bluuebunny/crossref_metadata_2025 huggingface dataset:
Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide
Authors: Black G W , Hazlewood G P , Xue G P , Orpin C G , Gilbert H J
Published: August 2015

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.
The primary structure of the Pol-RFamide neuropeptide precursor protein from the hydromedusa Polyorchis penicillatus indicates a novel processing proteinase activity
Authors: Schmutzler C , Diekhoff D , Grimmelikhuijzen C J P
Published: August 2015

Neuropeptides containing the C-terminal sequence Arg-Phe-NH2 (RFamide) occur throughout the Animal Kingdom and are abundant in evolutionarily ‘old’ nervous systems such as those of cnidarians. From the hydromedusa Polyorchis penicillatus we have previously isolated two neuropeptides, Pol-RFamide I (<Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH2) and Pol-RFamide II (<Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2). Here we describe the cloning of a common precursor protein for these peptides from P. penicillatus. The precursor protein contains one copy of Pol-RFamide I, 11 copies of Pol-RFamide II and one putative neuropeptide sequence. The Pol-RFamide I sequence is flanked by pairs of basic residues (Arg-Lys). At the C-termini of all Pol-RFamide II sequences, single basic residues (Arg) occur. Paired and single basic residues are established sites for post-translational precursor cleavage. At the N-termini of the Pol-RFamide II sequences, however, basic residues are lacking and, instead, either single Asp (in eight cases) or single Asn residues (in three cases) occur. This means that processing must take place at Asp and/or Asn residues. This is firm evidence for the presence of one or more unconventional processing enzymes. The first type of processing enzyme could be an endoproteinase or aminopeptidase hydrolysing at the C-terminal side of Asp residues. Proteolytic cleavage at acidic amino acid residues has previously been inferred from other cnidarian neuropeptide precursors. The second type of processing enzyme could be an endoproteinase or aminopeptidase hydrolysing at the C-terminal side of Asn residues.
Cell proliferation status, cytokine action and protein tyrosine phosphorylation modulate leukotriene biosynthesis in a basophil leukaemia and a mastocytoma cell line
Authors: Hagmann W
Published: August 2015

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.
Characterization of a basic serine proteinase (pI ∼ 9.5) secreted by virulent strains of Dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains
Authors: Kortt A A , Caldwell J B , Lilley G G , Edwards R , Vaughan J , Stewart D J
Published: August 2015

An extracellular serine proteinase with a PI approximately 9.5 (referred to as ‘basic proteinase’) was purified to homogeneity, from strains of Dichelobacter nodosus that cause virulent foot-rot, by gel filtration of concentrated culture supernatant on Sephadex G-100 and chromatography on sulphopropyl-Sephadex C-25 at pH 8.6 D. nodosus strains that cause benign foot-rot do not secrete a corresponding basic proteinase with a pI of approximately 9.5. Benign strains secrete a closely related, but distinct, proteinase which has the same molecular mass and N-terminal sequences as the ‘virulent’ basic proteinase, but a lower pI of approximately 8.6. The basic proteinases from both strains appear to interact with other proteins present in the culture medium, which results in anomalous behavior on gel filtration. Pure D. nodosus ‘virulent’ basic proteinase has a molecular mass of 36 kDa and showed a low solubility at I < 0.05 precipitating quantitatively from solution as microcrystals. The proteinase shows optimal activity at pH 8.0 and is stable to heating to 55 degrees C for 30 min, but at higher temperatures activity is rapidly lost. Bivalent-metal ions (e.g. Ca2+) are required to maintain the structural integrity and stability of the proteinase; in the presence of EDTA or conditions that cause protein unfolding, the proteinase undergoes rapid and complete autolysis. Cleavage of oxidized insulin A- and B-chain showed that the basic proteinase has a broad specificity, including cleavage at lysine and arginine bonds.
Identification and properties of a peptidyl dipeptidase in the housefly, Musca domestica, that resembles mammalian angiotensin-converting enzyme
Authors: Lamango N S , Isaac R E
Published: August 2015

