Characterization of the ribonuclease activity on the skin surface (original) (raw)
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Isolation of ribonuclease P activity from human epidermis and its regulation by retinoids in vitro.
Ribonuclease P (RNase P) is a key enzyme in tRNA biogenesis that catalyses the endonucleolytic cleavage of tRNA precursors and generates their mature 5' ends. The activity of this ribozyme has never been isolated from living human tissues and data about epidermal tRNA biogenesis are not available. The purpose of the present study was to isolate and purify RNase P from human epidermis and to investigate the in vitro effects of retinoids on its activity. Enzyme isolation and purification from homogenates of keratinocytes derived after trypsinization from dispase-separated human epidermis were carried out using phosphocellulose chromatography. The optimal activity of the enzyme was found at 100 mM NH4Cl and 5 mM MgCl2 at pH 7.5 and 37 degrees C. All-trans retinoic acid and acitretin revealed a dose-dependent inhibitory effect on RNase P activity. The isolation of RNase P activity from human epidermis, reported here for the first time, will enable the investigation of the possible involvement of this ribozyme in the regulation of epidermal differentiation and proliferation and the evaluation of its significance for the pathogenesis and gene therapy of various cutaneous disorders.
Ribonucleases and their Applications
Ribonuclease (RNase) is a type of nuclease that catalyzes degradation of RNA into smaller components. RNase can be classified into two broader categories namely endoribonucleases and exoribonucleases on the basis of their site of action. RNases play key roles in the maturation of all RNA molecules; endoribonucleases cleave the RNA molecules from the interior at 5′ end while exoribonucleases degrade RNA molecules in a 3′-5′ direction. With the advent of new frontiers in biotechnology, the applications of ribonucleases besides molecular biology have expended into many other fields like medicinal, clinical, and analytical chemistry. RNase A that belongs to pancreatic ribonucleases super family plays an important key role in structural, biochemical and evolutionary studies. Discovery of eukaryotic orthologues of the bacterial double stranded (ds) RNA-specific ribonuclease III (RNase III) suggests a central role for these enzymes in the regulation of ds-RNA and eukaryotic RNA metabolism. The more recent studies have shown that the mammalian and some fungal RNases are also bestowed with antiproliferative, antiangiogenic and/ or antitumor/ anticancer activities. Some of the members of RNase A superfamily such as RNase 6 and RNase 7 appears to be evolutionary conserved peptides with potent antimicrobial activities for upkeep of sterility in the urinary tract.
Molecular and Cellular Biochemistry, 1992
Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA : RNA hybrids under standard assay conditions (0.15 M NaC1, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed. (Mol Cell Biochem 117: 139-151, 1992) Key words." double-stranded RNA, ribonuclease(s), bovine RNase A, pancreatic-type RNases, nucleic acid helixdestabilizing proteins Abbreviations: RNase -Ribonuclease, dsRNA -Double-stranded RNA, ssRNA -Single-stranded RNA, poly(A) : poly(U), poly(I) : poly(C) -Double-stranded Homopolymers formed between Polyadenylate and Polyurydilate, and Polyinosinate and Polycytidylate, respectively, poly(dA) : poly (rU) -Double-stranded complex formed between Polydeoxyriboadenylate and Polyribouridylate, poly(A), poly(C) -Polyadenylate and Polycytidylate, respectively, poly[d(A-T)] -Double-stranded Homopolymers formed between Polydeoxyriboadenilate and Polydeoxyribothymidylate, poly(dA-dT) : poly (dA-dT) -Double-stranded alternating copolymers, SSC -0.15 M Sodium Chloride, 0.015 M Sodium Citrate pH 7
Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay
Nucleic Acids Research, 2006
Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5 0-fluorescein-labeled RNA hybridized to a complementary 3 0-quenchermodified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a k cat /K m of 330 000 M À1 s À1 , a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.
High-Level Soluble Production and Characterization of Porcine Ribonuclease Inhibitor
Protein Expression and Purification, 2001
type ribonucleases with 1:1 stoichiometry and competi-Ribonucleases can be cytotoxic if they retain their ribotively inhibits their ribonucleolytic activity (1-3). RI is nucleolytic activity in the cytosol. The cytosolic ribofound in the cytosol of mammalian cells and has been nucleolytic activity of ribonuclease A (RNase A) and other purified from many species and tissue types (4, 5). Its pancreatic-type ribonucleases is limited by the presence inhibition of ribonucleolytic activity and its cytosolic of excess ribonuclease inhibitor (RI). RI is a 50-kDa cytolocation has led to the suggestion that RI protects cellusolic scavenger of pancreatic-type ribonucleases that lar RNA from degradation by invading pancreatic-type competitively inhibits their ribonucleolytic activity. RI ribonucleases (3). had been overproduced as inclusion bodies, but its fold-Ribonucleases can be cytotoxic by entering the cytosol ing in vitro is inefficient. Here, porcine RI (pRI) was overand degrading cellular RNA. Although bovine pancreproduced in Escherichia coli using the trp promoter and atic ribonuclease (RNase A (6); EC 3.1.27.5) has high minimal medium. This expression system maintains pRI ribonucleolytic activity (6) and is capable of entering the in the soluble fraction of the cytosol. pRI was purified by cytosol (7), its ribonucleolytic activity there is limited affinity chromatography using immobilized RNase A and by the presence of excess RI (3, 8). The noncovalent by anion-exchange chromatography. The resulting yield interactions of the RI-RNase A complex are exceptionof 15 mg of purified RI per liter of culture represents a 60ally strong (K d ϭ 6.7 ϫ 10 Ϫ14 M (9)). Homologous ribofold increase relative to previously reported recombinant nucleases are able to evade inhibition by RI (10, 11). DNA systems. Differential scanning calorimetry was used Indeed, Onconase, which is a homolog of RNase A from to study the thermal denaturation of pRI, RNase A, and the Northern leopard frog (Rana pipiens), has Ͼ10 8the pRI-RNase A complex. The conformational stability of the complex is greater than that of the individual com-fold lower affinity for RI than does RNase A and is ponents.
