Febs Letters

Dick, Frederick A; Eisinger, Dominic P; Trumpower, Bernard L

doi: 10.1016/S0014-5793(97)01402-6pmid: 9426207

Qsr1p is a 60S ribosomal subunit protein that is necessary for joining of large and small ribosomal subunits and is also one of the last proteins assembled onto the 60S ribosomal subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large ribosomal subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S ribosomal subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S subunits.

Budnik, L.T; Mukhopadhyay, A.K

doi: 10.1016/S0014-5793(97)01408-7pmid: 9426208

The aim of this study was to determine whether luteal cells possess functional receptors for lysophosphatidic acid (LPA). We present evidence that [3H]LPA binds to a 38–40 kDa protein in a membrane fraction prepared from luteal cells and that this phospholipid is able to induce tyrosine phosphorylation of several proteins (65–125 kDa). Furthermore, LPA upregulates forskolin‐ and LH/GTP‐stimulated adenylyl cyclase activity by changing its Vmax. Although a pertussis toxin‐sensitive G‐protein has been reported to transmit the inhibitory signals between the LPA receptor and adenylyl cyclase, the observed upregulation of the enzyme activity in luteal cells is not abolished after pre‐treating the cells with the toxin, suggesting that a different mechanism is operative in these cells. According to the pharmacological regulatory pattern it is suggested that the modulated adenylyl cyclase isoform is the enzyme subtype V expressed in luteal cells.

Hoshino, Hideki; Konda, Yoshitaka; Takeuchi, Toshiyuki

doi: 10.1016/S0014-5793(97)01409-9pmid: 9426209

Furin, a member of the yeast Kex2 endoprotease family, converts a number of proproteins to their active forms. The liver produces a number of proproteins having a furin‐cleavable site; thus, furin may be involved in growth and differentiation both in the partially hepatectomized liver and in primary cultured hepatocytes. Furin mRNA levels are elevated in tissues regenerated from partially hepatectomized rat liver. In primary culture of rat hepatocytes, furin expression increases gradually with time, and its expression is greatly enhanced by transforming growth factor β1, whose processing from the precursor requires cleavage by furin. Thus, we suggest that the regeneration and differentiation of hepatocytes is dependent upon the co‐elevation of furin and transforming growth factor β1 mRNAs.

Lang, Jochen; Zhang, Hui; Vaidyanathan, Vadakkanchery-Viswanathan; Sadoul, Karin; Niemann, Heiner; Wollheim, Claes B

doi: 10.1016/S0014-5793(97)01411-7pmid: 9426210

We have investigated the effect of botulinum neurotoxin (BoNT) C1 light chain (LC) on insulin exocytosis from the clonal β‐cell line HIT‐T15. In streptolysin‐O permeabilized cells, the β‐cell impermeant BoNT C1 cleaved mainly syntaxin 1 and inhibited Ca2+ as well as GTPγS induced exocytosis. To study the effect of BoNTs in intact cells, we transiently coexpressed the BoNT LC together with a reporter gene for insulin release. BoNT C1 inhibited K+ induced insulin secretion by 95% but reduced insulin release stimulated by glucose only by 25%. Thus a component of glucose stimulated insulin release is insensitive to BoNT C1.

Mitsopoulos, Costas; Hashemzadeh-Bonehi, Lida; Broome-Smith, Jenny K

doi: 10.1016/S0014-5793(97)01413-0pmid: 9426211

Mature β‐lactamase was attached to the N‐terminus of human glycophorin C, an N‐out membrane protein lacking a cleavable signal peptide (an N‐tail membrane protein). When synthesised in Escherichia coli more than 30% of the intact mature β‐lactamase‐glycophorin C molecules assembled N‐out, C‐in into the cytoplasmic membrane. The N‐tail translocated β‐lactamase folded into an enzymatically active form, but it was more susceptible to proteolysis than the equivalent portion of β‐lactamase‐glycophorin C synthesised with an N‐terminal signal peptide. Its translocation was virtually abolished when the N‐out domain of glycophorin C was truncated or when the basic residues C‐terminally flanking the glycophorin C membrane‐spanning segment were replaced with neutral ones.

