bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells
Authors: van der Ende A , du Maine A , Schwartz A L , Strous G J
Published: August 2015

BeWo human choriocarcinoma cells normally grow as cytotrophoblast cells. However, in the presence of 100 microM-forskolin or 5 mM-theophylline, these cells form syncytia similar to morphologically well differentiated syncytiotrophoblasts. We have examined the effect of syncytia formation on transferrin-receptor activity and recycling. Although cellular proliferation stops upon growth in the presence of forskolin or theophylline, the number of cell-surface transferrin-receptors unexpectedly increased 2-fold, whereas the total cellular number increased at most 15%. The rate of biosynthesis of the transferrin receptor as well as class I MHC glycoprotein did not change measurably during syncytium formation. The biosynthesis of human chorionic gonadotropin increased 35-fold after 30 h of growth in the presence of theophylline. The redistribution of the transferrin receptor in syncytia is maintained by a decreased rate constant of endocytosis (0.141 min-1 compared with 0.231 min-1 for control cells) and an increased rate constant of externalization (0.122 min-1 compared with 0.060 min-1 for control cells). These altered rates of endocytosis and externalization resulted in an increased rate of iron accumulation in the syncytia. Furthermore, the recycling time of the transferrin receptor decreased in cells grown in the presence of theophylline (14.6 min compared with 21.2 min in control cells).
Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing
Authors: Johnson J A , Neal T L , Collins J H , Siegel F L
Published: August 2015

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.
Ultraviolet B radiation induction of ornithine decarboxylase gene expression in mouse epidermis
Authors: Rosen C F , Gajic D , Jia Q , Drucker D J
Published: August 2015

The cellular effects of u.v. radiation have been studied by using a hairless-mouse model in vivo. U.v. B radiation (u.v.B) induced the activity of the enzyme ornithine decarboxylase (ODC) in mouse epidermis. Maximal induction was noted after radiation with 90 mJ/cm2, and increased ODC activity was first detected 2 h after u.v.B exposure. U.v.B. also induced the expression of the ODC gene in a time- and dose-dependent manner, but did not induce the levels of actin mRNA transcripts. Cycloheximide treatment did not alter basal levels of ODC mRNA transcripts and had no effect on the u.v.B induction of ODC-gene expression. The results of these experiments demonstrate that u.v.B radiation induces both the expression of the ODC gene and the activity of the enzyme, and provides a useful ‘in vivo’ paradigm for the analysis of the molecular effects of u.v.B radiation.
Concurrent up-regulation of guanine-nucleotide-binding proteins Gi1α, Gi2α and Gi3α in adipocytes of hypothyroid rats
Authors: Milligan G , Saggerson E D
Published: August 2015

Rat white adipocytes express three distinct ‘Gi-like’ guanine-nucleotide-binding proteins (G-proteins) [Mitchell, Griffiths, Saggerson, Houslay, Knowler & Milligan (1989) Biochem. J. 262, 403-408]. We have previously noted elevated levels of Gi in membranes of adipocytes from hypothyroid rats [Milligan, Spiegel, Unson & Saggerson (1987) Biochem. J. 247, 223-227]. Using a series of anti-peptide antisera able to discriminate between the individual gene products we have examined levels of each Gi-like G-protein in adipocyte membranes of hypothyroid rats compared with euthyroid controls. We demonstrate that up-regulation of Gi in adipocytes of hypothyroid rats is not restricted to a single subtype of Gi but that each of Gi1 alpha, Gi2 alpha and Gi3 alpha is present at markedly higher levels compared with euthyroid animals. In contrast, levels of both the 45 and 42 kDa forms of Gs alpha were not altered substantially in the hypothyroid state.
Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action
Authors: Borthwick A C , Edgell N J , Denton R M
Published: August 2015

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide (‘I’-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.
Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region
Authors: Chakravarty R , Rong X H , Rice R H
Published: August 2015

The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.
Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification
Authors: Kanaya S , Kimura S , Katsuda C , Ikehara M
Published: August 2015

