THE JOURNAL OF BIOLOGICAL. CHEMISTRY. 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc . 264, No. 4, Issue of February 5, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc
. 264, No. 4, Issue of February 5, pp. 2024-2028.1989 Printed in U.S.A.
Amphomycin Inhibits MannosylphosphoryldolicholSynthesis by Forming a Complex with Dolichylmonophosphate* (Received for publication, September 6, 1988)
Dipak K. Banerjee From the Department of Biochemistry and Nutrition, School of Medicine, University of Puerto Rim, San Juan, PuertoRico 00936-5067
The inhibitory effect of the lipopeptide antibiotic to undecaprenylmonophosphate, the prokaryotic glycosyl-caramphomycin on the mechanism of mannosylphospho- rier lipid (2, 3). Those glycosylation reactions involving DolryldolicholbiosynthesisbycalfbrainroughendoP’ (the eukaryotic glycosyl-carrier lipid) in RER membrane plasmic reticulum membranes has been studied exten- preparations from pig aorta, plants, and rat parotid acinar sively. Calf brain rough endoplasmic reticulum mem- cells are also inhibited by amphomycin (4-9). However, spebranes when incubated with varying concentrations of cific interactions between the lipopeptide and the components GDP-mannose in the presence andabsence of ampho- of the enzymatic transfer reactions were not elucidated. We mycin showedno significant difference in apparent K , have demonstrated earlier (10) that inhibition of Man-P-Dol, for GDP-mannose (1.08 and 1.37 p ~ respectively). , However, the V,, was reduced to 0.17 pmol/mg pro- Glc-P-Dol, and GlcNAc-PP-Dol synthesis, “key” intermeditein/min in the presence of amphomycin as compared ates of the dolichol-cascade (11) is reversed by the addition with 1.86 pmol/mg protein/min in its absence. On the of exogenous Dol-P but not by sugar nucleotides or divalent otherhand,when mannosylphosphoryldolichol syn- cations. Furthermore, amphomycin also interfered with the * P not [3H]Manthase activity was measured in the presence of ampho- extraction of exogenous prelabeled D o ~ - ~but mycin and as a function of dolichylmonophosphate P-Dol or the major membrane phospholipids. These results (Dol-P) concentrations, the shape of the substrate ve- indeed suggested an interaction between the lipopeptide and locity curve changed froma rectangular hyperbola to dolichylmonophosphate. This paper presents kinetic evidence for the inhibition of a sigmoid. The Hill coefficients (n)for this reaction were calculated to be 2.02 and 1.22 in the presence Dol-P specific mannosyltransferase (Man-P-Dol synthase)by and absence of the antibiotic and the corresponding K , amphomycin which is primarily due to its interaction with values for Dol-P were found to be 333 and 47.3 p ~ , dolichylmonophosphate. It also provides evidence that comrespectively. In separate experiments when radiolaplexes are formed with polyprenylmonophosphates but not beled antibiotic was reacted with Dol-P in the presence with polyprenylpyrophosphates or polyprenols, the free alcoof Ca2+,a complex was formed. The complex formation hols. The complex formation is dependent on the presence of was dependent on both Ca2+ in the reaction mixture Ca2+ in the incubation medium and is not affected by the andfattyacidresidue on theantibiotic.Similar addition of phosphatidylserine. Ca2+dependence of the lipidcomplex formation was also observed with undeca- lipopeptide interaction encourages future studies to investiprenylmonophosphate. No such complex, however, was gate the possibility that themetal ion acts as bridge a between formed with dolichylpyrophosphate, with undecapren-Dol-P andamphomycin perhaps at thesubstrate-binding site ylpyrophosphate, or with their free alcohols (dolichol in a defined stoichiometry. or undecaprenol). Furthermore, when an equimolar mixture of Dol-P and phosphatidylserine was reacted MATERIALSANDMETHODS with the antibiotic under identical conditions, the complex formation was observed selectively with Dol-P. Dolichylmonophosphate, undecaprenylmonophosphate, andunThese data demonstrated thatamphomycin interacted decaprenol (C55-prenol) were purchased from Behring Diagnostics. with the active siteof the glycosyl-carrier lipid (Dol- Dolichol and Triton X-100 were purchased frorm Sigma. Phosphaand phosphatidylserine were the P), thereby preventing its participation at the enzy- tidicacid,phosphatidylcholine, products of Serdary Research Laboratories, Canada. Bio-Gel A-1.5 m matic reaction.
