- Email: [email protected]
[2] Utilization of labeled Escherichia coli as phospholipase substrate
24
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
[2] U t i l i z a t i o n o f L a b e l e d Escherichia coli as Phospholipase Substrate
By PETER ELSBACH and JERROLD W~ISS Introduction The inherent difficulties in the kinetic analysis of the hydrolysis by phospholipolytic enzymes of their hydrophobic substrates in an aqueous environment has led to the use of numerous methods of presentation of different phospholipids that vary greatly in their physicochemical properties, depending on fatty acyl and polar head group composition. How well these various assays, in which the phospholipids are presented in different physical forms, for example, as monomers, vesicles, miceUes in the presence or absence of detergents, or as monolayers, reflect the action of phospholipases on their natural substrates in the biological environment is not clear. Escherichia coli marked with 14C-labeled fatty acid have proved to be convenient for the assay of deacylating phospholipases under relatively more physiologic conditions. They have been used in two ways: (1) as intact bacteria and (2) after autoclaving.l-4 As is the case for phospholipids in the membranes of most unperturbed cells, the phospholipids of intact E. coli are refractory to the action of both endogenous and added deacylating phospholipases. Consequently, it has been possible to study the ability of various agents to activate selectively or indiscriminately phospholipolysis by a broad range of secretory and cellular phospholipases.5-7 The availability of E. coli mutants lacking one or both of the major phospholipases A 8-~2 allows exclusion of the bacterial enzymes and assessment of the I p. Elsbach, J. Weiss, R. C. Franson, S. Beckerdite-Quagliata, A. Schneider, and L. Harris, J. Biol. Chem. 254, 11000 (1979). 2 p. Patriarca, S. Beckerdite, P. Pettis, and P. Elsbach, Biochim. Biophys. Acta 280, 45
(1972). 3 R. Franson, P. Patriarca, and P. Elsbach, J. Lipid Res. 100, 380 (1974). 4 p. Elsbach and J. Weiss, in "Bacteria-Host Cell Interaction," (M. A. Horwitz, ed.), p. 47. Alan R. Liss, New York, 1988. 5 j. Weiss, S. Beckerdite-Quagliata, and P. Elsbach, J. Biol. Chem. 254, 11010 (1979). 6 S. Forst, J. Weiss, and P. Elsbach, J. Biol. Chem. 257, 14055 (1982). 7 p. Elsbach and J. Weiss, Biochim. Biophys. Acta 947, 29 (1988). 8 M. Ohki, O. Doi, and S. Nojima, J. Bacteriol. 110, 864 (1972). 9 0 . Doi and S. Nojima, J. Biochem. (Tokyo) 74, 667 (1973). l0 M. Abe, J. Okamoto, O. Doi, and S. Nojima, J. Bacteriol. 119, 543 (1974). II j. Weiss and P. Elsbach, Biochim. Biophys. Acta 466, 23 (1977).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[2]
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
25
role of added phospholipases in the phospholipolysis observed in a given setting. ~3 Alternatively, autoclaving of E. coli tagged with ~4C-labeled fatty acid inactivates all bacterial phospholipases and exposes the phospholipids in the E. coli membranes to all deacylating phospholipases tested so far. The use of autoclaved labeled E. coli as substrate has now been widely adopted. Advantages are the ease of preparation and the high degree of sensitivity of the assay (Fig. 1); moreover, the retention of the typical rod shape and hence of the gross envelope structure of the E. coli after autoclaving suggests that the presentation of the bacterial phospholipids is more similar to that of phospholipids in natural membranes than is the case for phospholipids in most of the other commonly used assays. Other advantages include the following: (1) The labeling of E. coli with ~4C-labeled fatty acid during several generations produces uniform labeling of the E. coli phospholipids so that the distribution of the label fairly reflects the bacterial phospholipid composition) 4,~5 In E. coli as well as most other Enterobacteriaceae, phosphatidylethanolamine is the most abundant phospholipid (-70%); phosphatidylglycerol and cardiolipin (diphosphatidylglycerol) represent, respectively, approximately 20 and 8% of the total bacterial lipid. Only trace amounts of other lipids have been recognized in E. coli. (2) Where examined the rate of hydrolysis of the phosphatidylethanolamine and phosphatidylglycerol is similar. 2,16(3) The rate of hydrolysis of the phospholipids of autoclaved E. coli is the same as ofphospholipids extracted and dispersed in the aqueous assay mixture, 6 suggesting that nonlipid bacterial components do not alter the access to the substrate. (4) The phospholipid and fatty acid composition of lipids extracted from autoclaved and untreated E. coli are the same, indicating that the exposure of the bacteria to the autoclave conditions (120 ° and 2.7 kg/cm 2 for 15 min) does not appreciably change the bacterial phospholipids. This must owe in part to the fact that E. coli do not contain (readily oxidizable) polyunsaturated fatty acids. (5) The incorporation of saturated fatty acids (palmitic acid) nearly exclusively into the sn-I position (>90%) and of unsaturated fatty acid (oleic or cis-vaccenic acid) into the sn-2 12p. de Geus, I. van Die, H. Bergmans, J. Tommassen, and G. H. de Haas, Mol. Gen. Genet. 19tl, 150 (1983). 13p. Elsbach, J. Weiss, G. Wright, and H. Verheij, in "Cell Activation and Signal Initiation: Phospholipase Control of Inositol Phosphate, PAF and Eicosanoid Production," (E. A. Dennis, T. Hunter, and M. Berridge, eds.), p. 323. Alan R. Liss, New York, 1989. 14 N. Wurster, P. Elsbach, J. Rand, and E. J. Simon, Biochim. Biophys. Acta 248, 282 (~1971). 15 p. Patriarca, S. Beckerdite, and P. Eisbach, Biochim. Biophys. Acta 260, 593 (1972). 16R. Franson, S. Beckerdite, P. Wang, M. Waite, and P. Elsbach, Biochim. Biophys. Acta 296, 365 (1973).
