IntroductionThe mammalian ribonuclease inhibitor (RI) is a 50-kDa cytosolic protein that binds to pancreatic-type ribonucleases with femtomolar affinity and renders them inactive (for other reviews, see (1-5)). Complexes formed by RI and its target ribonucleases are among the tightest of known biomolecular interactions. The three-dimensional structure of RI is likewise remarkable, being characterized by alternating units of α-helix and β-strand that form a striking horseshoe shape (Fig. 1A) (6). The repeating structural units of RI possess a highly repetitive amino acid sequence that is rich in leucine residues (7,8). These leucine-rich repeats (LRRs) are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein protein interactions (9-12). The unique structure and function of RI have resulted in its emergence as the central protein in the study of LRRs, as well as its widespread use as a laboratory reagent to eliminate ribonucleolytic activity (13).The biological role of RI is not known in its entirety. The ribonucleases recognized by RI are secreted proteins, whereas RI resides exclusively in the cytosol. Nevertheless, RI affinity has been shown to be the primary determinant of ribonuclease cytotoxicity: only ribonucleases that evade RI can kill a cell (for reviews, see (14)(15)(16)(17)). In addition, the complex of RI with human angiogenin (ANG), which stimulates neovascularization by activating transcription in the nucleus (18,19), is the tightest of known RI·ribonuclease complexes. Yet, a role for RI in angiogenesis is not clear. Also intriguing are the 30-32 cysteine residues of RI, all of which must remain reduced for the protein to retain activity (20). These observations have lead researchers to hypothesize multiple biological roles for RI: (1) to protect cells from invading ribonucleases, (2) to regulate or terminate the activity of ribonucleases with known intracellular functions, and (3) to monitor the oxidation state of the cell in response to factors such as aging and oxidative stress. Here, we review the salient features of RI biochemistry and structure and thereby provide a context for examining the roles of RI in biology. I. Biochemical PropertiesThe inhibitory activity of RI in guinea pig liver extracts was discovered in 1952 (21). This activity was inactivated by proteases, heat, or sulfhydryl-group modification, and was sensitive to changes in pH (for a review, see (22)). In addition, the inhibitory activity was isolated in the supernatant fraction during a high-speed centrifugation, indicative of cytoplasmic localization. In the 1970's, techniques were developed to purify RI to homogeneity, enabling its biochemical characterization (23,2). Since then, RI has been isolated from numerous mammalian sources, including brain (24-26), liver (27,28,26), testis (29), and erythrocytes (30). A. PurificationRI is particularly abundant in mammalian placenta and liver, which have served as the major source of RI for purification. Human placenta...

The Staudinger ligation between an azido-protein and a phosphinothioester-derivatized surface is demonstrated to be an effective means for the site-specific, covalent immobilization of a protein. Immobilization yields of >50% are obtained in <1 min, and immobilized proteins have >80% of their expected activity. No other method enables more rapid immobilization or a higher yield of active protein. Because azido-peptides and azido-proteins are readily attainable by synthesis, biosynthesis, or semisynthesis, the Staudinger ligation could be of unsurpassed utility in creating microarrays of functional peptides and proteins.

We report an investigation of the binding ability of a protein immobilized on surfaces with different orientations but in identical interfacial microenvironments. The surfaces present mixed self-assembled monolayers (SAMs) of 11-[19-carboxymethylhexa(ethylene glycol)]undecyl-1-thiol, 1, and 11-tetra(ethylene glycol) undecyl-1-thiol, 2. Whereas 2 is used to define an interfacial microenvironment that prevents nonspecific adsorption of proteins, 1 was activated by two different schemes to immobilize ribonuclease A (RNase A) in either a preferred orientation or random orientations. The binding of the ribonuclease inhibitor protein (RI) to RNase A on these surfaces was characterized by using ellipsometry and the orientational behavior of liquid crystals. Ellipsometric measurements indicate identical extents of immobilization of RNase A via the two schemes. Following incubation of both surfaces with RI, however, ellipsometric measurements indicate a 4-fold higher binding ability of the RNase A immobilized with a preferred orientation over RNase A immobilized with a random orientation. The higher binding ability of the oriented RNase A over the randomly oriented RNase A was also apparent in the orientational behavior of nematic liquid crystals of 4-cyano-4'-pentylcyanobiphenyl (5CB) overlayed on these surfaces. These results demonstrate that the orientations of proteins covalently immobilized in controlled interfacial microenvironments can influence the binding activities of the immobilized proteins. Results reported in this article also demonstrate that the orientational states of proteins immobilized at surfaces can be distinguished by examining the optical appearances of liquid crystals.

Fluorescent molecules are essential for basic research in the biological sciences and have numerous practical applications. Herein is described the synthesis and use of a new class of latent fluorophores based on a novel design element, the trimethyl lock, that confers distinct advantages over extant fluorophores and pro-fluorophores. A diacetyl version of the latent fluorophore is stable in a biological environment, but rapidly yields rhodamine 110 upon acetyl-group hydrolysis by pig liver esterase or endogenous esterases in the cytosol and lysosomes of human cells. This design element is general and, hence, provides access to an ensemble of useful latent fluorophores.

Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity.To stimulate blood-vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10 6 -fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.Angiogenin (ANG) is a potent inducer of blood vessel growth (1) and has been implicated in the establishment, growth, and metastasis of tumors (2,3). A homologue of bovine pancreatic ribonuclease (RNase A (4-6); EC 3.1.27.5), ANG is the only human angiogenic factor that displays ribonucleolytic activity. ANG was first isolated from the conditioned medium of human adenocarcinoma cells (1), and is present in normal human plasma (7) as well as numerous other tissues and organs (8). After receptor-mediated endocytosis (9), a nuclear localization sequence (NLS) directs ANG to the nucleus (10). The receptor-binding, nuclear localization, and ribonucleolytic activity of ANG are all required for angiogenic activity (9-11). In endothelial and smooth muscle cells, ANG induces a wide range of cellular responses, including transcriptional activation (12), differentiation (13), cell migration and invasion (14), and tube formation (13).The ribonuclease inhibitor (RI (15)), a cytosolic protein found in all mammalian tissues analyzed to date, binds to mammalian ribonucleases with extraordinary affinity. The RI·ANG complex ( Figure 1A) is among the tightest of known protein-protein interactions with K d = † This work was supported in part by Grants CA073808 (NIH) and M10749000231-08N4900-23110 (Korea Science and Engineering Foundation A known role for RI is to protect cellular RNA from invading ribonucleases (19,20). ANG, however, possesses <1% of the ribonucleolytic activity of its homologues with cytotoxic activity (21). Moreover, the IC 50 values for cytotoxic ribonucleases are ≥10 2 -fold greater than the concentration of ANG required to induce endothelial cell proliferation in vitro. Thus, a major role for RI as an antagonist of the cytotoxic activity of ANG is unlikely.Does RI play a role in ANG-induced angiogenesis? The exogenous addition of extracellular RI is known to antagonize angiogenesis (22,23). That experiment, however, puts RI in a nonnative location. We sought to determine whether endogenous, intracellular RI regulates ANG-induced neovascularization. We reasoned that we could do so by using a variant of ANG that evades RI.To disrupt its interaction with RI, we introduce...