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Biomedical subjects

U Brandt

Publications and source records attributed to U Brandt.

At least 73 records · Page 4Linked to original sources
PubMed1992

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.

Amino Acid Sequence↗
PubMed1992

Uncoupling activity and physicochemical properties of derivatives of fluazinam.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.

Amines↗
PubMed1992

A psychometric analysis of the Mental Adjustment to Cancer scale.

This paper describes a psychometric replication study of a British coping scale, the Mental Adjustment to Cancer scale, on 239 USA cancer patients. The internal consistency and construct validity were similar to the original validation study. However, the factor structure was different. Cultural differences between the British and USA patient populations are discussed.

Adaptation, Psychological↗
PubMed1992

Vascular bound recombinant extracellular superoxide dismutase type C protects against the detrimental effects of superoxide radicals on endothelium-dependent arterial relaxation.

Extracellular superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme that, in vivo, is bound to heparan sulfate proteoglycans in the glycocalyx of various cell types (e.g., endothelial cells) and in the connective tissue matrix. The aim of this study was to investigate the efficacy of vascular bound EC-SOD C in protecting arterial relaxation mediated by endothelium-derived relaxing factor (EDRF) against the inhibitory effects of superoxide radicals. For comparison, the effect of CuZn SOD was also studied. This SOD isoenzyme lacks affinity toward heparan sulfate and does not bind to cell surfaces. Rings from rabbit aorta were mounted in an organ bath and acetylcholine-induced endothelium-dependent relaxation was then studied in preparations precontracted with phenylephrine. Pyrogallol (10(-4) M), used to generate superoxide radicals, reduced the maximal relaxant effect of acetylcholine from about 65% to 25%. When present in the buffer throughout the experiment, CuZn SOD and EC-SOD C caused a concentration-dependent prevention of the pyrogallol effect on EDRF-mediated relaxation, with a half-maximal effect at about 100 units/ml (KO2 assay). In a second set of experiments, the arterial rings were preincubated with 8,000 units/ml CuZn SOD (50 micrograms/ml) or EC-SOD C (69 micrograms/ml) during 30 minutes, followed by washing, before the effect of pyrogallol on EDRF-mediated relaxation was studied in SOD-free buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1991

Single electron reduction of 'slow' and 'fast' cytochrome-c oxidase.

Evidence is presented that single electron reduction is sufficient for rapid electron transfer (k greater than 20 s-1 at pH 8.0 in 0.43 M potassium EDTA) between haem a/CuA and the binuclear centre in 'fast' oxidase, whereas in 'slow' oxidase intramolecular electron transfer is slow even when both CuA and haem a are reduced (k congruent to 0.01 s-1). However, while a single electron can equilibrate rapidly between CuA, haem a and CuB in 'fast' oxidase, it seems that equilibration with haem a3 is relatively slow (k congruent to 2 s-1). Electron transfer between cytochrome c and CuA/haem a is similar for both types of enzyme (k congruent to 2.4 x 10(5) M-1.s-1).

Animals↗
PubMed1991

Significance of the "Rieske" iron-sulfur protein for formation and function of the ubiquinol-oxidation pocket of mitochondrial cytochrome c reductase (bc1 complex).

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.

Acrylates↗
PubMed1991

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.

Bacillus subtilis↗
PubMed1991

Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.

Acrylates↗
PubMed1991

Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a 'catalytic switch' at the Qo center.

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi center, 2-n-nonyl-4-hydroxyquinoline N-oxide and for the Qo center (E)-beta-methoxyacrylate-stilbene (MOA-stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non-fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up. We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center. Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox-dependent. In contrast, the binding of MOA-stilbene to the Qo center is decreased after reduction of the iron-sulfur center and cytochrome c1, whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3-n-undecyl-2-hydroxynaphthoquinone. From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non-competitive one, we made the hypothesis of a 'catalytic switch' to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed 'switch' gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.

Acrylates↗
PubMed1990

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes. Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells.

The recent demonstration of a large cell surface-derived pool of insulin-sensitive glucose transporters, presumably concentrated in the microvilli of 3T3-L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3-L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle-specific transporter, GluT4, expressed for the first time 3-4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3-L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface-derived vesicle (LDSV) fraction of 3T3-L1 adipocytes most likely consisting of microvilli-derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.

Adipose Tissue↗
PubMed1990

Phospholipid specificity of bovine heart bc1 complex.

Bovine heart bc1 complex was reversibly inactivated by a new simple and effective chromatographic delipidation method. Upon phospholipid replenishment, catalytic activity increased from values near zero to values 2-6-times higher than those of the original preparation. Compared to original preparations maximally activated by additional phospholipid, the degree of reactivation was up to 100%. By this delipidation method, the 6.4-kDa protein subunit was removed with the phospholipid. The loss of this protein neither diminished electron transport activity nor abolished proton translocation. Two requirements were necessary to obtain quantitative data: (a) only bc1 complexes, homogeneously dissolved before and after relipidation had to be used and (b) the phospholipid bound to the complex had to be determined. The correlation of catalytic activity to bound phospholipid was studied in the range of low phospholipid/protein ratios, which had previously been insufficiently resolved. Catalytic activity increased linearly with added phospholipid up to a molar ratio of 80-100 lipid molecules/dimeric complex. This corresponds to the number of phospholipid molecules that complete a single bilayer annulus. The activating effect of phospholipid is not merely due to a hydrophobic phase effect, since it strongly depends on the nature of the polar head group of the added phospholipid. Of the three major phospholipids bound to the bc1 complex, only phosphatidylethanolamine and phosphatidylcholine activated when added as sole phospholipid. Tightly bound diphosphatidylglycerol was needed for preservation of the native complex structure.

Animals↗
PubMed1990

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes.

The recently proposed mechanistic concept of a receptor-regulated entrance compartment for hexose transport formed by microvilli on 3T3-L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B-binding technique was used to determine in basal and insulin-stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface-derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface-derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin-sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 mU/ml for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4-fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin-sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100-200 nm in diameter.

Adipose Tissue↗
PubMed1990

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes.

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗
PubMed1989

Purification of cytochrome-c oxidase retaining its pulsed form.

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.

Animals↗
PubMed1989

Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology.

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.

Actins↗
PubMed1988

Studies on the effect of stigmatellin derivatives on cytochrome b and the Rieske iron-sulfur cluster of cytochrome c reductase from bovine heart mitochondria.

Stigmatellin and its derivatives represent a third class of Qo site inhibitors besides the hydroxyquinone derivatives and the E-beta-methoxyacrylate (MOA) inhibitors [von Jagow and Link (1986) Methods Enzymol. 126, 253-271]. The stigmatellins consist of a chromone ring system connected to an substituted alkenyl side chain. Alterations in the side chain, i.e. saturation of the C = C double bonds, shift of a methoxy group or loss of the methyl groups, specifically affect the binding characteristics. Besides changing the red shift spectrum of reduced cytochrome b566 and the EPR spectrum of the Rieske iron-sulfur cluster, the side chain alterations diminish the binding affinity and the extent of the midpoint potential shift of the iron-sulfur protein. Thus, the side chain of the molecule makes an essential contribution to the binding energy and is not necessary solely for partitioning the molecule into the hydrophobic phase, as assumed so far.

Animals↗