26
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
position (>95%) of the E. coli phospholipids 17 provides an easy means of distinguishing positional preference of deacylating activities tested by analysis of labeled products of hydrolysis. (6) Since many deacylating phospholipases are more readily detected when phosphatidylethanolamine is the substrate than with phosphatidylcholine, the autoclaved E. coli assay is useful for initial screening of phospholipase activities. Procedures
Preparation of Escherichia coli with 14C-Fatty Acid-Labeled Phospholipids Fatty acid incorporation by a range of wild-type and mutant E. coli strains is brisk during exponential growth. Its rate parallels the division time and hence depends mainly on the composition of the growth medium. Fresh bacterial cultures grown overnight in triethanolamine buffered (pH 7.7-7.9) minimal salts medium, 18 or nutrient broth, are diluted 1 : 20 in fresh medium and subcultured at 37° for 3 hr (roughly six generations) in the presence of 1 mCi/ml of the appropriate fatty acid, usually either [~4C]oleic acid or [~4C]palmitic acid. The labeled fatty acid in organic solvent is added to the empty culture flask, and the solvent is removed by evaporation under a stream of nitrogen, after which the bacterial suspension is added. We have omitted adding albumin to complex and disperse the fatty acid adherent to the vessel wall, because incorporation by the bacteria is at least as good in the absence of albumin. At the end of incubation the bacteria are sedimented by centrifugation at 3000 g for 12 min at room temperature, resuspended in fresh growth medium, and reincubated for 30 rain at 37° to chase adherent unincorporated radiolabeled fatty acid into ester positions. Finally, the labeled bacteria are washed once with 1% (w/v) bovine serum albumin (commercial Cohn fraction V, e.g., from United States Biochemical Corp., Cleveland, OH) to remove unincorporated radiolabeled precursors. More than 50% of the added precursor is recovered in the E. coli phospholipids. The percentage of total bacterial radioactive lipid in free fatty acid should not exceed 5%. The lower this background radioactivity in the free fatty acid fraction, the greater the sensitivity of the measurement of subsequent hydrolysis. Bacterial concentrations are determined by measuring the OD550, followed by resuspension to the desired concentration 17 j. Weiss, R. Franson, K. Schmeidler, and P. Elsbach, Biochim. Biophys. Acta 436, 154 (1976). 18 N. Wurster, P. Elsbach, E. J. Simon, P. Pettis, and S. Lebow, J. Clin. Inoest. 50, 1091 (1971).
[9.]
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
27
in sterile physiologic (0.9%) saline. The labeled bacteria can be used either as live organisms to examine the effect of agents that may be able to activate bacterial and/or added phospholipases 5'7m or after autoclaving as a substrate for the quantitative assay of phospholipase activity. Measurement of Phospholipid Hydrolysis In Intact Bacteria. Since E. coli retain their viability and metabolic integrity under a broad range of environmental conditions, their use for the study ofphospholipase action on the envelope phospholipids of initially intact E. coli requires no prescription of the incubation conditions nor of the composition of the incubation medium. In our own investigations we have aimed generally at providing a reasonably physiologic setting with respect to electrolytes and pH. Assay of Phospholipase A Activity with Autoclaved Bacteria as Substrate. The reaction mixture (total volume 250/zl) is composed of 2.5 x 108 autoclaved E. coli labeled with 14C-fatty acid [a mix of labeled and unlabeled bacteria to provide ~ 10,000 counts per minute (cpm) of radioactivity] containing approximately 5 nmol of phospholipid, 40 mM Tris-HCl (pH 7.5) or appropriate other buffers for determination ofpH dependence or for assay of enzymes with known different pH optima, and 10 mM CaCh. For termination of the reaction, because in the live as well as in the autoclaved E. coli the substrate is particulate and because product free fatty acids and lysophospholipids are quantitatively trapped in the extracellular medium by added bovine serum albumin (BSA),2 the reaction is effectively arrested by addition of ice-cold 0.5% BSA (w/v) and prompt centrifugation of the reaction mixture in an Eppendorf microfuge at 10,000 g for 2 min at room temperature. The sedimented bacteria contain the undegraded phospholipids, and the albumin-complexed products of hydrolysis are in the supernatant. A measured sample of the supernatant is counted by liquid scintillation for quantitation of hydrolysis. If the identification of hydrolysis products is desired, reaction mixtures are extracted by the Bligh and Dyer procedure,19 and the lipid extracts are analyzed by thin-layer chromatography? Activity is expressed in arbitrary units: one unit equals 1% hydrolysis/hr, representing approximately 10 -6/.~mol/min (IU). Sensitivity of Phospholipase Assays Using Autoclaved Escherichia coli Our use of autoclaved E. coli has been largely restricted to assays of p h o s p h o l i p a s e s A 2 . Phospholipid hydrolysis is approximately linear with
increasing time and protein concentration until 20-25% of the substrate 19 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
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PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
has been hydrolyzed. The activity of a wide range of pure phospholipases A 2 is readily detected at protein concentrations of approximately 10-11 M, at a substrate concentration of about 2 x 10 -5 M (Fig. 1). The sensitivity of the assay is further exemplified by the detection of phospholipase A 2 activity in samples of extracts of rabbit granulocytes, representing 1-2 × 105 cells. 1The granulocyte phospholipase A 2 has been purified to homogeneity. 2° Based on recovery data we can therefore estimate the enzyme content of this number of cells at 100-200 pg (representing -0.001% of total granulocyte protein). This demonstrates that trace amounts of the typical low-abundance cellular phospholipases A 2 c a n be detected readily in small tissue samples. The use of autoclaved phospholipid-labeled E. coli for assay of other phospholipases (C and D) should also be explored.