[D-Ala2,Leu5]Enkephalin was readily metabolized by membranes (40,000 g pellet) prepared from heads of the housefly, Musca domestica, with Gly3-Phe4 being the major site of cleavage. This hydrolysis was only partially inhibited (40%) by 10 microM phosphoramidon, an inhibitor of endopeptidase-24.11, but was almost totally abolished in the presence of a mixture of 10 microM phosphoramidon and 10 microM captopril, a potent inhibitor of mammalian angiotensin-converting enzyme (ACE). An assay for ACE employing Bz-Gly-His-Leu as the substrate was used to confirm the presence of an ACE-like peptidyl dipeptidase activity in fly head membranes. The peptidase had a Km of 1.91 mM for Bz-Gly-His-Leu and a pH optimum of 8.2. The activity was inhibited by 100 microM EDTA and was greatly activated by ZnCl2 but not other bivalent metal ions. Captopril, lisinopril, fosinoprilat and enalaprilat, all selective inhibitors of mammalian ACE, were also good inhibitors of the insect enzyme with IC50 values of 400 nM, 130 nM, 16 nM and 290 nM respectively. An M(r) value of around 87,000 was obtained for this enzyme from gel-filtration chromatography, indicating that the insect enzyme is similar in size to mammalian testicular ACE (M(r) = 90,000-110,000) and not the larger form of the enzyme (M(r) = 150,000-180,000) found in mammalian somatic tissues. The fly peptidyl dipeptidase was released from membranes into a soluble fraction by incubating the head membranes at 37 degrees C but not at 0 degree C, suggesting that the insect ACE-like enzyme can be solubilized from cell surfaces through the activity of a membrane-bound enzyme activity. In conclusion, we have shown the existence of a peptidyl dipeptidase in membranes from the heads of M. domestica, which has similar properties to those of mammalian ACE.
Inhibitors of serine/threonine phosphatases enhance phosphorylation of the interferon-gamma receptor while selectively attenuating interferon-gamma-induced gene expression in human peripheral-blood monocytes.
Authors: Luong H , Winestock K D , Finbloom D S
Published: August 2015

Since many events following ligand-induced receptor clustering are controlled by serine and threonine (Ser/Thr) phosphorylation, we initiated an investigation into the role of Ser/Thr phosphatases in both phosphorylation of the interferon-gamma (IFN-gamma) receptor and IFN gamma-induced gene expression in human peripheral-blood monocytes. Whereas IFN gamma alone did not enhance phosphorylation of the IFN gamma receptor, treatment of monocytes with the Ser/Thr phosphatase inhibitors, okadaic acid and calyculin A, resulted in increased phosphorylation of the IFN gamma receptor. However, when these cells were analysed for IFN gamma-induced IP-10 gene expression, there was profound inhibition. Using three IFN gamma-induced early-response genes, IP-10, the Fc gamma receptor type I (Fc gamma RI) and ISG-54, we found selective sensitivity to pretreatment with okadaic acid and calyculin A. Whereas IFN gamma induction of IP-10 was blocked by both inhibitors, only calyculin A prevented Fc gamma RI-gene expression. Neither inhibitor prevented ISG-54 induction by IFN gamma. IFN-gamma-activated formation of the DNA-binding-protein complex FcRF gamma (which binds to the promoter of the Fc gamma RI gene) remained unaffected by okadaic acid or calyculin A. Therefore these data suggest that Ser/Thr phosphatases have no major part in IFN gamma-initiated signal transduction across the membrane, but selectively control the ultimate transcription of a set of early-response genes.
Structural requirements for the utilization of ascorbate analogues in the prolyl 4-hydroxylase reaction
Authors: Tschank G , Sanders J , Baringhaus K H , Dallacker F , Kivirikko K I , Günzler V
Published: August 2015

The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied. In experiments using the purified enzyme, variations of the compounds′ side chain were compatible with co-substrate activity. The presence of very large hydrophobic substituents or a positively charged group caused an increase in the observed Km values. A negative charge and smaller modifications did not change the affinity to the enzyme when compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower Km than the physiological reductant. Substitution at the -OH group in ring position 3 prevented binding to the enzyme. The same pattern of activity was observed when the full and uncoupled prolyl 4-hydroxylase reactions were studied. The Vmax. values with all compounds were similar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-bromo-6-deoxy-L-ascorbate gave a lower Km and a similar Vmax. value. L-Ascorbic acid 6-carboxylate produced substrate inhibition at concentrations above 0.3 mM. The Km and Vmax. values calculated from concentrations up to 0.2 mM were similar to those of L-ascorbate. The enzyme activity observed with 6-amino-6-deoxy-L-ascorbate was very low in the microsomal hydroxylation system. The calculated Vmax. value was lower than that of L-ascorbate, suggesting a restriction of the access of this compound to the enzyme.
Human glucocerebrosidase catalyses transglucosylation between glucocerebroside and retinol
Authors: Vanderjagt D J , Fry D E , Glew R H
Published: August 2015

