bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for an O‐glycan sialylation system in brain
Authors: BAUBICHON‐CORTAY Hélène , BROQUET Pierre , GEORGE Pascal , LOUISOT Pierre
Published: March 2005

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N‐glycans and monosialylated and disialylated O‐glycans. The varying effects of N‐ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N‐ethylmaleimide, was identified as the Neu5Acα2→3Galβ1→3GalNAc‐R:α2→6 sialyltransferase previously described [Baubichon‐Cortay, H., Serres‐Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209–223]. This enzyme was responsible for the synthesis of disialylated O‐glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O‐glycan. N‐ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N‐glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O‐glycan synthesis, we used another substrate: Galβ1→3GalNAc–protein obtained after galactosylation of desialylated ovine mucin by a GalNAc‐R:β1→3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N‐glycans and of NeuAc in α2→3 linkages on the galactose residue. When using N‐ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Acα2→3Galβ1→3GalNAc. The enzyme leading to synthesis of this monosialylated O‐glycan was identified as a Galβ1→3GalNAc‐R:α2→3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Acα2→3Galβ1→3GalNAc‐R:α2→6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the α2→3 sialyltransferase regulates the α2→6 sialyltransferase activity since it synthesizes the α2→6 sialyltransferase substrate.
Biosynthetic studies of several of the nuclear‐encoded subunits of mammalian NADH dehydrogenase
Authors: GIBB Graham M. , RAGAN C. Ian
Published: March 2005

An investigation into the biogenesis of several of the nuclear‐encoded subunits of the iron‐protein fragment of mitochondrial NADH dehydrogenase was undertaken utilising a bovine kidney cell line (NBL‐1). Inhibition of import was achieved by treating the cells with the uncoupler carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP) and it was demonstrated that the 75‐kDa, 51‐kDa and 49‐kDa components of the enzyme were synthesised as larger polypeptides of 76‐kDa, 52‐kDa and 53‐kDa, respectively. The precursors could subsequently be processed to the mature subunits by reversing the FCCP treatment and chasing for 45 min at 37°C. Subcellular localisation studies using the detergent digitonin illustrated that the 76‐kDa, 52‐kDa and 53‐kDa precursor forms were almost exclusively located in the soluble fraction of the cell, whereas the mature and pulse‐chased proteins fractionated with the particulate portion of the cell. Although the mature 30‐kDa and 24‐kDa subunits of NADH dehydrogenase could be visualised, their precursor forms went undetected in this system.
Conformational transmission in condensed lipid model compounds
Authors: MEULENDIJKS Gijsbert H. W. M. , de HAAN Jan W. , van GENDEREN Marcel H. P. , BUCK Henk M.
Published: March 2005

On changing the phosphorus coordination from four to five via a trigonal bipyramidal geometry in phospholipid model systems, a conformational change is initated around the C2‐C3 linkage caused by enhanced electrostatic repulsion between the atoms O3 and O2, situated in the axis of the trigonal bipyramid. This is supported by the absence of conformatinal change upon substitution of O2 by methylene. With solid‐state cross‐polarization and magic‐angle spinning 13C‐NMR it is shown that such a conformational transmission leads to an increased packing density of the lipid chains. It seems to us that changes in lipid packing caused by conformational transmission may also occur when pentacoordination is present transiently, as under biological conditions, thereby inducing an activation of the transport proteins that are embedded in the membrane.
Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis
Authors: ADHAM Ibrahim M. , KLEMM Uwe , MAIER Wolf‐Martin , HOYER‐FENDER Sigrid , TSAOUSIDOU Sofia , ENGEL Wolfgang
Published: March 2005

Complementary DNA clones for the boar preproacrosin have been isolated from a randomly primed testis cDNA library in λgt10 and from an oligo(dT)‐primed testis cDNA in λt11. The nucleotide sequence of the 1418‐bp cDNA insert includes a 46‐bp 5′‐untranslated region, an open reading frame of 1248 bp corresponding to 416 amino acids (45.59 kDa) and a 121‐bp 3′‐untranslated region. The deduced amino acid sequence includes the active‐site residues histidine, asparagine and serine of the catalytic triad of the serine proteinase super‐family and is colinear with that determined by amino acid sequencing of the boar acrosin light chain and of a small region of the NH2‐terminal sequence of the heavy chain. The preproacrosin cDNA contains at the 3′ end a 381‐bp sequence which codes for an amino acid sequence not yet found in any other serine proteinase. This amino acid sequence is rich in proline (42 out of 127 amino acids) and is suggested to be involved in the recognition and binding of the spermatozoa to the zona pellucida of the ovum. The mRNA for preproacrosin is synthesizd as an approximately 1.6‐kb‐long molecule only in the postmeiotic stages of boar and bull spermatogenesis.
Mathematical modelling of metabolic pathways affected by an enzyme deficiency
Authors: SCHUSTER Ronny , JACOBASCH Gisela , HOLZHÜTTER Hermann‐Georg
Published: March 2005

The effects of various forms of glucose‐6‐phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism.

