111

Bi~chimicu et Bi~$ryrkr Acta, 1046 (1990) 111-119 Elsevier BBALIP 53470

Acyl carrier protein interacts with melittin Mary Lou Ernst-Fonberg, De~~t~nt

Sande G. Williams and Lesa M.S. Worsham

of B~~hern~st~, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN (U.S.A.) (Received 1.5January 1990)

Key words: Acyl carrier protein; Melittin

Acyl carrier protein (ACP) from Escherichiu coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (M, 8843, acidic, Ca*+-binding protein, which possesses some characteristics resembling those of regulatory Ca2+-binding proteins induding interaction with me&tin. Compfexing between melittin and ACP which occurred both in the presence and absence of Ca2’ was evident by chemical cross-linking the two peptides, flmrescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Eugkwu. Also, antidpis meflifem antibodies which contained antibodies against met&tin specifically inhibited the same enzyme system activity relative to non-immune IgG.

Introduction Many proteins in the performance of their metabolic functions interact with other proteins in a manner which can be qualitatively regulated. Cat+-binding proteins exemplify such regulatory strategies. These proteins bind Ca*+ with subsequent conformational modifications which mask or unveil sites that are recognized by the proteins with which they interact. The regulatory protein calmodulin is the paradigm for this phenomenon and is the most intensely studied. The binding site which is revealed subsequent to conformational changes wrought by Ca*+ binding is recognized by a number of calmodulin’s target proteins and also several toxic peptides from Hymenoptera species, including melittin. Melittin, a 26-residue basic peptide from bee venom, binds to many c~cium-bin~ng proteins. The complexes between melittin and some of these proteins have been studied as models of interactions between calcium-binding proteins and their respective physiologic interactive proteins. Examples of the different interactions include those between melittin and calmodulin [l], melittin and myosin light chains [2], melittin and SlOOb protein [3],

Abbr~ations: ACP, acyl carrier protein; EGTA, ethylen~ycol bis(&uninoethyt ether)-N,N’-tetraacetic acid; Pipes, piperazine-N, N ‘-bis(2-ethanesulfonic acid); Tris, tris(hydroxymethyl)aminomethane; Tes, 2([2-hydroxy-l,l-bis(hydroxymethyl)4-ethylJamino) ethane sulfonic acid. Correspondence: M.L. Ernst-Fonberg, Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0002, U.S.A. ~5-27~/~/~3.50

and melittin and troponin C [4]. In support of the biological relevance of this approach, antibodies specific for melittin have been shown to recognize calmodulinbinding domains of calmodulin acceptor proteins [5]. Anti-me&tin antibodies also inhibit complement lysis, a finding which led to the identification of regions of amino acid sequence homology between the bee venom peptide and portions of the human complement system protein, C9 [6]. In these instances, melittin is apparently a structural replica of a physiologically significant topological site involved in protein interaction. Acyl carrier protein (ACP) occupies a pivotal position in fatty acid biosynthesis. All fatty acid synthetases contain ACP, but the molecular organizations of fatty acid synthetases vary depending upon the source. Fatty acid synthetases of plants and primitive bacteria are isolated as nona~egated enzymes and ACP. ~~glen~ gracilis is unique in having two de novo fatty acid synthetases [7]. A true multienzyme complex of great size is in the cytosol[8] while a nonaggregated fatty acid synthetase whose expression is light regulated is located in the chloroplasts [9]. The enzymatic orga~zation of the two fatty acid synthetases appears to reflect different evolutionary paths of the two enzyme systems. The activity of the nonaggregated fatty acid synthetase likely depends upon transient but crucial macromolecular interactions involving at least the constituent enzymes and ACP. ACP by virtue of its physiologic role as a carrier of substrate in fatty acid and lipid biosynthesis interacts with numerous proteins. ACP binding to proteins is not limited to enzymes for which it functions as a substrate carrier; it also inhibits (Ki 5 PM) an NADH-dependent acetoacetyl-CoA reductase from

6 1990 Elsevier Science Publishers B.V. (Biomedical Division)

112 Euglena that is not part of the fatty acid synthetase [lo]. The way that ACP and proteins to which it binds recognize one another is not known. In addition to interaction with a diversity of proteins, ACP binds Ca*+ [ll]. Although a regulatory role has not been shown, ACP Ca’+-binding does result in altered biological activity [12] and in pH-dependent conformational changes (131. ACP and other calcium-binding proteins share some common features, although ACP’s primary structure is not homologous with those of several EF hand calcium-binding proteins [14]. Both ACP and many EF hand calcium-binding proteins, including calmodulin, group in the same region of a hydrophobic moment plot [15]. Other common features include acidic pf, interaction with diverse proteins, the like~h~d of some amp~pat~c helical structure and conformational changes associated with calcium-binding. Whether or not ACP shares the melittin-binding feature with other calcium-binding proteins has not been examined. The interaction of ACP with the small peptide melittin, if it occurred, might help to frame specific questions about the currently unknown mechanisms of physiologically important interactions between ACP and other proteins.

