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Biomedical subjects

K H Winterhalter

Publications and source records attributed to K H Winterhalter.

At least 127 records · Page 7Linked to original sources
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↗
PubMed1983

Erythrocytic proteases: preferential degradation of alpha hemoglobin chains.

Proteolytic activity against native hemoglobin polypeptide chains is demonstrated, under strictly physiological conditions, in human reticulocytes of both normal subjects and individuals suffering from a variety of pathologic conditions involving erythrocytes, including beta-thalassemia. Two thirds of the activity are found in the cytoplasm and the remainder of it is associated with the reticulocyte membrane. That this proteolytic activity is due to contamination by WBC is excluded. The activity preferentially degrades the alpha-hemoglobin chains. An increase in this substrate within the erythroid cells, as observed in beta-thalassemia, does not enhance proteolysis. Protease inhibitors produce a variable decrease in proteolysis. None inhibit completely, thus showing that several enzymes, with different specificities, are involved.

Erythrocytes↗
PubMed1982

Control and pH dependence of ligand binding to heme proteins.

The recombination after flash photolysis of dioxygen and carbon monoxide with sperm whale myoglobin (Mb), and separated beta chains of human hemoglobin (beta A) and hemoglobin Zürich (beta ZH), has been studied as a function of pH and temperature from 300 to 60 K. At physiological temperatures, a preequilibrium is established between the ligand molecules in the solvent and in the heme pocket. The ligand in the pocket binds to the heme iron by overcoming a barrier at the heme. The association rate is controlled by this final binding step. The association rate of CO to Mb and beta A is modulated by a single titratable group with a pK at 300 K of 5.7. The binding of CO to beta ZH, in which the distal histidine is replaced by arginine, does not depend on pH. Oxygen recombination is independent of pH in all three proteins. Comparison of the binding of CO at 300 K and at low temperatures shows that pH does not affect the preequilibrium but changes the barrier height at the heme. The pH dependence and the difference between O2 and CO binding can be explained by a charge-dipole interaction between the distal histidine and CO.

Adult↗
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↗
PubMed1982

Basement membrane (type IV) collagen is a heteropolymer.

Type IV collagen was isolated in high yield from bovine kidney cortex. The protein revealed Mr = 380,000 and contained, in a 2:1 ratio, two different disulfide-linked polypeptide chains, C-1 and D-1 (Mr = 125,000). Carboxymethyl-cellulose chromatography before and after reduction proved that the two polypeptide chains are arranged in a single triple helical molecule with the chain composition (C-1)2(D-1). The disulfide bridges appear to be located 180 amino acid residues from the NH2 terminus of the chains.

Amino Acids↗
PubMed1981

Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin.

Native rat haemoglobins were found to bind simetryn sulphoxide to an extent 40-fold greater than human haemoglobin. This specific behaviour was studied by using only high-pressure ('performance') liquid chromatography for the preparative separation of globin chains and the isolation of peptides resulting from chemical and enzymic degradation. High recoveries (greater than 80%) of peptides throughout the procedures in combination with microsequence techniques, allow a definitive assignment of the residue undergoing modification. The haemoglobin beta-chain cystine-125 residue, with a stoichiometry of one per tetramer of rat haemoglobin, was found to be modified. Stereochemical implications of this finding are discussed. Simetryn sulphoxide would appear to be useful as a specific reagent for the mapping of exposed thiol residues in proteins.

Animals↗
PubMed1981

ATP prevents both hydroperoxide-induced hydrolysis of pyridine nucleotides and release of calcium in rat liver mitochondria.

The mechanism of the hydroperoxide-induced release of calcium from rat liver mitochondria was investigated. In calcium-loaded mitochondria both the hydroperoxide-induced hydrolysis of pyridine nucleotides and the release of calcium is inhibited by intramitochondrial ATP. In submitochondrial particles, in the absence of ATP a hydrolytic product of NAD+, probably ADP-ribose, binds covalently to a protein of the inner mitochondrial membrane. It is proposed that a compound deriving from NAD(P)+ is an important factor in the calcium release mechanism in rat liver mitochondria.

Adenosine Triphosphate↗
PubMed1980

Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria.

It has been previously reported (Lötscher, H. R., Winterhalter, K. H., Carafoli, E., and Richter, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4340-4344) that in Ca2+-loaded mitochondria hydroperoxides induce a release of Ca2+ from mitochondria and an irreversible oxidation of mitochondrial pyridine nucleotides. Here we show that in the presence of Ca2+ oxidized mitochondrial pyridine nucleotides are hydrolyzed inside mitochondria and that nicotinamide is released from mitochondria. The extent of the hydrolysis of NAD(P)+ is dependent on the amount of both hydroperoxide and Ca2+. The hydrolysis is reversible in the presence of added nicotinamide. The release of Ca2+ from mitochondria is electroneutral, and is directly or indirectly dependent on oxidized mitochondrial pyridine nucleotides. By contrast, the uptake of Ca2+ most probably does not require the present of reduced pyridine nucleotides. Control experiments show that even under the most drastic conditions employed in this study (100 nmol of Ca2+ and 85 nmol of t-butylhydroperoxide/mg of protein) mitochondria retain a considerable degree of functional integrity.

Animals↗
PubMed1980

Nonenzymatic glycosylation of proteins. A warning.

Radioactively labeled glucose from several manufacturers contains radioactive impurities of to date unknown structure. These contaminants bind covalently to proteins, e.g. collagen, fibronectin, basic myelin protein, bovine serum albumin, and hemoglobin, and thus simulate nonenzymatic glycosylation.

Animals↗
PubMed1980

The reaction of hemoglobin Zürich with oxygen and carbon monoxide.

The present paper reports a spectroscopic and kinetic study of the reaction with oxygen and carbon monoxide of (a) the abnormal hemoglobin Zürich (beta 63, E7 His leads to Arg), (b) its isolated abnormal chains (beta ZH), and (c) a reconstituted hybrid containing cobalt instead of iron on the normal alpha chains (Co alpha)2-(Fe beta ZH)2. The abnormal beta ZH chains, isolated from hemoglobin Zürich (HbZH) tetramer, display very peculiar spectral properties in the Soret region which were determined, for the oxy and deoxy derivatives, by kinetic difference spectra. The spectral properties of abnormal chains are maintained in the native and reconstituted hybrid tetramer. The rate constants for CO and O2 binding to isolated beta ZH chains were compared with those of the normal alpha and beta chains, It is confirmed that the CO combination rate constant to beta ZH is much higher than that of normal chains, whereas that for O2 is similar for the normal and abnormal chains. The CO binding rate constant for Fe beta ZH chains in the hybrid tetramer is the same as in the native HbZH molecule, i.e. much higher than that of normal chain. The new data are fully consistent with the sequential mechanism for CO binding previously proposed by us; on the other hand, on the basis of the spectral and kinetic results, and contrary to what has been suggested by other authors, a sequential binding of the ligand is excluded in the case of O2.

Carbon Monoxide↗