Inhibition of adenine nucleotide transport through the mitochondrial porin by a synthetic polyanion

The effect of a synthetic polyanion of Mr 10,000 (a copolymer of methacrylate, maleate and styrene in a 1:2:3 proportion) was studied on isolated rat liver mitochondria and on mitochondrial porin reconstituted into lipid bilayer membranes. Increasing concentrations of the polyanion inhibited the adenyl kinase located between both mitochondrial membranes in a dose-dependent fashion. Upon addition of the detergent digitonin in increasing concentrations the adenyl kinase activity was fully reversible. In reconstitution experiments with mitochondrial porin the polyanion increased the voltage dependence of the pore in such a way that the pore is switched into the closed state at much smaller voltages than in the absence of the polyanion. The asymmetric addition of the polyanion resulted in an asymmetric shift of the voltage-dependence of the pore. If the voltage is negative at the cis-side (the side of the addition of the polyanion) the pore closed rapidly whereas it was always open for potentials of opposite polarity. The results are discussed on the basis of a modification of the gate properties of the mitochondrial porin by the polyanion and by the assumption that the closed state of the pore is not permeable for nucleotides.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Occurrence of enteroviruses in marine sediment along the coast of Barcelona, Spain

A survey of the occurrence of enteroviruses in marine sediment was undertaken in an area receiving polluted effluents. Enteroviruses were detected in 21 out of 38 samples (55%). Viruses were found as far as 5 km from the shoreline and at a depth of 82 m. Multiple correlations between enteroviruses and bacteria, detected in the same samples, were computed. No correlation could be demonstrated between virus numbers and any other parameter in sediment samples collected south of Barcelona. This lack of correlation is probably due to the different decay rates shown by bacteria and viruses. In contrast, the cases where pollution resulted from a more recent deposition, as in sediment samples collected north of Barcelona, enterovirus levels were correlated with fecal streptococci levels.     

Acyl-CoA:dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method

In relation to the finding that human skin fibroblasts are capable of de novo either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

Dissociation between enhanced resistance and delayed hypersensitivity induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyl-dioctadecyl-ammonium bromide

In this study we investigated the relation between enhanced resistance and delayed hypersensitivity (DH) induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyldioctadecylammonium bromide (DDA). Ribosomal RNA as well as cell envelope fragments (fraction I) protected mice against lethal Listeria infection. However, only fraction I induced DH against killed Listeria. For the induction of protection with fraction I or RNA as well as for the induction of DH with fraction I, preparations had to be administered in combination with DDA. Fraction I elicited a DH response in mice immunized with viable Listeria, but RNA did not. These observations pointed to a dissociation between DH and enhanced resistance induced with RNA, and to a dissociation between fraction I and RNA with respect to their ability to induce or elicit DH. Also DH and enhanced resistance induced with fraction I could be dissociated. Intracutaneous administration of fraction I induced high levels of DH without concomitant induction of protection against lethal challenge with Listeria. On the other hand, intraperitoneal administration of fraction I fully protected mice against lethal infection, but only induced a moderate DH response. DH induced with fraction I was largely specific, whereas enhance resistance induced with this preparation was nonspecific. Finally, proteinase K-sensitive proteins were found to be essential for the induction of DH but not for the induction of protection with fraction I.     

Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli.

The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally. The results show that these gene clusters are closely related.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.