Materials and Methods Materials. Acetyl-CoA and malonyl-CoA were from P-L Biochemicals. [2-‘4C]Malonyl-CoA was from New England Nuclear. Bio-Rad was the source of silver stain kit, M, standards for electrophoresis, Bio-Gel A-15, DEAE Affi-Gel Blue, CM Affi-Gel Blue, zeta probe and goat anti-rabbit IgG conjugated enzymes. Dimethylsube~~date and bicinchoninic acid protein assay reagents were from Pierce and blotting media from Schleicher and Schuell. Gradient polyacrylamide gels (lo-20%) were from PGC Scientifics. Rabbit normal immuno~obulin and anti-Apis mel~i~era venom antibodies were from Sigma and were purified by chromatography on CM Affi-Gel Blue. Melittin was from either Serva or Sigma, a highly purified grade that had no detectable phospholipase A, activity. Escherichia co/i were from Grain Processing Corp. and ACP was purified from the bacteria [I6]. Rabbit antibodies against E. coli ACP were raised as described by Ernst-Fonberg et al. [17] and the IgG fraction of the immune serum was separated by interaction with DEAE Affi-Gel Blue. Euglena gracilis variety bacillaris were grown in light and harvested as described by Boehler and Ernst-Fonberg [18]. Cells were stored at - 80°C for up to 2 weeks prior to use. After thawing, all subsequent manipulations were at 4°C. For the preparation of soluble and insoluble cell fractions, EugIena were suspended in an equal volume 0.01 M Tes (pH 7.4) 0.15 M NaCl, 0.2 M sucrose, 0.002 M dithiothreitol and disrupted with ultrasonic irradiation. The mixture was centrifuged at

100000 X g for 60 min. The supernatant liquid was saved and the pellet was suspended in a volume of buffer roughly equal to the original volume of cells. For isolation of the nonaggregated fatty acid synthetase system, Euglena were processed as described to obtain the 100000 x g supernatant solution which was applied to a calibrated Bio-Gel A-15 column (24 x 2.5 cm) equilibrated and run in the above buffer. Fractions of 2 ml were collected and those containing nonaggregated fatty acid synthetase activity were pooled. Activity of the nonaggregated fatty acid synthetase was distinguished by its requirement for the addition of ACP to the assay [19,9]. The pool was divided into aliquots that were stored at - 80°C. Protein was 3.5 mg/ml. Cross-linking with dimethyisuberimidate. ACP, 18 FM, in 0.01 M Pipes (pH 6.8) 0.15 M NaCl, 0.002 M dithiothreitol was made 1 mM in either CaCl, or EGTA (pH 7.0). The ACP was then exposed to l/10 of its volume of 0.1 M dimethylsuberimidate both alone and with an equimolar amount of melittin. After 15 min at room temperature, l/10 volume of 1 M NH,(CH,COO) was added. The solutions were kept at - 80°C until analyzed. Electrophoreses. For immunoblot analysis, protein was precipitated with cold 10% trichloroacetic acid, washed twice with acetone, then suspended and boiled for 5 min in 0.125 M Tris-HCl (pH 6.8) 5% sodium dodecylsulfate, 0.002 M dithiothreitol, 10% glycerol, and 0.01% bromophenol blue. ACP and Eugiena protein mixtures were electrophoresed on either 12% or lo-20% acrylamide gradient gels using a Hoeffer Mighty Small apparatus [20]. ACP gels were blotted on to 0.1 (LM nitrocellulose for l/2 h or zeta probe for 1 h. The gel of Eugiena proteins was blotted overnight onto 0.2 PM nitrocellulose in a Bio-Rad Minitrans-Blotter following the basic procedures given in the instrument instructions. Samples were always cross-linked to the blotting medium by exposure to 0.025% glutaraldehyde f21]. Immunoblots were analyzed using a Hoeffer GS360 program and GS-300 scanning densitometer. Fluorescence spectroscopy. Fluorescence studies were done at room temperature in a Perkin-Elmer 650-40 with a cell compartment modified by C.N. Wood Manufacturing Company (Newton, PA) to accommodate automated entrance and exit Polaroid filters. Fluorescence emissions spectra were recorded with the excitation wavelength set at 295 nm so that tyrosine fluorescence was largely excluded. Excitation band widths were 8 mm; emission band widths were 2 mm. AzSO values were kept below 0.05 to obviate inner filter corrections. Samples were checked for photodecomposition. Concentrations of melittin were determined based on the extinction coefficient of 5570 M-i f cm-’ at 280 nm [22]. ACP concentrations were determined by bicinchoninic acid protein assay [23] normalized to the concentration of an ACP solution that was determined