The basal activity of human placental glucocerebrosidase is elevated 16-fold by n-pentanol when assayed using p-nitrophenyl beta-D-glucopyranoside (pNPGlc) as the beta-glucosidase substrate. This enhancement of activity is the result of the formation of a transglucosylation product, n-pentyl beta-D-glucoside, in rate-determining competition with the hydrolytic reaction. The transglucosylation product accounts for approximately 80% of the reaction product generated in the presence of n-pentanol (0.18 M) when either glucocerebroside or pNPGlc was used as the substrate. This stimulatory effect can be increased an additional 3-fold by the inclusion of phosphatidylserine (20 micrograms/ml) or sodium taurodeoxycholate (0.3%, w/v) in the incubation medium. In the presence of retinol, glucocerebrosidase also catalyses the synthesis of a novel lipid glucoside, retinyl glucoside, when either glucocerebroside or pNPGlc serves as the substrate. The reaction product was identified as retinyl beta-D-glucoside, based on its susceptibility to hydrolysis by almond beta-D-glucosidase and the subsequent release of equimolar amounts of retinol and glucose. The rate of retinyl-beta-glucoside formation is dependent on the concentration of retinol in the incubation medium, reaching saturation at approximately 0.3 mM retinol. Retinyl beta-D-glucoside is a substrate for two broad-specificity mammalian beta-glucosidases, namely the cytosolic and membrane-associated beta-glucosidases of guinea pig liver. However, retinyl beta-D-glucoside is not hydrolysed by placental glucocerebrosidase. These data indicate that the glucocerebrosidase-catalysed transfer of glucose from glucocerebroside to natural endogenous lipid alcohols, followed by the action of a broad-specificity beta-glucosidase on the transglucosylation product, could provide mammals with an alternative pathway for the breakdown of glucocerebroside to glucose and ceramide.
Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity
Authors: Calder P C , Yaqoob P , Harvey D J , Watts A , Newsholme E A
Published: August 2015

The fatty acid compositions of the neutral lipid and phospholipid fractions of rat lymph node lymphocytes were characterized. Stimulation of rat lymphocytes with the T-cell mitogen concanavalin A resulted in significant changes in the fatty acid composition of both neutral lipids and phospholipids (a decrease in the proportions of stearic, linoleic and arachidonic acids and an increase in the proportion of oleic acid). Membrane fluidity was measured using nitroxide spin-label e.s.r., and increased during culture with concanavalin A. Culturing the lymphocytes in the absence of mitogen did not affect fatty acid composition or membrane fluidity. The uptake and fate of palmitic, oleic, linoleic and arachidonic acids were studied in detail; there was a time-dependent incorporation of each fatty acid into all lipid classes but each fatty acid had a characteristic fate. Palmitic and arachidonic acids were incorporated principally into phospholipids whereas oleic and linoleic acids were incorporated in similar proportions into phospholipids and triacylglycerols. Oleic acid was incorporated mainly into phosphatidylcholine, palmitic and linoleic acids were incorporated equally into phosphatidylcholine and phosphatidylethanolamine, and arachidonic acid was incorporated mainly into phosphatidylethanolamine. Supplementation of the culture medium with particular fatty acids (myristic, palmitic, stearic, oleic, linoleic, alpha-linolenic, arachidonic, eicosapentaenoic or docosahexaenoic acid) led to enrichment of that fatty acid in both neutral lipids and phospholipids. This generated lymphocytes with phospholipids differing in saturated/unsaturated fatty acid ratio, degree of polyunsaturation, index of unsaturation and n - 6/n - 3 ratio. This method allowed the introduction into lymphocyte phospholipids of fatty acids not normally present (e.g. alpha-linolenic) or usually present in low proportions (eicosapentaenoic and docosahexaenoic). These three n - 3 polyunsaturated fatty acids replaced arachidonic acid in lymphocyte phospholipids. Fatty acid incorporation led to an alteration in lymphocyte membrane fluidity: palmitic and stearic acids decreased fluidity whereas the unsaturated fatty acids increased fluidity. It is proposed that the changes in lymphocyte phospholipid fatty acid composition and membrane fluidity brought about by culture in the presence of polyunsaturated fatty acids are responsible for the inhibition of lymphocyte functions caused by these fatty acids.
Refolding of denatured lactate dehydrogenase by Escherichia coli ribosomes
Authors: Chattopadhyay S , Das B , Bera A K , Dasgupta D , Dasgupta C
Published: August 2015

Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.
Stimulation of glucose uptake by insulin-like growth factor II in human muscle is not mediated by the insulin-like growth factor II/mannose 6-phosphate receptor
Authors: Burguera B , Elton C W , Caro J F , Tapscott E B , Pories W J , Dimarchi R , Sakano K , Dohm G L
Published: August 2015

Although the growth-promoting effects of insulin-like growth factor II (IGF-II) have been intensively studied, the acute actions of this hormone on glucose metabolism have been less well evaluated, especially in skeletal muscle of humans. We and other groups have shown that IGFs reduce glycaemic levels in humans and stimulate glucose uptake in rat muscle. The purpose of the present study was to evaluate the effect of IGF-II on glucose transport in muscle of normal and obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM), as well as to identify the receptor responsible for this action. 2-Deoxyglucose transport was determined in vitro using a muscle-fibre strip preparation. IGF-II were investigated in biopsy material of rectus abdominus muscle taken from lean and obese patients and obese patients with NIDDM at the time of surgery. In the lean group, IGF-II (100 nM) stimulated glucose transport 2.1-fold, which was slightly less than stimulation by insulin (2.8-fold) at the same concentration. Binding of IGF-II was approx. 25% of that of insulin at 1 nM concentrations of both hormones. Obesity with or without NIDDM significantly reduced IGF-II-stimulated glucose uptake compared with the lean group. In order to explore which receptor mediated the IGF-II effect, we compared glucose uptake induced by IGF-II and two IGF-II analogues: [Leu27]IGF-II, with high affinity for the IGF-II/Man 6-P receptor but markedly reduced affinity for the IGF-I and insulin receptors, and [Arg54,Arg55]IGF-II was similar to that of IGF-II, whereas [Leu27]IGF-II had a very diminished effect. Results show that IGF-II is capable of stimulating muscle glucose uptake in lean but not in obese subjects and this effect seems not to be mediated via an IGF-II/Man 6-P receptor.
M1 protein and protein H: IgGFc- and albumin-binding streptococcal surface proteins encoded by adjacent genes
Authors: Åkesson P , Schmidt K H , Cooney J , Björck L
Published: August 2015

M1 protein and Protein H are surface proteins simultaneously present at the surface of certain strains of Streptococcus pyogenes, important pathogenic bacteria in humans. The present study concerns the structure, protein-binding properties and relationship between these two molecules. The gene encoding M1 protein (emm1) was found immediately upstream of the Protein H gene (sph). Both genes were preceded by a promoter region. Comparison of the sequences revealed a high degree of similarity in the signal peptides, the C repeats located in the central parts of the molecules and in the C-terminal cell-wall-attached regions, whereas the N-terminal sequences showed no significant similarity. Protein H has affinity for the Fc region of IgG antibodies. Also M1 protein, isolated from streptococcal culture supernatants or from Escherichia coli expressing emm1, was found to bind human IgGFc. When tested against polyclonal IgG from eight other mammalian species, M1 protein and Protein H both showed affinity for baboon, rabbit and pig IgG. M1 protein also reacted with guinea-pig IgG, whereas both streptococcal proteins were negative in binding experiments with rat, mouse, bovine and horse IgG. The two proteins were also tested against other members of the immunoglobulin super family: human IgM, IgA, IgD, IgE, beta 2-microglobulin, and major histocompatibility complex (MHC) class-I and class-II antigens. M1 protein showed no affinity for any of these molecules whereas Protein H reacted with MHC class-II antigens. M1 protein is known to bind albumin and fibrinogen also. The binding sites for these two plasma proteins and for IgGFc were mapped to different sites on M1 protein. Thus albumin bound to the C repeats and IgGFc to a region (S) immediately N-terminal of the C repeats. Finally, fibrinogen bound further towards the N-terminus but close to the IgGFc-binding site. On the fibrinogen molecule, fragment D was found to mediate binding to M1 protein. The IgGFc-binding region of M1 protein showed no similarity to that of Protein H. Still, competitive binding experiments demonstrated that the two streptococcal proteins bound to overlapping sites on IgGFc.
Effect of increasing intravesicular pH on nitrite production and leishmanicidal activity of activated macrophages
Authors: Buchmüller-Rouiller Y , Corradin S B , Smith J , Mauël J
Published: August 2015

