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Biomedical subjects

K H Winterhalter

Publications and source records attributed to K H Winterhalter.

At least 109 records · Page 6Linked to original sources
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

Proteoglycan Lt from chicken embryo sternum identified as type IX collagen.

Proteoglycan Lt (PG-Lt), isolated from 17-day-old chicken embryo sterna, appeared to differ from its counterpart from tibia and femur (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The intact disulfide-bonded molecule of approximately 300 kDa was separable into three chains of 115, 84, and 68 kDa on reduction, the molecular masses being relative to those of collagen standards on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is in contrast to tibial cartilage PG-Lt, from which there was no observed release of a 68-kDa chain (100 kDa relative to globular protein standards) after reduction. The 115-kDa chain of sternum PG-Lt consists of a core 68-kDa polypeptide to which the chondroitin sulfate chains are attached. The ratio of 4-sulfated to 6-sulfated disaccharides released after either chondroitinase ABC or AC digestion is 3:1. Identity of PG-Lt with type IX collagen was indicated by their similar elution profiles on DEAE-Trisacryl and by the presence in both proteins of co-migrating 84- and 68-kDa bands on SDS-PAGE. This identity was confirmed by immunoblotting PG-Lt after SDS-PAGE, with affinity-purified polyclonal antibodies specific for a triple helical domain (HMW) of type IX collagen. The nonreduced high molecular mass material and all three bands of the reduced PG-Lt were immunoreactive, giving immunostaining patterns similar to autoradiographs from the [14C]glycine-labeled protein.

Animals↗
PubMed1985

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.

Animals↗
PubMed1985

High post-translational modification levels in type II procollagen are not a consequence of slow triple-helix formation.

The kinetics of triple helix formation of newly synthesized type II procollagen in chick embryo sternal cartilage cells were determined. The data obtained were in agreement with a model for triple helix formation in cellulo proposed recently (Bruckner et al., 1981). The time required to produce fully triple helical type II procollagen in cartilage cells was only 30% longer than for type I procollagen in tendon cells. Therefore, the several-fold increase in post-translational modification observed for type II collagen as compared to type I collagen cannot be attributed solely to slow helix formation but rather is a result of higher levels of enzymic activity of post-translational modification present in cartilage 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↗
PubMed1985

Type IX collagen from sternal cartilage of chicken embryo contains covalently bound glycosaminoglycans.

Type IX collagen was isolated as a native protein from chicken embryo sternal cartilages and purified to homogeneity. Chondroitin and/or dermatan sulfate were bound covalently to one of the three polypeptide chains present in this protein containing collagenous and noncollagenous domains. Type IX collagen could be metabolically labeled with both radioactive sulfate and glycine. The protein containing either of these labels was sensitive to digestion by bacterial collagenase as well as chondroitinase ABC. Besides the glycosaminoglycans, type IX collagen contains asparagine-linked carbohydrate chains because the protein could be labeled with radioactive mannose and no glycosaminoglycans other than those mentioned above were present. The melting curve indicated that, in contrast to interstitial collagens, this molecule contains at least two disulfide-bonded collagenous domains with distinct thermal stabilities.

Animals↗
PubMed1984

Studies on the oxidation of hemoglobin Zurich (beta 63 E7 Arg).

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.

Chemical Phenomena↗
PubMed1984

Proton nuclear magnetic resonance investigation of the heme cavity structure of liver fluke (Dicrocoelium dendriticum) methemoglobin.

The 1H nuclear magnetic resonance characteristics of met-cyano and met-aquo hemoglobin from the sheep bile duct parasite Dicrocoelium dendriticum have been compared to those of other monomeric hemoglobins and myoglobins. By varying temperature and pH, it was found that the studied material is a mixture of several isozymes differing slightly in their structural features around the heme cavity. The heme in-plane rhombic asymmetry, as indicated by the spread of the heme methyl hyperfine shifts, is intermediate between that of sperm whale myoglobin and leghemoglobin. The proximal histidine is present and its dynamic properties, as probed by the exchange of the ring NH with bulk solvent protons, point towards a cavity more stable than those of sperm whale myoglobin and leghemoglobin. In the met-cyano form, an exchangeable proton was detected close to the iron center that was tentatively assigned to an arginine residue located three amino acid residues closer to the C terminus than the proximal histidine. The transition from the met-aquo form to the met-hydroxy form occurring at pH 8.1 and previously detected by optical methods was observed. Furthermore, consideration of the mean heme methyl hyperfine shift average indicates that the iron remains six-co-ordinate down to below pH 4.5 irrespective of an acid-transition (pK approximately 5) in the protein. However, the presence of a "pseudo" six-co-ordinate (i.e. high-spin, in-plane, five-co-ordinate) iron at pH values below the acid-transition pK cannot be excluded on the basis of the presently available data. The pH dependence of several resonances in both the met-cyano and met-aquo forms of the protein reflect a pK value compatible with the titration of a heme propionate.

Animals↗
PubMed1984

Nonenzymatic glucosylation of proteins: a new and rapid solution for in vitro investigation.

The rates of nonenzymatic glucosylation of albumin, high density lipoprotein (HDL) and low density lipoprotein (LDL) were determined in vitro using [14C]glucose repurified by a new and rapid HPLC method. All commercial preparations were found to contain contaminants reacting 15-20-times faster with protein than the repurified [14C]glucose. Removal of contaminants was critical to the rate determinations and constitutes a substantial improvement over the widely used existing method. The initial rates of nonenzymatic glucosylation determined in vitro for albumin, HDL and LDL were used to predict normal in vivo levels of 0.40, 0.65 and 0.08 mol glucose per mol protein, respectively. This is within the range of values found in vivo for albumin and LDL, but low for HDL. These values would be expected to increase 2-4-fold in diabetes.