Mochimaru, Mari; Sakurai, Hidehiro

doi: 10.1016/S0014-5793(97)01421-Xpmid: 9426212

Tentoxin binding on chloroplast coupling factor 1 (CF1) was studied using a centrifugation column method followed by HPLC analysis. From non‐linear regression analysis of the results, the presence of three types of binding site with the following K d values was deduced: 6.9×10−8 M (first site), 1.4×10−5 M (second site), and 6.3×10−3 M (third site). The binding of one tentoxin inhibits, that of two tentoxins moderately restores, and that of three tentoxins greatly stimulates the ATPase activity of CF1. The forward rate constant of the binding of tentoxin on the first site was 6.3×103 M−1 s−1.

Shchyolkina, Anna K.; Borisova, Olga F.

doi: 10.1016/S0014-5793(97)01417-8pmid: 9426213

Oligonucleotide‐directed triplex formation attracts much attention due to its potential usefulness in diagnostic and biotechnological applications (for review, see [1, 2]). Among other aspects, the research embraces numerous studies probing the influence of intercalating ligands on triplex stability. The effect of the intercalator on triplex formation and stability is known to depend on nucleotide sequence, type of intercalator and solution conditions (for review, see [3]). The present work is aimed at determining the average number of intercalated ethidium bromide (EtBr) and acridine orange (AO) molecules leading to the most effective stabilization of triplexes. First, fluorescing complexes of intramolecular parallel (recombinant) triplex 5′‐d(CATGCTAACT)‐L‐d(AGTTAGCATG)‐L‐d(CATGCTAACT)‐3′ (parARB) and classical antiparallel 5′‐(dA)10‐L‐(dT)10‐L‐(dT)10‐3′(antiATT) (L=‐pO(CH2CH2O)3p‐) with EtBr and AO were characterized, binding constants were obtained and compared to those for homologous DNA duplexes. Then the total EtBr and AO concentrations corresponding to an average of one, two or three intercalated molecules per oligonucleotide were estimated. Thermal denaturation of parARB and antiATT complexes with an average of one, two or three bound molecules was carried out, thermodynamic parameters of the triplex‐to‐duplex and duplex‐to‐open‐strand transitions were evaluated using a three‐state model. The ability of EtBr and AO to stabilize or destabilize both parallel (recombinant) and classical antiparallel triplexes was found to depend strongly on the concentration of bound intercalator. The triplexes were shown to be stabilized by intercalation of the first and second EtBr or AO molecules, while binding of the third intercalator molecule to 10 nucleotide long triplex resulted in significant triplex destabilization.

Oakley, Aaron J; Lo Bello, Mario; Mazzetti, Anna Paola; Federici, Giorgio; Parker, Michael W

doi: 10.1016/S0014-5793(97)01424-5pmid: 9426214

The diuretic drug ethacrynic acid, an inhibitor of pi class glutathione S‐transferase, has been tested in clinical trials as an adjuvant in chemotherapy. We recently solved the crystal structure of this enzyme in complex with ethacrynic acid and its glutathione conjugate. Here we present a new structure of the ethacrynic‐glutathione conjugate complex. In this structure the ethacrynic moiety of the complex is shown to bind in a completely different orientation to that previously observed. Thus there are at least two binding modes possible, an observation of great importance to the design of second generation inhibitors of the enzyme.

Bracco, Enrico; Peracino, Barbara; Noegel, Angelika A; Bozzaro, Salvatore

doi: 10.1016/S0014-5793(97)01425-7pmid: 9426215

The main function of vacuolar H+ ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+ ATPase (vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54 874 Da. The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid‐phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.

Volpe, Filippo; Clatworthy, Jonathan; Kaptein, Allard; Maschera, Barbara; Griffin, Anne-Marie; Ray, Keith

doi: 10.1016/S0014-5793(97)01426-9pmid: 9426216

Following interleukin‐1 (IL1) stimulation, an IL1 receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1‐RI or if its recruitment is mediated by a different molecule. Using the yeast two‐hybrid system, we have analysed possible protein‐protein interactions between IRAK, IL1‐RI and IL1‐RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the IL1‐RAcP but is unable to interact with the cytoplasmic region of the IL1‐RI. Immunoprecipitation of the IL1‐RAcP followed by Western blot analysis using anti‐IRAK antibodies revealed that IRAK co‐precipitated with the IL1‐RAcP. We propose that, in non‐stimulated cells, IRAK is bound to the IL1‐RAcP and therefore, following IL1 stimulation, both molecules are recruited simultaneously to the IL1‐RI complex.