The antibiotic amphomycin is a straight chain undecapeptide with either 3-isododecenoic or 3-anteisotridecenoic acid attached to the NHn-terminal aspartic acid residue by an amide linkage (1).It inhibits the synthesis of peptidoglycan in Gram-positive bacteria by blocking the transfer of phospho-N-acetylmuramyl pentapeptide from its UDP derivative * This work was supported by United StatesPublic Health Service Grants HL35011 and S06RR08224 as well as Biomedical Research Support Grant from the University of Puerto Rico. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
(200-400 mesh) andBio-Gel P-2 (200-400 mesh) were obtained from Bio-Rad. [3H]KBH4(1.1Ci/mmol) and [y-32P]CTP(10-25 Ci/mmol) were supplied by ICN Pharmaceuticals Inc., and GDP-[U-“Clmannose (307 mCi/mmol)was supplied by Amersham Corp. Amphomycin (calcium salt) was a gift from BristolLaboratories and Dr. M. Bodanszky,Case Western Reserve University. [‘‘C]Undecaprenylpyrophosphateand [32P]dolichylmono- andpyrophosphates were kindly made availableby Dr. MalkaG. Scher, University of Maryland Medical School. All other chemicals and reagents were of analytical reagent grade. For use in enzymatic reactions as well as in binding studies, the lipopeptide was dissolved in 0.1 N acetic acid, and the solution was adjusted to 0.05 M sodium acetate, pH 7.0, with 0.2 N NaOH.
The abbreviationsused are: Dol-P, dolichylmonophosphate; ManP-Dol, mannosylphosphoryldolichol; Glc-P-Dol, glucosylphosphoryldolichol; GlcNAc-PP-Dol, N-acetylglucosaminylpyrophosphoryldolichol; Dol-PP, pyrophosphoryldolichol; Undeca-P, undecaprenylmonophosphate; Undeca-PP, undecaprenylpyrophosphate.
2024
Interaction between
Dol-P and Amphomycin
2025
Tritium Labeling of Amphomycin-Isotopic labeling of amphomycin was carried out at the free NHZ group of the diaminobutyric acid
inhibition of Man-P-Dol synthesis, a detailed kinetic study on the transferof mannose from GDP-mannose toexogenous residue. The method involves reductive methylation with formaldeDol-P in calf brain RER membranes was performed. When hyde and high specific activity [3H]potassium borohydride essentially the same as described before (12) for proteins. In most cases 100 pg Man-P-Dol synthesis was measured as a function of Dol-P of amphomycin wastaken up in 50 p1 of 0.2 M sodium borate, pH9.5, concentrations, it showed a simple substrate saturation curve and cooled in ice. To this ice-cold solution, 3 p1 of 20 mM formaldehyde in the absence of amphomycin, but the shape of the curve was added followed after 30 s by 1 pl (160 pCi) of J3H]KBH4 in 10 changed from a rectangular hyperbola to a sigmoid in the mM KOH. After 20 min, 2 p1 of unlabeled 10 mM NaBH4 were added presence of amphomycin (Fig. 2), indicating a biphasic reand the reaction mixture left foranother 20 min at 0 "C. The reaction sponse. Analysis of these data according to the Hill equation was stopped by the addition of 200 p1 of 0.4 M sodium phosphate, pH coefficients (n)were 1.22 in the absence 5.8, and 100 mM glycine. The pH of the solution was then adjusted indicated that the Hill to 3.0, and amphomycin was extracted with n-butanol. The organic and 2.02 in the presence of amphomycin, which suggested a phase was washed with waterand dried underNz. Finally, the labeled strong positive cooperative interaction between amphomycin lipopeptide antibiotic (26.7 mCi/mmol) was dissolved in 0.05 M so- and Dol-P. After the power of substrate concentrations was dium acetate, pH 7.0, and storedat -20 "C until use. raised to that of the Hill coefficients (n = 1.22 and 2.02) to SG-81 Paper