bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular-membrane characterization of [3H]ryanodine-binding sites in smooth muscle
Authors: Zhang Z D , Kwan C Y , Daniel E E
Published: August 2015

The plant alkaloid ryanodine, known to interact selectively with the intracellular Ca(2+)-release channel in skeletal and cardiac muscles, has been repeatedly reported to affect smooth-muscle contractile functions that are consistent with its intracellular action at the Ca(2+)-release channel sites. Direct evidence for the binding of [3H]ryanodine to smooth-muscle membranes is sparse. Following our recent detailed characterization of functional effects of ryanodine and a preliminary report on the presence of [3H]ryanodine binding sites in rat vas deferens smooth muscle, we now report in this study a detailed characterization of binding of [3H]ryanodine to smooth muscle at the subcellular-membrane level. The ryanodine receptor in rat vas deferens muscle layer is primarily of smooth-muscle origin and is localized at the subcellular membrane site that is consistent with its role as a Ca(2+)-release channel in the sarcoplasmic reticulum (SR). Ryanodine binding to its receptor is Ca(2+)-dependent, with half-maximal binding occurring within the physiologically relevant cytosolic Ca2+ concentration. It is also sensitive to many factors, including change in Mg2+ concentration, ionic strength and osmolarity across the membrane vesicles. Agents known to inhibit (Ruthenium Red, Mg2+) or enhance (caffeine, Na+, K+) the Ca(2+)-induced Ca2+ release also inhibit or enhance the binding of ryanodine. Quantitative differences in ryanodine receptors exist among smooth muscles and do not seem to parallel their SR contents. Results from the present study indicate both the need and the basis for future investigations of the functional role of the ryanodine receptor in different smooth muscles.
Effect of saposins on acid sphingomyelinase
Authors: Tayama M , Soeda S , Kishimoto Y , Martin B M , Callahan J W , Hiraiwa M , O'Brien J S
Published: August 2015

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes.
Kinetic behaviour of zymogen activation processes in the presence of an inhibitor
Authors: Varón R , Manjabacas M C , García-Moreno M , Valero E , Garcia-Canovas F
Published: August 2015

A global kinetic analysis of a general zymogen activation model, where not only the activating but also the activated enzyme suffer an irreversible inhibition is presented. A reaction in which the enzyme acts upon a substrate is coupled to monitor the process. In addition, we determined the corresponding kinetic equations for a number of particular cases of the general model studied. Finally, a kinetic data analysis and a procedure to discriminate among the different mechanisms considered, which are based on the kinetic equations obtained, are suggested.
Low-density lipoproteins are degraded in HepG2 cells with low efficiency
Authors: Lombardi P , Mulder M , de Wit E , van Berkel T J C , Frants R R , Havekes L M
Published: August 2015

In previous studies we have shown that in HepG2 cells, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor is only weakly down-regulated upon incubation of the cells with LDL. To explain this difference in down-regulation of the LDL-receptor activity, we studied simultaneously the intracellular processing of 125I-labelled LDL in both cell lines. Upon incubation of HepG2 cells with 125I-LDL, the appearance of degradation products started at 90 min, whereas in fibroblasts this lag time was only 30 min. The degradation efficiency (representing the ratio degradation/cell association of LDL) in HepG2 was less than 50% of that in fibroblasts up to 5h of incubation at 37 degrees C. The longer lag time and low efficiency of the degradation of LDL in HepG2 cells were independent of the cell density. Pulse-chase experiments indicated that the internalization rate of surface-bound LDL in HepG2 cells is similar to that of fibroblasts. Endosomal loading of 125I-LDL by incubation at 18 degrees C for 4.5 h, followed by a shift to 37 degrees C, resulted in degradation of LDL within 30 min in fibroblasts, whereas in HepG2 cells the lag time of the degradation was 90 min. In parallel experiments using subcellular fractionation by Percoll-gradient centrifugation of homogenized cells and 125I-tyramine-cellobiose-labelled LDL, we observed that in both cell types LDL is equally rapidly shifted from a low- to a high-density compartment (within 15 min), representing the endosomal and the late-endosomal plus lysosomal compartment respectively. We conclude that in HepG2 cells the cell-bound LDL, upon internalization, goes through the intracellular itinerary at the same rate as in fibroblasts, but that either the fusion between late endosomes and lysosomes or the lysosomal degradation itself is proceeding at a lower efficiency. A low degradation rate of LDL may contribute to explain the relatively weak down-regulation of the LDL-receptor activity in HepG2 cells by LDL.
Characterization of the bovine brain cytosolic phosphatidylinositol 3-kinase complex
Authors: Ruiz-Larrea F , Vicendo P , Yaish P , End P , Panayotou G , Fry M J , Morgan S J , Thompson A , Parker P J , Waterfield M D
Published: August 2015