113 by amino acid composition [9]. Solutions were in 0.01 M Pipes (pH 7.0), 0.15 M NaCl with either 1 mM CaCl, or 1 mM EGTA. To determine anisotropies, fluorescence intensities were determined at the maximum emission wavelength of each solution. To measure fluorescence quenching, 2-~1 aliquots of freshly prepared 4 M solutions of the respective quenchers (in buffers identical to those containing the proteins) were added to the protein solutions. Fluorescence intensity of the emission maximum wavelength was read 1 min after each addition. Assays. Fatty acid synthetase activity was measured as described by Worsham et al. [9]. Protein, except for ACP, was assayed according to Bradford [24]. RWlltS

Chemical cross-linking of melittin and ACP Interaction between melittin and ACP was examined by a variety of methods. During these experiments, the

ionic strength was maintained at 0.15-0.2 M in order to dampen ionic interactions between the two peptides. At these ionic strengths and neutral pH, melittin exists in solution mainly as monomer with a structure that, although still ‘unordered’, possesses a pronounced rigidity with an approximate compact prolate ellipsoid shape [25,26]. Equimolar mixtures of ACP and melittin and each peptide alone were exposed to dimethylsuberimidate. The resulting structures were analyzed by Western blots using antibodies raised against melittin or ACP. No cross-reaction between either peptide and the antibodies against the other was detected. Lanes 1, 2 and 3 of Fig. 1A show melittin, cross-linked melittin and cross-linked melittin-ACP mixture visualized via anti-melittin antibodies. The existence of oligomeric forms of melittin is well established and melittin with an IV, of 2840 and oligomers are evident at relatively low molecular weights in the Western Blot of both melittin and cross-linked melittin. As with ACP [27], melittin aggregates are not

Fig. 1. Immunoblots of cross-linked and non-cross-linked ACP and melittin. (A) ACP (5 pg), melittin (1.6 pg) and their cross-linked species were electrophoresed on a lo-20% gradient gel, then transferred onto zeta probe. Primary antibodies used were rabbit anti-melittin in lanes l-3 and rabbit anti-ACP in lanes 4-6. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used to detect the immune complexes. Lane 1, melittin; lane 2, cross-linked melittin; lane 3, ACP and melittin cross-linked in the presence of 1 mM EGTA; lane 4, same as lane 3; lane 5, ACP; Lane 6, cross-linked ACP; and lane 7, pre-stained standards: (a) bovine serum albumin (75000), (b) ovalbumin (SOOOO),(c) carbonic anhydrase (39000), (d) soybean trypsin inhibitor (27000). and (e) lysozyme (17000). (B) ACP (1.7 pg). cross-linked ACP, and ACP cross-linked with melittin were electrophoresed on a 12% gel and transferred onto 0.1 gg nitrocelhilose. Rabbit anti-ACP and horseradish peroxidase conjugated goat anti-rabbit IgG were used to detect ACP and ACP-containing species and amid0 black was used to detect standards. Lane 1, ACP exposed to 1 mM EGTA; lane 2, ACP exposed to 1 mM Ca *+. lane 3 ACP and melittin cross-linked in the presence of 1 mM EGTA; lane 4, ACP and melittin cross-linked in the presence of 1 mM CA *+., land 5, AC; cross-linked in 1 mM EGTA; lane 6, ACP cross-linked in 1 mM Ca*+; lane 7, high M, standards: (a) myosin (rabbit skeletal muscle) 200000, (b) &galactosidase (E. coli) 116250, (c) phosphorylase B (rabbit muscle) 92500, (d) bovine serum albumin 66200, and (e) ovalbumin (hen egg white) 45000, lane 8, low M, standards, (f) carbonic anhydrase (bovine) 31000 and (g) lysozyme (hen egg white) 14400.

