Ribonuclease protein inhibitors

PRI is also an inhibitor (K <0.1 nM) of both the angiogenic and ribonucleolytic activities of angiogenin, a blood vessel-inducing protein (20). PRI may therefore be called a ribonuclease/angiogenin inhibitor (RAI). Note that PRI does not inhibit some other RNases such as RNase Tl, nuclease SI, and RNase H. 

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

Leucine residues 2, 5, 7, 12, 20, and 24 of the motif are invariant in both type A and type B repeats of the ribonuclease inhibitor. An examination of more than 500 tandem repeats from 68 different proteins has shown that residues 20 and 24 can be other hydrophobic residues, whereas the remaining four leucine residues are present in all repeats. On the basis of the crystal structure of the ribonuclease inhibitor and the important structural role of these leucine residues, it has been possible to construct plausible structural models of several other proteins with leucine-rich motifs, such as the extracellular domains of the thyrotropin and gonadotropin receptors. 

Kobe, B., Deisenhofer, J. Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366 751-756, 1993. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Levitt, M. Sander, C. Stem, P.S., Protein normal-mode dynamics - trypsin-inhibitor, crambin, ribonuclease and lysozyme, 7. Mol. Biol. 1985,181, 423 47... 

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

A potent enzyme inhibitor (abbreviated DEP) that acts by ethoxyformylation of proteins, usually at histidine residues. DEP is an irreversible inhibitor of ribonuclease, and rinsing glassware with a 0.1% (weight/volume) DEP solution is recommended to render glassware nuclease-free. Aqueous solutions must be freshly prepared for maximal effectiveness, because DEP will hydrolyze in 6-12 hours at neutral pH. 

An example of enzyme depletion is the ribonuclease inhibitor isolated from human placenta by Blackburn, Wilson Moore. This protein forms a 1 1 complex with bovine pancreatic RNase A and is a noncompetitive... 

Figure 16.13 The free energy of denaturation AfjG as a function of temperature for a number of proteins Lys = lysozyme Rna = ribonuclease A Ct = a-chymotrypsin Cyt = cytochrome c Mb = metmyoglobin Tr = Trypsin and PTI2 = the dimer of pancreatic trypsin inhibitor. Reprinted with permission from P. L. Privalov, Stability of Proteins — Small Globular Proteins, Adv. Prot. Chem., 33, 167 (1979).

Leucine-rich repeats represent binding motifs found in a wide variety of both plant and mammalian proteins (Kobe and Kajava, 2001). These are involved in a multitude of protein-protein interactions. The sequence of porcine ribonuclease inhibitor, for example, displays a leucine-rich repeat (LRR) of length 27-29 residues that occurs 15 times in tandem (Fig. 9). Likewise, the family of small leucine-rich proteoglycans that includes biglycan, decorin, epiphycan, fibromodulin, keratocan, and lumican... 

Despite having a large number of ankyrin repeats (superseded only by the 24 repeats in ankyrin itself), a-LTX does not assume the monotonous arch-like shape so characteristic of proteins with multiple ankyrin repeats, such as the 12-repeat ankyrin fragment (Michaely et al. 2002), the human protein phosphatase HEAT (15 repeats), or the porcine ribonuclease inhibitor LRR (16 repeats) (Andrade et al. 

FIGURE 13 Plot of the logarithm of the retention volume (In VR) versus the concentration of the displacing salt, ammonium sulphate, in the HP-HIC mode with the proteins I, insulin B-chain 2, bovine trypsin inhibitor 3, bovine trypsinogen 4, insulin A-chain 5, ribonuclease 6, sperm whale myoglobin 7, horse heart cytochrome c. Data from Ref. 42. 

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

---