Receptor-linked phosphatidylinositol (PtdIns) 3-kinase may generate a second-messenger signal. Here a large-scale purification of the bovine brain enzyme, based on methods developed by Morgan, Smith and Parker [(1990) Eur. J. Biochem. 191, 761-767] and Fry, Panayotou, Dhand, Ruiz-Larrea, Gout, Nguyen, Courtneidge and Waterfield [(1992) Biochem. J. 288, 383-393] is described. The purified enzyme is shown to be a heterodimer of 85 kDa and 110 kDa protein subunits (p85 and p110). Labelling with 5′-p-fluorosulphonylbenzoyladenosine shows that p110 contains an ATP-binding site and confers catalytic activity to the complex. The purified complex is known to be highly phosphorylated on both p85 alpha and p110 subunits, and dephosphorylation generates a deactivated complex, indicating that phosphorylation is an important covalent modification of the complex and may modulate PtdIns 3-kinase activity.
The G9a gene in the human major histocompatibility complex encodes a novel protein containing ankyrin-like repeats
Authors: Milner C M , Campbell R D
Published: August 2015

The class III region of the human major histocompatibility complex spans approx. 1.1 Mbp on the short arm of chromosome 6 and is known to contain at least 36 genes. The complete nucleotide sequence of a 3.4 kb mRNA from one of these genes, G9a (or BAT8), has been determined from cDNA and genomic DNA clones. The single-copy G9a gene encodes a protein product of 1001 amino acids with a predicted molecular mass of 111,518 Da. The C-terminal region (residues 730-999) of the G9a protein has been expressed in Escherichia coli as a fusion protein with the 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26). The fusion protein has been used to raise antisera which, in Western-blot analysis, cross-react specifically with an intracellular protein of approx. 98 kDa. The function of the G9a protein is unknown. However, comparison of the derived amino acid sequence of G9a with the protein databases has revealed interesting similarities with a number of other proteins. The C-terminal region of G9a is 35% identical with a 149 amino acid segment of the Drosophila trithorax protein. In addition the G9a protein has been shown to contain six contiguous copies of a 33-amino acid repeat. This repeat, originally identified in the Notch protein of Drosophila and known as the cdc10/SW16 or ANK repeat, is also found in a number of other human proteins and may be involved in intracellular protein-protein interactions.
Different modulatory effects of elevated cyclic AMP on cytosolic Ca2+ spikes induced by phenylephrine or vasopressin in single rat hepatocytes
Authors: Sanchez-Bueno A , Marrero I , Cobbold P H
Published: August 2015

We show here, by aequorin measurements in single isolated rat hepatocytes, that elevation of cyclic AMP, by dibutyryl cyclic AMP, forskolin or glucagon, has different effects on oscillations in cytosolic concentration of free Ca2+ (‘free Ca’) induced by phenylephrine or vasopressin. Elevated cyclic AMP does not itself induce free Ca oscillations, but enhances both the peak free Ca and the frequency of spikes induced by phenylephrine. In contrast, elevated cyclic AMP has no effect on peak free Ca of vasopressin-induced spikes, but markedly prolongs the falling phase, with the result that spiking frequency (peak to peak) falls, although the period between spikes of resting free Ca is usually decreased. The data provide another example of receptor-specific information being retained in the oscillator mechanism, with implications for models of the hepatocyte calcium oscillator.
Intracellular Ca2+ stores of rat cerebellum: heterogeneity within and distinction from endoplasmic reticulum
Authors: Nori A , Villa A , Podini P , Witcher D R , Volpe P
Published: August 2015