Other Uses of Escherichia coli Labeled with t4C-Fatty Acid Identification of Structural Determinants of Functional Diversity among Highly Conserved Phospholipases A2. In contrast to the closely similar enzymatic activity of a wide range of phospholipases A 2 toward autoclaved E. coli, these enzymes differ markedly in their ability to act on the phospholipids of intact E. coli mutants, lacking activatable endogenous phospholipases A, treated with membrane-perturbing peptides or proteins 5,13,21(Fig. 1). Such differences are most pronounced when phospholipase A-deficient E. coli are treated with a purified antibacterial protein of polymorphonuclear leukocytes (PMN), the bactericidal/permeabilityincreasing protein (BPI). 7 The combination of structural comparison of enzymes active or inactive against BPI-treated E. coli, chemical modification of phospholipase A 2 ,6 and genetic manipulation of inactive enzymes resulting in conversion to active enzymes 22has allowed initial identification of properties of variable regions ofphospholipases A 2 that are determinants of BPI responsiveness.7 Thus, the availability of phospholipase A-less E. coli mutants and the ability of certain membrane-damaging agents to trigger selectively the action of added phospholipases A 2 provide a convenient means of exploring structural determinants of functional differences among these conserved enzymes. Role of Endogenous Phospholipase(s) in Biological Events. By genetic methods isogenic strains of E. coli have been generated that differ only in 2o G. Wright, C. E. Ooi, J. Weiss, and P. Elsbach, J. Biol. Chem. 26S, 6675 (1990). 21 p. Elsbach, J. Weiss, and S. Forst, in "Lipids and Membranes: Past, Present and Future" (J. A. F. Op den Kamp, B. Roelofsen, and K. W. A. Wirtz, eds), p. 259. Elsevier, Amsterdam, 1986. 22 G. Wright, J. Weiss, C. van den Bergh, H, Verheij, and P. Elsbach, Clin. Res. 37, 444A (1989).
29
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
12]
Autoclaved E. co//
Intact (untreated) E. co//
80
80
60
60
40
40
2o
20
8 "o
a
_. ~==,.~.----e •
0
2 ng PLA-2
3
0
100 ng PLA-2
200
BPI-Treated E. coil
Polymyxin B-Treated E. co// 80
•
i
1
..._._.4111-
8o
60
8 40
,o
20
20
/
J
a ..J ft.
o
,
0
100 ng PLA-2
.
200
100 ng PLA-2
-1
200
FIG. I. Action o f different phospholipases A 2 on variously treated E. co/i. Incubations (see also description in the text) were carried out with 2.5 × 108 autoclaved E. coli or with 107 intact E. co/i 1303 (pldA -), either untreated or treated with polymyxin B (0. I/~g) or BP1 (1-2 /xg), at 37 ° for either 15 rain (autoclaved E. co/i) or 60 min (intact E. coil). [], Pig pancreas; O, agkistrodon halys blomhoffii venom phospholipase A 2 (from Ref. 25); II, agkistrodon piscivorus piscivorus venom phospholipase A2 ; ~ , ascritic fluid phospholipase A2 (from Ref. 26).
their content of the pldA gene that encodes an outer membrane phospholipase A ofEo coli. Of three strains, one lacks the pldA gene and its product, the wild-type strain contains from 200 to 500 phospholipase A molecules/ cell, and the third strain contains a multicopy plasmid with the inserted pldA gene, raising the phospholipase content 20- to 200-fold. 12Comparison of the response of the three strains to agents that trigger bacterial phospholipolysis has shown the participation of the pldA gene product in the antibacterial action of intact PMN, purified BPI, bacteriophage infection, and bacteriocin release.