We examined the effect of bafilomycin A1 (BAF), an inhibitor of vacuolar-type H(+)-ATPases, on macrophages activation (measured as increased nitrite production and leishmanicidal activity) induced by interferon gamma alone or together with lipopolysaccharide or tumour necrosis factor alpha. BAF increased intravesicular pH and enhanced nitrite release by activated macrophages; however, the NO concentration necessary to kill parasites was higher in BAF-exposed than control macrophages, suggesting that microbicidal nitrogen derivatives were less active at alkaline pH. Antibody to tumour necrosis factor alpha inhibited BAF-induced nitrite production in interferon-activated cultures. To determine if enhanced NO synthesis was related to vesicular alkalinization, macrophages were incubated with the lysosomotropic bases NH4Cl and methylamine. These agents also increased intravesicular pH and nitrite production. Nitrite production was correlated with enhanced NO synthase activity in cytosolic extracts of the activated cells.
Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation
Authors: Chen H M , Ford C , Reilly P J
Published: August 2015

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.
Threonine-497 is a critical site for permissive activation of protein kinase Cα
Authors: Cazaubon S , Bornancin F , Parker P J
Published: August 2015

Phosphorylation of the region containing Thr-494, Thr-495 and Thr-497, present in the catalytic domain of protein kinase C alpha (PKC alpha), is a preliminary event necessary for subsequent PKC activation [Cazaubon and Parker (1993) J. Biol. Chem. 268, 17559-17563]. To define the essential residues in this region, various combinations of alanine substitutions for threonine residues 494, 495 and 497 have been tested. These mutations yielded expressed polypeptides of 76 and 80 kDa in ratios that vary from 100% 80 kDa (wild-type kinase, active) to 100% 76 kDa (AAA mutant, inactive) with the hierarchy being wild-type PKC alpha (TTT), ATT, AAT, TTA, ATA, TAA, AAA (the nomenclature indicates the location of alanine residues substituted for Thr-494, Thr-495 and Thr-497 respectively). Only the mutants retaining Thr-497 displayed kinase activity in vitro. The results overall indicate that Thr-497 plays the dominant role in the regulation of PKC alpha activity but that in the wild-type protein, Thr-495 may also be important. Consistent with the need for phosphorylation in this region, an intrinsically active PKC alpha could be produced in bacteria by exchanging Thr-495 for a glutamic acid residue.
Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms
Authors: Mezna M , Ahmad T , Chettibi S , Drainas D , Lawrence A J
Published: August 2015

The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface.
Dynamics of Ca2+ and guanosine 5′-[γ-thio]triphosphate action on insulin secretion from α-toxin-permeabilized HIT-T15 cells
Authors: Jonas J C , Li G , Palmer M , Weller U , Wollheim C B
Published: August 2015

The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond to Ca2+ after prolonged perfusion at low Ca2+ (run-down), nor to depletion of a particular secretory-granule pool. It was rather due to desensitization of the secretory machinery to Ca2+ that was not reversed by selective inhibition of the Ca2+/calmodulin-dependent kinase II with KN-62. However, an intermediate Ca2+ concentration (2 microM) increased insulin secretion to stable level without causing any desensitization. Imposed oscillations of Ca2+ (0.1-10 microM) produced phasic oscillations of insulin secretion, but did not prevent desensitization to Ca2+. Poorly hydrolysable GTP analogues increased insulin secretion at low Ca2+, whereas they strongly inhibited Ca(2+)-induced insulin secretion. By contrast, GTP did not affect basal secretion, and slightly increased Ca(2+)-evoked secretion. These results indicate the following. (1) Oscillations of insulin secretion are tightly coupled to cytosolic Ca2+ oscillations. (2) Oscillations of Ca2+ do not prevent high-Ca(2+)-induced desensitization to Ca2+; this result does not support the idea of a greater efficiency of oscillations compared with sustained Ca2+ rises in triggering exocytosis. (3) Activation of G-proteins modulates exocytosis in a bimodal manner.
Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation
Authors: Gardner R A , Travers M T , Barber M C , Miller W R , Clegg R A
Published: August 2015

