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Biomedical subjects

H Sandermann

Publications and source records attributed to H Sandermann.

At least 73 records · Page 4Linked to original sources
PubMed1987

Mutagenicity of chloroaniline/lignin metabolites in the Salmonella/microsome assay.

Chloroanilines are constituents of many agrochemicals and have been found to be metabolized to succinic acid conjugates, e.g., succinamides and succinimides. The mutagenic potential of five chloroanilines and their succinamides and succinimide derivatives have been tested with two strains of Salmonella typhimurium (TA98 and TA100) with and without rat hepatic microsomal fraction. None of the compounds produced a dose response effect with a two-fold increase in revertants indicating that these compounds are not mutagens or promutagens in these assays.

Aniline Compounds↗
PubMed1986

Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin.

Binding Sites↗
PubMed1985

N-Ethyl-17(R,S)-methyl-(6aR,10aR)-delta 8-tetrahydrocannabinol-18-oic amide. Brain pharmacokinetics in mice, triglyceride/phospholipid partitioning and generalization to the discriminative stimulus properties of delta 9-THC in rats.

The title compound, designed as a model for the affinity moiety of a cannabinoid affinity gel was synthesized in tritiated form (sp. act. 7.27 mCi/mmole). To validate the affinity approach to isolate the putative THC receptor, the properties of the amide were studied. Upon i.p. injection in mice the amide reaches peak brain levels of 0.13% of the total dose after 15 min. Following i.v. injection, maximal brain concentrations of 1.9% are observed at 5 min. Compared to delta 9-THC, which distributes almost equally between triglyceride and phospholipid phases (51:49) the amide exhibits a strong preference for phospholipids (5:95) that can be interpreted as high relative membrane affinity. In rats trained in a water maze to discriminate between i.p. injections of 3 mg/kg delta 9-THC (ED50 = 1.8 mg/kg) and its vehicle, the amide was generalized to the training drug, being five times less potent (ED50 = 8.7 mg/kg) than delta 9-THC. This demonstration of cannabis-like activity indicates that the amide retains affinity to the postulated receptor and justifies the choice for the affinity ligand.

Animals↗
PubMed1984

Beta-D-galactoside transport in Escherichia coli. Solubilization in organic solvent and reconstitution of binding.

The beta-D-galactoside transport protein (y-gene product) of Escherichia coli, strain T 206, was solubilized in 85-95% yield using the organic solvents hexamethylphosphoric triamide at pH 7.5 or butan-1-ol at pH 4.2. The transport protein obtained with the former solvent could be incorporated into a defined lipid/protein aggregate of density 1.12 g/ml, but no beta-D-galactoside binding was restored. Diacylglycerol kinase regained activity in the same lipid/protein aggregates. In control experiments, liposomes formed from hexamethylphosphoric triamide were found to be active in the valinomycin-mediated uptake of Rb+ ions. beta-D-Galactoside binding (3.6-5.7 nmol/mg protein) as well as diacylglycerol kinase activity [7 nmol min-1 (mg protein)-1] was reconstituted into proteoliposomes from butan-1-ol solution by adaptation of a published procedure [Wright, J. K. et al. (1982) Eur. J. Biochem. 124, 545-552]. A microparticulate nature of the butan-1-ol-solubilized transport protein could be excluded by gel permeation chromatography on a newly synthesized matrix, hydroxypropyl-Sephacryl S-300.

Butanols↗
PubMed1984

Plant biochemistry of xenobiotics. Purification and properties of a wheat esterase hydrolyzing the plasticizer chemical, bis(2-ethylhexyl)phthalate.

A soluble wheat esterase, catalyzing a cleavage of the mass-produced plasticizer chemical, bis(2-ethylhexyl)phthalate (DEHP), has been discovered. Although wheat plants and seeds as well as cultured wheat cells contained more than 12 non-specific esterase activities, only a single protein with a marked preference for a substrate chain-length of 6-8 carbon atoms was active with DEHP. This enzyme is shown to differ from all previously characterized plant lipases and esterases. The enzyme was purified 10-fold from wheat plants and 280-fold, to electrophoretic homogeneity, from cultured wheat cells. An apparent functional molecular mass of 38 000 Da and an apparent subunit molecular mass of 22 000 Da were determined. Inhibitor experiments pointed to the catalytic involvement of a serine residue. Cleavage of DEHP by the purified enzyme was about 10(4) times slower than cleavage of 4-nitrophenyl octanoate. This was consistent with previous evidence for a rate-limiting role of the esterase reaction in DEHP metabolism by intact wheat cells.

Carboxylic Ester Hydrolases↗
PubMed1984

Site--site interaction in the lipid activation of beta-hydroxybutyrate dehydrogenase.