114 entirely eliminated by exposure to sodium dodecyl sulfate. A shift to aggregated forms of melittin appears to occur upon exposure to dimethylsuberimidate (lane 1 compared to lane 2, Fig. 1A). With the addition of ACP to the cross-linking solution, higher M, melittin-containing bands appeared (arrows, lane 3). Lanes 5 and 6 are ACP and ACP cross-linked alone, while lane 4 is ACP and melittin exposed to dimethylsuberimidate together. Lanes, 4, 5 and 6 bands were visualized by reaction with anti-ACP antibodies. New bands appeared in lane 4 (arrows) compared to lanes 5 and 6; furthermore two of these distinct bands correspond to the novel bands visualized in lane 3 with anti-melittin antibody. A very faint new band in lane 4 is also evident that corresponds to a faint new band evident in lane 3. Fig. lB, an immunoblot using anti-ACP antibodies, also depicts the diverse oligomers of ACP alone and ACP-melittin complexes (lanes 3 and 4). The absence or presence of Ca2+ in the cross-linking medium did not affect the pattern of the oligomers generated from ACP alone or ACP and melittin; lanes 1, 3 and 5 and lanes 2, 4 and 6 were cross-linked without and with Ca2+, respectively. ACP alone, with or without cross-linking, showed multiple discrete bands on immunoblots, which

6

r

'i,

I

320

400 wavelength

320

I

I

400

(nm)

Fig. 2. Fluorescence emission spectra of melittin and melittin-ACP mixtures with and without Ca’+. The two less intense spectra in A and B with the higher wavelength peaks are those of 5 pM solutions of melittin. The two more intense spectra in A and B with the peaks at lower wavelengths are those of where 15 pM ACP was adde to the 5 gM melittin solutions. Experimental conditions are given in the text. Solutions in A contained 1 mM CaCl,; those in B contained 1 mM EGTA.

are not evident with less sensitive analytical techniques. This behavior of ACP has been investigated extensively by us and stems not from impurities but rather from ACP aggregates which are not entirely eliminated by exposure to sodium dodecylsulfate [27]. Again, three unique bands, ACP-melittin complexes, are seen when melittin was present in the cross-linking solution. The ACP-melittin complexes appeared at M, values 23400, 28 800 and 33000. The monomer of ACP appeared at about M, 21000; ACP from E. coli typically behaves on denaturing gels as though it were a peptide of M, about 20000 rather than its true size of 8847 [28,29]. According to the IV, values, the three unique bands probably each contain 1 mole of ACP and 1, 2 or 3 and 4 moles of melittin, respectively. The molecular architectures of the associations are not known. The existence of the tetrameric form of melittin is, however, well established and the existence of an intermediate dimer form has been shown in a study of the kinetics of melittin self-association [26]. The staining intensities of the three bands suggest that the complex of ACP-melittin tetramer is less prevalent than the other two types of complexes between ACP and melittin. The band corresponding to 1 mole each of ACP and melittin is the most intense, especially upon visualization with antimelittin antibodies. These findings are in agreement with the selection of conditions that were unfavorable to the formation of melittin tetramer. Fluorescence studies of melittin ACP complexes ACP contains no tryptophan and melittin contains a single residue at position 19. Excitation at 295 nm practically excludes tyrosine fluorescence from the intrinsic fluorescence spectra of proteins [30]. Changes in the intrinsic fluorescence of melittin can be used to assess alteration in the environment of tryptophan 19 upon complexing with other proteins such as ACP that contain no tryptophan. This experimental approach has been used to demonstrate binding between several tryptophan containing peptides and various proteins which contain no tryptophan [2,31]. The association between the peptides and, for example, calmodulin is accompanied by changes in the tryptophan relative fluorescence intensity and shifts of the emission maximum to shorter wavelengths [l]. The conditions of salt and melittin concentrations that we have used in fluorescence studies were outside the range of melittin self-association [25,26]. So any changes in melittin fluorescence in the presence of ACP stem from heterologous protein interactions rather than self-interaction. Addition of ACP to melittin solutions caused a blue-shift in the fluorescence of melittin tryptophan 19 and an increase in the relative fluorescence intensity. This occurred in both Ca2+ and EGTA-containing buffers although the effect was slightly more accentuated when Ca2+ was not present. With 1 mM Ca2+, the

