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Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 ,uM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNM. One potential physiological function ofPNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts. (5, 6). Ossowski and Reich (7) recently reported that anti-urokinase antibody inhibited the metastasis of human epidermoid carcinoma cells seeded onto chicken embryo chorioallantoic membranes. In view of the ability of PNI to inhibit urokinase and plasmin, the present investigation was undertaken to determine the effect of this inhibitor on tumor-cell-mediated destruction of extracellular matrix (ECM). Jones and DeClerk (8)

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Using oligonucleotide-mediated 'loop-in' mutagenesis strategies in Ml 3, a heat-inducible ubiquitin (Ub) gene was extended by sequences coding for the C-terminal 11 amino acids of Ha-RAS. The resulting gene was transformed into AR1 3 and production of the Ub-peptide extension was induced by heat treatment. After one-step purification, the fusion protein (Ub-cRAS) was used as a substrate for farnesyl-protein transferase. Ub-cRAS was farnesylated on incubation in Xenopus egg extract or rabbit reticulocyte lysate. In contrast, when serine was substituted for the last cysteine in the RAS extension, transfer of the [3H]farnesyl group from [3H] farnesyl pyrophosphate to the modified Ub-cRAS was not observed. Farnesylation of Ub-cRAS permitted us to develop an easy membrane-binding assay for farnesyl-protein transferase enzyme activity. Using this assay, we partially purified the enzyme from rabbit reticulocyte lysate. We also detected methylation of the farnesylated Ub-cRAS terminus in Xenopus egg extract. INTRODUCTIONAmong the enzymes left to be discovered and characterized, many catalyse reactions in which proteins are the substrate. This class of enzymes includes the ever-expanding families of protein kinases [1,2], enzymes that add lipids to proteins [3,4] and a wide variety of enzymes that modify specific amino acid side chains on cellular and secreted proteins [5][6][7]. Analysis of such enzymes requires peptides, either as potential substrates for measuring kinetic features of these catalysts or in the preparation of affinity matrices for purifying them. Recently, we described a method for synthesizing peptides as ubiquitin (Ub) extensions [8]. We have also demonstrated that Ub-peptide extensions can be used as substrates for a specific protein kinase [9]. In this regard, they offer distinct advantages over smaller peptide substrates commonly used to assay phosphotransferases.The RAS proteins, members of a large family of small guanine nucleotide-binding proteins, are present in all eukaryotic organisms. In order to function, they have to be localized at the plasma membrane [10]. This localization requires a series of posttranslational modifications which include the farnesylation of cysteine (the fourth residue from the C-terminus), proteolytic cleavage of the C-terminal three amino acids and the methylation of exposed COOH of cysteine [3,[11][12]. These modifications seem to require only the C-terminal consensus motif Cys-A-A-X (A, aliphatic amino acid; X, any amino acid) which is referred to as a CAAX box [13].Exploring further the utility of Ub-peptides as enzyme substrates, we have constructed a ubiquitin molecule extended by 11 amino acids from the C-terminus of Ha-RAS (PGCMSCKCVLS in the one-letter code). Here we describe the preparation and use of the Ub-peptide extension (Ub-cRAS) as a substrate for farnesyl-protein transferase (FPT) and carboxymethyltransferase. We also report on the partial purification and characterization of FPT from rabbit reticulocyte lysate.

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