Kinetic data for the activation of the beta-hydroxybutyrate dehydrogenase by long-chain lecithins [(1979) Biochemistry 18, 2420-2429; (1983) J. Biol. Chem. 258, 208-214] are analyzed. A previous kinetic model [(1982) Biochemistry 21, 3899-3908] is shown not to apply. Instead, the use of a two-site Adair equation points to a strongly cooperative interaction between the lecithin binding sites (delta G, -2.8 kcal/mol).

Binding Sites↗
PubMed1984

Use of plant cell cultures to study the metabolism of environmental chemicals.

The metabolism of the following environmental chemicals has been studied in cell suspension cultures of wheat (Triticum aestivum L.) and soybean (Glycine max L.):2, 4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), hexachlorobenzene, pentachlorophenol, diethylhexylphthalate , benzo [alpha] pyrene, and DDT. All chemicals tested, including the persistent ones, were partially metabolized. Polar conjugates predominated in all cases. A covalent incorporation into lignin could be demonstrated for 2,4-D and pentachlorophenol. A specific deposition in the cellular vacuole could be demonstrated for the beta-D-glucopyranoside conjugates derived from 2,4-D. A rapid assay procedure to evaluate the metabolism of a given 14C-labeled chemical in plant cell suspension cultures is described. This procedure requires about 1 week, and the reproducibility of the results obtained has been assessed.

Benzo(a)pyrene↗
PubMed1983

Cooperative lipid activation of (Na+ + K+)-ATPASE as a consequence of non-cooperative lipid-protein interactions.

Lipid activation data for (Na+ + K+)-ATPase (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113-131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123-128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 microM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity.

Animals↗
PubMed1983

Lipid dependence of diacylglycerol kinase from Escherichia coli.

Diacylglycerol kinase apoprotein was purified from membranes of Escherichia coli K 12. The protein was catalytically inactive, but regained activity upon recombination with phospholipids, certain neutral lipids, or fatty acids. Activation proceeded with positive cooperativity and was independent of the exact chemical structure, bilayer arrangement or electrical charge of the lipid. The apoprotein was activated by lysophosphatidylethanolamine but not by lysophosphatidylcholine. 1-Monooleoylglycerol was an effective activator and substrate at the same time. The fluidity and the polarity of lipids appeared to be generally important for activation. Lipid polarity was estimated by a triacylglycerol/phosphatidylcholine-partitioning procedure. All lipids showing preferential association with triacylglycerol failed to activate the kinase apoprotein even in the presence of detergent. It is concluded that a defined hydrophilic/lipophilic balance of the lipid was required for the formation of a functional lipoprotein complex.

Apoenzymes↗
PubMed1982

Diglyceride kinase from Escherichia coli. Modulation of enzyme activity by glycosphingolipids.

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.

Cardiolipins↗
PubMed1982

Lipid-dependent membrane enzymes. A kinetic model for cooperative activation in the absence of cooperativity in lipid binding.

The dependence of integral membrane enzymes on lipid activators in analyzed in terms of multiple binding site kinetics. Rate equations for an enzyme with n independent and indentical lipid binding sites are derived for the case that enzyme activity is proportional to the total amount of lipid bound, or that only fully substituted enzyme is active. A third equation applies to the case that lipids bind with infinite cooperativity to give fully substituted and active enzyme. None of the three models was entirely consistent with existing experimental data. The following kinetic model is shown to accommodate the degree of cooperativity observed in lipid activation experiments as well as the number of independent lipid-binding sites determined by electron-spin resonance measurements. The membrane enzyme is assumed to have n non-interacting and identical lipid-binding sites. Only fully substituted enzyme (ELn) and the next most highly substituted forms such as ELn-1 and ELn-2 may possess enzyme activity. These assumptions lead to cooperativity in activation. Cooperativity reaches a maximum when enzyme activity starts to appear with about 80% of the full lipid substitution. The increase in cooperativity is accompanied by a decrease in the lipid concentration required for half-maximal activation. Further kinetic aspects of a dynamic boundary lipid layer around integral membrane enzymes are discussed.

Allosteric Regulation↗
PubMed1979

Diglyceride kinase from Escherichia coli. Purification in organic solvent and some properties of the enzyme.

The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan-1-ol. The enzyme was purified in organic solvent by precipitation at -20 degrees C, chromatography on DEAE-cellulose and repeated chromatography on Sephadex LH-60. The final 1460-fold purified enzyme preparation gave a single protein band upon isoelectric focusing in the presence of Triton X-100 (pI, 4.0) and upon polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulphate. The latter method as well as gel chromatography on Sephadex LH-60 indicated a molecular weight of about 15400. The purified enzyme was devoid of lipid, and it required re-addition of lipid for activity. sn-1,2-Dipalmitate and ceramide were phosphorylated, whereas the C55-isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol-soluble C55-isoprenoid-alcohol kinase from Staphylococcus aureus did not phosphorylate sn-1,2-dipalmitate.

Cell Membrane↗