115

maximum A shifted from 352 to 342 upon addition of 15 PM ACP to 5 PM melittin (Fig. 2A). The same addition of ACP in the absence of Ca*+ shifted the X maximum from 351 to 337 (Fig. 2B). The change in the h max and quantum yield of fluorescence upon the addition of ACP indicated association between the peptides. Complex formation may be detected by an independent method, measurement of fluorescence anisotropy. This technique is based on M, changes and is responsive to molecular associations even when the fluorescence spectrum and relative fluorescence intensity are invariant. The measured anisotropy, r is related to the fluorescence lifetime 7 and to +, the rotational correlation time of the fluorophore, by the following equation in which r, is a property of the fluorophore

The value of + of the molecule to which the fluorophore is attached is directly related to the volume of the molecule (or the complex in which it is contained),V ]301.

Changes in the anisotropy of melittin upon mixing with other peptides stem mainly from altered rotational correlational times due to changes in the macromolecular size subsequent to complex formation. The addition of increasing amounts of ACP to a 5 PM solution of melittin caused an increase in the anisotropy of melittin tryptophan 19. Fig. 3 shows the increasing anisotropy as the proportion of ACP to melittin increased in the absence of Ca*+; similar results were obtained when 1 mM Ca*+ was present. The ACP complexed with melittin caused a change in the anisotropy similar in magnitude to the change in anisotropy seen when melittin or other small tryptophan containing peptides combine with calmodulin or troponin C [31]. Information about the topology of the tryptophan in the complex can be obtained by examining its interaction with quenchers, low molecular weight agents that decrease fluorescence intensity of tryptophan residues by physical contact with the excited indole ring. The ease with which a fluorophore is quenched depends upon its accessibility to the quencher. Ionic quenchers such as I- being charged and hydrated are generally regarded as quenchers of surface tryptophan residues. Acrylamide is a polar, uncharged molecule that quenches indole derivatives predominately by a collisional process and is sensitive to the exposure of tryptophan in proteins [32]. The quenching of melittin’s intrinsic fluorescence with and without ACP in both the presence and absence

’

6

0

4

8

12

16

ACP Fig. 3. Anisotropy of melittin with increasing ACP. ACP concentrations are PM. Relative fluorescence intensities were measured at the maximum emission wavelength of each of the following mixtures of 5 pM melittin and ACP: 0 ACP, 351 nm; 2.5 pM ACP, 346 nm; 5 pM ACP, 341 nm; 10 uM ACP, 338 nm; and 15 pM ACP, 337 nm. Measurements with four different arrangements of emission and excitation light polarizers were made at each wavelength. The respective anisotropies were calculated from each set of four relative fluorescence intensities. The buffer contained 1 mM EGTA; further description of solvent and experimental conditions is in the text.

of 1 mM Ca*+ was measured. The collisional quenching constants were calculated using the Stem-Volmer equation [32] and are listed in Table I. Acrylamide is the better and more discriminating quencher of the two. Quenching by I- was the same in the presence and absence of Ca*+ and the presence of ACP slightly decreased the accessibility of melittin tryptophan 19 to I-. Acrylamide was better able to interact with the tryptophan indole ring of free melittin than I-. The quenching constants were different depending on the presence or absence of 1 mM Ca*+ in the buffer. When Ca*+ was present, the accessibility of acrylamide to melittin’s tryptophan 19 was maximum. Absence of Ca*+ led to a decline in the quenching of melittin suggesting a Ca*+ -induced change of melittin’s conformation with a coincident change in the tryptophan environment. The acrylamide quenching constants for free melittin are close to the values of 21 and 17.7 reported by others for the dilute peptide in different buffers near neutrality [4,33]. Upon complexing with ACP, the melittin tryptophan was comparatively shielded from the acrylamide, where without Ca*+ the K,, changed from 15.8 to 9.6. A similar sized change was seen in the quenching of melittin compared to melittin-ACP complex in the presence of 1 mM Ca*+; constants of 19.0 and 14.0, respectively, were measured. The ACP-bound melittin also responded to the presence of Ca*+ like the free melittin with a Ca*+-induced increase in KS,, indicating that the

116 conformational was preserved

difference in melittin induced by Ca2+ when the melittin complexed to ACP.