Chromatography of PHIAmphomycin-Tritium-la- obey a rectangular hyperbola, the rate constants ( K , and beled and unlabeled amphomycinwere chromatographed on EDTAVmax)for Man-P-Dol synthesis were calculated. The results treated SG-81paper(13)anddevelopedwith (a) n-butanollacetic acid/water (3:l:l) and ( 6 ) n-propanollwater (7:3) (1). Radioactivity indicated thatK, and V,,, for transferring mannose to Dolwas detected by spraying the paper with En3hance spray (DuPont- P were 47.28 p~ and 13.31 pmol/mg protein/min (r = 0.99) New England Nuclear) followed by exposureto XAR-5 filmat -80 "C under normal circumstances, but in the presence of ampho(14) or by cutting a 1-cm area and counting in a liquid scintillation mycin the corresponding kinetic constants were changed to counter. For normal visualization, the paper was sprayed with 3% 332.93 p M and 50.83 pmol/mg protein/min ( r = 0.99), respecninhydrin in acetone. of Enzyme Preparation-RER membranes prepared from whole calf tively. Seven-fold increase in K , for Dol-P in the presence brain by the procedure described for white matter (15) were used as amphomycin clearly indicated that the interaction between enzyme. Protein concentration was determined by the Bio-Rad pro- amphomycin and Dol-P reduced the affinity of Dol-P for the tein assay (16)using bovine serum albumin as standard. enzyme. Assay for the Transfer of P4C]Mannose from GDP-p4C]Mannose Additional evidence that amphomycin interacted with DolintoCalf Brain EndogenousAcceptors-The enzymatic transfer of P rather than any other componentsof the reaction cascade ['4C]mannose from its nucleotide derivative into membrane-associwas obtained from the kinetic analysis as often followed for ated endogenous acceptors was assayed by the procedure reported allosteric enzymes. The equationfollowed here is earlier (17). Measurement of Radioactiuity-All radioactive samples were counted in Hydrofluor (National Diagnostics). RESULTS
were uo = initial velocity in the absence and u = velocity in Effect of Amphomycin on the Enzymatic Synthesisof Man- the presence of a modifier and Kl and K2 are dissociation P-Dol as a Function of GDP-Mannose-Earlier works (5, 10) constants of substrate (B) and modifier (M), respectively, which predicts thatdouble-reciprocal showed that thelipopeptide antibiotic amphomycin inhibited from the allosteric site, the transfer of ['4C]mannose from GDP-[14C]mannose to the plots of l / u - uo against (1/M) are linear inone combination endogenous acceptor, Dol-P. But there were no kinetic data and a parabola if combined twice (18, 19). In fact, the present results indicated that thedouble-reciprocal plot was linear in evaluatinginteraction between amphomycinwitheither effect of ampho- theabsence of amphomycin, where as in thepresence of GDP-mannose or Man-P-Dol synthase. The mycin on Man-P-Dol synthesis was therefore, studied as a amphomycin it was a parabola (Fig. 3). This strongly sugfunction of GDP-mannose concentrations, and the results aregested a direct interaction between amphomycin and Dol-P. Interaction between fHjAmphomycin and Dolichylmonoshown in Fig. 1. The substrate-saturationcurve displays Michaelis-Menten kinetics in the presence and absence of am- phosphate-To obtain a direct evidence for the dolichylmonphomycin, but the amount of Man-P-Dol synthesized was ophosphate-amphomycininteraction,the[3H]amphomycin substantially lower in presence of amphomycin for a given was reacted at room temperature with 50 pg of Dol-P in the GDP-mannose concentration. Analysis of these data accord- presence of 10 mM CaCL