Substrate specificity and regulation of activity of rat liver beta-D-glucuronidase.
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Animals↗
Biomedical subjects
E Buddecke
Publications and source records attributed to E Buddecke.
[The effect of benzarone on serum lipids and arterial wall of cholesterol fed rats].
Rats of BD X strain and SHR/NIH Montreal Ingelheim strain (genetic hypertension) received a diet containing 3.9% cholesterol or 3.7% cholesterol plus 0.6% benzarone, respectively, and libitum for 5 or 9 months. The following chemical and ultrastructural results were obtained. 1. The cholesterol-benzarone diet causes a body weight reduction of 10%, a relative increase of serum HDL and a corresponding decrease of serum LDL and VLDL, as compared with the effects of the cholesterol diet. No differences of total serum cholesterol and serum triglycerides between the two groups were observed. 2. The aorta of cholesterol fed animals shows a slight but statistically not significant increase of total cholesterol content. 3. No differences in the composition of connective tissue components (collagen, elastin, uronic acid content) between the cholesterol fed animals and a control group on normal diet could be detected. 4. Electron micrographs of several vessel wall areas from cholesterol fed hypertensive animals revealed fibrosis, necrosis of media muscle cells and an increase of matrix vesicles. Severe damages were found in coronary arteries and in the caudal arteries. 5. Cholesterol feeding of hypertensive rats increases the cholesterol content of liver 10fold and the triglycerides content 3fold as compared with liver lipids of control rats. Benzarone application to cholesterol fed rats effects a statistically significant decrease of liver cholesterol and triglycerides.
Animals↗
Sphingolipid-induced enhancement of receptor-mediated uptake of low density lipoproteins in normal and receptor-deficient human skin fibroblasts.
(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides GM1 or GD1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced suppression of [14C] acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of alpha 2-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.
Cholesterol↗
Phosphate ester groups in proteoglycans from bovine nasal cartilage.
1. Proteoglycan subunits isolated by standard procedures from bovine nasal cartilage, previously incubated in the presence of [32P]phosphate contain [32]-phosphate ester groups as a regular structural component. 2. Contamination of the proteoglycan subunit with 32P-labeled nucleic acids could be excluded by repeated cesium chloride density gradient centrifugation under associative and dissociative conditions, lanthanum chloride precipitation, gel filtration and by the resistance of the proteoglycan subunit associated 32P to phosphoric diester hydrolases. 3. The [32P]phosphate ester groups are associated to the chondroitin sulfate peptide fraction obtained by proteolytic digestion of the proteoglycan subunit molecule. Degradation of the chondroitin sulfate peptide by chondroitinase ABC resulted in a 32P-labelled oligosaccharide peptide fraction, that contains xylose, galactose, glucuronic acid and inorganic phosphate in a molar ratio 1 : 2 : 1 : 0.12. 4. 32P radioactivity is released as inorganic phosphate by treatment of the 32P-labelled oligosaccharide peptide with acid phosphatase or alkali.
Acid Phosphatase↗
Homologous low density lipoprotein does not affect proteoglycan metabolism of cultured skin fibroblasts and arterial smooth muscle cells.
The relevance of lipoprotein-glycosaminoglycan interactions on the proteoglycan metabolism was investigated. The following results were obtained: 1) Biosynthesis of [35S]proteoglycans by cultured human skin fibroblasts and their distribution to different compartments are neither affected by preincubation of the cells with homologous LDL nor by their presence. The internalisation of LDL was evidenced by a marked depression of [14C]cholesterol synthesis from [1-(14)C]-acetate. 2) Under the conditions of endocytosis experiments the formation of insoluble proteoglycan-LDL complexes is insignificant. Endocytosis and degradation of exogenous proteoglycans by skin fibroblasts or arterial smooth muscle cells proceed at normal rates in the presence of low or excess LDL concentrations. 3) From the results it may be concluded, that internalized LDL and their degradation products neither influence the synthesis and distribution of sulfated proteoglycans nor control expression and function of proteoglycan specific cell surface receptors.