The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.
Purification, properties and partial amino acid sequence of the blood-group-A-gene-associated α-3-N-acetylgalactosaminyltransferase from human gut mucosal tissue
Authors: Navaratnam N , Findlay J B C , Keen J N , Watkins W M
Published: August 2015

An alpha-3-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-N-acetylgalactosamine to H-active structures to form A determinants was purified to homogeneity from human gut mucosal tissue of blood-group-A subjects. The mucosa was homogenized, then treated with Triton X-100, and the solubilized enzyme was purified by affinity chromatography on UDP-hexanolamine-agarose and octyl-Sepharose CL-4B. Enzyme activity was recovered in 44% yield with a specific activity of approx. 7 mumol/min per mg. The only effective acceptor substrates for the transferase were those containing a subterminal β-galactosyl residue substituted at the O-2 position with L-fucose. The purified enzyme had a weak capacity to transfer D-galactose from UDP-D-galactose to similar acceptors to make blood-group-B determinants. H.p.l.c. and SDS/PAGE analysis indicated an Mr of 40,000 for the purified enzyme. For the first time a partial amino acid sequence Xaa-Ser-Leu-Pro-Arg-Met-Val-Tyr-Pro-Gln-Ile-Ser?-Val-Leu was obtained for the N-terminal region of the soluble alpha-3-N-acetylgalactosaminyltransferase.
The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes
Authors: Blackmore R S , Gadsby P M A , Greenwood C , Thomson A J
Published: August 2015

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the ‘non-reducing’ haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.
Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase
Authors: Blackmore R S , Brittain T , Gadsby P M A , Greenwood C , Thomson A J
Published: August 2015

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed ‘resting’ and ‘redox-cycled’. The nitrite reductase as initially isolated, termed ‘resting’, has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed ‘redox-cycled’, possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.
Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation
Authors: Linnekin D , Farrar W L
Published: August 2015

To elucidate the rapid events in signal transduction of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL 3), we examined phosphorylation of proteins on both serine and tyrosine residues in a cytokine-stimulated human myeloid cell line. We found increases in tyrosine phosphorylation within 30 s of stimulation with GM-CSF or IL 3, with peak responses occurring within 2 min. IL 3 and GM-CSF also induced serine phosphorylation, though 10 min of stimulation was required for maximum phosphate incorporation. Interestingly, both IL 3 and GM-CSF stimulated phosphate incorporation in identical substrates, a 68 kDa seryl-phosphoprotein (p68) and a 140 kDa tyrosyl-phosphoprotein (p140). Treatment of AML 193 cells with phorbol myristate acetate resulted in serine phosphorylation of p68; however, p140 was not phosphorylated on tyrosine. Depletion of protein kinase C isoenzymes with high concentrations of phorbol myristate acetate resulted in p68 phosphorylation, which was not further increased by IL 3 or GM-CSF. In contrast, cytokine-induced phosphorylation on tyrosine of p140 was observed after protein kinase C depletion. These data demonstrate the co-ordinate yet independent serine and tyrosine phosphorylation in IL 3- and GM-CSF-treated human myeloid cells, and thus suggest a common set of protein kinases stimulated by each separate ligand.
Glutathione transferase isoenzymes from human prostate
Authors: Di Ilio C , Aceto A , Bucciarelli T , Angelucci S , Felaco M , Grilli A , Federici G
Published: August 2015

By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.
Temperature modulation of oxygen transport in a diving mammal (Balaenoptera acutorostrata)
Authors: Brix O , Condò S G , Bardgard A , Tavazzi B , Giardina B
Published: August 2015

The functional properties of haemoglobin from the Lesser Rorqual whale (Balaenoptera acutorostrata) have been characterized as a function of the heterotropic effector concentrations and temperature. The results obtained suggest the existence of sophisticated modulation mechanisms based on the interplay of organic phosphates, carbon dioxide, lactate and temperature. These, together with the very small apparent heat of oxygenation (delta H) of oxygen binding, have been physiologically interpreted on the basis of the specific metabolic needs of this diving mammal.
Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium
Authors: Macrae M B , Davidson J S , Millar R P , van der Merwe P A
Published: August 2015

