Changes In Ribonuclease Research Articles

Calorimetric investigation of ribonuclease thermal denaturation.

Thermal denaturation of ribonuclease has been investigated by scanning microcalorimetric, spectrophotometric and polarimetric techniques. It is shown that the temperature changes of ribonuclease are of a complicated character and may be subdivided by the temperature region into two qualitatively different stages: (a) the predenaturational stage, at which neither abrupt changes in physico‐chemical properties nor heat absorption occur, but where the protein partial heat capacity considerably grows; (b) the denaturational stage which is accompanied by intensive heat absorption with the abrupt change in all physico‐chemical properties. Judging by the coincidence of the calorimetric and effective enthalpies, there is a transition at this stage between the two states without intermediate thermodynamically stable forms. Enthalpy values of denaturation, temperature of denaturation and denaturational changes of the heat capacity at various pH values are given.

The subgellular localization of the changes in ribonuclease and phosphatase activities of the mouse kidney produced by castration and testosterone

Male mice were castrated at 2 months of age and a pellet of testosterone propionate was implanted subcutaneously ten days later. After 14 days, normal, castrated and androgen treated mice were autopsied simultaneously. The kidneys of each group were homogenized, pooled and fractionated by centrifugation into the various subcellular components. The alkaline ribonuclease and phosphatase activities were localized in the microsomal fraction and changed in inverse relationship to the previously observed changes in the rate of protein biosynthesis and the concentration of microsomal and polysomal RNA induced by castration and androgen administration. Esterase activity also was localized in the microsomal fraction and its specific activity decreased after castration and was restored to normal by testosterone. The activities of acid ribonuclease, acid phosphatase and the several marker enzymes (glucose-6-phosphatase, nucleotidase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase) changed in direct proportion to the changes in weight of the kidney after castration and androgen stimulation.

Changes in ribonucleases during differentiation (spherulation) of Physarum polycephalum

Ribonuclease activity in Physarum polycephalum increased rapidly after the addition of mannitol for spherule induction. Most of the 12 ribonuclease isoenzymes detected by the gel isoelectric focusing technique increased, whereas one isoenzyme disappeared during spherulation. It is suggested that the rise in ribonuclease activity depends on protein synthesis de novo since it was prevented by cycloheximide.

Conformational Changes in Ribonuclease during Photodynamic Inactivation

Bovine pancreatic ribonuclease A was inactivated by photodynamic treatment at pH 8 with eosin Y, FMN and methylene blue as sensitizers. Enzyme inactivated with methylene blue showed partial aggrega...

A deuterium-hydrogen exchange study of inhibitor-induced conformational changes in ribonuclease A.

Deuterium-hydrogen exchange has been used to detect possible conformational changes accompanying the interaction of ribonuclease A with two competitive inhibitors: cytidine 3′-phosphate (Cyd-3′-P) and cytidine 2′-phosphate (Cyd-2′-P). An improvement of the cryosublimation technique was employed and combined with mass spectrometry measurements of sample deuterium content. Exchange kinetics were followed from 1 min to 24 h, at pH 6.5, and 38 °C (0.1 M NaCl). Two methods were used to present the data: a graphical exponential-curve-fitting and a numerical analysis. They both lead to the same conclusions. Four different classes of hydrogens were found. Among them, the one involving the instantaneously exchanged atoms is increased by about ten hydrogens after the binding of these inhibitors; the number of unexchanged hydrogens (within 24 h) is hardly altered in the presence of Cyd-3′-P whereas it is decreased by one third after the fixation of Cyd-2′-P. These exchange results suggest that both inhibitors induce in ribonuclease A conformational changes which may be correlated with inhibitor strength.

Conformational changes of ribonuclease A as seen by nuclear magnetic resonance line shifts

An approach to a quantitative experimental description of enzyme denaturations is considered. A parameter, Ω, derived from the first moment of the aromatic region of ribonuclease A nuclear magnetic resonance spectra, is used to follow thermal and lithium bromide denaturations. It is shown that Ω does not depend on widths of individual resonance lines; consequently, it can be defined from even poorly resolved protein spectra. The parameter Ω permits one to follow protein denaturations as shown by a well-defined number of aromatic residues. An interpretation of Ω is given assuming that a protein denaturation, like a homopolypeptide denaturation, is a continuous process within each macromolecule.

Ligand-induced Conformational Changes in Ribonuclease

The rate of formation of ribonuclease-S from ribonuclease A by the action of subtilisin was measured at pH 5.5 in the presence and absence of the strongly bound ribonuclease inhibitor, 2′-cytidylate. Presence of the inhibitor in the active center significantly reduced the rate of formation of ribonuclease-S. Since bond 20–21 (and, to a lesser extent, 21–22), the cleavage of which is responsible for the formation of this derivative, is well removed from the active center region, and thus from the binding site of the inhibitor, steric hindrance by the latter can be excluded as the cause for this reduced rate. Instead, it is concluded that the presence of the inhibitor in the active center caused a conformational change which affected a distant region of the molecule. Amino-terminal analysis confirmed cleavage at the expected major site, bond 20–21. The presence of the competitive inhibitors, 2′- and 3′-cytidylate, afforded considerable protection against inactivation of ribonuclease A by trypsin and by chymotrypsin, when digested at 60°. End group analysis confirmed that the bonds split were those expected from previous work of others. The presence of 2′-cytidylate completely prevented the temperature-induced optical rotatory change characteristic of ribonuclease, indicating a high degree of structural stabilization by the ligand. The data presented support the view that the presence of the competitive inhibitor in the active center region alters the proteolytic susceptibility of bonds at multiple sites, situated outside of this region.

Fluorescence and protein structure IX. Relationship between tyrosine fluorescence and various stages in denaturation of ribonuclease

Abstract 1. 1. Ribonuclease A and two modified forms, ribonuclease S and S-protein, were exposed to a variety of denaturing agents, i.e. , acid, heat urea, dioxane, dodecyl sulfate and cetyldimethylethylammonium bromide. (a) The observed order of stability was: ribonuclease A > ribonuclease S > S-protein. (b) Reversible changes of ribonuclease S in 8 M urea were by a 2-stage process: ribonuclease S → I S-protein → II extensive modification of the S-protein core, and Stage I was the rate-limiting step at 27°. (c) Dioxane (40%) at neutral pH produced reversible changes in ribonuclease S and S-protein, but denaturation of ribonuclease A occurred only below pH 4. (d) Actions of the cationic detergent cetyldimethylaammonium bromide on ribonuclease are complex but clearly involve a disulfide interchange and additional conformational changes from bound detergent. 2. 2. Fluoremetric titration curves showed marked differences for ribonuclease A, ribonuclease S and S-protein that are compatible with an expansion of the protein by electrostatic repulsive forces. 3. 3. Reduction of the disulfide bonds of denatured ribonuclease gave largeincreases in fluorescence. These results suggest that some hydrophobic regions persist as long as the disulfide bonds are intact. 4. 4. Perturbation of the fluorescence of native ribonuclease A by dioxane indicated that most, if not all, fluorescence came from the normal (exposed) tyrosyl residues.

The relationship between the densitometric and dilatometric volume changes of ribonuclease accompanying a change in pH.

The relationship between the densitometric and dilatometric volume changes of ribonuclease accompanying a change in pH.