Acetates↗
Age-related changes in the incorporation of [35S]sulfate into two proteoglycan populations from human cartilage.
From human hyaline cartilage (processus xyphoid) preincubated in the presence of [35S]sulfate, proteoglycans were extracted by 4M guanidinium chloride and divided into 6 age groups. Fractionation of proteoglycans by gel filtration under dissociative conditions resulted in two proteoglycan fractions (a and b) with different hydrodynamic volumes. The higher molecular weight fraction a contained chondroitin sulfate, the fraction b keratan sulfate as predominant glycosaminoglycan, the chondroitin sulfate/keratan sulfate ratio decreasing with increasing age in either fraction. The relative portion of proteoglycan fraction b and its 35S-labelling increased with increasing age. From the specific 35S radioactivities of the chondroitin sulfate and keratan sulfate preparations, the occurrence of two independent proteoglycan populations is suggested. A precursor-product relationship between proteoglycan fraction a and b could be excluded.
Adolescent↗
Desialized low-density lipoprotein regulates cholesterol metabolism in receptor-deficient fibroblasts.
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Acetates↗
Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.
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Acetylation↗
Acceptor-specific glucuronyl transfer catalyzed by beta-glucuronidase.
Highly purified rat liver microsomal or lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) catalyzes the specific transfer of glucuronly residues from phenyl-beta-D-[U-14C]glucuronide to acceptor sugars. Specificity requirements of acceptor sugars are found to be: pyranose structure, 4C1-conformation and equatorial position of C2 and C3 hydroxyl groups or pyranose structure, 1C4-conformation and equatorial position of C3 and C4 hydroxyl groups. The acceptor capacities of 30 monosaccharides and glycosides including di- and tri- saccharides conform to this prinicple. The specificity of the beta-glucuronidase catalyzed glucuronyl transfer is proved by the exclusive formation of beta-glucuronly (1--3)glycosidic linkages. Glucuronly transfer rates increase with increasing donor substrate and increasing acceptor sugar concentration. In the presence of 1 M acceptor sugar the ratio of the transfer rate to the rate of enzymatic hydrolysis is about 2:1. An 'acceptor substrate binding site' on the surface of the beta-glucuronidase molecule which brings the C3 hydroxyl function of the acceptor sugar close enough to the C1 atom of the glucuronyl residue, is postulated.
Animals↗
Sialic-acid content of low-density lipoproteins controls their binding and uptake by cultured cells.
The (high-affinity receptor)-mediated uptake of homologous low-density (low-rho) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low-rho lipoprotein particles. This conclusion is derived from the following results. 1. Gangliosides incubated with native low-rho lipoproteins associate with low-rho lipoprotein particles. Low-rho lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low-rho lipoproteins or those preincubated with desialized gangliosides. 2. The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low-rho-lipoprotein receptor. 3. Desialyzed low-rho lipoproteins are internalized by smooth muscle cells up to 100% faster than native low-rho lipoproteins, the enhancement of uptake corresponding to the degree of desialization.
Aorta↗
The effect of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of arterial tissue and cultured arterial smooth muscle cells.
Studies on the action of 2-ethyl-3-(4-hydroxybenzoyl)-benzofuran (benzarone) on the metabolism of cultured arterial smooth muscle cells and arterial tissue had the following results: 1. On incubation of calf arterial tissue in the presence of 0.03--0.1 mmol/l benzarone (8--26 micrograms/ml medium) the metabolic transformation of [14C]-glucose to [14C]-lactate and 14CO2 and the incorporation of 14C radioactivity into the total lipids is not significantly altered as compared with control values. In cultured human arterial smooth muscle cells 0.03 mmol/l benzarone stimulates the incorporation of [14C]-acetate and [3H]-palmitate into the cellular lipids while the receptor mediated uptake of homologous low-density lipoproteins (LDL) by the cells and their release are not influenced. 2. In concentrations greater than 0.2 mmol/l benzarone the glucose utilisation of arterial tissue is enhanced, while the labelling of lipids, in particular the labelling of the triglyceride fraction, is depressed. Under the same conditions the protein biosynthesis and the incorporation of [14C]-acetate and [3H]-palmitate into the total lipids of cultured arterial smooth muscle cells are decreased.
