The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis

A single cDNA of cytochrome c oxidase subunit VIa was characterised from liver, heart and the thermogenic organ of the partially endotherm tuna fish. The amino acid sequence revealed high identity with subunit VIa from carp and trout, but low identity to subunits VIaL (liver type) and VIaH (heart type) of mammalian cytochrome c oxidase. In reconstituted cytochrome c oxidase from bovine heart, the H ⁺/e⁻ stoichiometry is decreased from 1.0 to 0.5 at high intraliposomal ATP/ADP ratios via exchange of bound ADP by ATP at the matrix domain of the transmembraneous subunit VIaH. Reconstituted cytochrome c oxidase from bovine liver and kidney, containing subunit VIaL, revealed H ⁺/e⁻ ratios below 0.5, independent of the ATP/ADP ratio. The results suggest the evolution of three types of subunit VIa. Subunits VIaH and VIaL are postulated to participate in mammalian thermogenesis. Received 3 May 1999; received after revision 10 June 1999; accepted 29 June 1999     

Structure of gap junctions in cultures of normal and neoplastic bladder epithelial cells

Summary Normal rat urinary bladder epithelial cells contain small subunit (PF-1) and large subunit (PF-2) gap junctions, whereas carcinoma cells only contain PF-1 gap junctions. The absence of PF-2 gap junctions, which are composed of larger connexons with slightly larger ionic channels, may contribute to altered metabolic coupling between urinary bladder carcinoma cells.Acknowledgments. The authors wish to thank Ms Denise Wiler and Mr William Leonard for expert technical assistance. This work was supported by funds from the National Cancer Institute, CA-25034, and in part by the Otho SA Sprague Memorial Institute.     

The ubiquitin-proteasome system and chromosome 17 in cerebellar granule cells and medulloblastoma subgroups

Chromosome 17 abnormalities are often observed in medulloblastomas (MBs), particularly those classified in the consensus Groups 3 and 4. Herein we review MB signature genes associated with chromosome 17 and the relationship of these signature genes to the ubiquitin-proteasome system. While clinical investigators have not focused on the ubiquitin-proteasome system in relation to MB, a substantial amount of data on the topic has been hidden in the form of supplemental datasets of gene expression. A supplemental dataset associated with the Thompson classification of MBs shows that a subgroup of MB with 17p deletions is characterized by reduced expression of genes for several core particle subunits of the beta ring of the proteasome (β1, β4, β5, β7). One of these genes (PSMB6, the gene for the β1 subunit) is located on chromosome 17, near the telomeric end of 17p. By comparison, in the WNT group of MBs only one core proteasome subunit, β6, associated with loss of a gene (PSMB1) on chromosome 6, was down-regulated in this dataset. The MB subgroups with the worst prognosis have a significant association with chromosome 17 abnormalities and irregularities of APC/C cyclosome genes. We conclude that the expression of proteasome subunit genes and genes for ubiquitin ligases can contribute to prognostic classification of MBs. The therapeutic value of targeting proteasome subunits and ubiquitin ligases in the various subgroups of MB remains to be determined separately for each classification of MB.     

Aldolase C is localized in neuroendocrine cells

To elucidate the localization of the subunit C of aldolase (aldolase C) in peripheral neuroendocrine cells, we made an immunohistochemical study with monospecific antibodies against human aldolase C. Aldolase C was found to be localized in various types of neuroendocrine cells; in the pituitary gland, thyroid, pancreas, adrenal gland, bronchus, and gastrointestinal tract.     

Demonstration of fructose 1,6-bisphosphatase in human term placenta.

A proteolysed form of fructose 1,6-bisphosphatase (Fru-P2ase) has been detected and characterized in human term placenta. The extract was found to contain very low levels of activity with an alkaline pH optimum. Western blotting demonstrated a polypeptide of Mr 26,000, instead of the subunit of Mr 36,000 observed in native mammalian Fru-P2ases.     

Aldolase C is localized in neuroendocrine cells

Summary To elucidate the localization of the subunit C of aldolase (aldolase C) in peripheral neuroendocrine cells, we made an immunohistochemical study with monospecific antibodies against human aldolase C. Aldolase C was found to be localized in various types of neuroendocrine cells; in the pituitary gland, thyroid, pancreas, adrenal gland, bronchus, and gastrointestinal tract.     

Phosphorylation of human red cell and liver pyruvate kinase. Differences between liver and erythrocyte L-type subunits

Summary Purified PK from human erythrocyte was phosphorylated by cAMP-dependent protein kinase type I from human erythrocyte membrane; this phosphorylation affected only the heavy L subunit but not the L subunit. On the other hand, the L subunit of liver PK was highly phosphorylated. Thus it appears that the L subunits from erythrocyte and liver PK are not identical protein molecules.Acknowledgments. This work was supported by a grant from INSERM (ATP 52.77.84). We thank J. Marie and Dr A. Kahn for the gift of purified liver PK.     

Conversion of mammalian cyclic GMP-dependent protein kinase into modulator-dependent protein kinase (type II) in vitro

The spontaneous conversion of mammalian cyclic GMP-dependent protein kinase (G-PK) into modulator-dependent protein kinase (type II) (M-PKII) in the absence of cGMP or histone was observed in vitro. The findings, together with similarity in substrate protein specificity, suggest that M-PKII is the catalytic subunit of mammalian G-PK.     