30
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
Role o f lntramembrane Ca 2+ in Activation o f Ca 2 +-Dependent Phospholipases A. Mobilization of Ca 2+ to (cellular) sites of action by Ca 2+dependent phospholipases is generally considered to be an essential step in activation. Maximal activity of phospholipases Az toward autoclaved E. coli requires at least 1 mM Ca 2+, and our standard assay mixtures contain I0 mM Ca 2+ . The purified pldA gene product, which is the major envelope phospholipase A of E. coli, has the same Ca 2+ dependence in its action toward micellar lipid. In contrast, both the endogenous bacterial and the added deacylases maximally hydrolyze the phospholipids of polymyxin B- or BPI-treated E. coli at ambient Ca z+ concentrations of no higher than 0.03 mM. 23 In fact, hydrolysis is less if 5-10 mM C a 2+ is added, presumably because the added divalent cations reduce binding of polymyxin B and BPI. 4'23 The cationic, membrane-inserting polymyxin B and BPI compete for anionic sites on the outer membrane lipopolysaccharides that are normally occupied by Ca 2+ and Mg 2+ and thereby displace bound Ca 2+ near where the phospholipids are exposed to the phospholipases. 13,23This bacterial membrane Ca 2+ pool can be reversibly decreased or increased by incubating E. coli in Ca z+-depleted or Ca 2+-repleted media (containing Mg2+), generating reversible alterations in the sensitivity of polymyxin B or BPI-treated E. coli to CaZ+-dependent phospholipases A (but without effect on Ca2+-independent phospholipases). 13 Selection of One Substrate versus Another in Phospholipase Assays The ease of preparation of autoclaved E. coli labeled with ~4C-labeled fatty acid to serve as substrate in phospholipase (A2) assays and the high sensitivity of the assay, allowing detection of pmolar concentrations of enzyme, have led to the widespread use of this substrate. It has to be recognized, however, that different phospholipases, including the highly conserved phospholipases A2, have different preferences for the wide range of phospholipids, presented in disparate ways, employed by the many investigators studying phospholipases. Hence, the use of any single substrate is likely to result in the detection of some, but the exclusion or the underestimation of other phospholipolytic enzymes. This is evident, for example, in our experience that genetically altered phospholipases A 2 may have similar specific activity in the autoclaved E. coli assay but may exhibit reduced specific activity when other substrates are used (unpublished observations with H. Verheij and G. de Haas of the University of Utrecht). Thus, conclusions based on results obtained with a given substrate must be tempered by the fact that the choice of any substrate 23 p. Elsbach, J. Weiss, and L. Kao, J. Biol. Chem. 260, 1618 (1985).
[3]
NMR
SPECTROSCOPY FOR P H O S P H O L I P A S E K I N E T I C S
31
imposes certain limitations, and it points to the need to compare the properties of crude as well as pure enzyme preparations vis-a-vis more than one set of substrate conditions. For a detailed analysis of the strengths and limitations of the many assays now in use, readers are referred to the recent comprehensive treatise by Waite: 4 Acknowledgments These investigationsare supported by U.S. PublicHealth Service Grants 5R37DK 05472 and AI 18571.
24 M. Waite, "The Phospholipases." Plenum, New York, 1987. S. Forst, J. Weiss, P. Blackburn, B. Frangione, F. Goni, and P. Elsbach, Biochemistry 25, 4309 (1986). 26 S. Forst, J. Weiss, and P. Elsbach, Biochemistry 25, 8381 (1986).
[3] N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r o s c o p y to F o l l o w Phospholipase Kinetics and Products
By MARY F. ROBERTS Introduction
Nuclear magnetic resonance (NMR) spectroscopy has proved to be an excellent technique for describing phospholipid structures used as substrates for phospholipases. High-resolution 1H, 13C, and 31p NMR studies have been used to characterize lipid structure and dynamics in micelles and small unilamellar vesicles. 1-5 The same aggregates are often used as substrates for phospholipases. 6-9 Multilamellar vesicles, while less frequently used as substrates, have been extensively studied by 2H NMR. i A. G. Lee, N. J. M. Birdsall, Y. K. Levine, and J. C. Metcalfe, Biochim. Biophys. Acta 255, 43 (1972). 2 I. C. P. Smith, Can. J. Biochem. 57, l (1979). 3 A. A. Ribeiro and E. A. Dennis, Biochemistry 14, 3746 (1975). 4 R. A. Burns, Jr., and M. F. Roberts, Biochemistry 19, 3100 (1980). P. L. Yeagle, in "Phosphorus NMR in Biology" (C. T. Butt, ed.), p. 95. CRC Press, Boca Raton, Florida, 1978. 6 E. A. Dennis, Arch. Biochem. Biophys. 158, 485 0973). 7 M. Y. E1-Sayed, C. D. DeBose, L. A. Coury, and M. F. Roberts, Biochim. Biophys. Acta 837, 325 (1985). 8 C. A. Kensil and E. A. Dennis, J. Biol. Chem. 254, 5843 (1979). 9 N. E. Gabriel, N. V. Agman, and M. F. Roberts, Biochemistry 26, 7409 (1987).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
[2] U t i l i z a t i o n o f L a b e l e d Escherichia coli as Phospholipase Substrate