and 0.2% Triton X-100. When the ing to a "double-reciprocal plot" indicated that theK, values reaction mixture was analyzed on a Bio-Gel A-1.5 m column for GDP-mannose for Man-P-Dol synthesis in the absence (0.64 X 25 cm), it was observed that the bulk of the radioacand presenceof amphomycin were 1.37 and 1.08 p~ ( r = 0.99) tivity eluted after thevoid volume ( Vo)but much earlier than whereas the corresponding V,,, values were 1.86 pmol/mg amphomycin alone (Vi) (Fig. 4B), evidentlysuggesting an protein/minand 0.17 pmol/mg protein/min, respectively. interaction between the lipopeptide and the glycosyl-carrier These suggested that therewas no changein apparent K, for lipid. For further documentation,a dual-label experimentwas performed where both [32P]Dol-P and [3H]amphomycin were GDP-mannose for Man-P-Dol synthase, when measured in presence of amphomycin and a constant amount of endoge- reacted under identical conditions asdescribed above. Comigration of [3H]amphomycin with [32P]Dol-P presented strong nous acceptor lipid, but the Vmaxwas found to be -10-fold supporting evidence for a complex being formed (Fig. 4C). lower. This observation supported the notion that amphomycin primarily had no effect on the binding of GDP-man- However, when dolichol,the free alcohol, waspresent instead nose to Man-P-Dol synthase nor that amphomycin interacted of its monophosphate ester, nocomplex was formed between directly either with GDP-mannose or with the enzyme. On the lipopeptide and the polyprenol (Fig. 4A). The complex the contrary,a 10-foldlower V,,, suggested that amphomycin formation between the lipopeptide and Dol-P was also demight bind to the endogenous carrier lipid, Dol-P, thereby pendent upon divalent cation in the reaction mixture. This making it unsuitable to function as an acceptor for mannose. was examined by incubating [3H]amphomycin (10,000 cpm) Effect of Amphomycin on Enzymatic Synthesis of Man-P- with Dol-P (50 pg) in the presence of 10 mM each of CaC12, Dol as a Function of Dol-P-In order to answer the question MnC12, MgC12, or CdC12 andthen analyzing thereaction aboutthe involvement of Dol-Pinamphomycin-mediated mixture on the Bio-Gel A-1.5 m column (0.64 X 25 cm) as
Interaction between
2026
Dol-P and Amphomycin
0 0
I
I
I
I
as
I.o
1.5
2.0
0
1 , pM"
[GDP-Man]. JIM
[GDP-Man]
FIG. 1. Effect of increasing amounts of GDP-mannose on the inhibition of Man-P-Dol synthesis by amphomycin. A , mannolipid synthesis was assayed with membranes (900 pg of protein), 50 mM Tris-HC1, pH 7.0, 0.125 M sucrose, 0.5 mM EDTA, 10 mMCaC12, 0.25 mM sodium acetate, pH 7.0, and the indicated amount of GDP-['*C]mannose (634 cpm/pmol) in the presence and absence of 5 pg of amphomycin in a total volume of 0.2 ml. The incubationperiod was 5 min at 37 "C. 0, in the absence and 0, in the presence of 5 pg of amphomycin. B, double-reciprocal plot of the data presentedin A .
6Q-
lI[DOL-P]'~". @"
[DOL-PI. PM
l/IDOL-PJ'.oI $4"
FIG. 2. Effect of exogenously added dolichylmonophosphate on amphomycin inhibition of Man-PDol synthesis. A , mannolipid synthesis was assayed with membranes (2.1 mg of protein), 50 mM Tris-HCI, pH 7.0,0.125 M sucrose, 0.5 mM EDTA, 10 mM CaC12,0.5 mM sodium acetate, pH 7.0,0.04% Triton X-100, the indicated amount of exogenous dolichylmonophosphate, and 1.3 p~ GDP-['4C]mannose (310 cpm/pmol) in the presence and absence of 10 pg of amphomycin in a total volume of 0.2 ml. The incubation period was 5 min at 37 "C. 0 , in the absence, and 0, in the presence of 10 pg of amphomycin. B, double-reciprocal plot of the data presented in A .
