bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C
Authors: BOROWSKI Peter , HEILAND Max , KORNETZKY Lutz , MEDEM Sven , LAUFS Rainer 
Published: August 2015

The catalytic domain of p72syk kinase (CDp72syk) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72syk preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5´-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the ‘linker ’ region of p72syk. By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72syk. The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72syk autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72syk was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72syk. Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (α &gt; βI = βII &gt; γ) of CDp72syk phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.
Interaction of macrophage-migration-inhibitory factor with haematin
Authors: PENNOCK Joanne L. , WIPASA Jiraprapa , GORDGE Michael P. , MEYER David J. 
Published: August 2015

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione–agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Åman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2, 143–149]. The l-dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC50 100–200 nM). The inhibition was non-competitive and showed positive co-operativity (h = 5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.
Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes
Authors: BEYETTE Jill , MASON Grant G. F. , MURRAY Rachael Z. , COHEN Gerald M. , RIVETT A. Jennifer 
Published: August 2015

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially purified from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-fluoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in vitro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.
Translated anti-sense product of the Na/phosphate co-transporter (NaPi-II)
Authors: HUELSEWEH Birgit , KOHL Beate , HENTSCHEL Hartmut , KINNE Rolf K. H. , WERNER Andreas 
Published: August 2015

The homeostasis of Pi in marine teleosts is maintained by renal Pi secretion as well as by Pi reabsorption. A Na/Pi co-transport system belonging to the NaPi-II protein family is instrumental in tightly controlled renal Pi handling in mammals and fish. We have isolated an NaPi-II related cDNA from winter flounder. It was cloned from a female gonad cDNA library and is 624 bp long. The transcript is expressed in female and male flounder gonads as well as in kidney and intestine, although at very low levels. RNase H digestion experiments revealed an opposite orientation of the transcript with regard to NaPi-II-related mRNA. The anti-sense orientation was confirmed by genomic sequence analysis and Southern blotting. Alluding to the sense transcript, the anti-sense transcript was denoted IPAN. The open reading frame of IPAN encodes a basic protein of 68 amino acid residues. Immunohistochemistry confined the anti-sense related protein, Ipan, to a submembranous compartment of immature oocytes, suggesting a role in oocyte development. In kidney and intestine Ipan is partly co-localized with the Na/Pi co-transporter, implying a regulatory function for the anti-sense protein. However, direct protein–protein interaction could not be established. The existence of a putative open reading frame in other species extends the biological significance of the novel protein.
Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone
Authors: VILGRAIN Isabelle , CHINN Anna , GAILLARD Isabelle , CHAMBAZ Edmond M. , FEIGE Jean-Jacques 
Published: August 2015

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.
Elucidation of a promoter activity that directs the expression of acetyl-CoA carboxylase α with an alternative N-terminus in a tissue-restricted fashion
Authors: BARBER Michael C. , TRAVERS Maureen T. 
Published: August 2015

Previous studies in rats and humans have demonstrated that acetyl-CoA carboxylase α (ACC-α), the principal ACC isoenzyme in lipogenic tissues, is transcribed from two promoters, PI and PII, that operate in a tissue-specific fashion. Each promoter gives rise to ACC-α mRNA isoforms that differ in their 5´ untranslated regions but essentially encode the same protein product. In the present study we demonstrate that such a pattern of promoter usage is evident in sheep tissues but in addition we have detected the expression of a novel ACC-α mRNA isoform that is expressed in a variety of tissues including kidney, lung, liver and mammary gland, where it is markedly induced during lactation. This novel transcript differs from the previously described ACC-α mRNA in that exon 5, the primary coding exon in both PI and PII transcripts, is replaced by a 424-nt sequence that seems to represent the 5´ terminus of the mRNA. The 424-nt sequence encodes a 17-residue N-terminal region as the N-terminal residue in the deduced sequence is a methionine flanked by several in-frame stop codons. The 5´ terminal 424 nt are present as a single exon, which we have termed exon 5A, in the sheep ACC-α gene and this is located approx. 15 kb downstream of exon 5 and 5 kb upstream of exon 6. A 1.5 kb HindIII–BglII fragment encompassing the 5´ terminus and sequence immediately upstream of exon 5A demonstrates promoter activity when transiently transfected into HepG2 cells and HC11 mouse mammary cells and this is markedly enhanced when insulin is present in the culture medium. Promoter activity is also evident in primary sheep mammary epithelial cells. These results demonstrate the presence of a third promoter, PIII, in the ACC-α gene that results in the tissue-restricted expression of an ACC isoenzyme.
l-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain
Authors: ILLIAS Rosli Md. , SINCLAIR Rhona , ROBERTSON Douglas , NEU Achim , CHAPMAN Stephen K. , REID Graeme A. 
Published: August 2015