Rat cerebellum microsomes were subfractionated on isopycnic linear sucrose (20-42%)-density gradients. The distribution of endoplasmic reticulum (ER) markers (RNA, signal-sequence receptor alpha, calnexin, calreticulin, the immunoglobulin-binding protein Bip) and markers of intracellular rapidly exchanging Ca2+ stores [Ca2+ channels sensitive to either Ins(1,4,5)P3 or ryanodine) was investigated biochemically and immunologically. The comparison indicates that: (a) vesicles bearing the InsP3 receptor were separated from those bearing the ryanodine receptor; (b) ER markers, i.e. Bip, calnexin, signal-sequence receptor alpha, RNA, did not sediment as either InsP3 or ryanodine receptors did; (c) calreticulin, an intralumenal low-affinity high-capacity Ca(2+)-binding protein, had a widespread distribution, similar to that of Bip and calnexin, and was present in Purkinje, granule, Golgi and stellate neurons, as indicated by immunofluorescent labelling of cerebellum cortex cryosections. The present results show that the ER is not a homogeneous entity, and that Ca2+ stores are heterogeneous insofar as InsP3 receptors and ryanodine receptors are segregated, either to discrete intracellular organelles or to specialized ER subcompartments.
δ-Aminolaevulinate synthase expression in muscle after contractions and recovery
Authors: Takahashi M , McCurdy D T M , Essig D A , Hood D A
Published: August 2015

The synthesis of haem has been postulated to be a key regulatory step in muscle mitochondrial biogenesis. We examined the expression of delta-aminolaevulinate synthase (ALAs), the regulatory enzyme of haem metabolism, in 10 Hz electrically stimulated and non-stimulated control rat tibialis anterior (TA) muscle. ALAs activity and mRNA levels were measured at 0, 18 and 48 h of recovery after 3 h of acute stimulation, or after 7 days of stimulation (3 h/day). ALAs activity in control muscles averaged 7.8 +/- 0.8 nmol/h per g (n = 30). After 3 h of stimulation and during recovery, no change in ALAs activity occurred. ALAs mRNA during the same time was unchanged except at 48 h of recovery, when it increased 1.3-fold above control (P < 0.05). After 7 days of stimulation, ALAs activity was unchanged at 0 h, but increased at 18 and 48 h of recovery to 2.0- and 1.8-fold above control (P < 0.05). ALAs mRNA was also increased, but to a level averaging 1.6-fold above control (P < 0.05) at all times, indicating an increased mRNA stability or synthesis. No change in the haem-containing enzyme cytochrome c oxidase (CYTOX) activity occurred after 3 h of stimulation in the red section of the TA. After 7 days of stimulation, the increase in CYTOX activity averaged 1.7-fold above control (P < 0.05) at all times. Thus the induction of ALAs during recovery after 7 days was regulated by factors which not only change ALAs mRNA content, but which also affect ALAs mRNA at translational or post-translational steps. This induction occurred despite a 1.7-fold increase in CYTOX, implying that a precursor-product relationship does not always exist.
Quantitative determination of the steady-state kinetics of multienzyme reactions using the algebraic rate equations for the component single-enzyme reactions
Authors: Stoner C D
Published: August 2015

Methods are given whereby the steady-state kinetic characteristics of multienzyme reactions consisting of individual single-enzyme reactions linked by freely diffusible intermediates can be determined quantitatively from the experimentally determined complete algebraic rate equations for the individual reactions. The approach is based on the fact that a valid steady-state rate equation for such a multienzyme reaction, in terms of the rate equations for the individual reactions, can be obtained simply from knowledge of the relative rates of the individual reactions when the multienzyme reaction is in the steady state. A number of model multienzyme reactions, which differ as to structural arrangement of the individual reactions, are examined by this approach. Simple mathematical methods which are applicable to most of these models are given for direct calculation of dependent variables. It is either pointed out or demonstrated with Mathematica that the rate equations for all of these models can be handled very easily with the aid of a personal computer equipped with appropriate equation-solving software. Since the approach permits evaluation of all dependent variables for any specific combination of values for the kinetic parameters and independent variables, numerical values for the flux control coefficients of the individual enzymes can be obtained by direct calculation for a wide variety of conditions and can be compared with those obtained according to the methods of Metabolic Control Analysis. Several such comparisons have been made and in all cases identical results were obtained. The intuitive notion that the individual enzymes of a multienzyme reaction would be equally rate limiting if the total amount of enzyme were being used with maximum efficiency is tested and shown to be incorrect. In the course of this test the flux control coefficient for the individual enzymes were found to be appropriate indicators of relative rate limitation or control by the enzymes and to account properly for differences in specific activity among the enzymes.
Binding of phospholipase Cδ1 to phospholipid vesicles
Authors: Pawelczyk T , Lowenstein J M
Published: August 2015