described above. The results suggested that the complex formation was -80% in thepresence of Ca2+,4% in thepresence of Cd2+, and none in the presence of Mn2+or M e . Moreover, addition of a10-foldmolarexcess of EDTA over [Ca"] completely abolished the complex formation. Complex FormationbetweenfH1AmphomycinandUndecaprenylmonophosphatebutNotUndecaprenylpyrophosphate or Dolichylpyrophosphate-Amphomycin is known to inhibit the translocation of phospho-N-acetylmuramyl pentapeptide from itsUDP derivative t o undecaprenylmonophosphate, theglycosyl-carrier lipidin Gram-positive bacteria (2, 3). But the details of this inhibition have not yet been worked out. Undecaprenylmonophosphate, an a-unsaturated C,,-prenylphosphate (Undeca-P), is structurallyvery similar to a-saturated Cgo-prenylphosphate (Dol-P). Therefore, one as described for might expect that under identical conditions Dol-P amphomycin shouldalso form a complex with Undeca-
P. In order to test this hypothesis, [3H]amphomycin was reacted with 50 Fgof Undeca-P in the presence of 10 mM CaC12 and 0.2% Triton X-100 and analyzed over the Bio-Gel A-1.5 m column. As shown in Fig. 5A, Undeca-P formed a complex with amphomycin. To determine the absolutespecificity of thisinteraction, ['4C]undecaprenylpyrophosphate (Undeca-PP) and [32P]dolichylpyrophosphate(Dol-PP) were reacted separately with [3H]amphomycin and analyzed over the same Bio-Gel A-1.5 m column. Comigration of [3H]amphomycin was never observed either with [14C]Undeca-PP or with [32P]Dol-PP, thereby providing convincing evidencethat amphomycin didnot formcomplexes withpyrophosphate derivatives of the polyprenols (Fig. 5 , B and C ) . Separately, the questions were asked if amphomycin would form complexes with the glycerophospholipids as well and what happens when a mixture of phospholipids and Dol-P is present in the incubation mixture? To obtain an answer for these
2027
Interaction between Dol-P and Amphomycin
"0
(x006
0.01
0.015
o.0~
l/[DoLP],
0.025
0.03
0.036
pM-1
FIG. 3. Multiple sites involvement in amphomycinmediated inhibition of Man-P-Dol synthesis. The initial velocity data from Fig. 2 are transformedaccording to Equation 1 as described in the text. u = velocity in the presence of dolichylmonophosphate, uo = velocity in the absence of Dol-P. 0, in the absence of amphomycin, and 0, in the presence of amphomycin (10 pg).
questions, first a variety of glycerophospholipids were reacted with [3H]amphomycin as described for Dol-P and thenmonitored by the Bio-Gel A-1.5 m column chromatography. The column profiles for [3H]amphomycin in presence of phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylcholine indicate that all seem to have interacted with amphomycin to varying degrees and eluted out at positions different from the dolichylmonophosphate-amphomycincomplex (data not shown). However, when an equimolar mixture of phosphatidylserine and Dol-P was reacted with [3H]amphomycin, the radioactivity eluted out in thearea where dolichylmonophosphate-amphomycin complex would normally elute and notin the area of phosphatidylserine-amphomycin complex. Thus, the antibiotic has selective affinity for Dol-P.
5
15
25
35
45
FRACTION NUMBER
FIG. 4. Interaction between [3H]amphomycinand isoprenoid lipids. A total of 50 pg each of dolichol, Dol-P alone, and Dol-P plus Do~-[~'P] (7,600 cpm) was suspended by sonication in 20 mM Tris-HC1, pH 7.0, buffer containing 10 mMCaC12 and 0.2% Triton X-100 and mixed with 10,000 cpm of [3H]amphomycin.The mixture was then applied to a Bio-Gel A-1.5 m column (0.64 X 25 cm). The column was washed and equilibrated with the same buffer as mentioned above; 0.5-ml fractions were collected and counted. A , dolichol [3H]amphomycin;B , Dol-P + [3H]amphomycin;C , [32P]Dol-P+ [3H]amphomycin. VO = blue dextran; Vi = [''C]leucine.