‘Expressed’ and ‘total’ activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in ‘total’ PK-A activity. The ‘expressed’ activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel ‘total’ PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.
Biochemical properties of recombinant human β-glucuronidase synthesized in baby hamster kidney cells
Authors: Gehrmann M C , Opper M , Sedlacek H H , Bosslet K , Czech J
Published: August 2015

The cDNA sequence encoding human beta-glucuronidase [Oshima, Kyle, Miller, Hoffmann, Powell, Grubb, Sly, Troplak, Guise and Gravel (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 685-689] was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant in one step by use of immunoaffinity chromatography, the biochemical properties of the enzyme were examined. With a pH optimum of 4.0, a Km of 1.3 mM and thermal stability up to 68 degrees C, this protein has characteristics very similar to those described for beta-glucuronidase from human placenta [Brot, Bell and Sly (1978) Biochemistry 17, 385-391. However, the recombinant product has several structural properties not previously reported for beta-glucuronidase isolated from natural sources. First, recombinant beta-glucuronidase is synthesized as a tetramer consisting of two disulphide-linked dimers. As can be inferred from the cDNA sequence, the enzyme possesses five cysteine residues after cleavage of the signal peptide. By introducing a C-terminal truncation, we eliminated the last cysteine at position 644. In the mutant, covalent linkage between two monomers is no longer observed, indicating that Cys-644 is involved in intermolecular disulphide-bond formation. The functional role of the disulphide bond remains elusive, as it was shown that (i) intracellular transport of the mutant is not impaired and (ii) it is still able to form an enzymically active tetramer. A second feature that has not previously been observed for beta-glucuronidase from any origin is the existence of two enzymically active species for recombinant beta-glucuronidase, when examined by gel filtration on a TSK 3000 column. With apparent molecular masses of 380 kDa and 190 kDa we propose that they represent tetramers and dimers respectively. Partial N-terminal sequencing and electrophoresis under denaturing conditions revealed that the dimers consist of subunits that have been proteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall protein backbone as well as its activity are resistant to a number of proteases. Only the C-terminal portion is susceptible to protease action, and the disulphide-linked form is readily converted into non-disulphide-bonded subunits. Pulse-chase analysis shows that human beta-glucuronidase remaining intracellular in BHK cells after synthesis undergoes a similar proteolytic processing event, i.e. a reduction in mass of 3-4 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)
Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt
Authors: Kass G E , Webb D L , Chow S C , Llopis J , Berggren P O
Published: August 2015

In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.
Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c
Authors: Schejter A , Koshy T I , Luntz T L , Sanishvili R , Vig I , Margoliash E
Published: August 2015

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.
Synthesis and secretion of mucin by the human colonic tumour cell line LS180
Authors: McCool D J , Forstner J F , Forstner G G
Published: August 2015

Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [3H]threonine or [3H]glucosamine and chased in complete media without radiolabel for various lengths of time. From cell and media extracts obtained at each time point, mucin proteins were immunoprecipitated with specific anti-mucin antibodies and analysed by SDS/PAGE and fluorography. At short labelling times with [3H]threonine, without chase, a monomeric thiol-reduction-resistant mucin precursor of apparent molecular mass > 670 kDa was identified. The precursor, in contrast to oligomeric species, was not labelled by [3H]glucosamine but exhibited binding to Vicia villosa isolectin B4, suggesting the presence of some core GalNAc residues. Treatment with tunicamycin to inhibit N-glycosylation had no effect on the apparent mass of the precursor. Identity of the mucin antigen with MUC2 mucin was established by immunoprecipitation with antibodies specific for a MUC2 tandem repeat and C-terminal regions. With increasing chase time the precursor was replaced by thiol-reduction-sensitive mucin oligomers that reached peak intracellular radiolabelling with [3H]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [3H]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [3H]glucosamine incorporation, sialylation and baseline (non-regulated) mucin secretion without affecting initial [3H]threonine incorporation or oligomerization. Oligomerization and Golgi transport are therefore essential early steps in MUC2 mucin secretion. Oligomerization may follow some core O-glycosylation with GalNAc, but precedes elongation of oligosaccharide chains.
Application of simultaneous spleen and liver perfusion to the study of reverse cholesterol transport
Authors: Mindham M A , Mayes P A
Published: August 2015

