Bovine seminal ribonuclease precursor synthesized in vitro

Native bovine seminal ribonucelase is a dimeric protein, whose identical subunits (M_r 14 500), linked through two disulfide bridges, can be dissociated by a selective reduction procedure. Evidence is presented that the synthesis in vitro, under reducing conditions, of bovine seminal RNAase, directed by polyadenylated RNA isolated from bull seminal vesicles (where the enzyme is synthesized in vivo), occurs in the form of a precursor, 18 000-Da polypeptide. The precursor nature of this translation product was deduced by two criteria: (1) its specific immunoprecipitation with anti-bovine seminal RNAase antibodies; (2) its processing by dog pancreas microsomal membranes to produce a protein with a molecular weight similar to that of the subunit(s) of bovine seminal RNAase. Moreover, evidence is offered that the precursor polypeptide is able to form in vitro a dimeric molecule under conditions where no exogenous reducing agents were added.     

Bovine seminal ribonuclease: Non-hyperbolic kinetics in the second reaction step

Peroxisomes contain enzymes catalyzing the β-oxidation of fatty acids, which have been purified and partially characterized. Hypolipidemic drugs, including clofibrate, cause a marked proliferation of peroxisomes and a striking increase in the activity of their β-oxidation system. We have compared by sodium dodecyl sulfate—polyacrylamide gel electrophoresis the polypeptide patterns of normal and clofibrate-induced peroxisomes and the purified β-oxidation enzymes. The data allow a tentative identification of the β-oxidation enzymes among the peroxisomal polypeptides; these enzymes constitute only a small part of the protein of normal peroxisomes. A subset of peroxisomal polypeptides, including the β-oxidation enzymes, is preferentially increased by clofibrate.     

Bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism

Fifty years ago, the group of Tony Mathias and Bob Rabin at University College London deduced the first mechanism for catalysis by an enzyme, ribonuclease [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961) Nature 190, 781-784]. Here, we celebrate this historic accomplishment by surveying knowledge of enzymology and protein science at that time, facts that led to the formulation of the mechanism, criticisms and alternative mechanisms, data that supported the proposed mechanism, and some of the refinements that have since provided a more precise picture of catalysis of RNA cleavage by ribonucleases. The Mathias and Rabin mechanism has appeared in numerous textbooks, monographs, and reviews and continues to have a profound impact on biochemistry.     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

The ribbon of hydrogen bonds and the pseudomolecule in the three-dimensional structure of globular proteins. III. Bovine pancreatic ribonuclease A and bovine seminal ribonuclease

The model of the three-dimensional structure of globular proteins, which is based on a ribbon of hydrogen bonds along the whole of the backbone, is now applied to the comparison between monomeric bovine pancreatic ribonuclease A and dimeric bovine seminal ribonuclease. Some waters are involved in the hydrogen bonding of the ribbon, and the protein molecule plus these waters forms a pseudomolecule. The conformations of the three backbones are essentially identical and the three ribbons of hydrogen bonds are conserved with greater than 90% accuracy. We suggest that the conservation of the backbone conformations of the two molecules is a consequence of the conservation of the ribbons of hydrogen bonds. There are 16 simple mutations between the two molecules, of which 15 involve only side-chain groups with no more than one hydrogen bond to the backbone. Such mutations are not sufficient to change the ribbon of hydrogen bonds and hence there is no change in the backbone conformation. Generalizing this result, we suggest that the conservation of the ribbon is the reason why single point mutations rarely change the conformation of the backbone of the globular proteins.     

Cytotoxic potential of ribonuclease and ribonuclease hybrid proteins

Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.     

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.     

Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

Human pancreatic ribonuclease presents higher endonucleolytic activity than ribonuclease A

Analyzing the pattern of oligonucleotide formation induced by HP-RNase cleavage shows that the enzyme does not act randomly and follows a more endonucleolytic pattern when compared to RNase A. The enzyme prefers the binding and cleavage of longer substrate molecules, especially when the phosphodiester bond that is broken is 8-11 nucleotides away from at least one of the ends of the substrate molecule. This more endonucleolytic pattern is more appropriate for an enzyme with a regulatory role. Deleting two positive charges on the N-terminus (Arg4 and Lys6) modifies this pattern of external/internal phosphodiester bond cleavage preference, and produces a more exonucleolytic enzyme. These residues may reinforce the strength of a non-catalytic secondary phosphate binding (p₂) or, alternatively, constitute a new non-catalytic phosphate binding subsite (p₃).     

Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.