Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids.

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.

Consequences of the interaction of calcium with dioleoylphosphatidate-containing model membranes: changes in membrane permeability.

The permeability behaviour of dioleoylphosphatidate/dioleoylphosphatidylcholine (20:80, mol%) large unilamellar vesicles at low millimolar calcium concentrations is different for various solutes. Between 0.5 mM and 2.5 mM of calcium a selective influx of calcium and efflux of enclosed calcium chelating anions is observed. At higher calcium concentrations the membrane loses its barrier function for a large variety of solutes. These permeability increases are a specific consequence of calcium phosphatidate interactions, because control experiments in which calcium was replaced by magnesium or in which dioleoylphosphatidate was replaced by dioleoylphosphatidylglycerol showed under the same conditions no permeability changes. These results are discussed on the basis of various putative mechanistic models for phosphatidate-mediated calcium translocation across membranes. Furthermore a kinetical model is presented by which the observed selective calcium and calcium-chelator translocation can be explained.

Consequences of the interaction of calcium with dioleoylphosphatidate-containing model membranes: calcium-membrane and membrane-membrane interactions.

Calcium binds to dioleoylphosphatidate/dioleoylphosphatidylcholine (DOPA/DOPC) (20:80, mol%) multilamellar vesicles in the presence of a calcium ionophore with stoichiometry of about 0.6 nmol calcium per nmol phosphatidate and an apparent dissociation constant of about 1.7 mM. Experiments on the behaviour of monomolecular films at an air/water interface show that calcium-phosphatidate binding results in a decrease in the area of the polar region of the phosphatidate molecule, probably caused by headgroup dehydration and partial charge neutralization. At calcium concentration higher than about 3 mM calcium neutralizes the negatively charged membrane surface of DOPA/DOPC (20:80, mol%) large unilamellar vesicles, and vesicle aggregation is observed. At 10 mM of calcium this results in a low level of vesicle fusion. These observed processes are not attended with calcium-induced phosphatidylcholine transbilayer movement in the membranes of DOPA/DOPC (20:80, mol%) large unilamellar vesicles. When these findings are compared with the results of a previous study on the permeability behaviour of large unilamellar vesicles of the same phospholipid composition under comparable conditions (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108) the following conclusions can be drawn. At low millimolar calcium concentrations (less than 2.5 mM) calcium does not occupy all the binding sites of the membrane, no membrane-membrane interactions are observed and a selective translocation of calcium and calcium-chelating anions is appearing. The mechanism of this translocation may be explained by the formation of uncharged dehydrated complexes of calcium, phosphatidate and calcium chelator, which can pass the membrane via transient occurring non-bilayer structures. Between 3 and 10 mM of calcium an a selective permeability increase of the vesicular membrane is found, which is not a consequence of vesicle fusion but apparently of vesicle aggregation, possibly causing packing defects in the membrane.

2H-NMR, 31P-NMR and DSC characterization of a novel lipid organization in calcium-dioleoylphosphatidate membranes. Implications for the mechanism of the phosphatidate calcium transmembrane shuttle.

2H-NMR, 31P-NMR and DSC investigations are presented on the structure and dynamics of the Ca2+-dioleoylphosphatidate complex which is formed upon addition of calcium to dispersions of pure dioleoylphosphatidate or of dioleoylphosphatidate in mixtures with dioleoylphosphatidylcholine (DOPC). It is concluded that the phosphate region in the polar headgroup of dioleoylphosphatidate is immobilized, while the oleate chains remain liquid and have increased disorder. In mixtures of dioleoylphosphatidate and DOPC in the presence of calcium a dioleoylphosphatidate-rich phase is segregated, in which the molecular behaviour of phosphatidate is rather similar to that of the pure Ca2+-dioleoylphosphatidate complex. A hypothetical model is proposed for the structure of this complex and this is correlated with the dioleoylphosphatidate-mediated transmembrane transport of calcium (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108). Data indicate that this transmembrane shuttle is an inverted organization of phosphatidate molecules enclosing calcium ions in an anhydrous core.

Calcium-induced changes in permeability of dioleoylphosphatidylcholine model membranes containing bovine heart cardiolipin.

