Use of Iodo-Gen and Iodine-125 to label the outer membrane proteins of whole cells of Neisseria gonorrhoeae

The outer membrane proteins of Neisseria gonorrhoeae are specifically labeled by use of 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodo-Gen) and ¹²⁵I under the conditions described in this report. Use of this procedure with whole cells of N. gonorrhoeae produces a clear labeling pattern which can be visualized by electrophoretic separation of the proteins, followed by autoradiography. Electrophoretograms reveal some 70 polypeptide bands, while autoradiograms reveal only 5 or 6 labeled bands. The labeled polypeptide bands correspond to isolated outer membrane proteins, the most intensely labeled of which is the principal outer membrane protein. The method described in this report is both specific and gentle, as well as rapid and convenient.     

In vitro iodination of plant ribonucleic acids

The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 × 10⁴ to 10 × 10⁶ cpm/μg. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and ¹²⁵I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between ¹²⁵I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [³²P]chloroplast-rRNA. Thermal stability of DNA-¹²⁵I-rRNA hybrids was similar to the thermal stability of DNA-[³²P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties.     

Heat-shock DNA homology in distantly related species of Drosophila

Polytene chromosomes of D. melanogaster and D. virilis were hybridized in situ with ¹²⁵I labeled mRNA isolated from polysomes of D. melanogaster tissue culture cells incubated at 37° C. ¹²⁵I mRNA hybridized preferentially with subdivisions 87A and 87Cl of the D. melanogaster 3R chromosome; grains were also observed at regions 93D, 95D and over the chromocenter. A considerable cross hybridization of this mRNA with D. virilis polytene chromosomes was observed. The 29C region of the D. virilis second chromosome was the main site of hybridization. Significant grain numbers also appeared in region 20F of the same chromosome. The two regions mentioned belong to heat shock loci in the latter species. Based on label intensity we conclude that region 29C of D. virilis contains DNA sequences retaining molecular homology with those at subdivisions 87A and 87Cl of D. melanogaster. SDS-polyacrylamide gel electrophoresis revealed similar distributions of heat shock proteins in the two species studied.     

Transfection: enhancement by assembly protein of bacteriophage R17.

Assembly protein was isolated by DEAE cellulose chromatography from disrupted R17 bacteriophage and reconstituted with purified R17 phage RNA. Following reconstitution, ¹²⁵I labeled assembly protein co-sediments with 27S R17 phage RNA in a sucrose gradient. SDS-polyacrylamide gel analysis of the 27S ¹²⁵I labeled protein-RNA complex confirmed that assembly protein was the only phage protein associated with the RNA. The specific infectivity (PFU/μg RNA) of the R17 phage RNA-assembly protein complex was 35-fold greater than that of R17 phage RNA when assayed on $\text{Escherichia coli}$ spheroplasts. Infectivity of both preparations was destroyed by treatment with pancreatic ribonuclease A. Furthermore, the assembly protein-RNA complex was infectious for intact cells whereas phage RNA was not infectious. Infectivity of this 27S complex for intact cells was totally eliminated by pretreatment with ribonuclease.     

Evolution of 5S ribosomal RNA genes in the chromosomes of the virilis group of Drosophila

Drosophila melanogaster 5S ribosomal RNA labeled with ¹²⁵I was used as an in situ hybridization probe to localize complementary sequences in chromosomes of species in the Drosophila virilis group. Whereas virilis, the ancestral species, has two different 5S gene loci, the derived species show only one of these loci; in the two lines that have evolved from virilis it is the opposite locus that is conserved. The possible events leading to such an arrangement are discussed.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

125I-Ifenprodil: Synthesis and Characterization of Binding to a Polyamine-Sensitive Site in Cerebral Cortical Membranes

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by ¹²⁵I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. ¹²⁵I-ifenprodil was synthesized using Na¹²⁵I in the presence of chloramine-T and purified by paper chromatography. Binding of the ¹²⁵I-ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of ¹²⁵I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn²⁺, Ca²⁺, and Mg²⁺ inhibited specific binding of ¹²⁵I-ifenprodil in a concentration-dependent manner, with IC₅₀ values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (K_D) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (B_max) of 141 pmol/mg of protein. Binding of ¹²⁵I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of ¹²⁵I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of ¹²⁵I-ifenprodil, suggesting this site was not involved. The binding site labeled by ¹²⁵I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Effects of different batches of iodine on properties of I-hFSH and characteristics of radioligand-receptor assays

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCi carrier free Na ¹²⁵I (Amersham Corp., 15 mCi/μg I₂) in the presence of lactoperoxidase. Incorporation of ¹²⁵I into hFSH was determined by the method of [7.]Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20°C) using different calf testis membrane preparations having similar receptor characteristics. Each ¹²⁵I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of K_a and R_t. Incorporation of ¹²⁵I into FSH was relatively constant over the large number of experiments (62.4 ± 6.4 μCi/μg; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of ¹²⁵I-hFSH was related to the lot of ¹²⁵I utilized, and was significantly (P ≤ 0.01) lower and more variable (28.7 ± 10.5 μCi/μg). Maximum bindability of ¹²⁵I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH ¹²⁵I incorporation (r = −0.47; P ≤ 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na¹²⁵I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes. Received: 20 April 1999 / Accepted: 26 July 1999     

Hybridization of tRNAs of Drosophila melanogaster to the region of the 5S RNA genes of the polytene chromosomes

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled ¹²⁵I-tRNA^Asp ₂ occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNA^Asp ₂ labeled by introducing ¹²⁵I-5-iodocytidylyl residues into the 3-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to its other sites. The hybridization of tRNA^Lys ₂, tRNA^Gly ₃ and tRNA^Met ₃ at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNA^Lys ₂ no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNA^Asp ₂ in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.     

Hormone activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressivein vitro effects ofimmunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11–20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10⁻¹¹−10⁻⁷ M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strainSalmonella typhimurium 415. By using a¹²⁵I-labeled “addressing” fragment of ACTH {[¹²⁵I]ACTH (13–24)}, we showed that MT-4 cells express specific receptors for ACTH (K _d 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [¹²⁵I]ACTH-(13–24) to these receptors withK _i1 of 0.38 andK _i2 of 0.34 nM, respectively. Specific receptors for ACTH (K _d 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to compete with labeled ACTH-(13–24) for binding to these receptors (K _i=1.8 nM), and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Localization of the 5S RNA genes in Zea mays by RNA-DNA hybridization in situ

5S RNA was extracted from Zea mays tissue and iodinated in vitro with ¹²⁵I to a high specific activity. Acrylamide gel electrophoresis of the ¹²⁵I-5S RNA, 11/2 weeks after iodination demonstrated that most of the 5S RNA molecules were degraded to half-size or smaller. In situ hybridization with this iodinated RNA to pachytene microsporocyte chromosomes showed that the 5S RNA cistrons are located near the end of the long arm of chromosome 2. No obvious association of the 5S locus with the nucleolus was seen during pachytene or later stages.     

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

Summary A temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu ^-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Synthesis and biological evaluation of indole-chalcone derivatives as β-amyloid imaging probe

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aβ_1-42 aggregates versus [¹²⁵I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of AD transgenic mice. The radioiodinated probe [¹²⁵I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [¹²⁵I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aβ plaques specifically. However, experiments with normal mice indicated that [¹²⁵I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting β-amyloid plaques in the brains of AD patients.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.