Human granulocyte ribonuclease.

Human granulocytes contain an RNase which is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for the secondary phosphate esters of uridine 3′-phosphates. It has no action on uridine 2′: 3′-cyclic phosphates. Poly (A) and poly (G) are inert to its action. Its rate of hydrolysis of poly (C) is about 1% of that of poly (U). It differs from bovine pancreatic RNase and human serum RNase. Because of its unique specificity, this enzyme might serve as a biochemical marker in certain granulocyte disorders.     

The ribonuclease P database.

Ribonuclease P is responsible for the 5'-maturation of tRNA precursors. Ribonuclease P is a ribonucleoprotein, and in bacteria the RNA subunit alone is catalytically active in vitro , i.e., it is a ribozyme. The Ribonuclease P Database is a compilation of ribonuclease P sequences, sequence alignments, secondary structures, three-dimensional models, and accessory information, available via the World Wide Web (http: //www.mbio.ncsu.edu/RNaseP/home.html ).     

Preparation of allosteric ribonuclease.

A method for the preparation of allosteric ribonuclease from bovine pancreas is described. The effects of freeze-drying ribonuclease from acid and alkaline solutions on plots of velocity versus substrate concentration for the hydrolysis of 2':3'-cyclic CMP are examined. Comparison of these plots with the plots obtained with severeal commercial enzyme preparations indicates that the conformation of the enzyme is dependent on the method of preparation. Aging experiments demonstrate that further conformational changes occur at different rates, depending on the methods of storage. Results suggest that the allosteric behaviour of ribonuclease has not always been observed with commercial preparations, owing to variations in methods of preparation and storage of the enzyme.     

The ribonuclease P database.

Ribonuclease P is responsible for the removal of leader sequences from tRNA precursors. Ribonuclease P is a ribonucleoprotein, and in bacteria the RNA subunit alone is catalytically active in vitro, i.e. it is a ribozyme.The Ribonuclease P Database is a compilation of ribonuclease P sequences, sequence alignments, secondary structures, three-dimensional models, and accessory information, available via the World Wide Web.     

Dynamics of ribonuclease A and ribonuclease S: computational and experimental studies.

RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 21 in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system.     

The varieties of ribonuclease P.

Ribonuclease P is a ribozyme involved in tRNA processing that is present in all cells and organelles that synthesize tRNA. Most of our understanding of ribonuclease P derives from studies of the bacterial enzyme. This enzyme has been characterized biochemically and a secondary structure for the RNA subunit has been proposed. Isolation and characterization of ribonuclease P from diverse Archaea and Eukarya are now modifying and adding to our model of this unusual enzyme. The latter instances of RNase P differ from the bacterial version, but similarities are emerging.     

An allosteric model for ribonuclease.

Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2':3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve of velocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature.     

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1.

The folding and unfolding kinetics within the transition region were measured for RNase A and for RNase T1. The data were used to evaluate the theoretical models for the influence of prolyl isomerization on the observed folding kinetics. These two proteins were selected, since the folding reaction of RNase A is faster than prolyl isomerization, whereas in RNase T1, folding is slower than isomerization in the transition region. Folding of RNase T1 was investigated for three variants with different numbers of cis prolyl residues. The results indicate that in the transition region the folding rates are indeed strongly dependent on the number of prolyl residues. The variant of RNase T1 that contains only one cis prolyl residue folds about ten times faster than two variants that contain two cis prolyl residues. For both RNase A and RNase T1, the apparent rates of folding and unfolding as well as the corresponding amplitudes depend on the concentration of denaturant in a manner that was predicted by the model calculations. When refolding was started from the fast-folding species, additional kinetic phases could be observed in the transition region for both proteins. The obtained values could be used to calculate the microscopic rate constants of folding and isomerization on the basis of theoretical models.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity.

Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions, 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.     

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

Reaction of ethanol radicals with ribonuclease.

Ribonuclease was irradiated under conditions such that ethanol radicals were the main reactive species in solution. Sephadex gel filtration of the irradiated solution demonstrated that ethanol radicals had reacted with the ribonuclease and had become firmly bound to the enzyme molecule. The number of ethanol molecules bound to ribonuclease was a function of dose and correlated with the loss of enzymatic activity and with the changes in the molecular configuration of the enzyme molecule.     

KFERQ sequence in ribonuclease A-mediated cytotoxicity.

Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.