Ribonuclease inactivators

Selected entries from Methods in Enzymology [vol, page(s)] Inhibitory properties, 68, 212 inactivator, of ribonucleases, 65, 681 lipase modification, 64, 390 nuclease inactivation, 79, 63 ribonuclease inactivation, 79, 52, 112-113, 267. 

RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBONUCLEASE (or RNase) PSEUDOROTATION DIETHYL PYROCARBONATE RIBONUCLEASE F RIBONUCLEASE II RIBONUCLEASE III Ribonuclease inactivation,... 

Thio UMP Ribonuclease Inactivates, loss of amino acids 120,121... 

Although slightly different from complementation of fragments, the ancient experiment of Fruchter and Crestfield (1965) on dimers of ribonuclease is the first example of in vitro complementation and must be cited to introduce this section. By complementation of ribonuclease inactivated by carboxy-methylation of either His 16 or His 119, but not both, formation of an active dimer was obtained in which 1 mole with free His 12 associates with 1 mole with free His 119. 

Adams, G.E., Bisby, RH., Cundall, R.B., Redpath, J.L. and Willson, RL (1972). Selective free radical reactions with proteins and enzymes. The inactivation of ribonuclease. Radiat. Res. 49, 290-298. 

Zale, S. E. and Klibanov, A. M., Why does ribonuclease irreversibly inactivate at high temperature , Biochemistry, 25, 5432, 1986. 

Papain is readily inactivated by PAN (at 115 ppm for 40 min), provided that it is in the sulfhydryl form. The reaction of sulfhydryl groups of hemoglobin with PAN is very similar to the reaction with p-mercuricbenzoate there is more reaction at a pH of 4.5 than at a pH of 7. However, there is one striking difference between PAN and classic compounds that react with sulfhydryl groups egg albumen is resistant to reaction with PAN. Thus, enzymes that have no free sulfhydryl groups should be quite resistant to PAN. This is the case with pancreatic ribonuclease the native enzyme was not affected by a 300-fold molar excess of PAN. 

Ribonuclease A (RNase A) is dissolved at 1 mg/ml in water, and stored at -20°C. To inactivate the possible contaminated DNase completely, RNase A solution is sometimes boiled once when prepared. 

The essential—SH group in D-glyceraldehyde-3-phosphate dehydrogenase and the imidazol residues of the ribonuclease are also more reactive because of side-chain interactions in the active center. Such functional groups may have such extremely high reactivity that an equivalent amount of the reagent causes full inactivation of the enzyme. 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

Walters and Loring (88) have purified a 3 -nucleotidase about 50-fold from mung bean sprouts (Phaseolus aureus Roxb.). The enzyme hydrolyzes 3 -AMP, 3 -GMP, 3 -CMP in decreasing order and also hydrolyzes the 3 -phosphate group of coenzyme A. (89), but it has no significant activity for 2 - or 5 -ribonucleotides. For 3 -GMP, 3 -AMP, 3 -UMP, and 3 -CMP, Km values are 0.67, 1.1, 7.7, and 15 mM, respectively. The enzyme preparation also contained acid stable ribonuclease activity (89). Both 3 -nucleotidase and acid ribonuclease were inactivated reversibly at pH 5.0 and by dialysis and this inactivation could be prevented by Zn2+. The two activities were similarly inactivated by heat at pH 5 and 7.5. Such data indicate that the two are metalloproteins— probably zinc metalloproteins. These similarities and other kinetic data provide evidence that the 3 -nucleotidase and ribonuclease activities reside in the same protein. 

In a classic study on bovine pancreatic ribonuclease A at 90°C and pH conditions relevant for catalysis, irreversible deactivation behavior was found to be a function of pH (Zale, 1986) at pH 4, enzyme inactivation is caused mainly by hydrolysis of peptide bonds at aspartic acid residues as well as deamidation of asparagine and/or glutamine residues, whereas at pH 6-8, enzyme inactivation is caused mainly by thiol-disulfide interchange but also by fi-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. 

Why does ribonuclease irreversibly inactivate at high temperatures Biochemistry, 25, 5432-5444. 

The enzymes used to generate reactive quinone methides often undergo inactivation by addition of this electrophile to essential nucleophilic amino acid side chains of the protein catalyst. This is a type of suicide enzyme inhibition.80 This was observed for the acid phosphatase and ribonuclease catalysts used to generate 43.76 79 Alkaline phosphatase has been used to remove the phosphate protecting group from a derivative of an o-difluoromethyl phenyl phosphate that was covalently attached to a solid support. Breakdown of the immobilized 4-hydroxybenzyl difluoride gives an immobilized quinone methide that, in principle, will react irreversibly with proteins and lead to their attachment to the solid support.81... 

Residual RNA in a DNA preparation can be removed by treatment with ribonuclease (RNase). RNase A, which is free of DNase, is available commercially, or the contaminant DNase in the crude RNase A solution can be heat inactivated by heating RNase A solution (10 mg/mL in 10 mM Tris-Cl, pH 7.5, 15 mMNaCl) at 100°C for 15 minutes [4], DNA solution in TE at a concentration of at least 100 pg/mL is treated with RNase to a final concentration of 1 pg/mL followed by incubation at 37°C for 1 hour [3], RNase... 

Inhibition of phosphoglucomutase Ribonuclease and avidin inactivation Inhibition of acetylcholine esterase... 

The function, if any, of methionine sulfoxide residues in peptides or proteins is a matter of conjecture. There is no evidence that they are of any structural significance. Indeed, since various enzymes such as ribonuclease and chymotrypsin are either partially or completely inactivated by oxidation of the methionine residues (15, 16), one hesitates to suggest any functional role for the sulfoxide. However, a role in the maintenance of oxidation-reduction potential of a biological system, as suggested by Dent (3), is conceivable. 

---