By PETER ELSBACH and JERROLD W~ISS Introduction The inherent difficulties in the kinetic analysis of the hydrolysis by phospholipolytic enzymes of their hydrophobic substrates in an aqueous environment has led to the use of numerous methods of presentation of different phospholipids that vary greatly in their physicochemical properties, depending on fatty acyl and polar head group composition. How well these various assays, in which the phospholipids are presented in different physical forms, for example, as monomers, vesicles, miceUes in the presence or absence of detergents, or as monolayers, reflect the action of phospholipases on their natural substrates in the biological environment is not clear. Escherichia coli marked with 14C-labeled fatty acid have proved to be convenient for the assay of deacylating phospholipases under relatively more physiologic conditions. They have been used in two ways: (1) as intact bacteria and (2) after autoclaving.l-4 As is the case for phospholipids in the membranes of most unperturbed cells, the phospholipids of intact E. coli are refractory to the action of both endogenous and added deacylating phospholipases. Consequently, it has been possible to study the ability of various agents to activate selectively or indiscriminately phospholipolysis by a broad range of secretory and cellular phospholipases.5-7 The availability of E. coli mutants lacking one or both of the major phospholipases A 8-~2 allows exclusion of the bacterial enzymes and assessment of the I p. Elsbach, J. Weiss, R. C. Franson, S. Beckerdite-Quagliata, A. Schneider, and L. Harris, J. Biol. Chem. 254, 11000 (1979). 2 p. Patriarca, S. Beckerdite, P. Pettis, and P. Elsbach, Biochim. Biophys. Acta 280, 45
(1972). 3 R. Franson, P. Patriarca, and P. Elsbach, J. Lipid Res. 100, 380 (1974). 4 p. Elsbach and J. Weiss, in "Bacteria-Host Cell Interaction," (M. A. Horwitz, ed.), p. 47. Alan R. Liss, New York, 1988. 5 j. Weiss, S. Beckerdite-Quagliata, and P. Elsbach, J. Biol. Chem. 254, 11010 (1979). 6 S. Forst, J. Weiss, and P. Elsbach, J. Biol. Chem. 257, 14055 (1982). 7 p. Elsbach and J. Weiss, Biochim. Biophys. Acta 947, 29 (1988). 8 M. Ohki, O. Doi, and S. Nojima, J. Bacteriol. 110, 864 (1972). 9 0 . Doi and S. Nojima, J. Biochem. (Tokyo) 74, 667 (1973). l0 M. Abe, J. Okamoto, O. Doi, and S. Nojima, J. Bacteriol. 119, 543 (1974). II j. Weiss and P. Elsbach, Biochim. Biophys. Acta 466, 23 (1977).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[2]
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
25
role of added phospholipases in the phospholipolysis observed in a given setting. ~3 Alternatively, autoclaving of E. coli tagged with ~4C-labeled fatty acid inactivates all bacterial phospholipases and exposes the phospholipids in the E. coli membranes to all deacylating phospholipases tested so far. The use of autoclaved labeled E. coli as substrate has now been widely adopted. Advantages are the ease of preparation and the high degree of sensitivity of the assay (Fig. 1); moreover, the retention of the typical rod shape and hence of the gross envelope structure of the E. coli after autoclaving suggests that the presentation of the bacterial phospholipids is more similar to that of phospholipids in natural membranes than is the case for phospholipids in most of the other commonly used assays. Other advantages include the following: (1) The labeling of E. coli with ~4C-labeled fatty acid during several generations produces uniform labeling of the E. coli phospholipids so that the distribution of the label fairly reflects the bacterial phospholipid composition) 4,~5 In E. coli as well as most other Enterobacteriaceae, phosphatidylethanolamine is the most abundant phospholipid (-70%); phosphatidylglycerol and cardiolipin (diphosphatidylglycerol) represent, respectively, approximately 20 and 8% of the total bacterial lipid. Only trace amounts of other lipids have been recognized in E. coli. (2) Where examined the rate of hydrolysis of the phosphatidylethanolamine and phosphatidylglycerol is similar. 2,16(3) The rate of hydrolysis of the phospholipids of autoclaved E. coli is the same as ofphospholipids extracted and dispersed in the aqueous assay mixture, 6 suggesting that nonlipid bacterial components do not alter the access to the substrate. (4) The phospholipid and fatty acid composition of lipids extracted from autoclaved and untreated E. coli are the same, indicating that the exposure of the bacteria to the autoclave conditions (120 ° and 2.7 kg/cm 2 for 15 min) does not appreciably change the bacterial phospholipids. This must owe in part to the fact that E. coli do not contain (readily oxidizable) polyunsaturated fatty acids. (5) The incorporation of saturated fatty acids (palmitic acid) nearly exclusively into the sn-I position (>90%) and of unsaturated fatty acid (oleic or cis-vaccenic acid) into the sn-2 12p. de Geus, I. van Die, H. Bergmans, J. Tommassen, and G. H. de Haas, Mol. Gen. Genet. 19tl, 150 (1983). 13p. Elsbach, J. Weiss, G. Wright, and H. Verheij, in "Cell Activation and Signal Initiation: Phospholipase Control of Inositol Phosphate, PAF and Eicosanoid Production," (E. A. Dennis, T. Hunter, and M. Berridge, eds.), p. 323. Alan R. Liss, New York, 1989. 14 N. Wurster, P. Elsbach, J. Rand, and E. J. Simon, Biochim. Biophys. Acta 248, 282 (~1971). 15 p. Patriarca, S. Beckerdite, and P. Eisbach, Biochim. Biophys. Acta 260, 593 (1972). 16R. Franson, S. Beckerdite, P. Wang, M. Waite, and P. Elsbach, Biochim. Biophys. Acta 296, 365 (1973).