The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose‐6‐phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed.

It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose‐6‐phosphate dehydrogenase deficiency.
Conformational analysis of brominated pA2′‐5′A2′‐5′A analogs
Authors: van den HOOGEN Yvonne Th. , HILGERSOM Coen M. A. , BROZDA Danuta , LESIAK Krystyna , TORRENCE Paul F. , ALTONA Cornelis
Published: March 2005

NMR and model‐building studies were carried out on PA2′‐5′A2′‐5′A and analogs in which one or more of the A residues were replaced by 8‐bromoadenosine. Chemical shifts, coupling constants and NOE data were used to obtain structural information. The N/S equilibrium constant of the ribose rings as well as the phase angles and puckering amplitudes were determined from the experimental coupling constants with the aid of an improved version of the PSEUROT program. Chemical shifts in combination with NOE data were used to monitor basebase interactions and the orientation of the bases (syn or anti).

The combined data suggest that different types of stacking interactions are present in the various compounds. Bromination of the first or second residue in the trimers results in a preference for N‐type sugar and syn orientation of the base in these residues. When A(3) is brominated, an S‐type sugar conformation together with a syn orientation of the base is favoured at the 2′ terminus. Energy‐minimized models of the different stacking interactions are presented which fit the present collection of data. The possible correlation between biological activity of these compounds and their conformation is briefly discussed.
Mapping of four discontiguous antigenic determinants on horse cytochrome c
Authors: OERTLE Maja , IMMERGLUCK Karin , PATERSON Yvonne , BOSSHARD Hans Rudolf
Published: March 2005

The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibodybound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double‐labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen‐antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5‐fold to over 20‐fold lower reactivity in antibody‐bound cytochrome c. The present results are complemented by previous data obtained by cross‐reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformation‐dependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information.
Composition of the ternary protein complex of the red cell membrane cytoskeleton
Authors: PEKRUN Arnulf , PINDER Jennifer C. , MORRIS Stephen A. , GRATZER Walter B.
Published: March 2005

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to do binding of additional spectrin, either to the inner surface of ghost membranes or to lipid‐free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimer/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G‐actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G‐actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross‐linking these can be recovered from a gel‐filtration column. Complexes prepared starting from G‐actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F‐actin.
The primary structure of rat ribosomal protein L37a
Authors: TANAKA Tatsuo , AOYAMA Yuji , CHAN Yuen‐Ling , WOOL Ira G.
Published: March 2005

The amino acid sequence of rat ribosomal protein L37a was deduced from the sequence of nucleotides in recombinant cDNAs isolated in Yamagata and in Chicago and confirmed from the NH2‐terminal amino acid sequence of the protein. Ribosomal protein L37a contains 91 amino acids (the NH2‐terminal methionine is removed after translation of the mRNA) and has a molecular mass of 10143 Da.
Crystallin gene expression during rat lens development
Authors: AARTS Henk J. M. , LUBSEN Nicolette H. , SCHOENMAKERS John G. G.
Published: March 2005

The analysis of the developmental pattern of the αA‐, αB‐, βB1‐, βB2‐, βB3‐, βA3/A1‐, and βs‐crystallin genes during fetal and postnatal development of the rat shows that the differential regulation of crystallin synthesis relies on differential gene shutdown rather than differential gene activation; that is, all crystallin gene are active during early development but turn off at different stages. The only two exceptions to this rule are the αB‐ and βs‐crystallin genes. The αB‐crystallin gene transcript becomes first detectable at 18 days of fetal development, while the βs‐crystallin gene appears to be active only in the postnatal period.

