-[(Gly-Ala-Asp-Ser-Le~~),(Asp-Thr-Val-Gl~~),Gly,

4'-PIIOSPIIOPA~VTICTHEINE

SH2-Terminus

is absent

(Vagelos et al. ; Wakil et al.)

E. coli

Organism

SUMMAKY OF PROPOSED PRIMARY

-I

Glu, C;ly]Ile~l

Vala, Asps, Gino]

940

MATSUMURA

Lys differ by only one base. In addition, these data would strongly suggest two species of ACP inspinachleaf tissue. It isdifficult to suggest the relative amounts of S-5 and S-4 species in spinach ACP. Clearly all the S-6-B is derived from S-5 and not from S-4B. However, a qualitative judgement based on Fig. 1 would indicate about 70% S-4 and 30% of s-5.

AND

STUMPF

peptic peptides, S-5 and S-4-B, isolated by enzymic digestion, have in common the Group II peptide fragment as indicated in Table IV and Fig. 2. The amino terminus of the peptide S-5 and S-4 B are aspartic acid and serine, respectively. As has already been described in RESULTS, the peptic peptide S-5 has one more aspartic, glutamic, and alanine than the S-4-B peptide. The peptic peptides S-5 and S-4-B are thus of considerDISCUSSION able interest since these two peptic peptides may represent two different acyl carrier proThe structure and function of E. coli teins, one having S-5 as the prosthetic core ACP has been the subject of intensive study unit and the other having S-4-B as its core chiefly in the laboratories of Vagelos and unit. Wakil and their collaborators. 4’-PhosphoNothing can be said about the extent of pantetheine is the active prosthetic group amidation of the carboxyl groups of aspartic and is attached to the polypeptide chain and glutamic acids in the ACP molecule. through a phosphodiester linkage to the As has been already noted by Simoni et al. hydroxyl group of a serine residue. Majerus (14), when either spinach or avocado ACP et al. (26) and Pugh et al. (27) established replaces E. coli ACP in an E. coli synthetase the amino acid sequence of a pentapeptide and a tripeptide at the prosthetic binding system, a large number of 3-hydroxy fatty acids are formed from malonyl-CoA, ranging site, respectively. Since much is now known from CiZ to C, hydroxy fatty acids. In addiabout the primary structure and function of tion, marked differences in specific activit’ies E. coli ACP, it became of interest to examine of CO, exchange and total lipid synthesis the plant ACPs from a comparative apwere observed between the plant and bacproach with special emphasis on the comterial ACPs. The evidence cited above parative primary structure and function of strongly suggests that the nine amino acids the acyl carrier protein molecule. Thus, in closely associated with 4’-phosphopanteaddition to E. coli ACP, the ACPs from theine are identical with both sourcesof acyl Arthrobacter viscosusand spinach leaves were carrier protein. Therefore, the marked difstudied. The sequencedata are summarized ference in biochemical activity must in part in Table VI. Table VI indicates the pubbe related to differences in composition in lished sequence of the peptic peptide of E. peripherally located amino acids. In addicoli around 4’-phosphopantetheine. The Group II peptic peptides, E-l-B and S-6-B, tion, recent work of Majerus (28) suggests that various enzymes of fatty acid biosynhave identical RF values in two solvent systhesis may bind to different parts of the E. tems, identical amino acid composition, and coli ACP. If the binding sites on ACP from the same N-terminus, glycine. The Group different sources differs markedly in the III peptic peptides, E-l-Ca, A-I-Ca, and peripheral region of the protein, then the S-6-Ca, are also identical in many propresults of Simoni et al. (14) in reference to the erties, but because they have a glycine Naccumulation of 3-hydroxy fatty acids can terminus, they are interrelated to the Group be readily explained. II peptides as described under Structure of Group III peptic peptides. Based on these ACKNOWLEDGMENTS data and the partial Edman degradation of We wish to acknowledge the considerable help Group III peptide, it’ is suggested that the of Dr. R. Simoni, Mr. J. J. Dunning, and Mrs. amino acid core of 9 residues around the Barbara Weigt in preparing various acyl carrier prosthetic group, 4’-phosphopantetheine, ap- proteins, and to Mr. J. Zaya for his generous aspears to be identical in Arthrobacter ACP, sistance in the amino acid analyses reported in E. coli ACP, and spinach ACP. Two larger this paper.

FAT

METABOLISAf

IN

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