At calcium concentrations up to about 4 mM a selective permeability increase of cardiolipin/dioleoylphosphatidylcholine (50:50, mol%) membranes for calcium and its chelator arsenazo III is observed. Under these conditions calcium does not occupy all the binding sites of cardiolipin at the membrane interface and no vesicle-vesicle interactions are found. Lowering of the cardiolipin content of the vesicles to 20 mol% extends the calcium concentration range in which a selective permeability for calcium and arsenazo III is appearing up to about 12 mM. We suggest that the observed selective permeability increase is caused by transient formation of inverted micellar structures in the membrane with cardiolipin as translocating membrane component for calcium and arsenazo III. At calcium concentrations of 4 mM and higher for 50 mol% cardiolipin-containing vesicles a general permeability increase is found together with calcium-cardiolipin binding in a 1:1 stoichiometry, vesicles aggregation and, above 8 mM of calcium, vesicle fusion. The loss of barrier function of the membrane under these conditions is correlated with vesicle aggregation and may be explained by a transition from a bilayer into a hexagonal HII organization of the phospholipids.

Involvement of microbodies in penicillin biosynthesis.

Penicillium chrysogenum strains were constructed which express a mutant acyltransferase lacking the putative targeting signal for microbody proteins. The mutated enzyme was located in vacuoles and in neighbouring cytoplasm. Although acyltransferase was expressed in vivo and was active in vitro, the mutants did not produce penicillin. The results demonstrate the involvement of microbodies in penicillin production.

Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-acid-acyltransferase gene of Penicillium chrysogenum.

A gene, aat, encoding acyl-CoA: 6-aminopenicillanic acid acyltransferase (AAT), the last enzyme of the penicillin (Pn) biosynthetic pathway, has been cloned from the genome of Penicillium chrysogenum AS-P-78. The gene contains three introns in the 5'-region and encodes a protein of 357 amino acids with an Mr of 39,943. It complements mutants of P. chrysogenum deficient in AAT activity. The aat gene is expressed as a 1.15-kb transcript and the encoded protein appears to be processed post-translationally into two nonidentical polypeptides of 102 and 255 aa, with Mrs of 11,498 and 28,461, respectively. Three proteins of 40, 11, and 29 kDa (the last one corresponding to the previously purified AAT), were identified in extracts of P. chrysogenum. The aa sequence of the N-terminal end of the 11-kDa polypeptide matched the nucleotide (nt) sequence of the 5'-region of aat. The N-terminal end of the 29-kDa polypeptide corresponded to the sequence beginning at nt position 916 of the sequenced DNA fragment (nt 441 of aat gene). The aat gene of P. chrysogenum resembles the genes encoding Pn acylases of Escherichia coli, Proteus rettgeri and Pseudomonas sp., all of which encode two nonidentical subunits derived from a common precursor, encoded by a single open reading frame.

Ethylene glycol causes acyl chain disordering in liquid-crystalline, unsaturated phospholipid model membranes, as measured by 2H NMR.

2H NMR has been used to probe the effects of ethylene glycol at the level of the acyl chains in liposomes prepared from dioleoylphosphatidic acid or dioleoylphosphatidylcholine, labeled with 2H at the 11-position of both oleic acid chains. Increasing concentrations of ethylene glycol lead to a proportional and substantial decrease in the quadrupolar splittings, measured from the 2H NMR spectra of both liposomal systems, indicative of acyl chain disordering.

Isolation and purification of cardiolipin from beef heart.

A simple and efficient procedure is described for the preparation of cardiolipin sodium salt from beef heart. A crude phospholipid fraction is isolated by chloroform-methanol extraction of the homogenized tissue, followed by acetone precipitation and reprecipitation in 4% aqueous CaCl2-methanol. Cardiolipin is separated from the calcium salts of the acidic phospholipids by partition column chromatography on silica gel (Polygosil 60-63100) using 2-propanol-cyclohexane-water 50:43:7 (v/v/v) as eluent. Further purification of the cardiolipin is achieved by high performance liquid chromatography of the calcium salt on silica gel (Lichrosorb Si 60-5) with a neutral eluent (2-propanol-cyclohexane-water 45:50:5 (v/v/v], followed by quantitative conversion to the sodium salt. The yield of this procedure is 1.5-2.1 g of pure 99% sodium salt of cardiolipin per kg of moist ventricular tissue.

A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno-electron microscopical studies.

A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.