Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. II. Formation of the sporocyst and structure of the sporocyst wall

The ultrastructural changes observed during sporocyst formation and the structure of the sporocyst wall was examined in oocysts which had been allowed to sporulate for between 12 and 48 hours at 27 degrees C. As the spherical sporoblast developed into the sporocyst the cytoplasmic mass became ellipsoidal in shape although no change was noted in the organelle compliment, which cosisted of two nuclei plus a number of polysaccharide granules, lipid globules, mitochondria, Golgi bodies, and some rough endoplasmic reticulum. The sporocyst wall consisted of a thin outer layer (15-20 nm) which was formed from two limiting membranes of the sporoblast and an inner layer (40-50 nm) which was comprised of four curved plates. This inner layer was formed under the outer layer and, although no specific cytoplasmic organelle disappeared with its formation, some unit membranes were observed close to the plasmalemma during its formation. Each curved plate has a marginal swelling and an interposing strip of material is present between the margins of adjacent plates. The plates are joined to the interposing strip by a thin band of osmiophilic material. In oblique and tangential sections through the plates two types of cross banding were observed which differed in periodicity.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

On the nature of the energised state of submitochondrial particles; Investigations with N-aryl naphthalene sulphonate probes

1. A further investigation has been made of the way in which the fluorescent probes 1-anilino-naphthalene-8-sulphonate and 2-(N-methyl-anilino)naphthalene-6-sulphonate report on the energised state of bovine heart submitochondrial particles.2. A comparison of the probe responses to energisation with ATP or to a potassium diffusion potential has been made. The fluorescence enhancements seen in these two cases have different characteristics, and in view of this it is questioned whether a substrate generated energised state of a submitochondrial particle can be equated with a trans-membrane potassium diffusion potential.3. Substitution of ITP for ATP reduces the rate at which either of the probes respond to energisation. In contrast reducing the ATPase activity of the particles by treatment with the covalent ATPase inhibitors 4-chloro-7-nitrobenzofurazan or N,N′-dicyclohexyl-carbodiimide has no effect on this rate. This finding that the rate of the fluorescence changes is directly sensitive to events at the level of the ATPase, but not to the total ATPase activity, suggests that this rate may not be controlled by a delocalised energised state. Reduction of ATPase activity decreases the extent of the fluorescence enhancement and a relationship between the change in probe fluorescence and ATPase activity is given.4. The results in this paper are discussed in the context of the mechanisms which have been proposed to account for the fluorescence enhancements of N-aryl naphthalene sulphonate probes upon energisation of submitochondrial particles.