Effects of melittin and anti-venom on the biological activity of ACP Melittin has been shown to be a valid model for protein-protein interactions in some systems, but how relevant is its complexing with ACP to protein-protein interactions in a nonaggregated fatty acid synthetase? The interaction of ACP with the proteins of the nonaggregated fatty acid synthetase from Euglena chloroplasts and other proteins is a subject of investigation in our laboratory [9,10,19,34]. In order to see if melittin bore a structural relationship to a portion of any Euglena protein, an homogenate of greened Euglena was divided into membranous and soluble portions. The proteins were separated by acrylamide gel electrophoresis under denaturing conditions, then immunoblotted and probed with antLApis mellijera venom antibodies. Euglena possessed several proteins that were recognized by the anti-venom antibodies (Fig. 4). Four peptides from the membrane fraction, approximate M, values 60 300, 36 900, 27 800 and 17400, cross-reacted. The largest of these was identical in size to a faintly visualized cross-reacting band in the soluble proteins; also, a soluble protein of M, 16400 was evident after reaction with anti-venom antibodies. Since immunologic crossreactivity has already been shown between anti-melittin antibodies and calmodulin target proteins in vertebrates [5], perhaps it is not surprising that Euglena would also contain a few cross-reacting proteins. Calmodulin, an evolutionarily highly conserved protein, has been isolated from Euglena [35]. If the interaction between melittin and ACP has any validity as a representation of physiologic ACP-protein interactions, an effect of melittin on such physiologic interactions between ACP and other proteins may be expected. The activity of the nonaggregated fatty acid synthetase, which is dependent upon the addition of extraneous ACP as a substrate, was measured with

TABLE

I

Effects of quenching agents on the fluorescence ACP complexes

of meliiiin and melittin-

were conducted as Stem-Volmer constants are M- ‘. Experiments described in the text. The melittin concentrations were 5 PM and the ACP concentrations were 15 FM. Constants were determined at quencher concentrations ranging from 0 to 100 mM using linear regression analyses. Correlation coefficients were greater than 0.99. Quencher

KS, melittin-ACP

melittin

IAcrylamide

complex

1 mM Ca*+

0 Ca*+

1 mM Ca*+

0 Ca2+

13.0 19.0

13.0 15.8

10.0 14.0

10.3 9.6

Fig. 4. Immunoblot of Euglena membrane and soluble proteins. Proteins (90 pg) were electrophoresed on a 10-209~ gradient gel and transferred onto 0.2 pm nitrocellulose. Anti-venon and horseradish peroxidase conjugated goat anti-rabbit was used to probe for antimelittin antibody-reactive proteins. Lane 1, mehttin; lane 2, membrane fraction; lane 3, soluble fraction; and lane 6. pre-stained standards.

increasing portions of melittin. For controls, assays were done in which lysozyme, another cationic peptide, was present instead of melittin. Assays were done with 1 mM EGTA and 0.15 M NaCl present. The presence of melittin inhibited fatty acid biosynthesis, Fig. 5, whereas comparable amounts of lysozyme, a different basic peptide, had no effect on fatty acid biosynthesis. Identical results were obtained when cytochrome c, another basic peptide, was similarly investigated (data not shown). The inhibition by melittin of the fatty acid synthetase assay could stem from its binding to ACP, thus removing an essential substrate. The degree of inhibition of the assay would depend upon the comparative avidities with which ACP bound to melittin or fatty acid synthetase enzymes. This, in turn, would depend upon their respective binding constants and concentrations. If the melittin-ACP interactions occurred because melittin structure resembled a portion of the ACP binding site of one of the fatty acid synthetase enzymes, the enzyme binding site may be recognized by anti-melittin antibodies. Melittin is the major component of honey bee venom, about 50% by dry weight [36]. Increasing

117

10

0

peptide

20

30

40

50

concentration

Fig. 5. Inhibition of fatty acid synthetase by melittin. The nonaggregated fatty acid synthetase was isolated from Euglena gracilis as described and assayed with E. coli ACP as a substrate according to Worsham et al [9] with the following exceptions: 1. Either melittin or lysozyme was added to the assays in the concentrations shown. 2. All assays were adjusted to an ionic strength of 0.15 M with NaCl in order to prevent non-specific interactions between ACP and basic peptides. The specific radioactivity of the [2-‘4C]malonyl-CoA was 1 Ci/mol. Enzyme activity is dpm of [2-‘4C]malonyl-CoA incorporated into long-chain fatty acids. Peptide concentration is pM. Lysozymecontaining assays are shown by circles and melittin-containing assays are shown by triangles. Least-squares lines were drawn to illustrate the data trends.

amounts of antLApis mellifera venom antibody were added to a series of fatty synthetase assays. Dithiothreitol was omitted from the assays to avoid structural disruption of the immunoglobulin molecules. For this reason, the activities of the control enzyme always are about one-half of those shown in Fig. 5. Relative to

>I .Z

2000

‘E

1500.