Binding of phospholipase C delta 1 (PLC delta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLC delta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) + PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE + PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLC delta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE + PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLC delta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at a level of 96 nmol/ml SM. This is well below the concentration of SM that can be calculated to face the cytosol. Binding of PLC delta to LUVs decreased as the temperature was lowered from 37 degrees C to 0 degree C. Thus PLC delta shows a high degree of specificity for binding to PIP2 and SM. Under physiological conditions a considerable fraction of PLC delta may be bound to cellular membranes, either in an inactive form if bound to PIP2 at low resting Ca2+ concentrations, or in the inhibited form if bound to SM.
Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur ‘P’ clusters
Authors: Miller R W , Smith B E , Eady R R
Published: August 2015

Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the ‘P’ clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that ‘P’ clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.
8-vinyl reduction and chlorophyll a biosynthesis in higher plants
Authors: Whyte B J , Griffiths W T
Published: August 2015

A technique involving solid-phase extractions and polyethylene h.p.l.c. suitable for the routine compositional analysis of the total protochlorophyllide pool of plants is described. The resynthesis kinetics of the individual components of the pool have been studied in briefly illuminated etiolated tissue of wheat (Triticum aestivum) and cucumber (Cucumis sativus) during subsequent redarkening. The data are interpreted in terms of a precursor-product relationship between the di- and mono-vinyl analogues of protochlorophyllide during their reaccumulation in darkness. The interconversion is assumed to be catalysed by an 8-vinyl reductase, which shows greater activity in wheat than in cucumber. Analyses of the redox state of the nicotinamide nucleotide of the pool during the process are compatible with NADPH as the cofactor of the putative reductase.
Evidence that stimulation of plasma-membrane Ca2+ inflow is an early action of glucagon and dibutyryl cyclic AMP in rat hepatocytes
Authors: Bygrave F L , Gamberucci A , Fulceri R , Benedetti A
Published: August 2015

The ability of glucagon (1 nM) and of dibutyryl cyclic AMP (50 microM) to increase cytosolic free Ca2+ concentration ([Ca2+]i) in Fura-loaded rat hepatocytes was examined in a system wherein Ca2+ inflow was induced by the re-admission of excess Ca2+ to a nominally Ca(2+)-free medium. An increase in [Ca2+]i did not occur in the absence of either agonist, but did so after co-addition of either agonist with Ca2+. Increasing the time between addition of dibutyryl cyclic AMP (or of glucagon) and Ca2+ led to increases in [Ca2+]i; half-maximal and maximal increases were observed at 0 s (i.e. at co-addition) and 5-7 s respectively. Dibutyryl cyclic AMP and Ca2+ each exhibited a concentration-dependence when their respective concentrations were changed for a fixed time interval between additions. Half-maximal and maximal effects were obtained with 30 microM and 50 microM dibutyryl cyclic AMP and with 0.5 mM and approx. 1 mM Ca2+ respectively. The data demonstrate an early action of glucagon and dibutyryl cyclic AMP on [Ca2+]i. It is argued that the agonist-induced rise in [Ca2+]i results from an increase in plasma-membrane Ca2+ inflow, an effect that appears to occur much earlier than that on mobilization of internal stores of Ca2+.
Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester
Authors: Dory L
Published: August 2015