+
However, when the same reaction is studied as a function of exogenously added Dol-P andin the presence of fixed amount of GDP-mannose, the kinetics appeared to be more complex. The sigmoidal nature of the Dol-Pconcentration curve in the presence of amphomycin suggested a cooperative interaction DISCUSSION between the antibiotic and the acceptor lipid. To examine it The lipopeptide antibiotic amphomycin inhibits not only further, the dataare analyzed according to the Hill equation the elongation of Man5GlcNAcz-PP-Dol to MangGlcNAc2- as well as Equation 1. Both the criteria supportedfor a strong PP-Dol or to Glc3MangGlcNAc2-PP-Dolbut also the initia- positive cooperative interaction between Dol-P and amphotion of the glycosylation process, Le. GlcNAc-PP-Dol forma- mycin during the enzymatic reaction. tion (10). The present study with Man-P-Dol synthase as a In separate experiments,it hasalso been observed that [3H] model describes the mechanism by which the lipopeptide amphomycin formed a complex with Dol-P but not with Dolinhibited the synthesis of the essential monosaccharide-lipid PP or dolichol. The complex formation between Dol-P and intermediates involved in the oligosaccharide assembly. This amphomycin is a rapid process, and incubation of the reaction study has been conducted in two parts. Thefirst half provides mixture either in ice or at room temperature for 5 min did detailed kinetic measurements on amphomycin inhibition of not improve the efficiency of binding any further. Both hyMan-P-Dol synthase activity in calf brain membranes while drophobic and polar interactions are involved in stabilizing the second half deals with the direct demonstration of com- the complex. In case of amphomycin, the fatty acid moiety is plex formation between the lipopeptide and polyprenylmon- essential for the interaction. Treatment of the antibiotic with ophosphates. Measurements of Man-P-Dol synthase activity mild acid (0.25 N acetic acid at 100 "C for 2 h), a condition as a functionof GDP-mannose concentrationsin thepresence that was shown to liberate the acylated aspartic acid residue and absence of amphomycin indicated a first-order kinetics at the NH2 terminus (l),abolished the complex formation (Fig. 1).Identical K , for GDP-mannose for Man-P-Dol syn- with Dol-P (data not included). Although the antibiotic has thesis both inthe presence and in the absence of amphomycin stringent structural requirements for complex formation it, but low V,,, in the presence of the antibiotic suggested that however, does not discriminate between the a-saturated(dolthere are (i) no changes in the affinity for sugar nucleotide ichol) anda-unsaturated (undecaprenol) polyprenylmonoand (ii) no direct effects of amphomycin on the enzyme. phosphates. Amphomycin, though forms a complex with Un-
2028
Interaction betweenDol-P and Amphomycin phospholipids as possible contaminants in the polyprenylpyrophosphate preparations. Among the various divalent metal ions tested here, Ca2+ has been found to play a centralrole in thecomplex formation between the glycosyl-carrier lipid and the lipopeptide antibiotic. This, in fact, supports our earlier observations (10) that 2-3 pg of amphomycin was only required for 50% inhibition of Man-P-Dol/Glc-P-Dol synthesis in the presence of Ca2+ but much higher concentration of the antibiotic was needed when Ca2+was replaced by either Mn2+,Mp”’, Ni2+,or Cd”. Such a complex formation between amphomycin and Dol-P in the presence of divalent metal ion is analogous to the mechanism of action of bacitracin (20, 21) in preventing the conversion of Undeca-PP to Undeca-P. It would therefore, be of interest to see if the inhibition of bacterial phospho-Nacetylmuramyl peptapeptide translocase by amphomycin is also due to its direct interaction with undecaprenylmonophosphate. Further studies on the interaction of the lipopeptide antibiotic with Dol-P/Undeca-P are needed to understand the stoichiometric relationship between the lipopeptide and polyprenylmonophosphates in the complex and the central role played by Ca2+in the overall process. Acknowledgments-I gratefully acknowledge the discussion with Dr. C. J. Waechter during the initial phase of this work. I also thank Maria Rivera for her help in preparing the manuscript. 5
