Eckhart Buddecke

The complete enzymic degradation of glycopeptides containing O-seryl and O-threonyl linked carbohydrate

Abstract In recent years a considerable amount of information on the molecular structure of glycoproteins has been published. Little is known, however, on the biosynthesis of these compounds and no information is available on the enzymic breakdown of glycoproteins to their individual constituents. In the present paper the purification and properties of a β-N-acetylhexosaminidase and its action on glycopeptides containing O-seryl and O-threonyl linked N-acetylgalactosamine are described.

Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression

Abstract The design and use of minimally modified oligonucleotides for specific inhibition of gene expression is discussed. The “minimal” protection strategy is a combination of the end-capping technique and the protection of internal pyrimidine positions which are the major sites of endonuclease degradation. By reducing the number of phosphorothioate modifications needed to make the oligonucleotide resistant to nuclease degradation, non-sequence-specific effects, which are frequently observed with uniformly phosphorothioate-modified oligonucleotides, can be reduced.

Compartmentation and characterization of different proteoglycans in bovine arterial wall.

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

Mannosidosis. Storage of Mannose-containing Material in Cultured Human Mannosidosis Cells and Metabolic Correction by Pig Kidney α-Mannosidase

Skin fibroblasts from healthy individuals and a mannosidosis patient were cultured in the presence of [2-3H] mannose and the cell homogenates were fractionated by trichloroacetic acid precipitation into a precipitable and a non-precipitable portion. In uptake as well as in chase experiments the precipitable fractions show no significant difference in their content of radioactivity, while an increased level of radioactivity is found in the non-precipitable fraction of mannosidosis cells. This higher radioactivity content is due to a higher mannose content and is caused by a slower degradation of this fraction. The differences between the metabolisms of the two cell lines can be expressed by the ratio of radioactivity in the non-precipitable and the precipitable fractions. This value is about three times higher for mannosidosis than for control cells. Pig kidney alpha-mannosidase is taken up by both cell lines and is able to correct the impaired degradation of the non-precipitable material in mannosidosis cells, as shown by the normalization of the above defined ratio of radioactivity for this cell type.

Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

The glucosyltransferase (UDP-glucose galactosylhydroxylsine collagen glucosyltransferase, EC 2.4.1.?.) was purified 50-fold from calf arterial tissue by ammonium sulfate precipitation, gel filtration and electrofocusing. The purified enzyme has a molecular weight of 72 000 and a requirement for Mn2. It resolves into two activity peaks when submitted to electrofocusing (isoelectric point at pH 4.2 and 8.1) or disc electrophoresis and exhibits a double pH optimum (pH 8.3 and 9.9). The enzyme was found to transfer glucose from UDP-glucose to the denatured forms of citrate-soluble calf skin collagen (I), the alphal chain (II) and the beta12 component (III) derived from it, and of an acetic-acid-souble collagen preparation (IV) obtained from alkali-treated calf arterial tissue. The Km values for the substrates were 1.67 X 10(-4) (I), 6.3 X 10(-4) (II), 3.3 X 10(-4) (III) and 2.8 X 10(-4) mol/l (IV), indicating that the enzyme has the greatest affinity for the calf skin collagen. The glucose transferred to hydroxylysine-linked galactose residues may be released subsequently by the action of a specific alpha-glucosidase purified from bovine spleen. The results support the assumtion that the glucosylation step in the course of the (pro-)-collagen biosynthesis depends on special structural features of the substrate and may be controlled by a specific alpha-glucosidase.

Evidence for material from mannosidosis fibroblasts crossreacting with anti-acidic alpha-mannosidase antibodies.

Mannosidosis is a lysosomal storage disease [ 1] caused by the deficiency of a-mannosidase (EC 3.2.1.24) forms A and B with an acidic pH-optimum [2-41. The residual cll-mannosidase activity present in mannosidosis tissues and cultured cells is structurally and genetically different, it can be activated by Co” and is heat labile [2-61. At extremely high substrate concentrations a residual acidic cr-mannosidase activity with altered Km and thermal stability becomes detectable in mannosidosis fibroblasts [7]. This was interpreted as due to a structurally altered enzyme formed in mannosidosis instead of the normal acidic c+ma.nnosidase. We give here radioimmunological evidence for the existence of material in mannosidosis fibroblasts that reacts with antibodies specifically binding acidic cw-mannosidases from different sources.

