Biomedical subjects

M de Wolf

Publications and source records attributed to M de Wolf.

12 recordsLinked to original sources

PubMed1988

Modification of TSH-stimulated adenylate cyclase activity of bovine thyroid by manipulation of membrane phospholipid composition with a nonspecific lipid transfer protein.

The lipid composition of bovine thyroid plasma membranes was modified using the nonspecific lipid transfer protein from bovine liver. Incubation of plasma membranes with transfer protein and phosphatidylinositol-containing liposomes caused a strong, concentration dependent, inhibition of TSH-stimulated adenylate cyclase activity. Other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid were two to four times less effective as inhibitors of TSH-stimulation. The phosphatidylinositol-induced inhibition was not reversed when more than 80% of phosphatidylinositol incorporated was removed using phosphatidylinositol-specific phospholipase C. Incorporation of phosphatidylinositol in plasma membranes provoked no significant change in the fluorescence anisotropies of the fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(14-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), indicating that the inhibition was not due to changes in membrane fluidity. At phosphatidylinositol concentrations causing a 66% reduction in TSH-stimulated adenylate cyclase activity cholera toxin- and forskolin-stimulated activity as well as basal activity were decreased by maximally 10%. Since TSH binding to bovine thyroid plasma membranes was not affected it is suggested that phosphatidylinositol can act as a negative modulator of the TSH activation of adenylate cyclase and this probably by interfering with the coupling between the occupied TSH receptor and the stimulatory GTP-binding regulatory protein of the adenylate cyclase complex.

Adenylyl Cyclase Inhibitors↗

PubMed1988

Topography, purification and characterization of thyroidal 5'-nucleotidase.

1. Subcellular studies of bovine thyroid indicate that 5'-nucleotidase is predominantly associated with plasma membranes, although a considerable part of this ectoenzyme is also found internalized. 2. The enzyme displaying the features of a glycoprotein has been purified 1400 times by detergent solubilization and two subsequent affinity chromatographic steps. 3. Thyroidal 5'-nucleotidase can be classified as an unspecific metallo-dependent 5'-ribonucleotide phosphohydrolase. The native enzyme exists as a dimer (MW 150 kDalton), composed of two similar or identical subunits.

5'-Nucleotidase↗

PubMed1987

Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes.

Bovine thyroid membranes are able to dephosphorylate exogenous [1-3H]DMP as well as endogenous prelabeled [32P]DMP. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1-3H]dolichol from [1-3H]DMP and the residual amount of [32P]DMP. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. Triton X-100 is necessary for enzymatic activity with [1-3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [32P]DMP. Divalent cations are not essential for enzymatic activity, Ca2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO4(3-) are strongly inhibitory. The inhibition by dolichol and PO3-4 can be explained as the result of product inhibition. An apparent Km-value of 2.5 X 10(-5) M is computed for [1-3H]DMP. Bacitracin inhibits DMP-phosphatase in contrast with other reports. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.

Animals↗

PubMed1986

On the binding of dolichol by bovine liver supernatant.

Experimental evidence is presented that a bovine liver pH 5.1 supernatant possesses binding capacity towards dolichol. Optimal binding is found at physiological pH and at 5 degrees C. At higher temperature the binding is drastically reduced. After binding, the labelled ligand cannot be chased by unlabelled dolichol. Scatchard analysis indicates a single class of binding sites (Bmax = 3.6 pmol/mg protein) with an apparent Kd of 1.8 X 10(-11) M. Only dolichol and dolichyl derivatives drastically reduce the binding phenomenon. The involvement of a protein-like structure is inferred from ammonium sulphate precipitation and proteolysis experiments. Exclusion chromatography and gel electrophoresis under nondenaturating conditions indicate a high molecular weight of the binding complex. Upon SDS electrophoresis, bound [3H]dolichol comigrates with a single protein band (Mr approximately equal to 25,000).

Animals↗

PubMed1986

On the binding of dolichol by human serum.

The presence of a dolichol binding system is demonstrated in human serum. The dolichol binding exhibits normal saturation kinetics with an apparent affinity constant Kd of 6.9 X 10(-6) M. Optimal binding is obtained at pH 7.4 and 5 degrees C. After binding the [3H]dolichol cannot be chased by unlabelled dolichol. The selectivity is examined by competition studies showing that only dolichyl derivatives equally compete for binding sites. From buoyant density centrifugation and gel filtration it is deduced that the dolichol binding is due to a serum protein fraction, displaying the characteristics of VLDL.

Binding Sites↗

PubMed1985

Fluidity characteristics of bovine thyroid plasma membranes.

Highly purified plasma membranes of bovine thyroid were obtained by differential pelleting followed by discontinuous gradient centrifugation in a swing-out rotor. Subfractions of plasma membranes were prepared by affinity chromatography on Con A-Sepharose. The final membrane fractions were enriched 25-30-fold over homogenate in 5'-nucleotidase and alkaline phosphatase and displayed a protein to phospholipid ratio of 1.67 and a cholesterol to phospholipid molar ratio of 0.55. The phospholipid composition did not deviate appreciably from that of whole tissue except for the higher sphingomyelin level (22.5 vs. 14.0%). The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. The physical state of the membrane was studied by (i) calculation of the lipid structural order parameter SDPH from steady-state fluorescence anisotropy determinations of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH); (ii) estimation of the lateral diffusion coefficient of pyrene following excimer formation. These parameters were determined in native thyroid plasma membranes and in reconstituted vesicles, obtained by detergent dialysis from octylglucoside solubilized membrane components. The presence of membrane protein or neutral lipids induced more restraint on the movements of the fluorophores. The lipid order parameter, SDPH was mainly determined by the neutral lipids. Subfractions of plasma membrane enriched in luminal membranes have a slightly lower fluidity (higher SDPH and lower Ddiff values) than subfractions enriched in basolateral membranes. This difference appears to be due to both differences in lipid as well as protein composition. Under physiological conditions, no significant alterations in probe dynamics could be observed upon addition of thyrotropin or cholera toxin, even at micromolar concentrations.

Animals↗

PubMed1976

Phospholipases A1 and A2 in bovine thyroid.

In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.

Animals↗

PubMed1975