26
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
position (>95%) of the E. coli phospholipids 17 provides an easy means of distinguishing positional preference of deacylating activities tested by analysis of labeled products of hydrolysis. (6) Since many deacylating phospholipases are more readily detected when phosphatidylethanolamine is the substrate than with phosphatidylcholine, the autoclaved E. coli assay is useful for initial screening of phospholipase activities. Procedures
Preparation of Escherichia coli with 14C-Fatty Acid-Labeled Phospholipids Fatty acid incorporation by a range of wild-type and mutant E. coli strains is brisk during exponential growth. Its rate parallels the division time and hence depends mainly on the composition of the growth medium. Fresh bacterial cultures grown overnight in triethanolamine buffered (pH 7.7-7.9) minimal salts medium, 18 or nutrient broth, are diluted 1 : 20 in fresh medium and subcultured at 37° for 3 hr (roughly six generations) in the presence of 1 mCi/ml of the appropriate fatty acid, usually either [~4C]oleic acid or [~4C]palmitic acid. The labeled fatty acid in organic solvent is added to the empty culture flask, and the solvent is removed by evaporation under a stream of nitrogen, after which the bacterial suspension is added. We have omitted adding albumin to complex and disperse the fatty acid adherent to the vessel wall, because incorporation by the bacteria is at least as good in the absence of albumin. At the end of incubation the bacteria are sedimented by centrifugation at 3000 g for 12 min at room temperature, resuspended in fresh growth medium, and reincubated for 30 rain at 37° to chase adherent unincorporated radiolabeled fatty acid into ester positions. Finally, the labeled bacteria are washed once with 1% (w/v) bovine serum albumin (commercial Cohn fraction V, e.g., from United States Biochemical Corp., Cleveland, OH) to remove unincorporated radiolabeled precursors. More than 50% of the added precursor is recovered in the E. coli phospholipids. The percentage of total bacterial radioactive lipid in free fatty acid should not exceed 5%. The lower this background radioactivity in the free fatty acid fraction, the greater the sensitivity of the measurement of subsequent hydrolysis. Bacterial concentrations are determined by measuring the OD550, followed by resuspension to the desired concentration 17 j. Weiss, R. Franson, K. Schmeidler, and P. Elsbach, Biochim. Biophys. Acta 436, 154 (1976). 18 N. Wurster, P. Elsbach, E. J. Simon, P. Pettis, and S. Lebow, J. Clin. Inoest. 50, 1091 (1971).
[9.]
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
27
in sterile physiologic (0.9%) saline. The labeled bacteria can be used either as live organisms to examine the effect of agents that may be able to activate bacterial and/or added phospholipases 5'7m or after autoclaving as a substrate for the quantitative assay of phospholipase activity. Measurement of Phospholipid Hydrolysis In Intact Bacteria. Since E. coli retain their viability and metabolic integrity under a broad range of environmental conditions, their use for the study ofphospholipase action on the envelope phospholipids of initially intact E. coli requires no prescription of the incubation conditions nor of the composition of the incubation medium. In our own investigations we have aimed generally at providing a reasonably physiologic setting with respect to electrolytes and pH. Assay of Phospholipase A Activity with Autoclaved Bacteria as Substrate. The reaction mixture (total volume 250/zl) is composed of 2.5 x 108 autoclaved E. coli labeled with 14C-fatty acid [a mix of labeled and unlabeled bacteria to provide ~ 10,000 counts per minute (cpm) of radioactivity] containing approximately 5 nmol of phospholipid, 40 mM Tris-HCl (pH 7.5) or appropriate other buffers for determination ofpH dependence or for assay of enzymes with known different pH optima, and 10 mM CaCh. For termination of the reaction, because in the live as well as in the autoclaved E. coli the substrate is particulate and because product free fatty acids and lysophospholipids are quantitatively trapped in the extracellular medium by added bovine serum albumin (BSA),2 the reaction is effectively arrested by addition of ice-cold 0.5% BSA (w/v) and prompt centrifugation of the reaction mixture in an Eppendorf microfuge at 10,000 g for 2 min at room temperature. The sedimented bacteria contain the undegraded phospholipids, and the albumin-complexed products of hydrolysis are in the supernatant. A measured sample of the supernatant is counted by liquid scintillation for quantitation of hydrolysis. If the identification of hydrolysis products is desired, reaction mixtures are extracted by the Bligh and Dyer procedure,19 and the lipid extracts are analyzed by thin-layer chromatography? Activity is expressed in arbitrary units: one unit equals 1% hydrolysis/hr, representing approximately 10 -6/.~mol/min (IU). Sensitivity of Phospholipase Assays Using Autoclaved Escherichia coli Our use of autoclaved E. coli has been largely restricted to assays of p h o s p h o l i p a s e s A 2 . Phospholipid hydrolysis is approximately linear with
increasing time and protein concentration until 20-25% of the substrate 19 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
28
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
has been hydrolyzed. The activity of a wide range of pure phospholipases A 2 is readily detected at protein concentrations of approximately 10-11 M, at a substrate concentration of about 2 x 10 -5 M (Fig. 1). The sensitivity of the assay is further exemplified by the detection of phospholipase A 2 activity in samples of extracts of rabbit granulocytes, representing 1-2 × 105 cells. 1The granulocyte phospholipase A 2 has been purified to homogeneity. 2° Based on recovery data we can therefore estimate the enzyme content of this number of cells at 100-200 pg (representing -0.001% of total granulocyte protein). This demonstrates that trace amounts of the typical low-abundance cellular phospholipases A 2 c a n be detected readily in small tissue samples. The use of autoclaved phospholipid-labeled E. coli for assay of other phospholipases (C and D) should also be explored.