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.
Interferon-induced transcriptional and post-transcriptional modulation of factor H and C4 binding-protein synthesis in human monocytes
Authors: Lappin D F , Whaley K
Published: August 2015

Cultured human monocytes synthesize factor H (H) and C4 binding protein (C4-bp), as assessed by measuring their presence in culture fluids, and demonstrating the presence of their corresponding mRNAs in total monocyte RNA by Northern blot analysis and by nuclear run-on experiments. H mRNA (4.3 kb and 1.8 kb) could only be detected when cycloheximide (2.5 micrograms/ml) was present in monocyte culture fluid. Recombinant interferon-gamma (IFN-gamma) and lymphoblastoid interferon-alpha (IFN-alpha) produce dose-related increases in the synthesis of H and C4-bp and in the abundance of C4-bp mRNA (2.5 kb). Increased abundance of H mRNA was also seen when cycloheximide (2.5 micrograms/ml) was present in the cultures. Both cytokines increased the transcription rates of the H and C4-bp genes. These changes in transcription were rapid, with significant increases being observed within 30 min of exposure. Following the removal of the cytokines from the cultures the transcription rates of both genes returned to control levels within 4 h. The effects of combining IFN-alpha and IFN-gamma on H and C4-bp transcription rates, mRNA abundances and protein secretion rates were not quantitatively additive. The half-life of H mRNA in monocytes was 15 min, whereas that of C4-bp mRNA was 2 h 45 min. Neither half-life was altered by IFN-gamma.
Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells
Authors: Nagy L , Freeman D A
Published: August 2015

The present studies characterize the turnover of plasma membrane cholesterol in MA-10 Leydig tumour cells. Plasma membrane cholesterol of MA-10 cells was slowly internalized and converted into cholesteryl ester. Low-density lipoprotein (LDL) stimulated, in a dose- and time-dependent fashion, plasma membrane cholesterol conversion into intracellular esters. Stimulation of membrane internalization was not simply the consequence of accelerated uptake of membrane with LDL, since binding and internalization of epidermal growth factor and transferrin had no effect on turnover of plasma membrane cholesterol. The protein of LDL is unimportant as well, since delipidated LDL had no effect on membrane turnover. The action of LDL on cholesterol turnover was explained entirely by its contribution to cholesteryl ester stores. The degree of plasma membrane cholesterol internalization and esterification was directly proportional to the size of cellular ester stores.
Application of electrical analogues for control analysis of simple metabolic pathways
Authors: Sen A K
Published: August 2015

I have used electrical analogues for calculating the Flux Control Coefficients of metabolic pathways. An analogue circuit consists of resistances that are connected in series (or parallel) with a voltage (or current) source. In constructing the analogues, each of the enzymes in the pathway is associated with a resistance whose magnitude depends on the Elasticity Coefficients of the enzymes. These circuits can be designed in a heuristic fashion directly from the configuration of the pathway, without the necessity of writing down the governing equations with the use of Summation and Connectivity Theorems. The Flux Control Coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's Law. Results are given for (a) a simple linear pathway without feedback or feedforward regulation, and (b) a linear pathway with feedback inhibition. The analogue circuits are also convenient for assessing the relative importance of the various enzymes in flux control, and for simplifying the structure of a given pathway.
Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland
Authors: Madon R J , Martin S , Davies A , Fawcett H A C , Flint D J , Baldwin S A
Published: August 2015

