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C Richter

Publications and source records attributed to C Richter.

At least 199 records · Page 11Linked to original sources
PubMed1985

Mechanism of alloxan-induced calcium release from rat liver mitochondria.

The objective of the present work was to investigate the mechanism of alloxan-induced Ca2+ release from rat liver mitochondria. Transport of Ca2+, oxidation and hydrolysis of mitochondrial pyridine nucleotides, changes in the mitochondrial membrane potential, and oxygen consumption by mitochondria were investigated. Alloxan does not inhibit the uptake of Ca2+ but stimulates the release of Ca2+ from liver mitochondria, which is accompanied by oxidation and hydrolysis of pyridine nucleotides. Oxidation of mitochondrial pyridine nucleotides by alloxan is not mediated by glutathione peroxidase and glutathione reductase and may occur largely nonenzymatically. Measurements of the mitochondrial membrane potential in combination with inhibitors of Ca2+ reuptake indicate that Ca2+ release takes place from intact liver mitochondria via a distinct pathway. Limited redox cycling of alloxan by mitochondria is indicated by measurements of the membrane potential and O2 consumption in the presence of cyanide. It is concluded that alloxan can cause Ca2+ release from intact rat liver mitochondria. Redox cycling of alloxan is not significantly involved in the Ca2+ release mechanism. Oxidation and hydrolysis of pyridine nucleotides, possibly in conjunction with oxidation of critical sulfhydryl groups, seem to be key events in the alloxan-induced Ca2+ release. Disturbance of cellular Ca2+ homeostasis may partly explain alloxan toxicity.

Alloxan↗
PubMed1985

Quantitative and mechanistic aspects of the hydroperoxide-induced release of Ca2+ from rat liver mitochondria.

We have previously demonstrated in rat liver mitochondria a hydroperoxide-induced hydrolysis of pyridine nucleotides and release of Ca2+ [Lötscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1979) Proc. Natl Acad. Sci. USA 76, 4340-4344, and Lötscher, H. R., Winterhalter, K. H., Carafoli, E. & Richter, C. (1980) J. Biol. Chem. 255, 9325-9330]. Here we investigate pyridine nucleotide hydrolysis and Ca2+ release under conditions of minimized Ca2+ cycling and with smaller Ca2+ loads. The extent of pyridine nucleotide hydrolysis, measured by pyridine-nucleotide-derived nicotinamide release from intact mitochondria, and the Ca2+ release rate show a very similar sigmoidal dependence on the mitochondrial Ca2+ load. The hydrolysis of oxidized pyridine nucleotides is limited under non-cycling conditions. Whereas pyridine nucleotide hydrolysis as measured by nicotinamide release is extensive, net loss of mitochondrial pyridine nucleotides is observed only at relatively high Ca2+ loads. Our results indicate the ability of mitochondria to resynthesize pyridine nucleotides after hydrolysis. Neither a decrease of reduced, nor an increase of oxidized, mitochondrial glutathione favour Ca2+ release. From these and previous findings it is concluded that the hydroperoxide-induced Ca2+ release is triggered by a factor which is distal to the oxidation of mitochondrial pyridine nucleotides. Ca2+ release is stimulated when the movement of protons across the inner mitochondrial membrane is facilitated, giving evidence for the operation of the hydroperoxide-induced release pathway as a Ca2+/H+ antiport.

Animals↗
PubMed1985

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies.

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.

Animals↗
PubMed1985

Divicine induces calcium release from rat liver mitochondria.

Divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism, induces calcium release from intact rat liver mitochondria. Divicine-dependent calcium release is accompanied by oxidation and hydrolysis of intramitochondrial pyridine nucleotides. Inhibition of both mitochondrial glutathione peroxidase and glutathione reductase slows down divicine-induced calcium release. Cyanide-insensitive respiration indicates redox cycling of divicine in mitochondria. The results suggest that attention should be paid to the action of divicine in cells other than red blood cells.

Animals↗
PubMed1985

Kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 from rat liver microsomes: a simple bimolecular process.

The kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 (P-450PB) and to its enzymatically inactive form P-420PB have been investigated by both stopped-flow and flash-photolysis spectrophotometry. When the simultaneous presence of both forms of the enzyme is taken into account, the binding of CO to these two proteins can be described in terms of two bimolecular processes with rate constants of 4.5 X 10(6) M-1.S-1 and 4.7 X 10(5) M-1.S-1 for P-450PB and 1.7 X 10(7) M-1.S-1 and 1.5 X 10(6) M-1.S-1 for P-420PB. From kinetic studies of the binding of CO to P-450PB under different experimental conditions, investigations of the homogeneity of our P-450PB preparations, and comparative kinetic investigations of P-450s from different sources, we conclude that CO binding to reduced P-450PB is a simple bimolecular process and that the observed biphasic traces are due to heterogeneity of the proteins. This conclusion is in contrast with previous reports of complex reaction mechanisms for the binding of CO to P-450PB. Optical spectroscopy studies indicate the existence of an equilibrium between P-450PB and P-420PB, at least for the reduced carbonyl derivatives of the enzymes. The interconversion is strongly influenced by the aggregation state of the protein. Large differences between the CO binding properties of P-450PB and those of P-420PB are found. These are discussed in terms of possible effects of the proximal ligation state of the iron on heme reactivity.

