Placental Ribonuclease Research Articles

Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes.

Angiogenin is a potent inhibitor of cell-free protein synthesis. When incubated with rabbit reticulocyte lysate at a concentration of 40-60 nM, it completely abolishes the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its ribonucleolytic activity since it (i) generates limited cleavage products from reticulocyte RNA and (ii) is prevented from both cleaving RNA and inhibiting protein synthesis by placental RNase inhibitor. The ribonucleolytic activity of angiogenin toward the reticulocyte RNA system is highly specific. Thus, under conditions where angiogenin totally abolishes protein synthesis, an equivalent concentration of pancreatic RNase A inhibits it only partially. In contrast, RNase A is a much more effective enzyme than angiogenin using isolated RNA as substrate. Angiogenin inhibits protein synthesis by cleaving rRNA, thereby inactivating the protein synthesis machinery. Addition of isolated reticulocyte ribosomes to an angiogenin-treated lysate restores the capacity for protein synthesis, whereas addition of tRNA or mRNA does not. This potent effect on protein synthesis suggests a possible physiological function of angiogenin whose overall relevance and implications should become evident as the mechanisms of neovascularization are deciphered. The use of angiogenin may also further elucidate ribosome structure and its role in protein synthesis.

RNA is required for enzymatic conversion of glutamate to δ-aminolevulinate by extracts of Chlorella vulgaris

Formation of δ-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell. extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Baker's yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNA glu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs With glutamate.

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli.

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.

Interaction of ribonuclease A with estrogen receptor from rat mammary tumor MTW9.

Incubation of crude estrogen receptor preparations from mammary tumor cytosol with RNase A increases the sedimentation coefficient of the receptor from 9.7 S to 10.4 S. The effect is not obtained with other low molecular weight basic proteins (lysozyme, cytochrome c, or histone H2B). Nonenzymically active RNase A derivatives such as performic acid oxidized RNase A, fully reductively methylated RNase A, carboxymethyl-His-119-RNase A, and RNase S-protein were ineffective. RNase T1, an acidic endoribonuclease, was also without effect. However, enzymically active RNase S', prepared from a mixture of RNase S-protein and S-peptide, shifted the sedimentation to 10.4 S. The increased sedimentation is not accompanied by a change in the Stokes radius of the receptor (74 A) or buoyant density in metrizamide (1.24 g/ml). The effect of RNase A on the sedimentation of the receptor can be reversed by subsequent incubation with human placental RNase inhibitor or with rabbit anti-RNase A antibodies. Direct interaction was shown by chromatography of the receptor on RNase A Sepharose. Thus, the shift in sedimentation results from binding of RNase A to the receptor and, although this requires that the enzyme active site be available, enzymic activity is not responsible for the effect. The interaction of RNase A with the receptor occurs at low ionic strength; it does not occur at elevated ionic strength or after activation of the receptor by precipitation with ammonium sulfate.

Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli.

An endoribonuclease which cleaves only single-stranded RNA has been purified from nucleoli of Ehrlich ascites tumor cells. The molecular weight of the ribonuclease is 50,000 to 52,000 as estimated from sedimentation in glycerol density gradients and by gel filtration on Sephadex G-100. The endoribonuclease requires Mg2+ or Mn2+ (0.2 mM) for optimum activity. Monovalent cations including K+, Na+, and NH+4 are inhibitory. The ribonuclease gave an apparent Km for single-stranded RNA of 30 microM. Using ribohomopolymers, we found that the enzyme could digest single-stranded, poly(C), poly(U), and poly(A) equally well, but would not degrade duplex poly(C) . poly(I) or poly(A) . poly(U). The lack of base specificity was further demonstrated using RNA sequence analysis of partial digest products of yeast 5.8 S RNA. The ribonuclease activity is sensitive to EDTA and N-ethylmaleimide, but is not inhibited by human placental RNase inhibitor. The enzyme makes endonucleolytic cleavages which generate 5'-phosphate-terminated oligonucleotides.

Identification of lysine residues in the binding domain of ribonuclease A for the RNase inhibitor from human placenta.

Amidination of the available lysine residues of the complex between RNase A and human placental RNase inhibitor has been performed with methyl acetimidate; the conditions of the derivatization preserve the complex functionally intact. Resistance of epsilon-acetimidyllysine residues to hydrolysis by trypsin allowed, after peptide mapping, the identification of lysine residues 7, 31, 41, 61, and 91 as those which were fully protected by the inhibitor from amidination. Lysine residue 37 was partially protected from amidination. In the presence of poly(A), lysine residues 41 and 61 of RNase A were fully protected from amidination, while lysine residues 7, 31, 37, 91, and 104 were only partially protected; the enzyme retained full activity. The results permit identification of lysine residues located in the binding domain of RNase A for the inhibitor. This region is not identical with, but does overlap, the binding domain for poly(A).

Translation of vesicular stomatitis and Sindbis virus mRNAs in cell-free extracts of Aedes albopictus cells.

We have developed a cell-free system from Aedes albopictus (mosquito) cells which is able to carry out endogenous protein synthesis and is stable to freezing and thawing. Successful preparation of extracts was found to depend on the presence of purified placental RNase inhibitor during cell breakage. Micrococcal nuclease-treated extracts translated exogenously added Sindbis 26S or vesicular stomatitis virus mRNA with a high degree of fidelity, demonstrating that initiation of protein synthesis had occurred. Evidence is presented showing that when cell fractions containing intracellular membranes were used to translate vesicular stomatitis virus mRNA, the G protein was glycosylated and inserted into microsomal vesicles. Additional studies indicate that initiation of protein synthesis in this system is dependent on a capped and methylated 5'-terminal structure in the mRNA.

Effect of human placental ribonuclease inhibitor in cell-free ribosomal RNA synthesis

Exposure to purified placental ribonuclease inhibitor caused a marked reduction in ribonuclease activity associated with intact nucleoli. Addition of the placental RNase inhibitor to a nucleolar RNA synthesizing system, prior to the addition of ribonucleoside triphosphates, resulted in a significant increase in the size but did not alter the yield of the RNA products. The effect of the RNase inhibitor on the size of distribution of in, vitro transcriptional products could be reversed by the addition of N-ethylmaleimide after transcription was terminated. The properties of the ribonnclease(s) affected by the inhibitor were indicated by its resistance to N-ethylmaleimide and EDTA inhibition and by its limited endonucleolytic cleavage of the in, vitro transcript.

Role of mammalian RNase inhibitor in cell-free protein synthesis

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.

Effect of moderate maternal malnutrition on the levels of alkaline ribonuclease activity in the human placenta†

Ribonuclease (RNAse) activity was measured in placentas from a sample of 49 urban Guatemalan women from low (30) and high (19) socioeconomic status (SES). Low SES women had a larger proportion (40%) of high levels of RNAse (> 60,000 units/g) than women from the high SES group (5 %). A trend to inverse association (p = 0.06) was also observed between amount of supplemented food energy during pregnancy and RNAse activity in rural populations covered by two food supplementation programs (protein energy and energy). Two indicators of maternal nutrition, third trimester weight and height, also showed inverse associations with placental RNAse activity. It is inferred from these results that improved maternal nutrition decreases placental RNAse activity. Moreover, high levels of placental RNAse activity were associated, up to 36 months of age, with higher proportions of: physical growth retardation in weight, height and head circumference; below‐average psychological test performance; and infant deaths. These results deserve consideration from both the physiological and public health points of view.