Plasma membrane- and Golgi vesicle-enriched membrane fractions were prepared from day-10 lactating rat mammary glands. Each fraction was found to contain a single set of D-glucose-inhibitable cytochalasin B-binding sites: plasma membranes and Golgi vesicles bound 20 +/- 2 and 53 +/- 4 pmol of cytochalasin/mg of membrane protein (means +/- S.E.M.), with dissociation constants of 259 +/- 47 and 520 +/- 47 nM respectively. Anti-peptide antibodies against the C-terminal region (residues 477-492) of the rat brain/human erythrocyte glucose transporter labelled a sharp band of apparent Mr 50,000 on Western blots of both fractions. Treatment with endoglycosidase F before blotting decreased the apparent Mr of this band to 38,000, indicating that it corresponded to a glycoprotein. Confirmation that this immunologically cross-reactive band was a glucose transporter was provided by the demonstration that it could be photoaffinity-labelled, in a D-glucose-sensitive fashion, with cytochalasin B. Quantitative Western blotting studies yielded values of 28 +/- 5 and 23 +/- 3 pmol of immunologically cross-reactive glucose transporters/mg of membrane protein in the plasma membrane and Golgi vesicle fractions respectively. From comparison with the concentration of cytochalasin B-binding sites, it is concluded that a protein homologous to the rat brain glucose transporter constitutes the major glucose transport species in the plasma membranes of mammary gland epithelial cells. Glucose transporters are also found in the Golgi membranes of these cells, at least half of them being similar, if not identical, to the transporters of the plasma membrane. However, their function in this location remains unclear.
Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules
Authors: Thévenod F , Gasser K W , Hopfer U
Published: August 2015

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5′-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5′-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5′-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5′-[beta-thio]diphosphate (GDP[S]). Adenosine 5′-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.
Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues
Authors: Larrabee M G
Published: August 2015

A classic equation that has frequently been used to estimate the fraction of glucose metabolized by the pentose phosphate pathway, using 14CO2 data, is more simply re-derived with careful consideration of the assumptions involved and the conditions under which it is applicable. The equation is shown to be unreliable for non-homogeneous tissues, depending on the fraction of triose phosphate converted to CO2. The formula in question is as follows: ([1]CO2/G-[6]CO2/G)/(1-[6]CO2/G) = 3Fmet./(1 + 2Fmet.) where [1]CO2 and [6]CO2 are output rates of carbons 1 and 6 of glucose respectively to CO2, G is the rate of glucose uptake and Fmet. is the fraction of the glucose that is metabolized to CO2 and triose phosphate by the pentose phosphate pathway, allowing for recycling of an appropriate fraction of the fructose-6-phosphate produced by the pathway. This analysis illustrates the importance of suitably testing any equation that assumes homogeneity before application to non-homogeneous tissues.
Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region
Authors: Nikrad P V , Pearlstone J R , Carpenter M R , Lemieux R U , Smillie L B
Published: August 2015

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.
Arachidonic acid-induced calcium influx in human platelets. Comparison with the effect of thrombin
Authors: Alonso M T , Sanchez A , Garcia-Sancho J
Published: August 2015

The effects of arachidonic acid and thrombin on calcium movements have been studied in fura-2-loaded platelets by a procedure which allows simultaneous monitoring of the uptake of manganese, a calcium surrogate for Ca2+ channels, and the release of Ca2+ from intracellular stores. Arachidonic acid induced both Ca2+ (Mn2+) entry through the plasma membrane and Ca2+ release from the intracellular stores. The release of Ca2+ was prevented by cyclo-oxygenase inhibitors and mimicked by the prostaglandin H2/thromboxane A2 receptor agonist U46619. Ca2+ (Mn2+) entry required higher concentrations of arachidonic acid and was not prevented by either cyclo-oxygenase or lipoxygenase inhibitors. Several polyunsaturated fatty acids reproduced the effect of arachidonic acid on Ca2+ (Mn2+) entry, but higher concentrations were required. The effects of maximal concentrations of arachidonic acid and thrombin on the uptake of Mn2+ were not additive. Both agonists induced the entry of Ca2+, Mn2+, Co2+ and Ba2+, but not Ni2+, which, in addition, blocked the entry of the other divalent cations. However, arachidonic acid, but not thrombin, increased a Ni2(+)-sensitive permeability to Mg2+. The effect of thrombin but not that of arachidonic acid was prevented either by pretreatment with phorbol ester or by an increase in cyclic-AMP levels. Arachidonic acid also accelerated the uptake of Mn2+ by human neutrophils, rat thymocytes and Ehrlich ascites-tumour cells.
Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function
Authors: Andersson C , Morgenstern R
Published: August 2015