Ribonuclease P: an enzyme with an essential RNA component
Proceedings of the National Academy of Sciences, 1978
The activity of ribonuclease P on precursor tRNA substrates from Escherichia coli can be abolished by pretreatment of this enzyme with micrococcal nuclease or pancreatic ribonuclease A, as well as by proteases and by thermal denaturation. Highly purified RNase P exhibits one prominent RNA and one prominent polypeptide com nent when examined in polyacrylamide gels containing sodum dodecyl sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is characteristic of a protein-RNA complex. The activity of RNase P is inhibited by various RNA molecules. The presence of a discrete RNA component in RNase P appears to be essential for enzymatic function. A model is described for enzyme-substrate recognition in which this RNA component plays an important role.
Journal of Controlled Release, 2010
The purpose of the study was to demonstrate the feasibility of using transdermal iontophoresis to deliver a functional protein, ribonuclease A (RNAse; 13.6 kDa), non-invasively across the skin. Iontophoretic transport experiments were conducted using porcine skin in vitro and established the effect of current density and protein concentration on delivery kinetics. A methylene blue-based assay was used to quantify RNAse transport and to simultaneously demonstrate that protein functionality was retained post-iontophoresis. The results confirmed that intact functional RNAse was indeed delivered across the skin; cumulative permeation and steady state flux after 8 h iontophoresis at 0.3 mA/cm 2 were 224.37 ± 72.34 µg/cm 2 and 68.28 ± 23.87 µg/cm 2 h, respectively. Significant amounts of protein were also deposited within the membrane (e.g., 1425.13 ± 312.09 µg/cm 2 at 0.3 mA/cm 2). In addition to the evidence provided by the enzymatic assay with regards to RNAse integrity and functionality, SDS-PAGE gels and MALDI-TOF spectra were also used to characterize RNAse present in the receiver phase (MALDI-TOF spectra: RNAse control, 13.690 kDa cf. RNAse from permeation samples, 13.692 kDa). Co-iontophoresis of acetaminophen showed that, despite its molecular weight, electromigration was the predominant electrotransport mechanism, accounting for N 80% of RNAse total flux. Increasing RNAse concentration from 0.35 to 0.7 mM in the formulation did not result in a statistically significant increase in delivery. Iontophoretic transport of RNAse across human skin was statistically equivalent to that seen with porcine skin under the same conditions; cumulative permeation across human and porcine skin was 241.48 ± 60.01 and 170.71 ± 92.13 µg/cm 2 , respectively. Laser scanning confocal microscopy was used to visualize the distribution of rhodamine B-labelled RNAse in the epidermis and dermis as a function of depth following 8 h iontophoresis (results were compared to control experiments involving passive administration of the same formulation for 8 h). Although fluorescence was localized at the skin surface following passive administration, it was visible throughout the membrane after current application. In conclusion, the results demonstrate that non-invasive transdermal iontophoresis can be used to deliver significant amounts of a structurally intact, functional protein across skin.
Cancer research, 1996
The similarities and differences among members of the RNase A su perfamily provide an ideal opportunity to examine the molecular basis for differences in their pharmacokinetics and biodistribution. Plasma clear ances in BALB/c mice are similar among the five RNases studied: human pancreatic RNase, angiogenin, eosinophil-derived neurotoxin, onconase, and bovine seminal RNase. The average clearance is 0.13 mI/mm or 60% of the glomerular filtration rate (measured by [‘4C]inulin clearance dur ing continuous infusion from an i.p. implanted osmotic pump). Aagiogenin has a higher volume of distribution and plasma-to-muscle transport rate than the other RNases, suggestive of binding to endothelial cells. Organ distribution differs dramatically among these RNases. The RNase most toxic to tumor cells, onconase, exhibits the longest retention in the kid neys: at 180 mm, 50% ofthe injected dose is found in the kidneys, whereas only 1% or less of the other RNases is retained In the kidneys. Slower elimination of onconase from the kidneys may be due to a higher degree of binding in the kidney or a resistance to proteolytic degradation. To elucidate the molecular determinants involved in tissue uptake, we exam med the blodistribution of recombinant onconase and two onconase pancreatic RNase chimeric proteins. The tissue retention property of onconase appears to be located in at least two regions, one of which is in the N112-terminal 9-amino acid a-helix. The NH2-terminal pyroglutamate of onconase, a residue essential for ribonucleolytic activity and cytotoxic ity, does not play a role in kidney retention.