>

9-4-s-_________~_______~______~ *

: E ;

0

8 500 'oool 0’

Discussion

The demonstration of chemical cross-linking between ACP and melittin attested to the specificity of their interaction. Proteins do not cross-link indiscriminately under the conditions employed [37]. Melittin’s interactions with diverse proteins appears to have a degree of specificity; parvalbumin and serum albumin, even in high concentrations, do not associate with melittin [2]. Conversely, other basic peptides, (e.g., lysozyme, cytochrome c, salmine) do not behave like the venom peptides with Ca2+-binding proteins [38], including ACP as reported herein. The interaction between ACP and melittin detected by fluorescence techniques was slightly more pronounced in the absence of Ca2+ compared to when Ca2+ was present. The activity of the nonaggregated fatty acid synthetase was also more efficient in the absence of Ca2+. The effects of Ca2+ on the accessibility of melittin-binding sites differs among different Ca2+ binding proteins. Ca2+ stabilizes the melittincalmodulin complex [l] while the melittin-myosin light chain interaction is enhanced by the absence of Ca2+ PI.

\

0

equal amounts of nonspecific assays containing immunoglobulin, increasing the anti-venom antibodies increasingly inhibited the Euglena nonaggregated fatty acid synthetase (Fig. 6). Although melittin is not the principle allergen of bee venom even though it is the major component, the inhibitory effect of the anti-venom on fatty acid synthetase activity appeared to be due to anti-melittin antibodies. When, at constant concentrations of anti-venom in parallel assays, the concentration of melittin was increased, there was a protective effect on fatty acid synthetase activity. The higher melittin concentration was neutralized by the antibody and the reaction was not inhibited by melittin as much as a parallel reaction containing nonimmune IgG was inhibited by the same amount of melittin.

I

500

1000

micrograms

1500

2000

IgG

Fig. 6. Inhibition of fatty acid synthetase by anti-A@ me/lifers antibodies. The nonaggregated fatty acid synthetase was isolated from Euglena gracilis as descrbied and assayed with E. coli ACP as a substrate according to Worsham et al [9] with the following exceptions: 1. Dithiothreitol was omitted from the assays. 2. After the addition of all components to the assays except malonyl-CoA, the assays were incubated for 15 min at 4°C before reaction initiation with malonyl-CoA and incubation at 35 ‘C. The specific activity of the malonyl-CoA was 1 Ci/mol. Enzyme activity is dpm of [2-i4C]malonyl-CoA incorporated into long-chain fatty acids. Assays that contained none or normal immunoglobulin are designated by circles. Assays that contained anti-Apis mellifera immunoglobulin are designated by triangles. Least-squares lines were drawn to illustrate data trends.

The blue shift in fluorescence observed when ACP was added to melittin solutions stems from a change in the melittin tryptophan residue’s environment upon interaction of the two proteins. The amount of blue shift in the h max observed upon association of ACP and melittin is like that seen with melittin upon self association, upon association with calmodulin, myosin light chains, SlOOb and troponin C and upon interactions between still other peptides and calcium binding proteins [1,4,22,31]. For example, the melittin fluorescence maximum of 350 nm reported by Maulet and Cox [l] was shifted to 340 nm for the complex of melittin with calmodulin both with and without Ca2+. If upon binding to melittin, ACP shielded the tryptophan residue of melittin from the aqueous environment, the residue would then be in a region which could mimic the apolar