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.
A requirement for Zn2+ for the induction of thymidine kinase but not ornithine decarboxylase in 3T3 cells stimulated from quiescence
Authors: Chesters J K , Petrie L , Travis A J
Published: August 2015

In 3T3 cells stimulated from quiescence by serum, impaired thymidine incorporation caused by inadequate supply of Zn2+ was associated with both decreased thymidine kinase activity and a comparable decrease in its mRNA concentration. In contrast, the amount of mRNA for ribosomal protein S6 was not affected, nor was the earlier increase in the activity of ornithine decarboxylase.
Structures of the asparagine-linked oligosaccharides of guinea-pig factor B of the alternative complement pathway
Authors: Mizuochi T , Hamako J , Titani K , Matsushita M , Okada H
Published: August 2015

This paper describes the structures of the asparagine-linked oligosaccharides of two forms of guinea-pig Factor B of the alternative complement pathway with different Mr values. Oligosaccharides were quantitatively liberated from both glycoproteins by hydrazinolysis, fractionated by paper electrophoresis and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. Both glycoproteins were shown to have the same biantennary complex-type oligosaccharides but it is suggested that they contain different numbers of oligosaccharide chains.
Hepatic iodothyronine 5′-deiodinase. The role of selenium
Authors: Arthur J R , Nicol F , Beckett G J
Published: August 2015

Selenium (Se) deficiency decreased by 8-fold the activity of type 1 iodothyronine 5′-deiodinase (ID-I) in hepatic microsomal fractions from rats. Solubilized hepatic microsomes from rats injected with 75Se-labelled Na2SeO3 4 days before killing were found by chromatography on agarose gels to contain a 75Se-containing fraction with ID-I activity. PAGE of this fraction under reducing conditions, followed by autoradiography, revealed a single 75Se-containing protein (Mr 27,400 +/- 300). This protein could also be labelled with 125I-bromoacetyl reverse tri-iodothyronine, an affinity label for ID-I. The results suggest that hepatic ID-I is a selenoprotein or has an Se-containing subunit essential for activity.
Acetazolamide-insensitive carbonic anhydrase activities in liver and tonic skeletal muscle of adult male rats with streptozotocin-induced diabetes mellitus
Authors: Moynihan J B , Ennis S
Published: August 2015

One of the four discrete isoenzymes of carbonic anhydrase hitherto characterized, CA III, has the lowest turnover rate and the greatest resistance to inhibition by sulphonamides. Streptozotocin-induced diabetes mellitus resulted in a reduction in acetazolamide-resistant activity of carbonic anhydrase in the liver, but not in tonic skeletal muscle, of adult male rats. The hepatic activity declined with apparent first-order kinetics [calculated rate constant (k) 0.089 day-1] to a minimum of approx. 6% of control values; the reduction in activity was moderated by administration of insulin.
Correction of abnormal small intestinal cytosolic protein kinase C activity in streptozotocin-induced diabetes by insulin therapy
Authors: Wali R K , Dudeja P K , Bolt M J G , Sitrin M D , Brasitus T A
Published: August 2015