Nicotinoprotein (NAD+ -containing) alcohol dehydrogenase: structural relationships and functional interpretations

The primary structure of nicotinoprotein alcohol dehydrogenase (ADH) from Amycolatopsis methanolica was determined and used for modelling against known ADH structures, and for evaluation of the coenzyme binding. The results establish the medium-chain dehydrogenase/reductase nature of the nicotinoprotein ADH. Its subunit model and that of the human class Ibeta ADH subunit structure are similar, with mean a carbon deviations of 0.95 A, but they differ in seven loops. Nicotinoprotein ADH occupies a phylogenetic position intermediate between the dimeric and tetrameric ADH families. Two of the differing loops are important for coenzyme binding in the nicotinoprotein model, where one (with a Thr271Arg exchange towards the traditional enzyme) may suggest a slight rotation of the coenzyme adenine ring in the nicotinoprotein, and the other, with an Asn288 insertion, may suggest an extra hydrogen bond to its nicotinamide ribose, favouring stronger binding of the coenzyme. Combined with previous data, this suggests differences in the details of the tight coenzyme binding in different nicotinoproteins, but a common mode for this binding by loop differences.     

The coordination and function of the redox centres of the membrane-bound nitrate reductases

Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe–S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.     

The effect of sulfhydryl reagents upon the activity of 40S ribosomal subunits

Summary p-Chloromercuribenzoate inhibited the poly (U)-dependent binding of Phe-tRNA to the 40S ribosomal subunit but displayed no inhibitory effect on the binding of poly (U) to the ribosome. Other sulfhydryl reagents tested, likeN-ethylmaleimide and iodoacetamide, did not affect the binding of Phe-tRNA to the small ribosomal subunit.     

Demonstration of fructose 1,6-bisphosphatase in human term placenta

Summary A proteolysed form of fructose 1,6-bisphosphatase (Fru-P₂ase) has been detected and characterized in human term placenta. The extract was found to contain very low levels of activity with an alkaline pH optimum. Western blotting demonstrated a polypeptide of Mr 26,000, instead of the subunit of Mr 36,000 observed in native mammalian Fru-P₂ases.     

Purification of an L-fucose binding lectin fromUlex europeus by affinity column chromatography

Summary An L-fucose binding lectin fromUlex europeus was purified by affinity column chromatography using an L-fucose-starch complex. The lectin thus purified had a mol.wt of 60,000, and consisted of 2 glycoprotein subunit with mol.wt 29,000 and 31,000, respectively.     

A specific GT1 ganglioside-luteinizing hormone interaction induces conductance changes in lipid bilayers.

A specific interaction was demonstrated between GT1 gangliosides incorporated in bilayer membranes and luteinizing hormone. This interaction would allow the penetration of a hormone subunit in the membrane. The results are discussed in terms of adenylate cyclase activation.     

The N-terminal amino acid sequence of the small subunit of ribulose-1,5-diphosphate carboxylase fromNicotiana tabacum

Summary The N-terminal sequence of the small subunit of Fraction I protein isolated from tobacco was investigated, using an automatic protein sequencer. The amino acid sequence of the first 21 residues is presented.     

eIF2A,an initiator tRNA carrier refractory to eIF2α kinases,functions synergistically with eIF5B

The initiator tRNA (Met-tRNA _i ^Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA _i ^Met on the small ribosomal subunit. Eukarya contain the Met-tRNA _i ^Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA _i ^Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA _i ^Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B–eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.     

An isoform of the vacuolar (H+)-ATPase accessory subunit Ac45

The vacuolar (H⁺)-ATPase (V-ATPase) is the main regulator of intraorganellar pH and in neuroendocrine cells is controlled by its accessory subunit, Ac45. Here, we report the discovery of the first isoform of a V-ATPase accessory subunit, namely an Ac45-like protein, denoted Ac45LP. Phylogenetic analysis revealed a lineage-dependent evolutionary history: Ac45 is absent in birds, and Ac45LP is absent in placental mammals, whereas all other tetrapod species contain both genes. In contrast to Ac45, Ac45LP is not proteolytically cleaved, a prerequisite for proper Ac45 routing. Intriguingly, Xenopus Ac45LP mRNA was expressed in developing neural tissue and in neural crest cells. In adult Xenopus, Ac45 mRNA is widely expressed mostly in neuroendocrine tissues, while Ac45LP mRNA expression was found to be restricted to the kidney and the lung. This novel Ac45LP may provide additional possibilities for V-ATPase regulation during neurodevelopment as well as in kidney and lung cells.     

Glutamate synthase: a complex iron-sulfur flavoprotein

Glutamate synthase is a complex iron-sulfur flavoprotein that forms l-glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain. Received 10 November 1998, received after revision 10 December 1998; accepted 10 December 1998     

A specific GT1 ganglioside-luteinizing hormone interaction induces conductance changes in lipid bilayers

Summary A specific interaction was demonstrated between GT₁ gangliosides incorporated in bilayer membranes and luteinizing hormone. This interaction would allow the penetration of a hormone subunit in the membrane. The results are discussed in terms of adenylate cyclase activation.