Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

小麦及其近缘物种中小分子量核糖核酸酶的初步分析

应用ＲＮａｓｅ功能胶体系,在小麦及其相关物物中发现１组小分子量核糖核酸酶（ＲＮａｓｅ）。ＲＮａｓｅ的分子量变化范围在６．５～１４ｋＤａ间,低于已报道的大多数植物ＲＮａｓｅ的分子量。小分子量ＲＮａｓｅ在幼苗中大量表达,并且在降解ＲＮＡ底物时,随着缓冲液ＰＨ值及离子浓度的不同而有所变化。在休眠及发芽泪科种 现几种可能不同的小分子量ＲＮａｓｅ。在发芽过程中,其中２种ＲＮａｓｅ的少变化不明显,而另外２种Ｒ     

Ribonucleases and neoplasia.

Biochemical data provide good evidence of a lack of acid and alkaline RNase activities in ascites tumour cells. Analyses of whole solid tumours appear of doubtful value, but fractionation studies reveal RNase deficiencies in mitochondrial fractions whereas inconsistent results are reported for microsomal fractions. Nuclei, nucleoli, and ribosomes isolated from tumours show relatively weak activities. Large variations are noted in determinations on purified lysosomes. Histochemical analyses by two different approaches demonstrate a multifocal loss of RNase activities in preneoplastic tissues, a lack of activities in cancer cells, and the presence of appreciable activities in stromal tissue and necrotic areas of tumours. These results suggest that RNase activities found in homogenates and cellular fractions of heterogeneous tumours may derive mainly from stromal cells, phagocytes, and extracellular fluids of necrotic areas. A close correlation seems to exist between activation of RNases and tumour regression. A large variety of therapeutic agents induce increases in tumour RNase activities whereas ineffective agents do not. The activation of RNases precedes obvious regression and apparently represents de novo synthesis of RNases in cancer cells. It emerges from these studies that loss of RNase activities could represent a critical event in carcinogenesis, that RNase deficiencies would persist in cancer cells, and that RNase activation would be closely associated with tumour regression. Losses of RNase activities in preneoplastic tissues are followed by changes in the properties of cytoplasmic RNA probably due to alterations in ribosomes in areas of neoplastic transformation. Deficiencies in the RNase system could be the source of abnormalities in cellular RNA or RNA-containing particles that would lead to neoplasia.     

Messenger and ribosomal RNA hydrolysis by ribonucleases II. Changes in ribonuclease activities and ribosomes of barley leaves during the early stages of powdery mildew infection

We have monitored changes in the properties of two barley (Hordeumvulgare L.) leaf RNases with respect to their action on polysomalmessenger RNA (mRNA) and the RNA of isolated ribosomes duringthe early stages of infection by the powdery mildew fungus (Erysiphegraminis f. sp. hordei). The results presented support the followingconclusions. (i) At 48 hr after inoculation, the pH 5 insolubleRNase undergoes significant changes in its catalytic properties.This is evident from the finding that under limit digestionconditions, the enzyme from inoculated leaves hydrolyzes chloroplastpolysomal mRNA and produces far greater quantities of chloroplastmonosomes than does the corresponding enzyme from healthy leaves.(ii) The acid soluble oligonucleotide fragments produced bythe soluble RNase from healthy and inoculated leaves (at 48hr after inoculation) in the RNA of isolated ribosomes are quantitativelysignificantly different. This suggests a change in the propertiesof the soluble RNase during the initial stages of host-parasiteinteractions. (iii) As early as 24 hr after inoculation, thereis a dramatic change in the distribution of the pH 5 insolubleand soluble RNase cleavage sites in the RNA of ribosomes indicatinga readily detectable conformational change in the ribonucleoproteinparticles. (iv) These changes in the RNases and ribosomes areonly detectable in the susceptible cultivars of barley and notin a cultivar which is genetically resistant to race 3 of thepowdery mildew fungus. (Received January 31, 1980; )     

Regulatory phosphorylation of phosphoenolpyruvate carboxylase in protoplasts from Sorghum mesophyll cells and the role of pH and Ca2+ as possible components of the light-transduction pathway.