Other Uses of Escherichia coli Labeled with t4C-Fatty Acid Identification of Structural Determinants of Functional Diversity among Highly Conserved Phospholipases A2. In contrast to the closely similar enzymatic activity of a wide range of phospholipases A 2 toward autoclaved E. coli, these enzymes differ markedly in their ability to act on the phospholipids of intact E. coli mutants, lacking activatable endogenous phospholipases A, treated with membrane-perturbing peptides or proteins 5,13,21(Fig. 1). Such differences are most pronounced when phospholipase A-deficient E. coli are treated with a purified antibacterial protein of polymorphonuclear leukocytes (PMN), the bactericidal/permeabilityincreasing protein (BPI). 7 The combination of structural comparison of enzymes active or inactive against BPI-treated E. coli, chemical modification of phospholipase A 2 ,6 and genetic manipulation of inactive enzymes resulting in conversion to active enzymes 22has allowed initial identification of properties of variable regions ofphospholipases A 2 that are determinants of BPI responsiveness.7 Thus, the availability of phospholipase A-less E. coli mutants and the ability of certain membrane-damaging agents to trigger selectively the action of added phospholipases A 2 provide a convenient means of exploring structural determinants of functional differences among these conserved enzymes. Role of Endogenous Phospholipase(s) in Biological Events. By genetic methods isogenic strains of E. coli have been generated that differ only in 2o G. Wright, C. E. Ooi, J. Weiss, and P. Elsbach, J. Biol. Chem. 26S, 6675 (1990). 21 p. Elsbach, J. Weiss, and S. Forst, in "Lipids and Membranes: Past, Present and Future" (J. A. F. Op den Kamp, B. Roelofsen, and K. W. A. Wirtz, eds), p. 259. Elsevier, Amsterdam, 1986. 22 G. Wright, J. Weiss, C. van den Bergh, H, Verheij, and P. Elsbach, Clin. Res. 37, 444A (1989).
29
LABELED E. coli AS PHOSPHOLIPASE SUBSTRATE
12]
Autoclaved E. co//
Intact (untreated) E. co//
80
80
60
60
40
40
2o
20
8 "o
a
_. ~==,.~.----e •
0
2 ng PLA-2
3
0
100 ng PLA-2
200
BPI-Treated E. coil
Polymyxin B-Treated E. co// 80
•
i
1
..._._.4111-
8o
60
8 40
,o
20
20
/
J
a ..J ft.
o
,
0
100 ng PLA-2
.
200
100 ng PLA-2
-1
200
FIG. I. Action o f different phospholipases A 2 on variously treated E. co/i. Incubations (see also description in the text) were carried out with 2.5 × 108 autoclaved E. coli or with 107 intact E. co/i 1303 (pldA -), either untreated or treated with polymyxin B (0. I/~g) or BP1 (1-2 /xg), at 37 ° for either 15 rain (autoclaved E. co/i) or 60 min (intact E. coil). [], Pig pancreas; O, agkistrodon halys blomhoffii venom phospholipase A 2 (from Ref. 25); II, agkistrodon piscivorus piscivorus venom phospholipase A2 ; ~ , ascritic fluid phospholipase A2 (from Ref. 26).
their content of the pldA gene that encodes an outer membrane phospholipase A ofEo coli. Of three strains, one lacks the pldA gene and its product, the wild-type strain contains from 200 to 500 phospholipase A molecules/ cell, and the third strain contains a multicopy plasmid with the inserted pldA gene, raising the phospholipase content 20- to 200-fold. 12Comparison of the response of the three strains to agents that trigger bacterial phospholipolysis has shown the participation of the pldA gene product in the antibacterial action of intact PMN, purified BPI, bacteriophage infection, and bacteriocin release.
