The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.     

Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.     

A Novel Cationic Ribonuclease with Antimicrobial Activity from Rana dybowskii

A novel ribonuclease (RNase) A superfamily gene (Rdronc) has been cloned from the frog Rana dybowskii. The deduced amino acid sequence shows that it belongs to the ribonuclease A superfamily, with the highest identity, 73%, to Rana pipiens onconase. Adaptive evolution analysis based on maximum likelihood models of codon substitution has been conducted on 10 members of the Rana RNases of subcluster B. Rapid adaptive evolution and multiple positive selection sites have been detected, which indicates that these genes may be evolving under positive selection pressure. Functional assay demonstrates that the recombinant Rdronc protein possesses antimicrobial activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa and weaker antimicrobial activity against Gram-positive Staphylococcus aureus and yeast Candida albicans. Our findings support the hypothesis that ribonuclease A superfamily members may function in host defense of early-diversified vertebrates.     

Evolution of oligomeric proteins. The unusual case of a dimeric ribonuclease.

The model system made up of a monomeric and a dimeric ribonuclease of the pancreatic-type superfamily has recently attracted the attention of investigators interested in the evolution of oligomeric proteins. In this system, bovine pancreatic ribonuclease (RNase A) is the monomeric prototype, and bovine seminal ribonuclease (BS-RNase) is the dimeric counterpart. However, this evolutionary case is unusual, as BS-RNase is the only dimeric member of the whole large superfamily comprising more than 100 identified members from amphibia, aves, reptilia and mammalia. Furthermore, although the seminal-type RNase gene can be traced back to the divergence of the ruminants, it is expressed only in a single species (Bos taurus). These unusual findings are discussed, as well as previous hypotheses on the evolution of seminal RNase. Furthermore, a new 'minimalist' hypothesis is proposed, in line with basic principles of structural biology and molecular evolution.     

Glycosylation of ribonuclease A catalysed by rabbit liver extracts.

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.     

Enzyme specificity: base recognition and hydrolysis of RNA by ribonuclease A

The substrate specificity of pancreatic ribonuclease A is discussed in light of observations based on accurate X-ray structure analysis of several enzyme-nucleotide complexes. A hypothesis for protein-nucleic acid recognition is presented which proposes that: (a) pyrimidine bases in RNA are recognised by ribonuclease due to the charge complementarity of two groups (the amide nitrogen and the side chain oxygen (OG) of threonine 45) of the protein and relevant atoms in the heterocyclic base (O2 and N3 in pyrimidine nucleotides); (b) interaction of the protein with the ribose moiety of the nucleotides is non-specific; and (c) conformational flexibility in the region of the scissile P-O bond is provided by different locations of the phosphoryl oxygens, rather than by an overall translation of the phosphate moiety.     

Microsomal mixed-function oxidase-dependent renaturation of reduced ribonuclease

A purified hepatic microsomal mixed-function drug oxidase (EC 1.14.13.8) catalyzes oxidation of cysteamine to cystamine. Since cysteamine is a normal intracellular metabolite, this reaction could provide an enzymic mechanism for the continuous generation of disulfides required for formation of disulfide bonds in newly synthesized proteins. This hypothesis was tested by studying the renaturation of reduced ribonuclease in media containing glutathione reductase, purified microsomal oxidase, an NADPH-generating system, and physiological concentrations of glutathione and cysteamine. Under these conditions renaturation of reduced-disorganized ribonuclease is completely dependent upon the microsomal oxidase, and optimal renaturation rates are obtained when the relative activities of glutathione reductase and cysteamine oxidase approximate levels present in whole liver homogenates.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

The return of pancreatic ribonucleases

A decade after losing favor as an 'uninteresting' digestive enzyme, pancreatic ribonuclease has been found to be homologous to a series of extracellular proteins that may influence tumor cell growth, neurological development and biological differentiation. One surprising outcome of these discoveries has been the confirmation of the hypothesis that extracellular 'communicator RNA' is a messenger important in cell growth and differentiation. The only question is: why wasn't this recognized earlier?     

The antitumor action of seminal ribonuclease tested with the plasmacytoma spleen colonization assay

The antitumor action of bovine seminal ribonuclease was evaluated with a quantitative assay based on the production of tumor foci in the spleens of mice injected with plasmacytoma cells. The antitumor action depended on the integrity of the catalytic site, and on the dimeric structure of the enzyme. A working hypothesis is proposed, based on these results, and on previous results obtained studying the antitumor action of seminal RNAase in vitro on cell cultures. According to this hypothesis, the antitumor action is based on the ability of seminal RNAase to interact at specific receptor sites on the tumor cell membrane, as well as on its RNA degrading ability.     