The light-dependent phosphorylation of the photosynthetic phosphoenolpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. It was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i.e. an increase in Vmax and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4Cl and methylamine (agents which increase cytosolic pH), but not by KNO3, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca(2+)-selective ionophore A23187 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2+, to the Ca(2+)-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activity/PyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.     

Endocytotic internalization as a crucial factor for the cytotoxicity of ribonucleases

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.     

Activities of RNases in different cell compartments in different regions of pea roots

RNase activity in three different regions of pea roots—thetip, middle and basal regions—was analyzed. There werethree types of RNases differing from each other in their intracellularlocalization; the enzymes in a soluble form and two bound formsassociated with unknown, small particles or ribosomes and withthe microsomal membrane. The top region showed a high activityper DNA content of RNase in the microsomal membrane and lowactivities for the other two RNases, as compared with the otherregions. The middle region contained a low amount of RNA perDNA and showed a higher activity per DNA content of RNase inthe unknown particles or ribosomes than in the basal region.The activity of RNase in the unknown particles or ribosomesvaried greatly among the regions, but that in microsomal membranevaried only slightly. ¹ Present address: Okitsu Branch, Fruit Tree Research Station,Okitsu, Shizuoka, Japan. ² Present address: Asahi Denka Co. Ltd., 7–1 Higashiogu,Arakawa, Tokyo, Japan. (Received July 25, 1974; )     

RNase T affects Escherichia coli growth and recovery from metabolic stress.

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.     

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.     

Messenger and ribosomal RNA hydrolysis by ribonucleases I. The action of two barley leaf ribonucleases on the messenger and ribosomal RNA of isolated polysomes

When barley (Hordeum vulgare L.) leaf polysomes are incubatedwith two RNase fractions (the pH 5 insoluble and soluble RNases)under limit digestion conditions, the two enzymes exhibit characteristicpreference for messenger and ribosomal RNA (mRNA and rRNA) hydrolysis.The pH 5 insoluble RNase from a cultivar of barley, Prior, andthe corresponding enzyme from two near-isogenic lines (M1622and M1623) cleave polysomal mRNA at specific sites and generatepolysome profiles that are unique to the cultivar. By contrast,the soluble RNase from barley leaves, although a typical endoribonuclease,catalyzes no detectable hydrolysis of polysomal mRNA. Both of these barley leaf RNases hydrolyze rRNA when eitherpolysomes or monosomes are treated with these enzymes. Withpolysomes as substrate, the pH 5 insoluble RNase hydrolyzesthe high molecular weight RNA component of both large and smallsubunits of chloroplast and cytoplasmic ribosomes. The solubleRNase preferentially hydrolyzes the high molecular weight RNAcomponent of the small subunit of chloroplast and cytoplasmicribosomes. Analytical gel electrophoresis of the RNA of theRNase-treated monosomes has revealed that both enzymes hydrolyzerRNA into very small fragments. However, despite scission inrRNA at multiple sites, the RNase-treated monosomes remain activein polyuridylate-directed polyphenylalanine synthesis. (Received January 31, 1980; )     

Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.     

RNase 3 (ECP) is an extraordinarily stable protein among human pancreatic-type RNases

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.     