118 interior region of a protein. This change in environment would create a blue shift. Quay and Condie [22] indicated that melittin tryptophan with an emission X max near 352 nm is in a fully solvent exposed environment, class I, whereas an emission maximum near 341 nm defines a ‘surface’ tryptophan (class II). In contrast (class III) ‘buried’ tryptophans exhibit maxima at 331 nm which is near the emission maximum of 335 nm reported for melittin bound to egg phosphatidylcholine vesicles [39]. The change in the melittin tryptophan’s fluorescence upon complexing with ACP was consistent with the creation of a more hydrophobic environment for the residue. Another possible cause of tryptophan blue fluorescence is, however, unrelated to the polarity of the environment and is seen when the residue is sandwiched in a rigid position in a protein [32]. The increased anisotropy observed upon association of melittin and ACP does not distinguish between the two possible explanations for the blue shift of the tryptophan residue. The changes in the acrylamide quenching constant, however, indicated a decrease in the accessibility of the tryptophan residues upon complexing of the two proteins. This finding indicated that the other fluorescence changes seen when melittin and ACP interacted stemmed from the environment of the tryptophan residue becoming more apolar. Melittin complexed with troponin C has an acrylamide quenching constant of 6.5 [4] and melittin bound to egg phosphatidylcholine vesicles and cardiolipin has quenching constants of 3.8 and 2.6, respectively [39]. The change in the quenching constants of melittin upon complexing with ACP (e.g., 9.6 in the absence of Ca*+) suggests that ACP shielding of melittin tryptophan 19 from acrylamide was comparable to similar shielding effected by troponin C. ACP and melittin clearly interact. The ACP-melittin interaction has some characteristics resembling those described for melittin interaction with EF hand calcium-binding proteins, complexes which have been productively studied as models of biologically significant bindings between Ca2+ effector proteins and their target proteins. Extending the resemblance, antibodies raised against melittin identified regions of similarity among calmodulin acceptor proteins, a common calmodulin-binding domain [5], and also a conformational epitope on human C9 [6]. Anti-Apis mellifera antibodies extensively inhibited the activity of the nonaggregated fatty acid synthetase, the component enzymes of which interact with the substrate-carrying protein ACP. Were the anti-venom antibodies recognizing a specific ACP-binding site or sites on one or more of the fatty acid synthetase proteins? If they were, then it would be likely that the melittin-ACP interaction had features in common with physiologically significant complexing between ACP and its target proteins. The sizes of the soluble proteins detected with anti-venom

antibody were identical in size to two of the protein bands visualized using anti-fatty acid synthetase antibodies (Worsham, L., unpublished data). Similarity of size does not mean that they are identical proteins, but it does mean that identicalness is plausible. The binding between melittin and ACP, is more specific than simple interaction of ACP with a linear hydrophobic surface, since ACP which lacks an acyl group does not bind to octyl-Sepharose. Nor do peptides charged similarly to melittin complex with ACP under the conditions where melittin and ACP readily interact. It is a recognized motif in biology that structurally similar protein domains may be used in diverse proteins for related or even unrelated functions. One example is the variety of proteins that are expressed as eye lens crystallins; these include heat shock proteins, alcohol dehydrogenase, hydroxyacyl-CoA dehydrogenase. lactate dehydrogenase, glutathione S-transferase and others [40]. Possibly melittin structure resembles the topology of a common binding domain present in a diversity of proteins in which binding to other proteins is a requirement for metabolic function. Perhaps proteins that are interactive with ACP in their physiologic roles possess such a domain. Both the melittin-ACP interaction and immunologic responsiveness of the nonaggregated fatty acid synthetase to anti-melittin antibodies support the merit of this hypothesis. This hypothesis may be useful in designing experiments to explore the binding between ACP and the enzymes of a nonaggregated fatty acid synthetase and to delineate ACP-binding sites on the enzymes. Acknowledgements We thank Ms. Raymonde Cox for assistance in preparing the manuscript and Mr. Gerry Philpott for his help in preparing the photographs. Support was from National Science Foundation Grants DMB-8519016 and DC8-8818931. References 1 Maulet, Y. and Cox, J.A. (1983) Biochemistry 22, 5680-5686. 2 Malencik, D.A. and Anderson, R. (1988) Biochemistry. 27, 19411949. 3 Baudier, J., Mochly-Rosen, D., Newton, A., Lee, S., Koshland. D.E. and Cole, R.D. (1987) Biochemistry 26, 2886-2893. Steiner, R.F. and Norris, L. (1987) Arch. B&hem. Biophys. 254, 342-352. Kaetzel, M.A. and Dedman, J.R. (1987) J. Biol. Chem. 262, 3726-3729. Laine, R.O., Morgan, B.P. and Esser, A.F. (1988) Biochemistry 27, 5308-5314. Delo, J., Ernst-Fonberg, M.L. and Bloch, K. (1971) Arch. Biothem. Biophys. 143, 384-391. Worsham, L. MS., Jonak, Z.L.P. and Ernst-Fonberg, M.L. (1986) B&him. Biophys. Acta 876, 48-57. Worsham, L.M.S., Tucker, M.M. and Ernst-Fonberg, M.L. (1988) B&him. Biophys. Acta 963, 423-428.

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