Selection Against Glycosylation in Ruminant Pancreatic Ribonucleases by Replacements in the Ancestral Carbohydrate Attachment Site

A hypothesis, proposed 25 years ago, that there is selection against glycosylation in ruminant pancreatic ribonucleases by replacement of methionine to leucine in the ancestral carbohydrate attachment site Asn-Met-Thr at residues 34-36, was experimentally confirmed. The replacement of leucine at position 35 by methionine in bovine ribonuclease resulted in a three-fold relative increase in glycosylation when expressed in Chinese hamster ovary cells.     

Effect of nucleic-acid-binding compounds on the hydrolytic activity of various ribonucleases.

Studies were conducted on the stimulatory effect that various nucleic-acid-binding compounds have on the hydrolysis of RNA and polyribonucleotides by pancreatic ribonuclease A and by other ribonucleases. The stimulatory activity of chloroquine on tRNA hydrolysis by pancreatic ribonuclease was due to the formation of oligonucleotides of a wide range of sizes and was not due to the formation of very short ( n greater than 5) oligonucleotide fragments of tRNA. The dextrorotatory and levorotatory isomers of chloroquine did not differ in their ability to stimulate the hydrolysis of tRNA by pancreatic ribonuclease A. In addition to chloroquine and primaquine, other nucleic-acid-binding compounds (e.g., quinacrine, lucanthone, and proflavin) stimulated the hydrolysis of tRNA by pancreatic ribonuclease A. Chloroquine did not alter the rate of hydrolysis by pancreatic ribonuclease A of low-molecular-weight substrates (cytidine cyclic 2':o'-monophosphate, uridine cyclic 2':3'-monophosphate, cytidylyl-adenosine, or uridylyl-uridine). Furthermore, chloroquine and primaquine did not affect the hydrolysis of poly(A) by high concentrations of pancreatic ribonuclease A. In studies on the hydrolysis of tRNA by other endoribonucleases, several of the nucleic-acid-binding compounds (e.g., quinacrine and ethidium) exhibited appreciable inhibition of both ribonuclease N1 and ribonuclease T1. None of the compounds tested stimulated the activity of ribonuclease T1, and only chloroquine, and perhaps lucanthone, stimulated the hydrolysis of tRNA by ribonuclease N1.     

An evidence for the equilibrium unfolding intermediates of ribonuclease A by tritium labeling method

A formation of a molten globule in the unfolding of ribonuclease A could be considered as an evidence supporting a hypothesis on the existence of such intermediates on the pathway of a protein folding. Using a novel technique (tritium labeling method) we have showed that the ribonuclease A equilibrium unfolding in urea and guanidinium chloride (GuCl) solutions proceeds through a formation of intermediates whose properties (compactness, retention of the larger part hydrophobic core, secondary structure, and native-like folding pattern) correspond to the fundamental characteristics of the molten globule state. The both intermediates are the “wet” molten globules (the globule interior contains the water molecules). The results reveal the noticeable distinctions in intermediates structure, first of all, in the extent of their compactness. The urea intermediate is less compact than that in GuCl. It is shown that the refolding of the protein denatured by GuCl results in the formation of the intermediate which enzyme activity is virtually the same as the activity of the native protein.     

Effect of stagnant hypoxia on acid ribonuclease activity in the rat prosencephalon during ontogenesis.

In the rat prosencephalon it proved possible to differentiate lysosomal ribonuclease from alkaline ribonuclease activity, which could be detected only in the presence of p-chlormercuribenzoate. Acid RNase activity related to the amount of protein in the prosencephalon fell during ontogenesis. It was not significantly affected by four hours' stagnant hypoxia induced by ligation of both carotids. Its release from the lysosomes rose, however (when isotonic homogenates were spun at 20,000 g, acid ribonuclease activity in the supernatants was elevated). The absence of correlation between this activation and the degree of maturity of the nervous tissue refutes the hypothesis that regulation of this enzyme is per se responsible for the known changes induced by hypoxia in the RNA content of the prosencephalon of rats of different ages. On the contrary, the results indirectly support studies which demonstrate changes in the extent of RNA synthesis after hypoxia.     

The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.     

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.     

Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.

Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.