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1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [³H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/ or long-term labelling with [35S]sulfate and [³H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]