| 61. |
- Kass, G E, et al.
(författare)
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Receptor-mediated Mn2+ influx in rat hepatocytes : comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt
- 1994
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 302, s. 5-9
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Tidskriftsartikel (refereegranskat)abstract
- In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.
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| 62. |
- Knorpp, Carina, et al.
(författare)
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Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria.
- 1995
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 310 ( Pt 2)
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Tidskriftsartikel (refereegranskat)abstract
- We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.
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| 63. |
- Kolm, Rüdiger H., et al.
(författare)
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Isothiocyanates as substrates for human glutathione transferases : structure-activity studies
- 1995
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 311, s. 453-459
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.
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| 64. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.
- 1994
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 304, s. 723-730
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Tidskriftsartikel (refereegranskat)abstract
- The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.
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| 65. |
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| 66. |
- Liao, W, et al.
(författare)
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Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression
- 1996
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 313313 ( Pt 3), s. 873-878
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Tidskriftsartikel (refereegranskat)abstract
- Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 μg/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83–136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63–65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats.
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| 67. |
- Lidholt, Kerstin, et al.
(författare)
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Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate.
- 1995
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 311:1, s. 233-238
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs), biosynthetically labelled with [S-35]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the S-35-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [S-35]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these S-35-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [S-35]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) similar to 30000) than the CS chains in the NA-PGs (M(r) similar to 25000). CS chains in the hybrid PGs were slightly smaller (M(r) similar to 20000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20 %) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [S-35]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.
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| 68. |
- Lindahl, P, et al.
(författare)
-
Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin
- 1992
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Ingår i: Biochemical Journal. - 0264-6021. ; 281:1, s. 49-55
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.
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| 69. |
- Lindkvist, B, et al.
(författare)
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Glutathione transferase mimics: micellar catalysis of an enzymic reaction
- 1997
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 323323 ( Pt 1), s. 39-43
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Tidskriftsartikel (refereegranskat)abstract
- Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as α,β-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium-co-dodecylmethyldiallylammon ium) bromide (86/14) ≫cetyltrimethylammonium bromide > zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) > zwittergent 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) ≈ N,N-dimethyl-laurylamine N-oxide > N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to α,β-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding.
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| 70. |
- Liu, G, et al.
(författare)
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Interaction of size-fractionated heparins with lipoprotein lipase and hepatic lipase in the rat.
- 1992
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 285 ( Pt 3), s. 731-6
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Tidskriftsartikel (refereegranskat)abstract
- Heparin and heparin partially depolymerized by enzymic digestion were separated into six size fractions. Hep 1 (tetrasaccharides), with a mean M(r) of 1200, did not release significant amounts of either lipoprotein lipase (LPL) or hepatic lipase (HL) on intravenous injection into rats. Hep 2 (mainly octa- and deca-saccharides), with a mean M(r) of 2400-3000, released both lipases. To evoke the same plasma activity of LPL and HL required about 10 times more by weight, or about 40 times more molecules, of this heparin than of hep 5 (mean M(r) 12,000, similar to conventional heparin). Hep 5 impeded binding and degradation of 125I-labelled bovine LPL by perfused rat livers. In contrast, hep 2 had no detectable effect on these processes. This demonstrates a difference between the sites in the liver that mediate binding, uptake and degradation of LPL, and the extrahepatic sites that bind functional LPL, and the hepatic sites that bind functional HL. After injection of 3.25 mg of hep 5/kg body weight, plasma LPL activity rapidly rose and then remained high for at least 1 h. With hep 2, plasma LPL also rose rapidly, but then decreased to almost basal by 1 h. When a labelled triacylglycerol emulsion was injected 1 h after the heparins, the fractional catabolic rate was enhanced in the rats that had received conventional heparin, as expected from the high plasma LPL activity, but decreased compared with controls in rats that had received hep 2, indicating that available LPL had been depleted through enhanced transport to and uptake in the liver.
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