Phorbol ester-mediated differentiation of THP-1 cells (a human monocytic cell line) into mature macrophages is associated with a transcriptional induction of apolipoprotein E (apoE) expression [Auwerx, Deeb, Brunzell, Peng and Chait (1988) Biochemistry 27, 2651-2655]. Endotoxin, on the other hand, which may also act through activation of protein kinase C, is a potent inhibitor of apoE expression in mouse macrophages [Werb and Chin (1983) J. Biol. Chem. 258, 10642-10648]. The present experiments examine the effect of phorbol ester, an activator of protein kinase C, on the apoE expression in mouse thioglycollate-elicited peritoneal macrophages. Phorbol ester inhibits apoE expression in a specific, time- and dose-dependent manner. A 75% inhibition in the rate of apoE secretion, but not that of total protein, was observed following a 4.5 h incubation with 160 nM phorbol ester, although nearly full inhibition was obtained with 40 nM. The changes in apoE secretion were paralleled by similar changes in apoE synthesis, indicating synthesis as the primary site of action. The decreased rates of apoE synthesis are shown not to be due to increased apoE degradation. The profound inhibition of apoE synthesis was not accompanied by significant changes in apoE mRNA levels at any concentration of phorbol ester (up to 16 microM), or length of treatment (up to 24 h), suggesting a post-transcriptional locus of regulation of apoE expression. Although the early changes in apoE synthesis correlate with increased microsomal protein kinase C activity, the suppression of apoE expression persists even during conditions of nearly complete (> 95%) loss of protein kinase C activity, suggesting that the direct or indirect effect of protein kinase C on apoE expression is mediated by a stable phosphorylated protein, or that the observed effects are mediated through a protein kinase C species that is not readily downregulated by phorbol esters. The presented studies clearly demonstrate the potential importance of the translational regulation of apoE expression through the protein kinase C signal transduction pathway.
Phospholipase D activity in phagocytic leucocytes is synergistically regulated by G-protein- and tyrosine kinase-based mechanisms
Authors: Dubyak G R , Schomisch S J , Kusner D J , Xie M
Published: August 2015

The regulation of phospholipase D (PLD)-type effector enzymes by G-proteins and protein kinases/phosphatases was characterized in the U937 human promonocytic leucocyte line. PLD activity was assayed by measuring (in the presence of 1% ethanol) the accumulation of phosphatidylethanol in cells permeabilized with beta-escin, a saponin-like detergent. Basal PLD activity was very low when cells were permeabilized and incubated in cytosol-like medium containing micromolar [Ca2+]. When this medium was supplemented with exogenous MgATP or guanosine 5′-[gamma-thio]triphosphate (GTP[S]), PLD activity increased by 9- and 14-fold respectively. Cells permeabilized in the absence of exogenously added MgATP, but in the presence of 1 microM vanadate/100 microM H2O2, also exhibited a modest 12-fold increase in PLD activity. However, the simultaneous presence of either GTP[S] plus exogenous MgATP or GTP[S] plus vanadate/H2O2 (and endogenous MgATP) induced similar 60-75-fold increases in the rate and extent of phosphatidylethanol accumulation. These latter effects of vanadate/H2O2 were strongly correlated with the very rapid accumulation of multiple tyrosine-phosphorylated proteins. Other studies utilized cells which were permeabilized in the presence of GTP[S] and then washed before assay of PLD. These cells retained approximately 60% of the MgATP-regulatable PLD activity (EC50 approximately = to 100 microM MgATP) observed in freshly permeabilized non-washed cells. In the absence of GTP[S] pre-treatment, washed cells retained minimal PLD activity. Genistein, a tyrosine kinase inhibitor, significantly attenuated the ability of MgATP to stimulate PLD activity and accumulation of tyrosine-phosphorylated proteins in the washed GTP[S]-treated cells. These data suggest that PLD activity in myeloid leucocytes involves co-ordinate regulation by both G-protein(s) and tyrosine phosphorylation.
Hypoglycaemic activity of Coccinia indica and Momordica charantia in diabetic rats: depression of the hepatic gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase and elevation of both liver and red-cell shunt enzyme glucose-6-phosphate dehydrogenase
Authors: Shibib B A , Khan L A , Rahman R
Published: August 2015

Coccinia indica leaves were extracted with 60% ethanol, solvents were evaporated and the residue was suspended in water. This suspension was administered orally at a dose of 200 mg/kg body wt. after 18 h of fasting to normal fed and streptozotocin-induced male diabetic rats (180-250 g). After 90 min the rats were killed, and blood-glucose, hepatic glucose-6-phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase (G6PDH) and red-cell G6PDH were assayed. Blood sugar was depressed by 23% (P < 0.01) and 27% (P < 0.001) in the normal fed and streptozotocin-diabetic rats respectively compared with controls which were given distilled water. Hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase activities were depressed by 32% (P < 0.001) 30% (P < 0.05) respectively in the streptozotocin-diabetic rats, compared with 19% (P < 0.02) and 20% (P < 0.01) depression in the normal fed controls, whereas both the red-cell and hepatic G6PDH activities were found to be elevated by feeding the extract in the streptozotocin-diabetic and in the normal fed controls. Similar results were obtained with the 95%-ethanolic extract of Momordica charantia. Taken together, these results indicate that Coccinia indica and Momordica charantia extracts lowered blood glucose by depressing its synthesis, on the one hand through depression of the key gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase and on the other by enhancing glucose oxidation by the shunt pathway through activation of its principal enzyme G6PDH.
Hydrazine and hydroxylamine as probes for O2-reduction site of mitochondrial cytochrome c oxidase
Authors: Kubota T , Yoshikawa S
Published: August 2015

