Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP P MgADP + ADP) from 1. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were 2. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by 3. Thc N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. 4. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages.5. The enzyme is a monomer of 220 amino acids with M , 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putativc activesite region of the enzyme. After position 11 1, however, there is an insertion of 32 residues in thc yeast species, similar to the adenylate kinase and the GTP : AMP phosphotransferase from beef heart mitochondria.baker's yeast has been determined. sequenced with either a solid-phase sequencer or a gas-phase sequencer. [5] and of two isozymes from beef heart mitochondria [6, 7] have been reported. An X-ray diffraction analysis of porcine adenylate kinase at 0.3 nm in conjunction with the sequence yielded the spatial structure [8]. Whereas the active-site region of the enzyme could be assigned unambiguously by localization of substrate analogues at 0.6-nm resolution 191, the exact binding positions of the substrates are still not known. However, the observed binding sites were challenged by nuclear magnetic resonance analyses [lo]. This controversy is likely to be resolved by study of adenylate kinase from yeast (AKy), because it cocrystallizes with the potent enzyme inhibitor Ap,A as well as with the natural substrate MgATP (Noda and Tomasselli, unpublished results). These crystals are presently being analyzed by X-ray diffraction. This paper presents the complete amino acid sequence of AKy, which together with the forthcoming X-ray analysis should yield the exact substrate binding geometry, leading us a step closer to the elucidation of the reaction mechanism. MATERIALS AND METHODS MaterialsBaker's yeast (Red Star Yeast, Universal Foods Corporation, Milwaukee, WI) was kindly donated by the manufacturer. Clostripain from Clostridiurn histolyticurn, endoproteinase Lys-C from Lysohacter enzyrnogenes, pronase from Streptornyces griseus (Boehringer Mannheim), Staphylococcus aureus V 8 protease (Miles Laboratories Inc.) and trypsin pretreated with L-3-tosyl-amido-2-phenylethylchloromethylanc (Worthington) were used for proteolytic cleavages. Cyanogen bromide (Fluka) and 2-iodosobenzoic acid (Serva) were employed for chemical cleavages. Reagents and solvents for the gas-phase sequencer were from Applied Biosystems. All other chemicals were of analytical grade. Protein preparation and chemical mod(ficationsAKy, prepared and assayed as in [l 11, had a specific activity of 1833 U/mg and was homogeneous by sodium dodecyl sulfate/pol...