Diabetes was induced in rats by administration of a single intraperitoneal injection of streptozotocin (50 mg/kg body wt). After 7 days, one group of diabetic animals was treated with insulin for an additional 5 days. Control, diabetic and diabetic + insulin rats were then killed, their distal small intestines were removed and the epithelial cells were examined and compared with respect to polyphosphoinositide turnover, total protein kinase C activity and cellular distribution, and 1,2-diacylglycerol mass and production. The results of these experiments demonstrated that, compared with their control counterparts, the intestines from diabetic rats had a decreased turnover of polyphosphoinositides, but an increase in 1,2-diacylglycerol mass which was a result, at least in part, of an increase in the synthesis of this lipid de novo. Total protein kinase C activity was decreased in the diabetic rats due to a decrease in cytosolic activity, with no significant change in particulate activity. Moreover, insulin administration for 5 days to diabetic animals did not affect their lowered intestinal polyphosphoinositide turnover, but did further accentuate their increased 1,2-diacylglycerol mass and synthesis de novo; this treatment also corrected total protein kinase C activity by increasing the cytosolic activity of this enzyme. These results indicate that signalling mechanisms involving polyphosphoinositides, 1,2-diacylglycerol and protein kinase C are abnormal in the intestines of diabetic rats and that some of these biochemical parameters can be modulated by insulin administration in vivo.
Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations
Authors: Morimoto B H , Brady J F , Atkinson D E
Published: August 2015

Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate.
Age-related thermal stability and susceptibility to proteolysis of rat bone collagen
Authors: Danielsen C C
Published: August 2015

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The ‘melting’ profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen ‘melted’ in the transition peak at 35.9 degrees C (pre-transition), and the ‘melting’ temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic ‘melting’ profile.
Increased association of ribosomes with myofibrils during the skeletal-muscle hypertrophy induced either by the β-adrenoceptor agonist clenbuterol or by tenotomy
Authors: Horne Z , Hesketh J
Published: August 2015

Ribosome distribution in skeletal-muscle myofibres was investigated by immunohistochemistry and microdensitometry by using anti-(60 S ribosomal subunit) antibodies. Administration of the beta-adrenoceptor agonist clenbuterol caused an increase in the staining of the myofibrillar region with this antibody relative to that found in the subsarcolemmal cytoplasm. A similar effect was observed during hypertrophy of the plantaris muscle following severance of the tendon to the gastrocnemius. The results suggest that increased association of ribosomes with the myofibrils occurs during muscle hypertrophy.
Comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in Acinetobacter calcoaceticus and from the TOL-plasmid-encoded toluene pathway in Pseudomonas putida. N-terminal amino acid sequences, amino acid compositions and immunological cross-reactions
Authors: Chalmers R M , Keen J N , Fewson C A
Published: August 2015

1. N-Terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus N.C.I.B. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53 and yeast K(+)-activated aldehyde dehydrogenase. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50161 (5 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby. West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273. 5. The extent of sequence similarity suggests that the benzyl alcohol dehydrogenases are related to each other and also to established members of the family of long-chain Zn2(+)-dependent alcohol dehydrogenases. Benzaldehyde dehydrogenase II from Acinetobacter appears to be related to the Pseudomonas TOL-plasmid-encoded benzaldehyde dehydrogenase. The yeast K(+)-activated aldehyde dehydrogenase has similarity of sequence with the mammalian liver cytoplasmic class of aldehyde dehydrogenases but not with any of the Acinetobacter or Pseudomonas enzymes. 2. Antisera were raised in rabbits against the three Acinetobacter enzymes and both of the Pseudomonas enzymes, and the extents of the cross-reactions were determined by immunoprecipitation assays with native antigens and by immunoblotting with SDS-denatured antigens. Cross-reactions were detected between the alcohol dehydrogenases and also among the aldehyde dehydrogenases. This confirms the interpretation of the N-terminal sequence comparisons and also indicates that benzaldehyde dehydrogenase I from Acinetobacter may be related to the other two benzaldehyde dehydrogenases. 3. The amino acid compositions of the Acinetobacter and the Pseudomonas enzymes were determined and the numbers of amino acid residues per subunit were calculated to be: benzyl alcohol dehydrogenase and TOL-plasmid-encoded benzyl alcohol dehydrogenase, 381; benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II, 525; TOL-plasmid-encoded benzaldehyde dehydrogenase, 538.