30
PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES
[2]
Role o f lntramembrane Ca 2+ in Activation o f Ca 2 +-Dependent Phospholipases A. Mobilization of Ca 2+ to (cellular) sites of action by Ca 2+dependent phospholipases is generally considered to be an essential step in activation. Maximal activity of phospholipases Az toward autoclaved E. coli requires at least 1 mM Ca 2+, and our standard assay mixtures contain I0 mM Ca 2+ . The purified pldA gene product, which is the major envelope phospholipase A of E. coli, has the same Ca 2+ dependence in its action toward micellar lipid. In contrast, both the endogenous bacterial and the added deacylases maximally hydrolyze the phospholipids of polymyxin B- or BPI-treated E. coli at ambient Ca z+ concentrations of no higher than 0.03 mM. 23 In fact, hydrolysis is less if 5-10 mM C a 2+ is added, presumably because the added divalent cations reduce binding of polymyxin B and BPI. 4'23 The cationic, membrane-inserting polymyxin B and BPI compete for anionic sites on the outer membrane lipopolysaccharides that are normally occupied by Ca 2+ and Mg 2+ and thereby displace bound Ca 2+ near where the phospholipids are exposed to the phospholipases. 13,23This bacterial membrane Ca 2+ pool can be reversibly decreased or increased by incubating E. coli in Ca z+-depleted or Ca 2+-repleted media (containing Mg2+), generating reversible alterations in the sensitivity of polymyxin B or BPI-treated E. coli to CaZ+-dependent phospholipases A (but without effect on Ca2+-independent phospholipases). 13 Selection of One Substrate versus Another in Phospholipase Assays The ease of preparation of autoclaved E. coli labeled with ~4C-labeled fatty acid to serve as substrate in phospholipase (A2) assays and the high sensitivity of the assay, allowing detection of pmolar concentrations of enzyme, have led to the widespread use of this substrate. It has to be recognized, however, that different phospholipases, including the highly conserved phospholipases A2, have different preferences for the wide range of phospholipids, presented in disparate ways, employed by the many investigators studying phospholipases. Hence, the use of any single substrate is likely to result in the detection of some, but the exclusion or the underestimation of other phospholipolytic enzymes. This is evident, for example, in our experience that genetically altered phospholipases A 2 may have similar specific activity in the autoclaved E. coli assay but may exhibit reduced specific activity when other substrates are used (unpublished observations with H. Verheij and G. de Haas of the University of Utrecht). Thus, conclusions based on results obtained with a given substrate must be tempered by the fact that the choice of any substrate 23 p. Elsbach, J. Weiss, and L. Kao, J. Biol. Chem. 260, 1618 (1985).
[3]
NMR
SPECTROSCOPY FOR P H O S P H O L I P A S E K I N E T I C S
31
imposes certain limitations, and it points to the need to compare the properties of crude as well as pure enzyme preparations vis-a-vis more than one set of substrate conditions. For a detailed analysis of the strengths and limitations of the many assays now in use, readers are referred to the recent comprehensive treatise by Waite: 4 Acknowledgments These investigationsare supported by U.S. PublicHealth Service Grants 5R37DK 05472 and AI 18571.
24 M. Waite, "The Phospholipases." Plenum, New York, 1987. S. Forst, J. Weiss, P. Blackburn, B. Frangione, F. Goni, and P. Elsbach, Biochemistry 25, 4309 (1986). 26 S. Forst, J. Weiss, and P. Elsbach, Biochemistry 25, 8381 (1986).
[3] N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r o s c o p y to F o l l o w Phospholipase Kinetics and Products
By MARY F. ROBERTS Introduction
Nuclear magnetic resonance (NMR) spectroscopy has proved to be an excellent technique for describing phospholipid structures used as substrates for phospholipases. High-resolution 1H, 13C, and 31p NMR studies have been used to characterize lipid structure and dynamics in micelles and small unilamellar vesicles. 1-5 The same aggregates are often used as substrates for phospholipases. 6-9 Multilamellar vesicles, while less frequently used as substrates, have been extensively studied by 2H NMR. i A. G. Lee, N. J. M. Birdsall, Y. K. Levine, and J. C. Metcalfe, Biochim. Biophys. Acta 255, 43 (1972). 2 I. C. P. Smith, Can. J. Biochem. 57, l (1979). 3 A. A. Ribeiro and E. A. Dennis, Biochemistry 14, 3746 (1975). 4 R. A. Burns, Jr., and M. F. Roberts, Biochemistry 19, 3100 (1980). P. L. Yeagle, in "Phosphorus NMR in Biology" (C. T. Butt, ed.), p. 95. CRC Press, Boca Raton, Florida, 1978. 6 E. A. Dennis, Arch. Biochem. Biophys. 158, 485 0973). 7 M. Y. E1-Sayed, C. D. DeBose, L. A. Coury, and M. F. Roberts, Biochim. Biophys. Acta 837, 325 (1985). 8 C. A. Kensil and E. A. Dennis, J. Biol. Chem. 254, 5843 (1979). 9 N. E. Gabriel, N. V. Agman, and M. F. Roberts, Biochemistry 26, 7409 (1987).
METHODS IN ENZYMOLOGY, VOL. 197
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