1. A new method to isolate and perfuse the rat spleen and liver simultaneously with a common blood perfusate at high haematocrit was developed. The spleen was pre-labelled with [3H]cholesterol, enabling reverse cholesterol transport from an extrahepatic tissue to the blood and thence to the liver and bile to be studied in a single preparation in vitro. 2. The presence of the liver significantly increased the release of [3H]cholesterol from the spleen by 15%, compared with experiments where the spleen was perfused alone. 3. There was a substantial release of [3H]cholesterol and cholesterol mass from the spleen to serum lipoproteins, the majority (80%) to high-density lipoprotein (HDL), in which cholesteryl ester accumulated. 4. The HDL subfractions HDL2 and HDL3 (d 1.085-1.250) were most important for removal of cholesterol from the spleen, whereas HDL1 and HDL2 (d 1.050-1.125) were important for delivery of cholesterol to the liver, a net uptake of cholesteryl ester occurring only from these fractions. 5. Approximately half of the [3H]cholesterol released by the spleen was recovered in erythrocytes. Also, in experiments utilizing a lipoprotein-free perfusate containing erythrocytes, a substantial quantity of [3H]cholesterol was transported and/or exchanged into the liver and bile, indicating that erythrocytes play an important role in the equilibration of unesterified cholesterol between the tissues.
Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans
Authors: Moreau A , Roberge M , Manin C , Shareck F , Kluepfel D , Morosoli R
Published: August 2015

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.
Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: effects of retinoic acid and differentiation
Authors: Antras-Ferry J , Franckhauser S , Robin D , Robin P , Granner D K , Forest C
Published: August 2015

3T3-F442A adipocytes express the gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK). Retinoic acid (RA) caused a 5-fold induction of PEPCK mRNA within 6 h in these cells with a half-maximal effective concentration of approximately 75 microM. This effect was independent of cycloheximide and inhibited by actinomycin D. In vitro run-on experiments using isolated nuclei confirmed that the RA-induced increase was mainly due to an increased rate of transcription of the gene. Stable transfectants bearing either the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) or -600 to +69 fused to CAT (pPL9-CAT) were used to study PEPCK gene regulation during differentiation. The same transfected cells were used to analyse the RA effect. Preadipocytes containing pPL1-CAT expressed a much lower level of CAT activity than did adipocytes. pPL9-CAT was not expressed in either preadipocytes or adipocytes. RA induced the expression of CAT activity in preadipocytes and adipocytes transfected with pPL1-CAT, but had no effect in cells transfected with pPL9-CAT. These results suggest that one or more DNA sequences located between -2100 and -600 bp of the PEPCK promoter is required for adipocyte-specific expression of this gene. RA action is independent of the state of differentiation and appears to require different elements in fat cells from those required in liver.
Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor
Authors: Feener E P , Shiba T , Hu K Q , Wilden P A , White M F , King G L
Published: August 2015

Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR.
Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated
Authors: Teerink H , Kasperaitis M A M , De Moor C H , Voorma H O , Thomas A A M
Published: August 2015

The majority of cellular mRNAs have relatively short and unstructured 5′ untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5′ UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5′ UTR. We investigated the translational efficiency in vitro of this 5′ UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5′ UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IGF-II 5′ UTR RNA construct. No translational stimulation was found when cell extract from differentiated P19 EC cells was added. In contrast with the beta-globin 5′ UTR, translation initiation on the IGF-II 5′ UTR was not dependent on the presence of a cap structure. The results imply that only in undifferentiated P19 EC cells and not in their differentiated derivatives is a factor present that specifically stimulates IGF-II RNA translation, thereby suggesting translational regulation of IGF-II production during early embryonic development. A mechanism for translation initiation on the 5′ UTR of IGF-II is discussed.