The l-mandelate dehydrogenase (l-MDH) from the yeast Rhodotorula graminisis a mitochondrial flavocytochrome b2 which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala, with about 40% identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b2, Leu-230, is replaced by Gly in l-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant l-MDH. In addition, we have expressed and purified the flavin-containing domain of l-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both l-MDH and its independently expressed flavin domain have been carried out. These indicate that l-MDH is efficient with both physiological (cytochrome c, kcat = 225 s-1 at 25 °C) and artificial (ferricyanide, kcat = 550 s-1 at 25 °C) electron acceptors. Kinetic isotope effects with [2-2H]mandelate indicate that H–C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 °C were measured as -120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b2 (l-lactate dehydrogenase) from S. cerevisiae.
Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of l-mandelate dehydrogenase activity
Authors: SINCLAIR Rhona , REID Graeme A. , CHAPMAN Stephen K. 
Published: August 2015

Flavocytochrome b2 from Saccharomyces cerevisiaeis an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198 → Gly and Leu-230 → Ala results in an enzyme with a kcat value (25 °C) with l-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of l-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198 → Gly and Leu-230 → Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with l-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.
Effects of dietary Pi on the renal Na+-dependent Pi transporter NaPi-2 in thyroparathyroidectomized rats
Authors: TAKAHASHI Fumie , MORITA Kyoko , KATAI Kanako , SEGAWA Hiroko , FUJIOKA Ai , KOUDA Tomoko , TATSUMI Sawako , NII Tomoko , TAKETANI Yutaka , HAGA Hiromi , HISANO Setsuji , FUKUI Yoshihiro , MIYAMOTO Ken-ichi , TAKEDA Eiji 
Published: August 2015

Dietary Pi and parathyroid hormone (PTH) are two most important physiological and pathophysiological regulators of Pi re-absorption in the renal proximal tubule. Effects of dietary Pi on Na+/Pi co-transporter NaPi-2 were investigated in thyroparathyroidectomized (TPTX) rats. NaPi-2 protein and mRNA in the kidney cortex of TPTX rats were increased ≈ 3.8- and 2.4-fold in amount respectively compared with those in the sham-operated animals. Administration of PTH to the TPTX rats resulted in a decrease in the amount of NaPi-2 protein, but not in the abundance of NaPi-2 mRNA. Deprivation of dietary Pi in the TPTX rats did not affect the amount of NaPi-2 mRNA and protein. In the Pi-deprived TPTX rats, feeding of a high-Pi diet resulted in marked decreases in Pi transport activity and the amount of NaPi-2 protein in the superficial nephrons. Immunohistochemical analysis demonstrated that administration of PTH to TPTX rats resulted in a decrease in NaPi-2 immunoreactivity from both superficial and juxtamedullary nephrons within 4 h. Switching TPTX animals from a low-Pi diet to the high-Pi diet decreased NaPi-2 immunoreactivity from superficial nephrons, but not from juxtamedullary nephrons, within 4 h. These results suggest that dietary Pi could regulate the amount of NaPi-2 protein in the superficial nephrons in a PTH-independent manner.
Secretory-granule dynamics visualized in vivo with a phogrin–green fluorescent protein chimaera
Authors: POULI Aristea E. , EMMANOUILIDOU Evaggelia , ZHAO Chao , WASMEIER Christina , HUTTON John C. , RUTTER Guy A. 
Published: August 2015

To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 β-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200–1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine β-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 β-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5–10 s and mean displacement &lt; 1 µm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5–10 µm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin–EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule–cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.
Human mitochondrial phosphoenolpyruvate carboxykinase 2 gene
Authors: MODARESSI Said , BRECHTEL Katja , CHRIST Bruno , JUNGERMANN Kurt , localization chromosomal 
Published: August 2015

The mitochodrial (mt) phosphoenolpyruvate carboxykinase 2 (PCK2) gene was isolated by screening a human genomic library with a rat cytosolic (cy) PCK1 cDNA probe comprising sequences from exons 2–9 and by PCR amplification of human genomic DNA spanning consecutive exons with known primer pairs from mtPCK2 cDNA containing sequences from two putative neighbouring exons. The mtPCK2 gene spans approx. 10 kb and consists of ten exons and nine introns. All exon–intron junction sequences match the classical GT/AG rule. Northern blot analysis of poly(A)+ and total RNA from various tissues revealed one mRNA species of approx. 2.4 kb. The gene is expressed in a variety of human tissues, mainly in liver, kidney, pancreas, intestine and fibroblasts. In contrast with the cytosolic isoenzyme, the mitochondrial form might not have a purely gluconeogenic function. The mtPCK2 gene maps to chromosome 14q11.2–q12, in contrast with the cyPCK1 gene located on 20q13.2–q13.31.
Refolding of urea-denatured adenylate kinase
Authors: ZHANG Hong-jie , SHENG Xiang-rong , PAN Xian-ming , ZHOU Jun-mei 
Published: August 2015

The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20–80-fold dilution of 4 M urea-denatured enzyme at 10 °C, the formation of the secondary structure is a fast process with a rate constant of &gt; 0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0.029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.
Cloning and thermostability of TaqI endonuclease isoschizomers from Thermus species SM32 and Thermus filiformis Tok6A1
Authors: CAO Weiguo , LU Jing , WELCH Simon G. , WILLIAMS Ralph A. D. , BARANY Francis 
Published: August 2015

Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 °C in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself.
The nucleotide sequence data reported in this paper for TfiTok6A1I and Tsp32IR appear in the GenBank Database under the accession numbers U86869 and U86870 respectively.