Chemical Phenomena↗
PubMed1984

Electron paramagnetic resonance properties of liver fluke (Dicrocoelium dendriticum) nitrosyl hemoglobin.

The electron paramagnetic resonance properties of the nitric oxide derivative of liver fluke (Dicrocoelium dendriticum) hemoglobin (DD-Hb) have been investigated in the pH range from 4.8 to 7.8. In the neutral and alkaline regions the spectra have a rhombic shape, with gx = 2.09, gy = 1.99 and gz = 2.009, and a triplet hyperfine structure of 2.2 mT, due to the nitrogen of the bound NO molecule, in the center resonance. No superhyperfine lines in the gz region, related to the interaction of the iron with the proximal histidine, are detected, suggesting a large distance between the metal and the N epsilon of the imidazole. By lowering the pH the EPR spectrum undergoes a reversible change showing a 3-line pattern in the high-field region. Such a spectrum is fully formed at pH 4.8 and is interpreted in terms of a dissociation of the proximal histidine from the heme iron.

Animals↗
PubMed1984

Nonenzymatic glycosylation of basement membrane collagen in diabetes mellitus.

For a better understanding of the processes leading to diabetic microangiopathy, type IV collagen from kidneys of patients with long-term diabetes was compared with the collagen from kidneys of sex- and age-matched controls. Type IV collagen from diabetic kidneys revealed no abnormalities in amino acid composition, hydroxylation of proline and lysine, enzymatic glycosylation of hydroxylysine, and immunological reactivity with several monoclonal and polyclonal, anti-type IV collagen antibodies. However, ketoamine-linked hexose, resulting from the nonenzymatic condensation of glucose with lysyl or hydroxylysyl residues, was 1.7-fold higher in diabetic type IV collagen. The stoichiometry of this modification was estimated to be 1-2 residues of hexose per triple helical molecule (Mr 380,000). This small amount of ketoamine-linked hexose might hardly have an effect on the function and turnover of type IV collagen, unless it is bound to a crucial site along the collagen molecule. The nonenzymatic glycosylation of collagen might therefore be a mere consequence of the metabolic disturbances, rather than the primary cause for the late complications of diabetes mellitus.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies to human type IV collagen: useful reagents to demonstrate the heterotrimeric nature of the molecule.

Monoclonal antibodies (mAbs) have been prepared against type IV collagen isolated from human kidney. Two mAbs, designated CIV 22 and CIV 16, were extensively characterized. CIV 22 reacted only with native type IV collagen, whereas CIV 16 also bound to fragments derived from the alpha 1(IV) chain after reduction and alkylation of the molecule. Therefore, CIV 22 recognizes a conformational epitope on the triple helical type IV collagen, whereas CIV 16 binds to a sequential determinant in the carboxyl-terminal half of the alpha 1(IV) chain. By immunofluorescence, typical basement membrane structures were stained with both mAbs on frozen sections of different human organs. The mAbs were used to investigate the chain composition of type IV collagen. Radiolabeled type IV collagen bound to CIV 22, proving its triple helical configuration. These native probes, containing both the alpha 1(IV) and the alpha 2(IV) chains, also bound to CIV 16. Since CIV 16 does not react with the isolated alpha 2(IV) chain, both chains must be arranged in a single triple helical molecule (heterotrimer).

Antibodies, Monoclonal↗
PubMed1983

Spectral properties and reactivity towards azide of Dicrocoelium dendriticum met-hemoglobin.

The spectroscopic properties of Dicrocoelium dendriticum met-hemoglobin, investigated between pH 3.8 and 10.5, display two proton-induced transitions with apparent pK values of 8.1 and 4.7. The spectral changes, over the pH region 6.5 to 10.5, correspond to the high to low spin transition usually observed in ferric hemoproteins. The relaxation time (tau = 3.2 ms at the pK) associated with this transition is closely similar to that observed in Aplysia limacina met-myoglobin, but approximately 1000-fold slower than that of sperm whale met-myoglobin. The spectral changes associated with the more acid transition are in the opposite direction and have not been resolved by the temperature jump method. The rate constants for the reaction of azide with Dicrocoelium dendriticum met-hemoglobin were measured between pH 3.8 and 6.7 by the temperature jump method. Two kinetic schemes, both consistent with the pH dependence of the apparent rate constant for binding of azide, were identified. No objective way of discriminating between the two models is available. However, one of them is amenable to a physical interpretation based on a comparison with the structural, kinetic and spectral properties of other monomeric hemoproteins.

Animals↗
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

Quaternary structure and spin equilibria in ferric hemoglobins. A room temperature study.

The effective magnetic moments for a number of human and carp methemoglobin derivatives were determined in solution at room temperature. The data permit us to confirm the dependence of the spin-state equilibrium of azide methemoglobin on the quarternary state of the hemoglobin and to demonstrate a similar dependence for both human and carp aquomethemoglobin. In addition, the pH dependence of the effective magnetic moment and the Soret spectrum of carp azidemethemoglobin are compared.

Animals↗
PubMed1983

Kinetic properties of cobalt--iron hybrid hemoglobins.

The replacement of O2 with CO was studied on cobalt-iron hemoglobin hybrids. Both proto- and mesocobalt hemes were used for the reconstitution. In the oxy quaternary conformation no difference is observed between alpha- and beta-subunits when only proto hemes are present in the hybrid (k4 = 30 s-1, k'4/l'4 = 2.5). If Co-meso heme is present on the beta-chains the binding properties of the partner subunit are modified (k'4/l'4 = 4).

Carbon Monoxide↗