Reactions of hydrazine and hydroxylamine with bovine heart cytochrome c oxidase in the fully reduced state were investigated under anaerobic conditions following the visible-Soret spectral change. Hydrazine gave a sharp band at 575 nm with 20% decrease in the alpha band at 603 nm, and hydroxylamine induced a 2 nm blue-shift for the alpha band without any clear splitting. The Soret band at 443 nm was decreased significantly in intensity, with the concomitant appearance of a shoulder with hydrazine or a peak with hydroxylamine, both near 430 nm. The dependence on pH of the affinity of these reagents for the enzyme indicates that only the deprotonated forms of these reagents bind to the enzyme, suggesting a highly hydrophobic environment of the haem ligand-biding site. These spectral changes were largely removed by addition of cyanide or CO. However, detailed analysis of these spectral changes indicates that hydrazine perturbs the shape of the spectral change induced by cyanide and hydroxylamine perturbs that induced by CO. These results suggest that these aldehyde reagents bind to haem a3 iron as well as to a second site which is most likely to be the formyl group on the haem periphery, and that these two sites bind these reagents anti-cooperatively with each other.
Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane
Authors: Oppong S Y , Hooper N M
Published: August 2015

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.
Luminal Ca2+ increases the affinity of inositol 1,4,5-trisphosphate for its receptor
Authors: Oldershaw K A , Taylor C W
Published: August 2015

Luminal Ca2+ has been proposed to regulate the sensitivity of intracellular Ca2+ stores to InsP3 and perhaps thereby to contribute to the mechanisms responsible for regenerative intracellular Ca2+ signals. Since Ca2+ release from intracellular stores is accompanied by K+ influx into the stores, InsP3-stimulated Ca2+ mobilization can be inhibited by substantially reducing the [K+] of incubation media. By measuring [3H]InsP3 binding to permeabilized hepatocytes in K(+)-deficient media, thereby preventing InsP3-stimulated Ca2+ mobilization, we have examined the effects of Ca2+ within the intracellular stores on equilibrium binding of InsP3 to its receptor. Our results demonstrate a small, but significant (P < 0.05, n = 8-9), effect of luminal Ca2+ on InsP3 binding; the concentration of InsP3 causing half-maximal displacement of [3H]InsP3 (1.25 nM) was 3.5 nM for empty stores and 2.1 nM for stores containing Ca2+. Our results suggest that at least part of the stimulatory effect of luminal Ca2+ on InsP3-stimulated Ca2+ mobilization may result from an effect of luminal Ca2+ on InsP3 binding to its receptor.
The isolation and identification of 6-hydroxycyclohepta-1,4-dione as a novel intermediate in the bacterial degradation of atropine
Authors: Bartholomew B A , Smith M J , Long M T , Darcy P J , Trudgill P W , Hopper D J
Published: August 2015

Growth of Pseudomonas AT3 on the alkaloid atropine as its sole source of carbon and nitrogen is nitrogen-limited and proceeds by degradation of the tropic acid part of the molecule, with the metabolism of the tropine being limited to the point of release of its nitrogen. A nitrogen-free compound accumulated in the growth medium and was isolated and identified as 6-hydroxycyclohepta-1,4-dione. This novel compound is proposed as an intermediate in tropine metabolism. It served as a growth substrate for the organism and was also the substrate for an NAD(+)-linked dehydrogenase present in cell extracts. The enzyme was induced during the tropine phase of diauxic growth on atropine or during growth on tropine alone.
Studies on secondary structure of caldesmon and its C-terminal fragments
Authors: Czuryło E A , SYu Venyaminov , Dabrowska R
Published: August 2015

