G. Van Dessel

Identification and characterization of dolichyl dolichoate, a novel isoprenoic derivative in bovine thyroid.

Inspection of the TLC pattern of the neutral lipid fraction of bovine thyroid reveals, in addition to cholesteryl esters and dolichyl fatty acid esters, the presence of a not yet identified compound in the most apolar lipid region. This unknown compound was purified on a preparative scale by silicic acid column chromatography, Lipidex-5000 gel-filtration chromatography and preparative thin-layer chromatography. By chemical (hydrolysis and reduction) and spectroscopic (ultraviolet, infrared, NMR, mass spectroscopy) methods this lipid was identified as dolichyl dolichoate. The homologue pattern was analyzed, by HPLC and FDMS, respectively.

Structural features of the binding site of cholera toxin inferred from fluorescence measurements.

The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed.

Characterization of the in vitro conversion of dolichol to dolichoate in bovine thyroid.

The enzymic conversion of dolichol into dolichoic acid has been studied in bovine thyroid subcellular fractions using [1-3H]dolichol as a substrate. The presence of conversion activity could be demonstrated in both the mitochondrial- and supernatant fractions. Investigation of cofactor requirements revealed that NAD+ was essential for reaching optimal activity. From kinetic studies Km-values of 3.5-4 microM and 0.29 mM could be calculated for, respectively, dolichol and NAD+ using the mitochondrial fraction as an enzyme source. No inhibitory effects from ethanol or pyrazole were detected suggesting that alcohol dehydrogenase is not involved in the dolichol-->dolichoate conversion as observed in a bovine thyroid mitochondrial fraction. From inhibitor studies the conversion system behaves distinctly differently from the NADP(+)-depending microsomal oxidoreductase as well as from catalase. The conversion activity in the supernatant on the other hand must be ascribed, at least partially, to a side activity of alcohol dehydrogenase.

Modification of the adenylate cyclase activity of bovine thyroid plasma membranes by manipulating the ganglioside composition with a nonspecific lipid transfer protein

Gangliosides (GM1, GT1b, GD3) were incorporated in bovine thyroid plasma membranes using the nonspecific lipid transfer protein from beef liver. The transfer of GT1b or GD3 in the presence of 16 units of transfer protein was twice as high as that of GM1. However, taking into account the spontaneous exchange (approximately 8% for GT1b or GD3 and 1% for GM1) the transfer protein seemed to be more effective for GM1. Incorporation of these gangliosides in bovine thyroid plasma membranes caused a concentration dependent inhibition of the TSH-stimulated adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was not significantly affected by ganglioside modification of the plasma membranes, indicating that the gangliosides do not act at the level of the catalyst of adenylate cyclase. Binding experiments on the other hand revealed that TSH binding to bovine thyroid plasma membranes was inhibited with the same order of efficacy (GT1b greater than GD3 greater than GM1) and to the same extent as their inhibitory effect on TSH stimulation. Therefore, this indicates that the ganglioside induced drop in TSH binding might be an important factor in the decrease in TSH-stimulated adenylate cyclase activity. Incorporation of GT1b or GD3 (approximately 11 nmol) in bovine thyroid plasma membranes, however, also induced a substantial decrease in cholera toxin-stimulated adenylate cyclase activity (approximately 30%) and to a lesser degree a decrease in NaF-stimulated activity (approximately 17%), whereas GM1 incorporation did not significantly affect these stimulated activities. These latter inhibitory effects were paralleled by changes in fluorescence steady-state anisotropy: GT1b modification of the plasma membranes provoked a slight increase in TMA-DPH anisotropy, whereas the anisotropy of DPH was substantially enhanced after incorporation of GD3 or GT1b. These results suggest that gangliosides might also interfere with the coupling between the alpha-subunit of the stimulatory GTP-binding regulatory protein and the catalyst of the adenylate cyclase system by affecting the membrane fluidity.

Intracellular and Extracellular Flow of Dolichol

Dolichols belong to a family of polymeric lipids with the isoprene unit as the repeating building block. This lipid class represents a relative large group of natural products displaying a wide variety of biological functions (Table I) (Rip et al., 1985). The existence of dolichols was first reported by Hemming and coworkers (Pennock et al., 1960; Burgos et al., 1963).

Spectrophotometric determination of dolichol and dolichyl derivatives using the Chugaev color reaction

A simple colorimetric method for the assay of microamounts of dolichol is described. It is based essentially on the Chugaev color reaction. The procedure allows the rapid (1 h after purification of the sample, less than half a day for the whole procedure) and reliable (% SE less than or equal to 5%) determination of dolichol and dolichyl derivatives in the microgram range. Color production is linear within the concentration range 2 to 20 micrograms dolichol. With biological samples, prior to colorimetric assay, dolichol and dolichyl derivatives are isolated by solvent extraction and TLC. Recoveries are monitored by isotope dilution analysis. The method has been used for the analysis of dolichol and dolichyl ester levels in bovine thyroid tissue. The procedure can also be applied for the analysis of other isoprenoids.

DNA-Dependent rna polymerases from bovine thyroid: Catalytic properties and template specificities

1. DNA-dependent RNA polymerases I and II have been purified starting from bovine thyroid nuclei yielding a purification factor of 230 for the RNA polymerase I and a purification factor 3212 for RNA polymerase II. RNA polymerase II was further characterized by gel electrophoresis and amino-acid analysis. 2. Kinetics and optimal assay conditions for both RNA polymerases were studied. 3. The template efficiency of a number of DNA preparations was investigated. 4. Rifamycin AF 013 and heparin act as initiation inhibitors. 5. Polyamines were shown to enhance the rate of chain elongation.

Isolation and identification of polyprenols from bovine thyroid gland.

The presence of polyprenols in bovine thyroid was demonstrated. After preparative isolation, the structure was elucidated by chemical and spectroscopic techniques. The main polyprenol homologue has a molecular weight of 1380 corresponding to the presence of 20 isoprene units. From NMR studies it appears that 18 units have the cis configuration and that the 2 others are trans isoprene units. The dolichol content amounts to 0.2 mg/g wet weight. About 5% was found in the esterified form.

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.

The Subcellular Biochemistry of Thyroid

In this review the subcellular localization of enzymes and constituents in thyroid is discussed. Conditions and results of differential pelleting and gradient centrifugation studies are described with special attention to the validity of the markets used (Table VI). Special approaches to the isolation and characterization of thyroid organelles and membranes are extensively reviewed (Table VII). Subcellular fractionation of thyroid tissue has been shown to be an arduous task. Classic approaches for differential pelleting and gradient centrifugation, which have been proved successful for rat liver, are not always equally satisfactory for thyroid. The major problem is the toughness of the tissue requiring rather traumatizing homogenizing procedures. Nevertheless, the fractionation procedures did allow the subcellular localization of some enzymes and constituents to be established with a high degree of certainty. Furthermore, enriched subcellular fractions have been isolated which have been useful for biochemical studies concerning the specific function of this tissue.