Immunological cross-reactivity between α-mannosidases from different sources

Abstract 1. 1. α-Mannosidase with acidic pH-optimum was purified from pig kidneys more than 2000-fold to a specific activity of 13.2U/mg protein. The final product contains the multiple forms A and B and has a quarternary structure. 2. 2. The γ-globulin fraction of rabbit antiserum against the pure pig kidney α-mannosidase leads in immunodiffusion and precipitation experiments to precipitation not only with α-mannosidases from pig tissues but also (though with lower affinity) from bovine liver and human placenta, urine and skin fibroblasts.

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.

Vergleichende Untersuchungen über die Wirkung freier und konjugierter Östrogene auf den Lipidstoffwechsel der ovarektomierten Ratte

Summary12 weeks old female Wistar rats were ovarectomized and divided into 4 groups, 3 of them obtained from the 21.–48. day after castration by oral administration 15–180 μg estrogensulfate (Presomen), 10–120 μg estradiol and 1–12 μg 17-ethinyl-estradiol respectively dissolved in 0.2 ml olive oil. 0.2 ml olive oil were given to a control group. At the 49. day all animals received an intraperitoneal injection of 300 μC14C-acetate and were sacrified 4 hrs later. Lipid analyses of serum, liver aorta, spleen, kidney and brain had the following results:1.The ratio of the specific radioactivities of total lipids isolated from liver, spleen, kidney, aorta and brain of control animals was 10∶7∶4∶4∶0.7.2.Estrogensulfate decreases serum cholesterol level statistically significant to half of the control values, due to a selective fall of esterified cholesterol. 17-ethinylestradiol does not influence the serum total cholesterol but increases the percentage of free cholesterol.3.Total lipids of liver are decreased by estrogensulfate treatment but their specific radioactivity was increased, all changes being statistically significant. Analyses of phospholipids, triglycerides, cholesterol and free fatty acids revealed an effect on all lipids subfractions. No changes of total lipid content are observed after 17-ethinylestradiol and estradiol treatment, though the former causes an increase of specific radioactivity of total lipids.4.Estrogensulfate and estradiol do not influence content, distribution and specific radioactivity of aortic lipids while 17-ethinylestradiol diminishes content of total lipids and specific radioactivity of the triglyceride and diglyceride fraction.5.Content and specific radioactivity of lipids in spleen, kidney and brain are in the range of control group after treatment with free or conjugated estrogens.6.Onin vitro incubation of arterial tissue in the presence of 10−5–10−4 M estrogen and testosteron respectively the incorporation of14C-acetate into total lipids and lipids subfractions is markedly increased, while estrogensulfate under otherwise same conditions has no effect.ZusammenfassungOvarektomierte Wistarratten erhielten 21 Tage täglich Östrogensulfat, Östradiol-17β bzw. 17α-Äthinylöstradiol und 4 Std vor Versuchsende eine intraperitoneale Injektion von 300 μCi [1-14C]Acetat. Lipidanalysen des Serums und verschiedener Organe wurden mit einer Kontrollgruppe unbehandelter ovarektomierter Ratten verglichen und hatten folgendes Ergebnis:1.Der Serumcholesterinspiegel (Gesamtcholesterin) sinkt unter der Behandlung von Östrogensulfat (Presomen®) statistisch signifikant auf die Hälfte des Wertes der Kontrollgruppe ab. Östradiol-17β und 17α-Äthinylöstradiol haben keinen Einfluß auf die Konzentration des Gesamtcholesterins im Serum.2.Der Gehalt der Gesamtlipide der Leber ist nach Östrogensulfatbehandlung signifikant erniedrigt, die spezifische Radioaktivität der Gesamtlipide mit gesicherter statistischer Wahrscheinlichkeit auf das Doppelte erhöht. Östradiol-17β besitzt keinen Einfluß auf den Gehalt und die spezifische Radioaktivität der Leberlipide. Unter 17α-Äthinylöstradiol-Behandlung ist bei unveränderter Lipidkonzentration die spezifische Radioaktivität der Gesamtlipide der Leber gesteigert.3.Östrogensulfat und Östradiol-17β beeinflussen Gehalt, Zusammensetzung und spezifische Radioaktivität der Lipide der Aorta im Vergleich zur Kontrollgruppe nicht. Dagegen sind nach 17α-Äthinylöstradiol-Behandlung Gehalt und spezifische Radioaktivität der Gesamtlipide verringert. Die beschriebenen Versuche lassen lipidstoffwechselspezifische Wirkungen des Östrogensulfats erkennen, die Östradiol-17β bzw. 17α-Äthinylöstradiol nicht besitzen.