Evaluation of the secondary structure of caldesmon from c.d. spectra revealed that it contains 51% helix, 9% beta-strand and 40% of remainder structures. These values agree well with the predicted ones from amino acid sequence, assuming an extended chain structure for caldesmon. The estimates of the secondary-structure elements in C-terminal 34 kDa and 19 kDa fragments are: 11 and 12% helix, 22 and 20% beta-strand, 13 and 17% beta-turns and loops, and 54 and 50% of remainder structure respectively. The best fit of experimental data was obtained assuming the globular state of the fragments. On the basis of structural analysis and fragmentation by proteolytic and chemical cleavages the six-domain model of caldesmon is proposed.
The Ca2+-transporting ATPases of rabbit and trout exhibit different pH- and temperature-dependences
Authors: Chini E N , de Toledo F G , Albuquerque M C , de Meis L
Published: August 2015

The phosphorylation of the trout sarcoplasmic-reticulum Ca(2+)-ATPase by Pi differs in its temperature- and pH-dependence from the rabbit ATPase. In the trout enzyme, the apparent affinity for Pi and maximum phosphoenzyme values do not vary over a pH and temperature ranges that have a pronounced effect on the rabbit enzyme. The lack of temperature-dependence for phosphorylation is observed at pH 6.8. At pH 8.0, the temperature profile for phosphorylation of the trout enzyme resembles that of the rabbit at pH 6.8. The rabbit ATPase is no longer phosphorylated by Pi after solubilization with the detergent C12E9. In contrast, the trout enzyme can be phosphorylated by Pi after solubilization with C12E9, and the same levels of phosphoenzyme were obtained with the soluble and membrane-bound ATPase at both 0 degrees and 25 degrees C. In the range of 0-20 degrees C, the rates of ATP synthesis and of Ca2+ uptake by the trout ATPase are less temperature-dependent than for the rabbit enzyme. However, both isoenzymes catalyse ATP hydrolysis with similar temperature-dependences. The results raise the possibility that protonation of specific amino acid residues may contribute to the lack of temperature-dependence for phosphorylation of the trout Ca(2+)-ATPase.
Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II
Authors: Francis G L , Aplin S E , Milner S J , McNeil K A , Ballard F J , Wallace J C
Published: August 2015

Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.
Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the ‘de novo’ pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake
Authors: Tronchère H , Tercé F , Record M , Chap H
Published: August 2015

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the ‘de novo’ pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the ‘de novo’ pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).
Reduced sulphydryl groups are required for DNA binding of Ku protein
Authors: Zhang W W , Yaneva M
Published: August 2015

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.
Phenylalaninylargininylarginine: a novel tripeptide exerting Zn2+-dependent, insulin-mimetic inhibitory action on myocardial proteolysis
Authors: Zhang L , Lockwood T D
Published: August 2015

A novel tripeptide, Phe-Arg-Arg, was found to exert a potent, insulin-mimetic inhibitory action on lysosomal proteolysis in the Langendorff-perfused rat heart. This tripeptide was synthesized based upon its partial structural analogy to the biguanide anti-hyperglycaemic agent, phenformin (phenylethylbiguanide), which has previously been found to exert a Zn(2+)-dependent inhibitory action on lysosomal proteolysis. Hearts were biosynthetically labelled with [3H]leucine in vitro. The percentage change in subsequent release of [3H]leucine (2 mM non-radioactive leucine) was determined in non-recirculating perfusate. The background Zn2+ content of the perfusate was determined to be 20 nM. Major endogenous Zn2+ buffers were present in molar excess of Zn2+: 0.1 mM citrate, 0.2% BSA, and complete physiological amino acids. Infusion of maximally effective levels of chloroquine (30 microM) or insulin (5 nM) caused a 38% inhibition of total proteolysis, which corresponds to the lysosomal subcomponent. In the presence of background levels of perfusate Zn2+ the infusion of Phe-Arg-Arg (10 microM), insulin (5 nM), or phenformin (2 microM) maximally caused a 39% inhibition of [3H]leucine release. Combined infusion of maximally effective levels of insulin and Phe-Arg-Arg, or maximal levels of chloroquine and Phe-Arg-Arg did not cause additive inhibition of [3H]leucine release greater than the 39% inhibition caused by either agent alone, regardless of the order of infusion. Addition of physiological concentrations of Zn2+ (1 microM) to the background perfusate Zn2+ accelerated the insulin-mimetic action of submaximally effective levels of Phe-Arg-Arg, and increased its potency. Prior chelation of background Zn2+ by a 3 h perfusion with CaNa2 EDTA (2 microM) reversibly delayed the time course of Phe-Arg-Arg action and decreased its potency at submaximal concentrations.
Restoration in vitro of normal rates of very-low-density lipoprotein triacylglycerol and apoprotein B secretion in hepatocyte cultures from diabetic rats
Authors: Duerden J M , Gibbons G F
Published: August 2015

