Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The invasive property of trophoblast cells is dependent on the activity of proteolytic enzymes of the metallo- and serine proteases family. Interleukin-1 (IL-1) was found to be involved in the regulation of these proteases in various systems, serving as an important modulator in trophoblast physiology (e.g. induction of hCG beta, cytokines, and others). Therefore, consideration is given in this report to the role of IL-1 in the regulation of metalloprotease activity in human trophoblasts. Human trophoblast cells were isolated from first trimester placentas by trypsin degradation and Percoll fractionation. Primary cell cultures of first trimester trophoblasts constitutively elaborated two species of collagenase type IV (92 and 72 kDa), as assessed in gelatin matrix. Treatment with IL-1 further augmented the 92-kDa type IV collagenase secretion in a dose-dependent manner. Furthermore, IL-1 significantly (P < 0.01) increased 92-kDa collagenase gene expression by trophoblast cells, as determined by solution hybridization/ribonuclease protection assay. Both the increase in gene expression and protein biosynthesis of the 92-kDa collagenase type IV were neutralized by the soluble IL-1 receptor, indirectly suggesting a receptor-mediated response. Interestingly, transforming growth factor-beta a putative modulator of IL-1 induced effects, was shown to induce the 92-kDa collagenase type IV secretion as well. These results provide indirect evidence supporting the idea that IL-1 and transforming growth factor-beta may play an intermediary role in trophoblast invasion at the feto-maternal interface by regulating trophoblast expression of 92-kDa type IV collagenase, a protease of prime importance in trophoblast invasion.
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PMID:Cytokine-mediated regulation of type IV collagenase expression and production in human trophoblast cells. 876 80

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.
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PMID:Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor. 877 78

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.
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PMID:Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells. 906 4

The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam'. The chimeric Pam residue integrates the heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position. The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme structure/ function relationships in a native-like peptidyl architecture.
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PMID:Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics. 927 83

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.
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PMID:A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. 940 79

In cultured rat hepatocytes, glucagon increased phosphoenolpyruvate carboxykinase mRNA transiently. Insulin, given at the maximal increase, enhanced the degradation by 3-fold. The levels of beta-actin mRNA and ribosomal RNA, which served as a control, remained unchanged. The transcriptional inhibitor, actinomycin D, or the serine/threonine phosphatase IIA inhibitor, okadaic acid, prevented the degradation of phosphoenolpyruvate carboxykinase mRNA. This indicated that the degradation of phosphoenolpyruvate carboxykinase mRNA requires the de novo synthesis of a bona fide destabilizing factor and/or active protein phosphatase. In vitro RNA degradation assays were developed in order to investigate whether insulin-treated cells contained enhanced ribonuclease activity. Fractionated cytosolic extracts were prepared by removing cell organelles by differential centrifugation and thereafter part of the cytosolic proteins by heat treatment. These extracts were incubated with exogenously added total RNA and the degradation of phosphoenolpyruvate carboxykinase mRNA, beta-actin mRNA and 28S ribosomal RNA was studied. In this assay, phosphoenolpyruvate carboxykinase mRNA and the otherwise stable beta-actin mRNA and ribosomal RNA were degraded 3-fold faster by extracts from insulin-treated, than from untreated, cells. The increase in RNase activity induced by insulin could be prevented by treatment of cultured rat hepatocytes with actinomycin D, indicating that ongoing gene transcription was required. The 'in vivo' specificity of the insulin effect on PCK mRNA degradation in cultured hepatocytes seemed to be lost in the in vitro assay in cytosolic extracts due to the disruption of the intracellular environment. Also in whole cell lysates, which were obtained by hypo-osmotic shock of the cells, and which contained the disrupted particulate and all soluble cellular components, PCK mRNA as well as beta-actin mRNA and ribosomal RNA, was degraded. The increase in ribonuclease activity due to insulin paralleled the insulin-induced acceleration of phosphoenolpyruvate carboxykinase mRNA degradation in cultured hepatocytes, which might indicate a functional correlation.
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PMID:Parallel acceleration of phosphoenolpyruvate carboxykinase mRNA degradation and increase in ribonuclease activity induced by insulin in cultured rat hepatocytes. 970 51

ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.
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PMID:Zonal induction of mixed lineage kinase ZPK/DLK/MUK gene expression in regenerating mouse liver. 973 Dec 38

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.
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PMID:Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. 981 11

Tonins are serine proteinases mainly found in the rat submandibular gland, which are capable of generating the pressor octapeptide angiotensin II (Ang II) not only from the classical substrate angiotensin I but also from the synthetic tetradecapeptide (AG(1-14)) and from angiotensinogen. In this work, tonin expression levels were evaluated in astrocytes and brain areas of the rat. By two different techniques (ribonuclease protection assay and reverse transcription-polymerase chain reaction), we could verify the presence of tonin mRNA in astrocytes and in the thalamus of the rat brain. Sequencing of the amplified brain cDNA determined it to be identical to that found in the submandibular gland. Central microinjection of tonin produced a transient (10-20 min) elevation of blood pressure and heart rate and induced water and saline intake within the first 10 min after injection. Urinary volume and salt excretion increased within 7 h after tonin injection. These effects were partially blocked by previously administered losartan, indicating that tonin effectively induced a central Ang II formation. Our data suggest that tonin may be an alternative pathway to Ang II generation in the brain and could participate in the physiological effects exerted by Ang II such as water and saline intake and blood pressure elevation.
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PMID:Tonin expression in the rat brain and tonin-mediated central production of angiotensin II. 1204 7

Onconase (ONC) is a ribonuclease isolated from amphibian oocytes that is cytostatic and cytotoxic to numerous tumor lines. ONC shows in vivo anti-tumor activity in mouse tumor models and is currently in Phase III clinical trials. Previous studies indicated that ONC induces apoptosis of the target cells most likely along the mitochondrial pathway involving caspase-9 as the initiator caspase. We have recently developed an approach to detect the activation of serine (Ser) proteases during apoptosis. The method is based on affinity labeling of Ser protease active centers with fluorochrome-tagged inhibitors. The aim of the present study was to reveal whether Ser proteases are activated during apoptosis induced by ONC. Human leukemic HL-60 cells were treated with ONC for up to 72 h and then exposed to 5(6)-carboxyfluoresceinyl-L-phenylalanylchloromethyl ketone (FFCK) or 5(6)-carboxyfluoresceinyl-L-leucylchloromethyl ketone (FLCK), the fluorescing green reagents reactive with active centers of the chymotrypsin-like enzymes that cleave proteins at the Phe (FFCK) or Leu (FLCK) site. Activation of caspases was assayed in the same cells using sulforhodamine-labeled (fluorescing red) pan-caspases inhibitor (SR-VAD-FMK). Administration of 1.67 microM ONC into cultures of HL-60 cells led to the appearance of cells that bound SR-VAD-FMK as well as FFCK and FLCK. Most labeled cells had features characteristic of apoptosis. We interpret the binding of these ligands, which was irreversible and withstood cell fixation, as revealing activation of caspases and chymotrypsin-like Ser proteases. Because the induction of binding of each of the three ligands occurred at approximately the same time, the data suggest that during apoptosis caspases and Ser proteases may transactivate each other. The intercellular and subcellular pattern of binding SR-VAD-FMK vs FFCK or vs FLCK was different indicating a variability in abundance and localization of these enzymes within individual apoptotic cells. The FFCK- and FLCK-reactive proteins were of similar molecular mass, approximately 59 and approximately 57 kDa, respectively.
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PMID:Activation of caspases and serine proteases during apoptosis induced by onconase (Ranpirnase). 1212 58


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