Human cytochrome c oxidase isoenzymes from heart and skeletal muscle; purification and properties

Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme.     

Hepatic Adaptation Compensates Inactivation of Intestinal Arginine Biosynthesis in Suckling Mice

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass ^fl and Villin-Cre mice. Unexpectedly, Ass ^fl/fl /VilCre ^tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass ^fl/fl /VilCre ^tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass ^fl/fl /VilCre ^tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.     

On the origin of the cyanogenic polymorphism in Trifolium repens L.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium. The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens . We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens . The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens , as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li . As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens .     

The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum-Free Medium

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na⁺,K⁺-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm² in the presence of serum to 1,500 Ω· cm² after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na⁺,K⁺-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

Expression of the Arabidopsis metallothionein 2b enhances arsenite sensitivity and root to shoot translocation in tobacco

We expressed the AtMT2b gene under the 35 S cauliflower mosaic virus promoter in Nicotiana tabacum (Sr1), using leaf disc transformation. Arsenite tolerance and uptake, as well as arsenite-induced phytochelatin (PC) accumulation in roots were measured in transgenic lines, and compared to untransformed (‘wild type’) tobacco. Measured after 5 days of exposure, arsenite tolerance was slightly but significantly decreased in the transgenic lines compared to wild type. The highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots, but an increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged, suggesting that AtMT2b expression enhanced the arsenic root to shoot transport. The same transformant line also exhibited a decreased rate of phytochelatin accumulation in the roots, but the phytochelatin-SH to As molar ratio was higher than in wild type, suggesting that the lower arsenite tolerance in the transformant lines was not due to a potential shortage of cysteine for PC synthesis, imposed by expression of the transgene.     

Upstream and intronic regulatory sequences interact in the activation of the glutamine synthetase promoter.

Glutamine synthetase (GS) is expressed at high levels in subsets of cells in some tissues and at low levels in all cells of other tissues, suggesting that the GS gene is surrounded by multiple regulatory elements. We searched for such elements in the 2.5-kb upstream region and in the 2.6-kb first intron of the GS gene, using FTO-2B hepatoma and C2/7 muscle cells as representatives of both cell types and transient transfection assays as our tools. In addition to the entire upstream region and entire intron, an upstream enhancer module at -2.5 kb, and 5', middle and 3' modules of the first intron were tested. The main effects of the respective modules and their combinatorial interactions were quantified using the analysis of variance (anova) technique. The upstream enhancer was strongly stimulatory, the middle intron module strongly inhibitory, and the 3'-intron module weakly stimulatory in both hepatoma and muscle cells. The 5'-intron module was strongly stimulatory in muscle cells only. The major new finding was that in both cell types, the upstream enhancer and 5'-intron module needed to be present simultaneously to fully realize their transactivational potencies. This interaction was responsible for a pronounced inhibitory effect of the 5'-intron module in the absence of the upstream enhancer in hepatoma cells, and for a strong synergistic effect of these two modules, when present simultaneously in muscle cells. The main difference between hepatoma and muscle cells therefore appeared to reside in tissue-specific differences in activity of the respective regulatory elements due to interactions rather than in the existence of tissue-specific regulatory elements.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.