A naturally occurring monodisperse Cu-thionein was prepared using ammonium sulfate precipitation followed by ion exchange (DEAE 23) and gel chromatography (Sephadex G-75). The Chromatographie steps were repeated at least twice, or until the Cu-thionein remained homogeneous when subjected to analytical polyacrylamide disc electrophoresis. The molecular weight of this copper protein was 9500 ± 500. Up to 24.3 % cysteine residues were determined, indicating the relationship to the metallothioneins. Aromatic amino acids were virtually absent, while there were about three times as many acidic amino acid residues, including aspartate and glutamate, as in metallothioneins. 10 g atoms of Cu were measured per mole of protein. The copper binding strength of thionein was extremely high. Displacement by protons (pH 1.5) and gel chromatography or dialysis employing EDTA were not effective. Dialysis against diethyldithiocarbamate produced a protein essentially free of copper. Both the ultraviolet properties and the circular dichroism measurements proved identical with those properties reported for artificially prepared Cu-thionein (see refJ 1 ). The major absorption was in the far ultraviolet region with a weak shoulder at 270 nm attributable to copper charge-transfer transitions. 6 Cotton extrema were seen at 213, 283 and 302 nm (negative) and 245, 328 and 359 nm (positive). The possible role of Cu-thionein as an electron transport system was discussed. Isolierung von Cu-Thionein aus Saccharomyces cerevisiaeZusammenfassung: Ein natürlich vorkommendes monodisperses Cu-Thionein konnte aus Saccharomyces cerevisiae isoliert werden. Die aufgeschlossenen Zellen wurden mit (NH 4 ) 2 SO 4 versetzt und der suspendierte Niederschlag chromatographisch (DEAE-23-Cellulose und Sephadex G-75) aufgetrennt. Die chromatographischen Schritte wurden wiederholt, bis das Cu-Thionein sich als homogen erwies. Es zeigte bei der analytischen Polyacrylamid-Disk-Elektrophorese nur eine einzige Bande. Das Molekulargewicht dieses Cu-Proteins war 9 500 ± 500. Bis zu 24.3% Cysteinreste wurden bestimmt. Dieser hohe Anteil ließ eine Beziehung zu den Metallothioneinen

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.

After an initial lag phase, tunicamycin (0.12-3.6 pM) inhibits the secretion of P-N-acetylglucosaminidase by human skin fibroblasts. The inhibition persists for up to 30 h. Thereafter a more than fivefold-increased rate of secretion is observed for at least 4 days. The intracellular P-N-acetylglucosaminidase activity decreases within 9 days in the presence of 0.36 pM tunicamycin to 55 % of that of controls. ,8-N-Acetylglucosaminidase formed in the presence of tunicamycin is only poorly recognized by fibroblasts, suggesting that tunicamycin interferes with the formation of the phosphomannosyl residues that function on b-N-acetylglucosaminidase as a recognition marker for the cell surface receptors present on fibroblasts. Tunicamycin (0.36 -3.6 pM) inhibits endocytosis of exogenous lysosomal enzymes only after a preincubation of 24 h in the presence of the drug. Control experiments indicated that this inhibition is not due to a general inhibition of receptor-mediated endocytosis. Incubation with tunicamycin leads furthermore to a loss of cell-surface-associated lysosomal enzymes.These findings indicate that tunicamycin interferes with the expression of phosphomannose receptors on the cell surface of fibroblasts and the synthesis of the phosphomannosyl residues on lysosomal enzymes after an initial lag phase. These alterations are associated with an increased accumulation of P-N-acetylglucosaminidase extracellularly, a loss of that enzyme from intracellular sites and from the cell surface.

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