We also determined the absolute numbers of the αA‐, αB‐, βB1‐, βB2‐, βB3‐, βA3/A1‐, βs‐, and γ‐crystallin gene transcripts present in the lens at various times after birth. Comparison of these RNA data with the published protein data shows that the αB‐ and βB2‐crystallin RNAs are relatively overrepresented, suggesting the possibility that these two RNA species are not used as efficiently as other crystallin mRNAs. Examination of the known (hamster) αB‐crystallin sequence and elucidation of the (rat) βB2‐crystallin sequence yielded no evidence for aberrant codon usage. These two RNAs have one sequence motif in common: they are the only crystallin mRNAs in which the translation initiation codon is preceded by CCACC.
NMR analysis of site‐specific mutants of yeast phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , WALKER Philip A. , MINARD Philippe , LITTLECHILD Jennifer A. , WATSON Herman C. , WILLIAMS Robert J. P.
Published: March 2005

Site‐specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the ‘basic‐patch’ residues, arginine 168 and histidine 170. The fully‐conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non‐conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500‐MHz 1H‐NMR spectra of the mutant proteins with that of wild‐type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native emzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence‐specific resonance assignments to histidines 62, 167 and 170.

NMR studies of 3‐phosphoglycerate binding show that decreasing the positive charge in the sequence 168–170 reduces the binding of this substrate (by about 15‐fold and 4‐fold for mutants R168M and H170D respectively). Mutant R168K binds 3‐phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild‐type emzyme. This indicates that this residue is not of major importance in the binding or reaction of 3‐phosphoglycerate. The observation is in agreement with results obtained for the wild‐type enzyme, which indicate that 3‐phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X‐ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3‐phosphoglycerate‐induced conformational changes.
Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes
Authors: ANTONENKOV Vasily D.
Published: March 2005

Subcellular distribution of pentose‐phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+‐dependent dehydrogenases of the pentose‐phosphate pathway (glucose‐6‐phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl‐α‐p‐chlorophenoxysobutyrate) caused a 1.5–2.5‐fold increase in the amount of glucose‐6‐phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased of phosphogluconate dehydrogenase, but did not alter glucose‐6‐phosphate dehydrogenase activity in the cytosolic fraction.

The results obtained indicate that the enzymes of the non‐oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose‐phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose.

Similar to catalase, glucose‐6‐phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose‐6‐phosphate and 0.10–0.12 mM for 6‐phophogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose‐6‐phosphate dehydrogenase was inhibited by palmitoyl‐CoA, and to a lesser extent by NADPH. Peroxisomal glucose‐6‐phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose‐phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.
O‐Glycosylation of leukosialin in K562 cells
Authors: PILLER Véronique , PILLER Friedrich , KLIER F. George , FUKUDA Minoru
Published: March 2005

The O‐glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O‐linked chains has been followed in pulse‐chase experiments with [35S]methionine by immunoprecipitation with an anti‐peptide antiserum as well as with a lectin from Salvia sclarea seeds (SSA) specific for GalNAc‐Ser/Thr and the peanut (Arachis hypogaea) agglutinin (PNA) which recognizes Galβ1 → 3GalNAc‐Ser/Thr structures. An O‐glycan‐free precursor was converted into the fully O‐glycosylated mature form within the 10‐min labeling period and no intermediates carrying only GalNAc‐Ser/Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O‐glycan synthesis. The durg partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell‐surface expression of leukosialin. It was found to have a marked effect on the synthesis of O‐linked carbohydrate structures of leukosialin since the amount of O‐glycans containing only GalNAc or NeuNAcα2 → 6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N‐glycan on leukosialin was completely arrested in an endoglycosidase‐H‐sensitive step of processing, whereas the O‐glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin‐treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc‐Ser/Thr‐bearing glycoproteins in the RER as well as in the Golgi. In contrast Galβ1 → 3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis‐Golgi. Together these results lead us to the following conclusions: N‐glycan transfer occurs in the RER before the initiation of O‐glycans which takes place at the entrance of the protein into the Golgi; further elongation of O‐glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N‐glycans to complex‐type structures.
The purification and characterization of a dextranase from Lipomyces starkeyi
Authors: KOENIG David , DAY Donal
Published: March 2005

Dextranase produced by Lipomyces starkeyi was purified 43‐fold, by carboxymethyl‐Sepharose chromatography followed by agarose gel‐filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The‐dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55°C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40°C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto‐oligosaccharides from glucose to isomaltotetrose.
Cloning of cDNA encoding the membrane‐bound form of bovine β1,4‐galactosyltransferase
Authors: D'AGOSTARO Giacomo , BENDIAK Brad , TROPAK Michael
Published: March 2005

UDPgalactose: N‐acetyl‐d‐glucosamine 4‐β‐d‐galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi‐membranebound enzyme that participates in the biosynthesis of the oligosaccharide structures of glyscoproteins and glycolipids.

Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine‐liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206‐base‐pair open reading frame that codes for 402 amino acids, including a presumptive N‐terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non‐overlapping amino acid residues which were positively identified by N‐terminal sequencing of two polypeptides isolated form the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones.

The finding of an N‐terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C‐terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51‐kDa and 42‐kDa soluble forms of the enzyme which encompass the C‐terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.
Human phosvitin/casein kinase type II. Molecular cloning and sequencing of full‐length cDNA encoding subunit beta
Authors: JAKOBI Rolf , VOSS Hartmut , PYERIN Walter
Published: March 2005

Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits α, α′ and β. Subunit β, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a λgt10 HeLa cell cDNA library for clones encoding subunit β. A full‐length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino acid sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human β subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537‐bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1‐kb subunit‐β transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the β‐subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genomic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene.
An actin‐interacting heptapeptide in the cofilin sequence
Authors: YONEZAWA Naoto , NISHIDA Eisuke , OHBA Masataka , SEKI Mariko , KUMAGAI Hiromichi , SAKAI Hikoichi
Published: March 2005

Cofilin, a 21‐kDa actin‐binding protein, has a hexapeptide sequence DAIKKK which is identical to the N‐terminal portion (residues 2–7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122–128 of cofilin, inhibited the binding of cofilin to F‐actin in a dose‐dependent manner. The heptapeptide cosedimented with F‐actin, decreased the fluorescence intensity of pyrene‐labeled F‐actin, and increased the rate of polymerization of G‐actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F‐actin and affected the fluorescence intensity of pyrene‐labeled F‐actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin‐binding domain of cofilin.
Insulin‐induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress
Authors: BURGER Matthias , LAWEN Alfons , MARTINI Oskar H. W.
Published: March 2005

Insulin treatment of HeLa S3 cells activates an S6‐phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease‐activated kinase II and histone‐4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin‐induced increase in S6‐kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin‐induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6‐kinase activities from serum‐ and from insulin‐stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitro transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphorylate the (principal) S6 site recognized by the cAMP‐dependent protein kinase.
Initiation of DNA synthesis by microtubule disruption in quiescent rat 3Y1 cells
Authors: SHINOHARA Yukiko , NISHIDA Eisuke , SAKAI Hikoichi
Published: March 2005

Disruption of cytoplasmic microtubules by colchicine, colcemid or vinblastine induced the initiation of DNA synthesis and cell division in quiescent cultures of rat embryo fibroblast cell line, 3Y1, in the absence of growth factors. The microtubule‐disruption‐induced DNA synthesis was so marked that we could initiate analysis of the mechanism. Incubation of quiescent 3Y1 cells with high concentrations of vinblastine or those of vinblastine plus colcemid, which formed large tubulin paracystals concomitant with the depolymerization of cytoplasmic microtubules, initiated DNA synthesis in the cells. Because these treatments did not increase the free tubulin level in the cells, but rather decreased it, an increase in the free tubulin level may not be required for initiation of DNA synthesis induced by microtubule‐disrupting agents. Culture fluid of the 3Y1 cells incubated with colchicine did not stimulate the initiation of DNA synthesis when added to other quiescent 3Y1 cells after conversion of the colchicine in the culture fluid to lumicolchicine by ultraviolet irradiation. This suggested that colchicine treatment did not induce secretion of any mitogenic factors from 3Y1 cells. These results suggest that disruption of normal microtubules may elicit intracellular signals leading to cell proliferation.
The third ribosomal tRNA‐binding site, the E site, is occupied in native polysomes
Authors: REMME Jaanus , MARGUS Tõnu , VILLEMS Richard , NIERHAUS Knud H.
Published: March 2005

The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in á buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37°C in the presence of 3H‐labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post‐translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post‐translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three‐site model for the elongation cycle but disagree with the two‐site model.
Evaluation of the use of S1 nuclease to detect small length variations in genomic DNA
Authors: BROOKES Anthony J. , SOLOMON Ellen
Published: March 2005