Animals↗
PubMed1984

The role of hydroperoxides as calcium release agents in rat brain mitochondria.

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.

Animals↗
PubMed1983

Chemical modification of microsomal cytochrome P450: role of lysyl residues in hydroxylation activity.

Cytochrome P450 purified from phenobarbital-induced rat liver microsomes was acetylated at 3 lysyl residues. When reconstituted with purified NADPH-cytochrome P450 reductase, the modified cytochrome showed full activity and substrate-induced spectral changes with d-benzphetamine. With 7-ethoxycoumarin, neither enzymic activity nor binding was detected. It is concluded that the positively charged lysine residues of cytochrome P450 are important for metabolism of 7-ethoxycoumarin by cytochrome P450.

Acetylation↗
PubMed1983

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking.

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).

Animals↗
PubMed1983

Purification and properties of a mitochondrial NAD+ glycohydrolase.

A 60- to 70-fold purification of an NAD+ glycohydrolase from the inner membrane of rat liver mitochondria to apparent homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide slab gel is described. The minimum molecular weight of the enzyme on polyacrylamide gels in the presence of SDS is around 62,000. The enzyme splits NAD+ to ADP-ribose and, presumably, nicotinamide. No phosphatase or phosphodiesterase activity is detected in the purified enzyme preparation. The enzyme shows high activity with NAD+ and moderate activity with NADP+ as substrates NAD(P)Hs are poor substrates. ATP and nicotinamide inhibit the enzyme. A possible participation of the enzyme in the mechanism of calcium release from rat liver mitochondria is discussed.

Animals↗
PubMed1983

ADP-ribosylation in inner membrane of rat liver mitochondria.

NAD+ glycohydrolase activity is found at high levels in submitochondrial particles. It leads to the reaction products ADP-ribose, nicotinamide, and small amounts of 5'-AMP. Furthermore, submitochondrial particles catalyze the exchange reaction: [adenosine-14C]ADP-ribose + NAD+ in equilibrium [adenosine-14C]-NAD+ + ADP-ribose. When submitochondrial particles are incubated with NAD+, mono(ADP-ribosyl)ation of protein molecules migrating with an apparent molecular weight of 30,000 in sodium dodecyl sulfate/polyacrylamide gel electrophoresis is demonstrable. Inhibitor studies suggest attachment of ADP-ribose to arginine residues. ADP-ribose bound to submitochondrial particles is rapidly turning over. The release of ADP-ribose from the protein is probably enzyme catalyzed. The rapid turnover, the specificity of the modification, and the inhibition of ADP-ribosylation by ATP and nicotinamide suggest a regulatory role of mono(ADP-ribosyl)ation of a protein in the inner mitochondrial membrane.

Adenosine Diphosphate Ribose↗
PubMed1982

The hydroperoxide-induced release of mitochondrial calcium occurs via a distinct pathway and leaves mitochondria intact.

The hydroperoxide-induced net release of Ca2+ from rat liver mitochondria is stimulated by the Ca2+ uptake inhibitor ruthenium red. At moderate Ca2+ loads the release takes place with preservation of a high mitochondrial membrane potential. During and after Ca2+ release mitochondria remain intact. The hydroperoxide-induced release of Ca2+ might therefore be of physiological relevance.

Animals↗
PubMed1982

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles.

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.

Animals↗
PubMed1982

Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

Rotation of cytochrome P-450 was examined in both liver microsomes and reconstituted and phospholipid vesicles. Purified cytochrome P-450 was incorporated into lipid vesicles composed of phosphatidylcholine-phosphatidylethanolamine-Phosphatidylserne. Rotational diffusion was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme. CO complex by a vertically polarized laser flash. No contribution of vesicle tumbling to r(t) was observed over the experimental time range of 0-500 mus for samples in 60% sucrose. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450. In vesicles of a high lipid to protein ratio (=30 by weight), the residual time-independent normalized anisotropy, r (infinity)/r (0), reached a limiting low value, implying that all cytochrome P-450 was rotating. The mean rotational relaxation time, phi 1, was about 95 mus. In contrast, about 35% of cytochrome P-450 was immobilized in vesicles of a low lipid to protein ratio (=1), with phi 1 of about 95 mus for the mobile fraction. The immobile fraction is presumably due to self-aggregation of cytochrome P-450. In rat liver microsomes, 0-50% of cytochrome P-450 was mobile with phi 1 of about 120 mus at 20 degrees C, and the rest was immobile. A significant temperature dependence of r(t) was observed in microsomes. All cytochrome P-450 was immobile below 7 degrees C, and about 50% of the enzyme was mobile at 37 degrees C with phi 1 approximately 60 mus. From the limiting value of r(infinity)/r(0) congruent to 0.12, the tilt angle of the heme plane of cytochrome p-450 from the membrane plane was calculated to be about 40 degrees.

Animals↗