Febs Letters

doi: 10.1016/S0014-5793(96)90839-Xpmid: N/A

doi: 10.1016/S0014-5793(96)90841-8pmid: N/A

Goñi, Félix M.; Requero, M.Asun; Alonso, Alicia

doi: 10.1016/0014-5793(96)00603-5pmid: 8706815

Palmitoylcarnitine is a well‐known intermediate in mitochondrial fatty acid oxidation. Less known are its properties as a surfactant, with a capacity to solubilize biological membranes similar to that of many synthetic detergents used in the biochemical laboratory. Some of the physico‐chemical properties of palmitoylcarnitine may help to explain the need for coenzyme A‐carnitine‐coenzyme A acyl exchange during mitochondrial fatty acid import. The amphiphilic character of palmitoylcarnitine may also explain its proposed involvement in the pathogenesis of myocardial ischemia.

Wlassoff, W.A.; Dymshits, G.M.; Lavrik, O.I.

doi: 10.1016/0014-5793(96)00479-6pmid: 8706830

On the basis of recent results, we propose a model for DNA polymerase translocation along DNA. Human immunodeficiency virus reverse transcriptase is taken as an example. According to the model, movement of the enzyme is the result of transition of the enzyme‐bound DNA from the A‐ to B‐form which is accompanied by lengthening of DNA within the binding channel. The driving force of this transition is the increase in water accessibility to the DNA‐binding cleft after dNTP binding. dNTP hydrolysis proceeding during the following chemical step supplies the energy for the reverse B → A transition of DNA. Translocation is considered to be an integral part of the stage of conformational change preceding catalysis and can be described as a worm‐like movement of DNA within the DNA‐binding cleft.

Araya, Zufan; Norlin, Maria; Postlind, Hans

doi: 10.1016/0014-5793(96)00617-5pmid: 8706816

A mitochondrial cytochrome P450 fraction catalyzing 1α‐ and 27‐hydroxylation but not 24‐hydroxylation of 25‐hydroxyvitamin D3 was purified from pig kidney. The ratio between the 1α‐ and 27‐hydroxylase activities was the same in all purification steps including a side fraction. Attempts to separate the 1α‐ and 27‐hydroxylase activities were unsuccessful. A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M r and immunoprecipitated both the 1α‐ and 27‐hydroxylase activities towards 25‐hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25‐hydroxyvitamin D3 hydroxylases in kidney mitochondria. Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1α‐hydroxylation but not 24‐hydroxylation of 25‐hydroxyvitamin D3 in kidney. In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25‐hydroxyvitamin D3 1α‐hydroxylase.

Kimura, Tominori; Nishikawa, Masao; Fujisawa, Jun-ichi

doi: 10.1016/0014-5793(96)00614-Xpmid: 8706820

The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS‐1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half‐life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (<0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS‐1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus.

Erttmann, K.D.; Domeyer, A.; Gallin, M.

doi: 10.1016/0014-5793(96)00620-5pmid: 8706821

Here we show that E1, an ankyrin‐related, potentially protective, neuronal protein of the human filarial nematode Onchocerca volvulus contains a death domain (DD), most similar to that of human Mort1/FADD (39% identity). In addition, sequence comparison of E1 to its homologue from Litomosoides sigmodontis and to Caenorhabditis elegans ankyrin defines two further putative functional domains. One represents the end of the spectrin‐binding domain of ankyrins, the other an unique domain, most highly conserved between these nematodes, containing a calpain sequence motif. Thus, E1 may be involved in apoptosis, raising the possibility that protection against this parasitic helminth may be induced by apoptotic processes.

Messana, Irene; Orlando, Mario; Cassiano, Loredana; Pennacchietti, Lucia; Zuppi, Cecilia; Castagnola, Massimo; Giardina, Bruno

doi: 10.1016/0014-5793(96)00624-2pmid: 8706822

The metabolic behaviour of human erythrocytes has been investigated with particular regard to the effect of their oxygenation state. Experiments performed at high phosphate concentration (80 mM) within the pH range 7.0–7.8 on erythrocytes at high (HOS) and low (LOS) oxygen saturation showed that at any pH value: (1) glucose consumption was independent of the oxygenation state; (2) pentose phosphate pathway (PPP) flux was about 2 times higher in the HOS than in the LOS state. At low phosphate concentration (1.0 mM) the PPP flux doubled in HOS as well as in LOS erythrocytes, whereas the decrease in glucose consumption was more marked in the HOS state. Metabolism of LOS erythrocytes approached that of HOS erythrocytes under the following conditions: (1) erythrocytes having band 3 modified by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid; (2) CO‐saturated erythrocytes. These data support the hypothesis of a modulation of the relative rates of PPP and glycolysis achieved through competition between deoxy‐hemoglobin (deoxy‐Hb) and glycolytic enzymes for the cytoplasmic domain of band 3.

Murata, Taku; Taira, Masato; Manganiello, Vincent C.

doi: 10.1016/0014-5793(96)00410-3pmid: 8706823

PDE3 or cGMP‐inhibited cyclic nucleotide phosphodiesterase (cGI PDE) activity was detected in homogenates of HepG2, Hep3B and HuH7, but not SK‐Hep‐1, human hepatoma cells. In HepG2 and Hep3B cells PDE3 activity was found predominantly in particulate fractions; in HuH7, in both particulate and supernatant fractions. cDNAs encoding two human PDE3s (an ‘adipocyte’ type, HcGIP1),and a ‘cardiovascular’ type, HcGIP2) have been cloned. HcGIP1 cDNA hybridized strongly with poly(A)+ RNA species from HepG2 and Hep3B. Both HcGIPI and HcGIP2 mRNAs were expressed in Hep3B and HuH7 cells. The nucleotide sequence of an ∼300‐bp cDNA fragment, isolated after RT‐PCR cloning from HepG2 RNA, was identical to a sequence within the conserved domain of HcGIP1 cDNA, consistent with the presence of HcGIPI mRNA in HepG2 cells.

Yamada, Hiroshi; Moriyama, Yoshinori; Maeda, Masatomo; Futai, Masamitsu

doi: 10.1016/0014-5793(96)00621-7pmid: 8706824

Escherichia coli H+‐ATPase subunit a is a hydrophobic F0 subunit. To investigate the topology of the subunit in the membrane, we prepared site‐specific polyclonal antibodies against amino‐terminal (Ser‐3 to Leu‐16), middle loop (Lys‐167 to Gln‐181), and carboxyl‐terminal (Thr‐259 to His‐271) peptide segments. Enzyme‐linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside‐out membrane vesicles, but not with that of right‐side‐out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X‐100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.