Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.4 (ribonuclease)
 6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.
 ...
PMID:Kinetic analysis of the mechanism of insulin degradation by glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase). 117 Aug 76

With the glutathione system that leads to rapid regeneration of reduced lysozyme (Saxena, V. P., and Wetlaufer, D. B. (1971) Biochemistry 9, 5015), reduced pancreatic ribonuclease (RNase) regenerated activity in high yield (greater than 90%) but at a considerably lower rate (t1/2 approximately 75 min). Systematic examination of the effects upon regeneration of the concentrations and ratios of reduced and oxidized glutathione (GSH and GSSG) showed the same broad optima for RNase as were earlier found for lysozyme: [GSSG] = 5 X 10(-4) M, [GSH] = 5 X 10(-3) M. Regeneration of reduced RNase by air oxidation was shown to be inhibitable by 10(-4) M EDTA, whereas the glutathione regeneration was unaffected by EDTA. In addition the air-oxidative regeneration showed a strong temperature dependence, in contrast with the glutathione system. The mechanisms of these two kinds of regenerations are therefore different. Six potentially catalytic metal ions were tested in the air-oxidative regeneration of RNase: Cu2+, Co2+, Mn2+, Fe3+, Zn2+, and Ni2+. Of these, only Cu2+ enhanced the rate of regeneration of RNase activity, although both Cu2+ and Co2+ catalyzed thioloxidation of reduced RNase. The rates and yields of RNase regenerations were independent of protein concentration from 3 X 10(-7) M to 1.2 X 10(-5) M in the glutathione system. Preincubation of freshly dissolved reduced RNase under nonoxidizing conditions before adding glutathione did not change the rate or extent of regeneration. Studies of its pH dependence showed that the glutathione regeneration depends on the deprotonation of prototropic groups with 7.5 less than pK less than 8.0. The major ion exchange chromatographic peaks from glutathione and air-oxidative regenerations appeared to be identical with native RNase, by the criteria of specific activity, chromatographic mobility, and circular dichroic spectra. The glutathione system permits regeneration at much higher RNase concentration than the air regeneration, with rates and yields comparable to the greatest reported for air regeneration.
 ...
PMID:Nonenzymic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by glutathione oxidoreduction buffers. 119 63

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.
 ...
PMID:A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease. 157 42

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.
 ...
PMID:Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. 198 50

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.
 ...
PMID:Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase. 198 51

The protein disulfide isomerase catalyzed reduction of insulin by glutathione is inhibited by peptides of various length and amino acid composition. Peptide inhibitors are competitive against insulin and noncompetitive against GSH, consistent with a sequential rather than a double displacement mechanism. Peptides of unrelated primary sequence that do not contain cysteine inhibit the GSH-insulin transhydrogenase activity of PDI, and the affinity of these peptides toward the enzyme is largely dependent on the peptide length rather than composition, hydrophobicity, or charge. Cysteine-containing peptides are 4-8-fold better inhibitors than non-cysteine-containing peptides of the same length, suggesting a cysteine-specific component to the interaction with the enzyme. Oxidized insulin chain B also inhibits the oxidative folding of reduced ribonuclease in a glutathione redox buffer with an inhibition constant that is comparable to that observed for the inhibition of insulin reduction, suggesting a similar if not identical binding site for the catalysis of oxidative protein folding and the reduction of insulin.
 ...
PMID:Effect of protein and peptide inhibitors on the activity of protein disulfide isomerase. 203 65

GSH, but not GSSG, inhibits the reactivation by phosphate ion of ribonuclease activity inactivated by urea or guanidine. The effects of GSH are rather slow and pretreatment of ribonuclease with urea is a requisite for the inhibitory action of GSH on enzyme reactivation. GSH is more effective in urea than in guanidine and its action is greatly enhanced by EDTA. An optimum pH of about 9.0 was found for the inhibitory effect of GSH. Titration of the thiol groups formed after inactivation of ribonuclease by GSH strongly suggests that the reduction of only one disulphide linkage is involved. The reduction of this bond is sufficient to completely abolish the enzymic activity.
 ...
PMID:Effect of glutathione on ribonuclease. 496 74

1. Bovine pancreatic ribonuclease is not reduced by GSH at near-physiological concentrations and pH. 2. Disruption of the structure of ribonuclease by proteolytic enzymes leads to products that can be reduced by GSH. 3. At higher temperatures the disulphide bonds of ribonuclease are completely reduced by GSH in a coupled system. The T(tr) is 51 degrees and this has been found to be lower than the T(tr) for the abnormal tyrosine residues under the same conditions.
 ...
PMID:The reactivity of the disulphide bonds of bovine pancreatic ribonuclease with glutathione. 586 27

1. The mechanism of the reaction between ribonuclease and GSH at elevated temperatures has been studied by using N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide to label the reduced ribonuclease. 2. After incubation for 2hr. at 35 degrees , enzymically active ribonuclease was recovered; at 50.8 degrees half of the initial ribonuclease was recovered as enzymically active ribonuclease and half as reduced labelled ribonuclease; at 55 degrees all of the initial ribonuclease was recovered in the labelled form. 3. It was inferred that the rate-limiting step was the reduction of the first disulphide bond in any one molecule. This was followed by rapid reduction of the other bonds in the same molecule.
 ...
PMID:Reduction of ribonuclease by glutathione at elevated temperatures: the molecular mechanism. 604 89

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.
 ...
PMID:Purification and some properties of bovine liver cytosol thioltransferase. 646 49


1 2 Next >>