Evidence that the zymogen of phospholipase A2 binds to a negatively charged lipid-water interface

Evidence is presented that the zymogen of porcine pancreatic phospholipase A2 (prophospholipase A2) interacts with a lipid-water interface provided that the interface has a net negative surface charge. Fluorescence spectroscopy and non-equilibrium gel filtration indicate that binding of prophospholipase A2 (proPLA) to mixed detergent micelles is dependent on the presence of an anionic detergent. Prophospholipase binding is accompanied by a change in the environment of the single tryptophan residue qualitatively similar to that observed when the active enzyme, phospholipase A2 (PLA), binds to micelles. In addition, the rate of tryptic activation of prophospholipase is significantly reduced in the presence of negatively-charged mixed micelles, whereas no change in rate occurs when neutral micelles are present. These observations suggest that the lack of catalytic activity of the zymogen toward organized substrates carrying a negative surface charge cannot be explained by a failure to bind at the lipid-water interface.     

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual kinetic behavior of porcine pancreatic (pro)phospholipase A2 on negatively charged substrates at submicellar concentrations

The negatively charged detergents S-n-alka-noylthioglycol sulfates (C8, C9, and C10) are substrates for porcine pancreatic phospholipase A2 and its zymogen. At pH 6.0 and detergent concentrations up to 0.08 X critical micelle concentration (cmc), the activities of active enzyme and zymogen are similar and very low. From 0.08 X cmc to 0.12 X cmc a tremendous increase in activity is observed for phospholipase A2, but not for the zymogen. Concomitant with this increase in activity there is a sharp rise in molecular weight of the substrate-enzyme complex, from 15 000 to 95 000, and in detergent to protein molar ratio of 1:1 to about 7:1. This indicates both substrate and enzyme aggregation. Most probably a lipid-water interface is formed inside the aggregated protein particle by which the enzyme is activated. Although the zymogen also forms high molecular weight complexes with similar molar ratios, no activation is observed probably because of distortion of its lipid binding domain.     

Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier.

In many proteins fluorescence from single tryptophan exhibits a nonexponential decay function. To elucidate the origin of this nonexponential decay, we have examined the fluorescence decay function and time-resolved fluorescence anisotropy of a fluorophore covalently bound to a macromolecule by solving a rotational analogue of the Smoluchowski equation. An angular-dependent quenching constant and potential energy for the fluorophore undergoing internal rotation were introduced into the equation of motion for fluorophore. Results of numerical calculations using the equations thus obtained predict that both the fluorescence decay function and time-resolved anisotropy are dependent on rotational diffusion coefficients of fluorophore and potential energy for the internal rotation. The method was applied to the observed fluorescence decay curve of the single tryptophan in apocytochrome c from horse heart. The calculated decay curves fit the observed ones well.     

Side-chain dynamics of two aromatic amino acids in pancreatic phospholipase A2 as studied by deuterium nuclear magnetic resonance

The flexibility of individual amino acid side chains of pancreatic phospholipase A2 in aqueous and micellar solutions was studied with deuterium nuclear magnetic resonance (2H NMR). Bovine pancreatic phospholipase A2 was selectively deuterated at the aromatic ring systems of Trp-3 and Phe-5 and porcine pancreatic phospholipase A2 at Trp-3 only. Solid-state 2H NMR spectra of the lyophilized enzymes exhibited quadrupole splittings on the order of 130 kHz, indicating almost complete immobilization of the aromatic ring systems. Exposure to a water-saturated atmosphere did not remove these steric constraints. However, side-chain mobility could be induced for the tryptophyl residue of the bovine enzyme by dissolving this enzyme in aqueous buffer or micellar solution whereas the phenyl ring always remained immobile and served as a probe for the protein's overall rotation. Typical correlation times for the tryptophyl and phenyl aromatic ring systems in aqueous solution were 7 ps and 13 ns (at 20 degrees C), respectively. The correlation time of the phenyl ring was longer than expected for the monomeric protein (approximately 6 ns), suggesting some aggregation of the protein at the high concentrations used for the NMR measurements. Addition of a micellar solution of oleoylphosphocholine had no influence on the motional freedom of the tryptophyl residue but approximately doubled the correlation time of the phenyl ring, indicating an increase of the effective volume of the tumbling particle due to lipid-protein interaction. A different behavior was observed for the Trp-3 residue of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic behavior of porcine pancreatic phospholipase A2 on zwitterionic and negatively charged double-chain substrates