Hepatocytes derived from diabetic rats were cultured in serum-free Waymouth's medium containing various supplements, after an initial 4 h period during which the cells were allowed to attach to the culture dish in the presence of foetal-bovine serum (10%). After removal of serum, these cells secreted much less very-low-density lipoprotein (VLDL) apoprotein B (apoB) and triacylglycerol than those derived from normal rats when cultured for 24 h in the basal medium. Inclusion of oleate (0.75 mM) in the medium initially increased the output of apoB and triacylglycerol, but the rates remained lower than those observed in normal hepatocytes and declined to zero after 72 h. This time-dependent decline in VLDL output was prevented by addition of dexamethasone to the oleate-containing medium. Levels of apoB and triacylglycerol output characteristic of normal hepatocytes could only be completely restored, however, by further addition of a mixture of lipogenic substrates (lactate plus pyruvate) to the medium. Restoration of normal levels of VLDL secretion in diabetic hepatocytes in vitro by this means was accompanied by a normal inhibitory response of apoB and triacylglycerol output to short-term (24 h) treatment with insulin or glucagon. Exposure of the cells to insulin for 72 h enhanced the secretion of VLDL, whereas treatment with glucagon for the same period potentiated the original inhibitory effect. The defective secretion of VLDL apoB observed when diabetic hepatocytes were cultured in the basal medium for 24 h could also be rectified by inclusion of a mixture of oleate (0.75 mM), lactate (10 mM), pyruvate (1 mM), dexamethasone (1 microM) and insulin (78 nM) in the medium during the 4 h attachment period in the presence of serum. Under these conditions, the increase in the secretory response of triacylglycerol was not so pronounced.
Species-specific induction of cytochrome P-450 4A RNAs: PCR cloning of partial guinea-pig, human and mouse CYP4A cDNAs
Authors: Bell D R , Plant N J , Rider C G , Na L , Brown S , Ateitalla I , Acharya S K , Davies M H , Elias E , Jenkins N A , Gilbert D J , Copeland N G , Elcombe C R
Published: August 2015

PCR was used to demonstrate the presence of a conserved region and to clone novel members of the cytochrome P-450 4A gene family from guinea pig, human and mouse cDNAs. This strategy is based on the sequences at nucleotides 925-959 and at the haem binding domain (nucleotides 1381-1410) of the rat CYP4A1 gene. Murine Cyp4a clones showed high sequence identity with members of the rat gene family, but CYP4A clones from human and guinea pig were equally similar to the rat/mouse genes, suggesting that the rat/mouse line had undergone gene duplication events after divergence from human and guinea-pig lines. The mouse Cyp4a-12 clone was localized to chromosome 4 using interspecific backcross mapping, in a region of synteny with human chromosome 1. The assignment of the human CYP4A11 gene to chromosome 1 was confirmed by somatic cell hybridization. An RNAase protection assay was shown to discriminate between the murine Cyp4a-10 and Cyp4a-12 cDNAs. Treatment of mice with the potent peroxisome proliferator methylclofenapate (25 mg/kg) induced Cyp4a-10 RNA in liver, and to a lesser extent in kidney; there was no sex difference in this response. Cyp4a-12 RNA was present at high levels in male control liver and kidney samples, and was not induced by treatment with methylclofenapate. However, Cyp4a-12 RNA was present at low levels in control female liver and kidney RNA, and was greatly induced in both organs by methylclofenapate. Guinea pigs were exposed to methylclofenapate (50 mg/kg), but there was no significant induction of the guinea-pig CYP4A13 RNA. These findings are consistent with a species difference in response to peroxisome proliferators between the rat/mouse and the guinea pig.