A method which utilises S1 nuclease to detect small length variations in cloned and genomic DNA has been evaluated. The methodology of this technique is simple and robust, permitting the rapid analysis of 104 base pairs. By employing defined sequence variants, this method is shown to have a sensitivity which should enable the detection of length variations of only a few base pairs in heterozygous individuals.
Inactivation of the Escherichia coli heat‐labile enterotoxin by in vitro mutagenesis of the A‐subunit gene
Authors: HARFORD Stephen , DYKES Colin W. , HOBDEN Adrian N. , READ Melanie J. , HALLIDAY Ishbel J.
Published: March 2005

In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat‐labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity. One inactive A‐subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity. A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.
Modulation of arginine decarboxylase activity from Mycobacterium smegmatis
Authors: BALASUNDARAM David , TYAGI Anil K.
Published: March 2005

Arginine decarboxylase (arginine carboxy‐lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5′‐phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km= 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5′‐phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A lowaffinity (Kd= 22.5 μM) binding to apoenzyme at lower pyridoxal‐5′‐phosphate concentrations and a high‐affinity (Kd= 3.0 μM) binding to apoenzyme at high pyridoxal 5′‐phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5′‐phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH‐dependent modulation of coenzyme function. Based on the pH‐dependent changes in the polarity of the active‐site environment, pyridoxal 5′‐phosphate forms different Schiff‐base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff‐base confer different catalytic efficiencies to the enzyme.
Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol‐OT/isooctane microemulsions
Authors: LARSSON Karin M. , OLDFIELD Christopher , FREEDMAN Robert B.
Published: March 2005

Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol‐OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH‐NADH and by determination of Km,app and Vmax,app values for cyclohexanone.

The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48‐h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged.

If no co‐enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co‐enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co‐enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.
Interaction of staphylococcal δ‐toxin and synthetic analogues with erythrocytes and phospholipid vesicles
Authors: ALOUF Joseph Elie , DUFOURCQ Jean , SIFFERT Odile , THIAUDIERE Eric , GEOFFROY Christiane
Published: March 2005

Staphylococcal δ‐toxin, a 26‐residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26‐residue analogues and two shorter peptides corresponding to residues 1–11 and 11–26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the α‐helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26‐residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11–26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1–11 did not bind lipids and was hemolytically inactive. All peptides except the latter crossreacted in immunoprecipitation tests with the natural toxin. The study of a 26‐residue analogue by circular dichroism revealed and α‐helical configuration in both the free and lipid‐bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc‐1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.
Protein kinase C stimulates adenylate cyclase activity in prolactin‐secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP‐binding protein Gi
Authors: GORDELADZE Jan O. , BJÖRO Trine , TORJESEN Peter A. , ØSTBERG BjöRN C. , HAUG Egil , GAUTVIK Kaare M.
Published: March 2005

The phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5′‐[β,γ‐imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin‐enhanced, 2.2 mM) for free Mg2+. The TPA‐mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half‐maximal activation) was 60 μM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA‐stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4α‐phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberinsensitive adenylate cyclase remained unaffected.

Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin‐stimulated enzyme activities, while absolute TPA‐enhanced adenylate cyclase was maintained. Association of extracted GTP‐binding inhibitory protein, Gi, from S49 cyc− murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone‐stimulated adenylate cyclase activities, while net inhibition of the cyclase by somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone‐elicited adenylate cyclase activities in the Gi‐enriched membranes. Finally, TPA completely abolished the somatostatin‐induced inhibition of adenylate cyclase in both hybrid and non‐hybrid membranes.

These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the nαi subunit of the inhibitory GTP‐binding protein, leaving the nβ subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP‐binding protein, Gs.
Intestinal‐like alkaline phosphatase expressed in normal human adult kidney
Authors: HIRANO Kazuyuki , KUSANO Kazutomi , MATSUMOTO Yoshikazu , STIGBRAND Torgny , IINO Shiro , HAYASHI Kyozo
Published: March 2005

Human adult kidney was found to contain not only the ‘tissue‐unspecific alkaline phosphatase’ but also another alkaline phosphatase isozyme. By use of monoclonal antibodies specific for human intestinal alkaline phosphatase, this kidney isozyme was purified to homogeneity by immunoaffinity chromatography. The structural and kinetic properties of the enzyme were compared with those of the other alkaline phosphatase isozymes expressed in normal human tissues, i.e. the placental, intestinal, meconial (fetal), liver and kidney isozymes. The new kidney isozyme was clearly different from both the tissue‐unspecific and the adult intestinal alkaline phosphatase as regards isoelectric point, molecular mass and peptide maps after cyanogen bromide cleavage, but it was found to be identical to the meconial alkaline phosphatase. The results demonstrate simultaneous expression of two alkaline phosphatase isozymes in human kidney, one of which is normally related only to the fetal intestine.
Structural characterization of the lipid A component of Bacteroides fragilis strain NCTC 9343 lipopolysaccharide
Authors: WEINTRAUB Andrej , ZÄHRINGER Ulrich , WOLLENWEBER Horst‐Werner , SEYDEL Ulrich , RIETSCHEL Ernst Th.
Published: March 2005