Electrochemical Potential Gradients of H+, K+, Ca2+, and Cl- across the Tonoplast of the Green Alga Eremosphaera Viridis

Using ion-selective microelectrodes, we measured the activity of H+, K+, Ca2+, and Cl- and the electrical potential both in the vacuole and in the cytoplasm of the unicellular green alga Eremosphaera viridis to obtain comparable values of the named parameters from the same object under identical conditions. The cytosol had a pH of 7.3, and activities of the other ions were 130 mM K+, 160 nM Ca2+, and 2.2 mM Cl-. We observed only small and transient light-dependent changes of the cytosolic Ca2+ activity. The vacuolar K+ activity did not differ significantly from the cytosolic one. The Ca2+ activity inside the vacuole was approximately 200 [mu]M, the pH was 5.0, and the Cl- activity was 6.2 mM. The concentrations of K+, Ca2+, and Cl- in cell extracts were measured by induction-coupled plasma spectroscopy and anion chromatography. This confirmed the vacuolar activities for K+ and Cl- obtained with ion-selective microelectrodes and indicated that approximately 60% of the vacuolar Ca2+ was buffered. The tonoplast potential was vanishingly low ([less than or equal to][plus or minus]2 mV). There was no detectable electrochemical potential gradient for K+ across the tonoplast, but there was, however, an obvious electrochemical potential gradient for Cl- (-26 mV), indicating an active accumulation of Cl- inside the vacuole.     

The role of electrostatic interactions in the antitumor activity of dimeric RNases

The cytotoxic action of some ribonucleases homologous to bovine pancreatic RNase A, the superfamily prototype, has interested and intrigued investigators. Their ribonucleolytic activity is essential for their cytotoxic action, and their target RNA is in the cytosol. It has been proposed that the cytosolic RNase inhibitor (cRI) plays a major role in determining the ability of an RNase to be cytotoxic. However, to interact with cRI RNases must reach the cytosol, and cross intracellular membranes. To investigate the interactions of cytotoxic RNases with membranes, cytotoxic dimeric RNases resistant, or considered to be resistant to cRI, were assayed for their effects on negatively charged membranes. Furthermore, we analyzed the electrostatic interaction energy of the RNases complexed in silico with a model membrane. The results of this study suggest that close correlations can be recognized between the cytotoxic action of a dimeric RNase and its ability to complex and destabilize negatively charged membranes.     

Evidence for a delta pH-driven Ca2+ uptake in EGTA-treated bovine spermatozoa

Calcium efflux from ejaculated bovine spermatozoa occurred upon incubation in Ca2+/EGTA buffers with Ca2+ ion concentrations ranging from 0.1 microM to 1 nM. Both total cellular calcium and cytosol free Ca2+ concentrations, the latter measured with Quin 2, were inversely correlated with the Ca2+ activity of the medium. An influx of radioactive 45Ca2+ parallel to a net efflux of calcium took place in spermatozoa incubated in 45Ca2+/EGTA buffers with 45Ca2+ activity of 0.01 microM or 0.1 microM. The uptake of the radioactive isotope was higher in spermatozoa incubated at pH 7.8 than that found at pH 6.8, increased in the presence of acetate or amiloride but decreased when ammonium chloride or monensin was added to the incubation mixture. Addition of acetate produced a decrease of the cytoplasmic pH, determined with the indicator carboxyfluorescein, whereas addition of NH4Cl or monensin caused a pH increase. Addition of either nigericin or monensin to spermatozoa suspended in a choline medium containing low concentrations of Na+, K+ and Ca2+ produced a cytosolic acidification, the subsequent addition of Ca2+ caused a cytosolic alkalinization parallel to an increase of the cytosolic free Ca2+. Addition of CaCl2 to EGTA-pretreated spermatozoa resuspended in a poorly buffered medium induced an evident decrease of extracellular pH suggesting a cellular proton extrusion. Both monensin and nigericin caused an increase of the calcium transport in spermatozoa suspended in a choline medium containing a physiological concentration of 1.5 mM CaCl2. Taken together the present results indicate that, under the experimental conditions used, a delta pH-driven Ca2+ uptake occurs in ejaculated bovine spermatozoa and suggest that Ca2+ is taken up in exchange with H+.     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Phosphatidylinositol-specific phospholipase C of murine lymphocytes

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.