Acetates↗
Endocytosis of sulphated proteoglycans by cultured skin fibroblasts.
1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 X 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 X 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.
Cell Count↗
Correlation of lysozyme activity with proteoglycan biosynthesis in epiphyseal cartilage.
Pig epiphyseal cartilage (proximal ulna epiphysis) previously incubated into vitro in the presence of sodium [35S]sulfate or [3H]thymidine was either analyzed by autoradiography or separated into 9 morphologically defined consecutive layers and investigated for 35S-incorporation into the guanidinium chloride-extractable proteoglycans and for lysozyme activity. The lowest 35S incorporation and lysozyme activity were determined in the zone of resting cells, but there is a consecutive increase in the rate of proteoglycan synthesis and lysozyme activity toward the diaphyseal cartilage-bone junction, with the maximum at the lower columnar cell zone and a sharp reduction of both parameters at the hypertrophic zone. The maxima of 35S incorporation and [3H]thymidine incorporation do not coincide. The guanidinium chloride-soluble proteoglycans exhibit macromolecular polydispersity. Fractions excluded from as well as retarded by Sepharose 2B gel could be separated and were detected in all zones. The results indicate a correlation of proteoglycan biosynthesis and lysozyme activity in epiphyseal cartilage.
Animals↗
Amount and properties of uptake forms in preparations of alpha-mannosidase from pig kidney.
alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from pig kidney has been shown to exist in multiple forms differing in their capability to be endocytosed by alpha-mannosidase deficient cultured cells. A method is presented to evaluate the amount of "uptake" forms in different preparations of the enzyme. Preparations with different rates of uptake were shown to contain different amount of "uptake" forms and "non-uptake" forms. The content of "uptake" forms in a preparation was identical with that of enzyme molecules bearing a phosphorylated carbohydrate group necessary for the recognition by cell surface receptors.
Alkaline Phosphatase↗
Differences in intracellular corrective activity in cultured mannosidosis cells of acidic alpha-mannosidases from various sources.
The ability of acidic alpha-mannosidases from pig kidney, bovine liver, human urine and placenta, and jack bean to correct the deranged metabolism of cultured skin fibroblasts from manosidosis patients is studied. One cause for the different corrective abilities are, as shown for other systems, the different rates of endocytosis of the enzyme forms into the cells. Besides this, it is demonstrated for the first time, that there are differences in the intracellular corrective activity of the internalized enzymes, which can be explained by different specificities of the enzymes against the storage material.
Animals↗
Morphological and autoradiographical characterization of membrane-like layers surrounding cultured fibroblasts.
Two membrane-like layers of approximately 7 nM thickness were observed in ultrathin cross-sections of flat-embedded cultured fibroblasts. One "membrane", underlying the cell-monolayer, possibly established contacts between cells and substratum. The other "membrane" covered the whole cell culture. These "membranes" were at least partially synthesized by the cells themselves, as was shown by high resolution autoradiography. With regard to our biochemical findings, the autoradiographical results showed that components of these "membranes" consisted of sulfated proteoglycans and may be the morphological equivalent to the biochemically well-defined cell surface associated material, the so-called pericellular pool of glycosaminoglycans.
Animals↗
[Serum lysozyme concentrations in patients with morbus Crohn before and after bowel resection (author's transl)].
Serum lysozyme (muramidase) concentrations were determined in five patients with Morbus Crohn before and after resection of inflammated bowel areas. The serum lysozyme activity which was elevated before surgical treatment in all patients fell to normal values after bowel resection within a few hours. Our findings suggest, that the elevated serum lysozyme reflects an increased lysozyme production in the inflammated bowel tissue.
Colitis, Ulcerative↗
Evidence for material from mannosidosis fibroblasts crossreacting with anti-acidic alpha-mannosidase antibodies.
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Animals↗