The chemical structure of Bacteroides fragilis NCTC 9343 lipid A was characterized by using conventional chemical procedures, methylation analysis, and laser desorption mass spectrometry. It was found that B. fragilis lipid A consists of a β‐D‐glucosaminyl‐(1–6)‐D‐glucosaminyl‐1‐O‐phosphate backbone whose hydroxyl groups in positions 4, 4′ and 6′ are free, the latter serving as the attachment site for the polysaccharide component in lipopolysaccharide. This backbone molecule carries up to of five molecules of ester‐ and amide‐bound long chain non‐hydroxylated and (R)‐3‐hydroxy fatty acids. With regard to the distribution of the fatty acids on the lipid A backbone, a considerable heterogeneity was revealed by laser desorption mass spectrometry. Despite this heterogeneity, a major species of B. fragilis lipid A could be defined in which the hydroxyl group at position 3′ of the distal GlcN carries (R)‐3‐hydroxyhexadecanoic acid and the hydroxyl group at position 3 of the reducing GlcN is acylated by (R)‐3‐hydroxypentadecanoic acid. The amino group of the distal GlcN residue carries (R)‐3‐(13‐methyltetradecanoyloxy)‐15‐methylhexadecanoic acid and that of the reducing GlcN group (R)‐3‐hydroxyhexadecanoic acid. The absence of ester‐bound phosphate and ester‐linked 3‐acyloxyacyl groups, the presence of not more than five acyl residues and the predominance of fatty acids possessing 15–17 carbon atoms are unique features of B. fragilis lipid A which differentiate it from enterobacterial and other lipids A and which are likely to be related to its low endotoxic activity.
Characterization of distinct mRNAs coding for putative isozymes of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase
Authors: CREPIN Karine M. , DARVILLE Martine I. , HUE Louis , ROUSSEAU Guy G.
Published: March 2005

Three distinct clones encoding full‐length 6‐phosphofructo‐2‐kinase (PFK‐2)/fructose‐2,6‐bisphosphatase (FBPase‐2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic‐AMP‐dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N‐terminal portion of 10 amino acids.

Poly(A)‐rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N‐terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK‐2/FBPase‐2 mRNAs. There were a 6.8‐kb mRNA typical of cardiac tissue, a 2.1‐kb mRNA typical of liver, corresponding to clone 22c, and a 1.9‐kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5′‐untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK‐2/FBPase‐2 isozymes.
Streptomyces griseus aminopeptidase is a calcium‐activated zinc metalloprotein
Authors: SPUNGIN Anya , BLUMBERG Shmaryahu
Published: March 2005

A heat‐stable aminopeptidase with an N‐terminal Ala‐Pro‐Asp‐Ile‐Pro‐Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel‐exclusion and anion‐exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12.

Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine‐4‐nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25°C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two‐stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions.
Differential induction of peroxisomal and microsomal fatty‐acid‐oxidising enzymes by peroxisome proliferators in rat liver and kidney
Authors: SHARMA Rajesh K. , LAKE Brian G. , MAKOWSKI Richard , BRADSHAW Tony , EARNSHAW Dave , DALE Jeremy W. , GIBSON G. Gordon
Published: March 2005

The induction of renal fatty‐acid‐oxidising enzymes has been investigated following short‐term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P‐450‐IVA1‐dependent ω‐hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid β‐oxidation pathway (measured as cyanide‐insensitive palmitoyl‐CoA oxidation and enoyl‐CoA hydratase).

Cytochrome P‐450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the ω‐hydroxylation of lauric acid. This activity was induced 2–3‐fold by peroxisome proliferators such as clofibrate, di‐(2‐ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P‐450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic ω‐hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome‐P‐450‐IVA1‐related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P‐450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P‐450 IVA1) and a minor one of 52 kDa. The kidney‐supported fatty acid ω‐hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P‐450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue.

Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid β‐oxidation, mirroring the increased ω‐hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty‐acid‐oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the kidney to the toxicity of peroxisome proliferators.