Ribonuclease inhibitors

Figure 4.11 Schematic diagram of the structure of the ribonuclease inhibitor. The molecule, which is built up by repetitive P-loop-a motifs, resembles a horseshoe with a 17-stranded parallel p sheet on the inside and 16 a helices on the outside. The P sheet is light red, a helices are blue, and loops that are part of the p-loop-(x motifs are orange. (Adapted from B. Kobe et al.. Nature 366 7S1-756,...

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. 

Figure 4.12 Schematic diagram illustrating the role of the conserved leucine residues (green) in the leucine-rich motif in stabilizing the P-loop-(x structural module. In the ribonuclease inhibitor, leucine residues 2, 5, and 7 from the P strand pack against leucine residues 17, 20, and 24 from the a helix as well as leucine residue 12 from the loop to form a hydrophobic core between the P strand and the a helix.

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

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

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... 

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... 

Fig. 9. The conformation adopted by a leucine-rich repeat (LRR) is that of a /9-strand followed by an o-helix. In porcine ribonuclease inhibitor, a /9-strand (residues 2-8) is connected to an o-helix (residues 14—27) by a connecting loop (residues 9-13). A horseshoe-shaped structure is formed and is exemplified in the crystal structure of ribonuclease inhibitor (PDB 1A4Y Kobe and Deisenhofer, 1993). This has an inner concave surface formed by curved /9-sheets and an outer convex surface formed by oh el ices. The leucines and other large apolar residues form the hydrophobic core of the structure.

Human placentyl ribonuclease inhibitor Narrow spectrum of specificity commercially available [139]... 

Blackburn, P., G. Wilson, and S. Moore. 1977. Ribonuclease inhibitor from human placenta Purification and properties. J Biol Chem 252 5904. 

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. 

Papageorgiou, A. C., Shapiro, R., and Acharya, K. R. (1997). Molecular recognition of human angiogenin by placental ribonuclease inhibitor—an X-ray crystallographic study at 2.0 A resolution. EMBO J. 16, 5162-5177. 

Figure 4.6 Ribonuclease inhibitors, a) Analogs of different nucleotides adopt distinct binding modes, b) Closely related analogs that differ only in the position of two phosphate groups and bind in very similar conformations show significant potency differences.

Human placental ribonuclease inhibitor (HPRI) (Amersham, E2310Z). 

L5. Little, B. W., and Meyer, W. H., Ribonuclease inhibitor system abnormality in dystrophic mouse skeletal muscle. Science 170, 747-749 (1970). 

As mentioned in a previous section, we generally find the use of diethylpyrocarbonate as a ribonuclease inhibitor superfluous. However, if glassware is shared with labs that use RNase A in their solutions, problems may arise. Therefore, beakers, tubes, etc. that are to be used for RNA work can be filled with a 0.1% (v/v) of diethylpyrocarbonate in water and left for a couple of hours at 37°C in a fume-hood. Rinse thoroughly with distilled water, and remove the final traces of diethylpyrocarbonate by autoclaving. 

Cy tidy lie Acid. Cyridine-3 -monophosphate cytidylic acid b 3 -cytidinephosphoric acid cytidine-3 -phosphate 3 -cytosylic acid 3 -CMP, CjH NjOjP mol wt 323.19. C 33.44% H 4-37%, N 13.00%, O 39-61%. P 9 58% Ribonuclease inhibitor. Prepn from yeast ribonucleic acid see refs under 2 -Cytidylic Acid. Prepn by pancreatic-ribonuclease-catalyzed ring opening of dicyclohexy] -guanidinium cytidine 2 ,3 -cyclic phosphate Lohrmann,... 

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