A number of isomeric diacylglycerophosphocholines and diacylglycero sulfates containing O-acyl and/or S-acyl ester bonds were investigated as substrates for porcine pancreatic phospholipase A2 and its zymogen. A comparison is made with the kinetic properties of the enzyme toward the corresponding glycol detergents previously described [van Oort, M. G., Dijkman, R., Hille, J. D. R., & de Haas, G. H. (1985) Biochemistry (preceding paper in this issue)]. Hydrolysis of the secondary ester bond in the 1,2-diacylglycero-3-type lipids proceeds much faster than the splitting of the primary ester function present in the isomeric 1,3-diacylglycerol and 1-acylglycol derivatives. In sharp contrast to the glycol detergents, the substitution of the cleavable oxygen ester by a thio ester bond in the glycerol lipids results in 5 times lower kcat values. At alkaline pH and above the critical micelle concentration, the anionic sulfates are much better substrates than the corresponding phosphocholine-containing detergents. At very low detergent concentrations, below the critical micelle concentration, the anionic sulfates induce protein aggregation such that phospholipase A2, as well as its zymogen, is present in high molecular weight complexes containing several protein molecules. In these aggregates, protein-protein and/or lipid-protein interactions strongly activate phospholipase but not the zymogen.     

Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2

A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A2, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission at 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A2, the emission maximum shifted to about 470 nm. PPC showed a critical micellar concentration around 5 microM, with evidence of premicellar aggregation above 1 microM. Binding of PPC to Crotalus adamanteus phospholipase A2 was evidenced by an increase in emission at 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 microM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants for PPC complexes with phospholipases A2 from Naja naja naja and porcine pancreas and the prophospholipase A2 from porcine pancreas were 0.509, 0.107, and 0.114 microM, respectively. PPC was shown to inhibit the activity of pancreatic phospholipase A2 in thio-PC-sodium cholate mixed micelles. Inhibition studies were complicated because PPC can also serve as an activator of the snake venom enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium ion binding to pancreatic phospholipase A2 and its zymogen: a 43Ca NMR study

Calcium ion binding to phospholipase A2 and its zymogen has been studied by 43Ca NMR. The temperature dependence of the band shape of the calcium-43 NMR signal has been used to calculate the calcium ion exchange rate. The on-rate was calculated to be 5 X 10(6) M-1 s-1, which is 2 orders of magnitude less than the diffusion limit of the hydrated Ca2+ ion in water. The 43Ca quadrupole coupling constant for calcium ions bound to phospholipase, chi = 1.4 MHz, is significantly larger than those found for EF-hand proteins, indicating a less symmetric site. For prophospholipase A2, we found chi = 0.8 MHz, indicating a calcium binding site, which is somewhat more symmetric than the EF-hand sites. The dependence of the 43Ca NMR band shape on the calcium ion concentration showed that there are two cation binding sites on the phospholipase A2 molecule: K1 = 4 X 10(3) M-1 and K2 = 20 M-1. The strong site was found to be affected by a pKa = 6.5 and the weak site by pKa = 4.5.     

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.     

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.     

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.     

Saturation transfer electron spin resonance study on the rotational diffusion of calcium- and magnesium-dependent adenosine triphosphatase in sarcoplasmic reticulum membranes.

The technique of saturation transfer electron spin resonance has been applied to study the rotational diffusion of spin-labeled Ca2+, Mg2+-dependent ATPase molecules in the membranes of sarcoplasmic reticulum vesicles. Comparison of the present data with those for spin-labeled hemoglobin undergoing isotropic rotation leads to a value of 2 X 10(-4) s for the apparent rotational correlation time at 20 degrees C for the membrane-bound protein. Consideration of the anisotropy of the Brownian rotation of the membrane-bound ATPase suggests that the true correlation time for the expected axial rotation may be somewhat smaller than the apparent value. An Arrhenius plot of the rotational motion shows a break, which is interpreted as indicating the occurrence of a conformational change of the ATPase molecule at about 15 degrees C.     

Characterization of the overall and internal dynamics of short oligonucleotides by depolarized dynamic light scattering and NMR relaxation measurements

The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The hydrodynamic diameter of the octamer and dodecamer is 20.5 +/- 1.0 A, assuming a rise per base of 3.4 A. The tridecamer, d(CGCGTTGTTCGCG), adopts a hairpin structure with nearly spherical dimensions and a diameter of 23.0 +/- 2.0 A. The DDLS relaxation measurements provide a powerful method for distinguishing between different conformations of the oligonucleotides (e.g., DNA double-helix versus hairpin structure). Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between internal motion and emission kinetics of tryptophan residues in proteins

Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.     

Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae. Use of the prepro sequence of the alpha-mating factor

The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.