135 5 25 FRACTION NUMBER
REFERENCES
45
1. Bodanszky, M., Sigler, G. F. & Bodanszky, A. (1973) J. Am. 95,2352-2357 Chem. SOC. FIG. 5. Interaction of [SH]amphomycinwith undecaprenyl2. Tanaka, H., Twai, Y., Oiwa, R., Shinohara, S., Shimizu, S., Oka, monophosphate, [‘4C]undecaprenylpyrophosphate, and [32P] T. & Omura, S. (1977) Biochim. Biophys. Acta 497,633-640 dolichylpyrophosphate. Each of undecaprenylmonophosphate (50 3. Tanaka, H., Oiwa, R. & Matsukara, S. (1979) Biochem. Biophys. pg), 10,000 cpm of [14C]undecaprenylpyrophosphate, and 19 pgof Res. Commun. 86,902-908 dolichylpyrophosphate plus [32P]dolichylpyrophosphate(14,000 cpm) 4. Kang, M. S., Spencer, J. P. & Elbein, A. D. (1978) Biochem. was suspended in 20 mM Tris-HC1, pH 7.0, buffer containing 10 mM Bwphys. Res. Commun. 82, 568-574 CaC12 and 0.2% Triton X-100 and mixed with 10,000 cpm of [3H] 5. Kanp. M. S.. SDencer, J. P. & Elbein, A. D. (1978) J. Biol. Chem. amphomycin. The mixture was then applied to a Bio-Gel A-1.5 m 253,8860-8866 column (0.64 X 25 cm). The column was washed and equilibrated 6. Ericson. M. C.. Gafford. J. T. & Elbein, A. D. (1978) Arch. with the same buffer as mentioned above; 0.5-ml fractions were Biochkm. Bio&ys. 191,698-704 collected and counted. A, Undeca-P [3H]amphomycin; B, [“C] 7. Kang, M. S. & Elbein, A. D. (1979) Arch. Biochem. Biophys. 198, Undeca-PP [3H]amphomycin; C, [32P]Dol-PP [3H]amphomycin. 304-313 VO = blue dextran; Vi = [3H]amphomycin. 8. Banejee, D. K., Kousvelari, E. E. & Baum, B. J. (1985) Biochem. Biophys. Res. Commun. 126,123-129 9. Elbein, A. D. (1987) Methods Enzymol. 138 (Part E), 661-709 deca-P, it however, does not interact with the pyrophosphate 10. Baneriee. D. K.. Scher, M. G. & Waechter, C. J. (1981) Biochemderivative or the corresponding free alcohol (Fig. 5 , A and B ) . istry 20,1561-1568 Since, it is beyond the scope of this paper, the details of the 11. Kornfeld. R. & Kornfeld.. S. (1985) . . Annu. Reu. Biochem. 54,631interaction between the lipopeptide and glycerophospholipids 664 such as phosphatidylcholine, phosphatidylserine, and phos- 12. Kumarasamy, R. & Symons, R. H. (1979) Anal. Biochem. 95, 359-363 phatidic acid have not been studied. However, these interac13. Steiner, S. M. & Lester, R. L. (1972) J. Bacteriol. 109, 81-88 tions are believed to be weak and could even be nonspecific 14. Banerjee, D. K. (1987) J. Biosci. 11,311-319 because, when an equimolar mixture of Dol-P and phospha- 15. Waechter, C. J. & Harford, J. B. (1977) Arch. Biochem. Biophys. 181, 186-198 tidylserine is reacted with [3H]amphomycin,the radioactivity migrated only with Dol-P and not with phosphatidylserine. 16. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 C. J., Kennedy, J. L. & Harford, J. B. (1976) Arch. This could also explain the anomalous behavior of [3H]am- 17. Waechter, Biochem. Biophys. 174, 726-737 phomycin when present in a mixture with either [14C]Undeca- 18. Worcel, A., Goldman, D. S. & Cleland, W. W. (1965) J. Biol. PP or [32P]Dol-PP(Fig. 5 , B and C ) . In neither case did [3H] Chem. 240,3399-4307 amphomycin comigrate with [14C]Undeca-PPor [32P]Dol-PP. 19. Sanwal, B. D., Stachow, C. S. & Cook, R. A. (1965) Biochemistry 4, 410-421 On the contrary, the radioactivity eluted in the areas corre20. Stone, K. J. & Strominger, J. L. (1971) Proc. Natl. Acad. Sci. sponding to phosphatidylserine-amphomycin (Fig. 5 B ) and U. S. A. 68, 3223-3227 phosphosphatidylinositol-amphomycin(Fig. 5 C ) complexes, 21. Storm, D. R. & Strominger, J. L. (1973) J. Biol. Chem. 248, 3940-3945 respectively, suggested the presence of a trace amount of these
+
+
+
