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.

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the presence of different divalent cations and the kinetics of CL vesicle fusion induced by these cations have been investigated. (31)P-NMR measurements of equilibrium cation-CL complexes showed the lamellar-to-hexagonal (L(alpha)-H(II)) transition temperature (T(H)) to be 20-25 degrees C for the Sr(2+) and Ba(2+) complexes, whereas in the presence of Ca(2+) or Mg(2+) the T(H) was below 0 degrees C. In the presence of Sr(2+) or Ba(2+), CL large unilamellar vesicles (LUVs) (0.1 microm diameter) showed kinetics of destabilization, as assessed by determination of the release of an aqueous fluorescent dye, which strongly correlated with the L(alpha)-H(II) transition of the final complex: at temperatures above the T(H), fast and extensive leakage, mediated by vesicle-vesicle contact, was observed. On the other hand, mixing of vesicle contents was limited and of a highly transient nature. A different behavior was observed with Ca(2+) or Mg(2+): in the temperature range of 0-50 degrees C, where the H(II) configuration is the thermodynamically favored phase, relatively nonleaky fusion of the vesicles occurred. Furthermore, with increasing temperature the rate and extent of leakage decreased, with a concomitant increase in fusion. Fluorescence measurements, involving incorporation of N-NBD-phosphatidylethanolamine in the vesicle bilayer, demonstrated a relative delay in the L(alpha)-H(II) phase transition of the CL vesicle system in the presence of Ca(2+). Freeze-fracture electron microscopy of CL LUV interaction products revealed the exclusive formation of H(II) tubes in the case of Sr(2+), whereas with Ca(2+) large fused vesicles next to H(II) tubes were seen. The extent of binding of Ca(2+) to CL in the lamellar phase, saturating at a binding ratio of 0.35 Ca(2+) per CL, was close to that observed for Sr(2+) and Ba(2+). It is concluded that CL LUVs in the presence of Ca(2+) undergo a transition that favors nonleaky fusion of the vesicles over rapid collapse into H(II) structures, despite the fact that the equilibrium Ca(2+)-CL complex is in the H(II) phase. On the other hand, in the presence of Sr(2+) or Ba(2+) at temperatures above the T(H) of the respective cation-CL complexes, CL LUVs rapidly convert to H(II) structures with a concomitant loss of vesicular integrity. This suggests that the nature of the final cation-lipid complex does not primarily determine whether CL vesicles exposed to the cation will initially undergo a nonleaky fusion event or collapse into nonvesicular structures.

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SummaryThe addition of Ca 2÷ to small unilamellar vesicles of an equimolar mixture of egg phosphatidylcholine and cardiolipin induces fusion of these vesicles in association with the appearance of lipidic particles on the fusion sites.Membrane fusion clearly requires that participating lipids adopt transitory non-bilayer configurations during the intermediate stages. The nature of the possible intermediate structures (such as micellar [1] or inverted micellar [2,3] ) and their relation to the physical properties of membrane lipids remain, however, a matter of some speculation. Recently, it has been argued that endogenous lipids which preferentially adopt the hexagonal (HII) phase under certain conditions may be directly involved in fusion events [4]. In this regard it has been established that the presence of Ca 2÷ is vital to the fusion process [ 5] and that the addition of Ca 2÷ can trigger formation of the hexagonal (HII) phase in model membrane systems consisting of certain pure [6] or mixed [7] species of naturally occurring phospholipid. It is therefore of interest to first establish that Ca 2÷ can also induce fusion between model systems partly comprised of such phospholipids, and secondly to establish that such fusion events proceed via the non-bilayer structures engendered by the presence of Ca 2.. In this work we present results obtained employing model systems comprised of an equimolar mixture of bovine heart cardiolipin and egg yolk phosphatidylcholine in conjunction with