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H J Hilderson

Publications and source records attributed to H J Hilderson.

At least 19 recordsLinked to original sources
PubMed1993

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.

Alcohol Dehydrogenase↗
PubMed1993

The shuttling of dolichol between VLDL and HDL: involvement of a protein factor from lipoprotein-deficient human serum.

The occurrence of a dolichol transfer factor in LPDS has been demonstrated using three different transfer assays. Applying a three step purification procedure, the transfer factor could be enriched 4000-5000-fold with a recovery of 1-2%. SDS-gel electrophoresis revealed a molecular weight of 64 kDa. Kinetics as well as the influence of a series of effectors were studied. Transfer was not accompanied by a concurrent esterification and the HDL3 subpopulation showed the highest acceptor capacity. The transfer factor also affected liposomal stability based on calcein fluorescence dequenching upon release. The characteristics of this dol-TP are discussed in view of these other plasma LTPs. Dol-TP might play a role in dolichol transfer from VLDL to HDL, observed in vivo.

Carrier Proteins↗
PubMed1990

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.

Adenylyl Cyclases↗
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↗
PubMed1987

pH-induced transitions in cholera toxin conformation: a fluorescence study.

Determination of the ratio of intrinsic fluorescence with dibrominated Bry 96 (F) relative to that with unbrominated Bry 96 (F0), at neutral pH and in the presence of 0.2 M NaCl, reveals that the A subunit of cholera toxin (CT A) has a somewhat higher affinity for this mild detergent than intact cholera toxin (CT) and its B subunit (CT B). Receptor (GM1 or oligo-GM1) binding has no influence on the very low detergent binding of CT and CT B. Activation of CT A by treatment with dithiothreitol (20 mM) also does not affect detergent binding. The weak hydrophobic nature of CT A is also reflected by the negative modulatory action of anionic phospholipids and deoxycholate on its mono-ADP-ribosyltransferase activity and the ability of the former to decrease its intrinsic fluorescence intensity in a salt-resistant way. Detergent binding of CT A is only slightly pH dependent whereas, upon lowering the pH, detergent binding to CT or CT B becomes significant. In the pH range 6.5-4.2 a gradual increase in detergent binding to CT and CT B occurs. In the narrow pH range 4.2-4.0 a sharp and time-dependent enhancement of brominated Bry 96 quenching is observed. The increase in detergent binding upon lowering the pH is fully reversible, salt dependent, and complete within 10 min (t1/2 = 2 min at 25 degrees C). Solute quenching experiments with the neutral polar quencher acrylamide reveal that upon lowering the pH to 5.0 a marked increase in the exposure of the lone Trp-88 residue in each beta-polypeptide chain of CT B occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗
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

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.

Animals↗
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↗
PubMed1985

Topography, purification and characterization of thyroidal NAD+ glycohydrolase.

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.

5'-Nucleotidase↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1982

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.

Animals↗
PubMed1980

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.

Animals↗
PubMed1979

Structure of the major gangliosides from bovine thyroid.

After preparative isolation, the carbohydrate, long chain base, and fatty acid composition of the major gangliosides from bovine thyroid have been analyzed. The structures were elucidated by determining the molar ratio of the building blocks, permethylation analysis, and enzymatic degradation studies. The following structures are identified: N-Acetylneuraminyl(2,3)-galactosyl(1,4)glucosyl(1,1)ceramidie; N-glycolyneuraminyl(2,3)galactosyl(1,4)glucosyl(1,1)ceramide; galactosyl(1,3)N-acetylgalactosaminyl[(3,2)N-acetylneuraminyl](1,4)galactosyl(1,4)glucosyl(1,1)ceramide; fucosyl(1,2)galactosyl(1,3)N-acetylgalactosaminyl[(3,2)N-acetylneuraminyl](1,4)galactosyl(1,4)glucosyl(1,1)-ceramide. The structures were confirmed by direct inlet mass spectrometry of the permethylated gangliosides and the corresponding asialo derivatives. Structures are proposed for common ions in the different mass spectra.

Animals↗
PubMed1979

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.

Animals↗
PubMed1979

Lipolytic enzymes in bovine thyroid tissue. III. Lysophospholipase activity.

Lysophospholipids are formed during phospholipid breakdown as a result of the action of phospholipases A. At certain concentrations these lysoderivatives destabilise biological membranes. Therefore, their concentration is of critical importance for membrane integrity. Prevention of lysophosphoglycerides accumulation may be the important role for lysophospholipases and is probably the explanation for their widespread occurrence in nature. Lysophospholipase activities were found in molds (Fairbairn, 1948), rice bran (Contardi & Ercoli, 1933), several microorganisms (Brockerhoff & Jensen, 1974), snake and bee venoms (Doery & Pearson, 1964; Mohamed et al., 1969; Shiloah et al., 1973), insects (Khan & Hodgson, 1967; Rao & Subrahmanyam, 1969), fish muscle (Yurkovski & Brockerhoff, 1965; Cohen et al., 1967) and in various animal tissues (Marples & Thompson, 1960). In mammalian tissue the enzyme was first described in beef pancreas (Shapiro, 1953). Relatively high levels were detected in intestine, lung, spleen, liver and pancreas, while lower levels were present in muscle, kidney, testes, brain and blood (Marples & Thompson, 1960). The presence of lysophospholipase activity in both supernatant and sediment of bovine thyroid was reported previously in relation to possible interference of this enzyme with the phospholipase A activity assay (De Wolf et al., 1976). The subcellular localization of bovine thyroid lysophospholipase and some properties of the membrane bound enzyme activity are discussed in this paper.

Animals↗