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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.
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PMID:[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. 825 Sep 78

Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1. The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined. The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein. The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity. Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme. CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes. CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain. CaM kinase I appeared to be encoded by a single gene. RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined. High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level. CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.
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PMID:Calcium/calmodulin-dependent protein kinase I. cDNA cloning and identification of autophosphorylation site. 825 80

Poly C preferential RNase previously reported by Levy and Karpetsky [J. Biol. Chem. 255, 2153-2159 (1980)] and Miura et al. [Chem. Pharm. Bull. 32, 4053-4060 (1984)] was extensively purified from chicken liver to homogeneity as determined by SDS-PAGE (RNase CL2). The poly C preference over poly U was slightly higher than that of bovine pancreatic RNase A. However, the kinetic constants for 8 dinucleoside phosphates, CpY and UpY (Y = one of A, G, U, and C) as substrates showed that RNase CL2 was preferential for cytidylic acid, but less so than RNase A, and the influence of Y base on the rate of hydrolysis of CpY or UpY was less marked than in the case of RNase A. The primary structure of RNase CL2 was determined. The molecular weight calculated from the sequence was 13,420. Comparison of the amino acid sequence of RNase CL2 with those of other vertebrate RNases showed that RNase CL2 is a member of the RNase A family, but is not a non-secretory RNase. It retains 3 disulfide bridges of RNase A, but Cys65-Cys72 of RNase A is missing. As for the active site, the amino acid residues of the P0 and P1 sites of RNase A are completely conserved. Among the B1 site components, Thr45 (RNase A numbering) is conserved, but Phe120 and Ser123 are substituted by Leu and Thr, respectively. Among the B2 site residues, Gln69, Asn71, and Glu111 are substituted by other amino acids.
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PMID:Characterization of poly C preferential ribonuclease from chicken liver. 840 69

Pax-8, a member of the paired box-containing gene family, was shown to be coexpressed with Pax-2 in several human kidney carcinoma cell lines. Four different Pax-8 mRNA isoforms, a to d, were cloned from one of these cell lines by polymerase chain reaction amplification, and the Pax-8 gene was isolated from a human cosmid library. Analysis of the exon-intron structure of Pax-8 revealed that the four mRNA isoforms arise by alternative splicing, resulting in inclusion or exclusion of exon 7 and/or exon 8 sequences. All four Pax-8 proteins retain the paired domain as their DNA-binding motif and recognize DNA in the same manner as do the closely related Pax-2 and BSAP (Pax-5) proteins. The Pax-8a and Pax-8b isoforms end in a serine/threonine/tyrosine-rich sequence, while the C terminus of Pax-8c and Pax-8d is translated in a different, proline-rich reading frame. Transient transfection experiments revealed that Pax-8 isoforms a and b, but not c and d, strongly stimulate transcription from a promoter containing six copies of a paired-domain recognition sequence. The same four mRNA variants were also detected by RNase protection analysis in the mouse embryo and adult kidney, thus indicating evolutionary conservation of Pax-8 mRNA splicing. A different splice pattern was observed in the developing placenta, which expresses two new variants, Pax-8e and Pax-8f, instead of transcripts b to d. Expression of these mRNAs is high at embryonic day 9.5 and is gradually reduced until Pax-8a is the predominant transcript in the 12.5-day placenta. In the embryo, however, the synthesis of mRNAs b to d is initially low and then increases relative to that of Pax-8a. Hence, alternative splicing of Pax-8 gene transcripts not only generates six different Pax-8 variants but is also temporally and spatially regulated during early mouse development.
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PMID:Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties. 841 5

Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.
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PMID:Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. 844 77

An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.
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PMID:Structural characterization of glycoprotein digests by microcolumn liquid chromatography-ionspray tandem mass spectrometry. 845 19

Gametophytic self-incompatibility, a mechanism that prevents inbreeding in some families of flowering plants, is mediated by the products of a single genetic locus, the S-locus. The products of the S-gene in the female sexual tissues of Nicotiana alata are an allelic series of glycoproteins with RNase activity. In this study, we report on the microheterogeneity of N-linked glycosylation at the four potential N-glycosylation sites of the S2-glycoprotein. The S-glycoproteins from N.alata contain from one to five potential N-glycosylation sites based on the consensus sequence Asn-Xaa-Ser/Thr. The S2-glycoprotein contains four potential N-glycosylation sites at Asn27, Asn37, Asn38 and Asn 150, designated sites I, II, IV and V, respectively. Site III is absent from the S2-glycoprotein. Analysis of glycopeptides generated from the S2-glycoprotein by trypsin and chymotrypsin digestions revealed the types of glycans and the degree of microheterogeneity present at each site. Sites I (Asn27) and IV (Asn138) display microheterogeneity, site II (Asn37) contains only a single type of N-glycan, and site V (Asn150) is not glycosylated. The microheterogeneity observed at site I on the S2-glycoprotein is the same as that observed at the only site, site I, on the S1-glycoprotein (Woodward et al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylation consensus sequence at site I is conserved in all S-glycoproteins from other species of self-incompatible solanaceous plants, glycosylation at this site may be important to their function. No other post-translational modifications (e.g. O-glycosylation, phosphorylation) were detected on the S2-glycoprotein.
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PMID:Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata. 856 38

Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern.
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PMID:Complex alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms. 862 5

RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.
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PMID:Structural determinants of the uridine-preferring specificity of RNase PL3. 870 17

We previously investigated the role of the Lys108 residue of ribonuclease (RNase) Rh from Rhizopus niveus, and suggested that Lys108 probably acts to stabilize the pentacovalent intermediate, and that an Arg residue could replace the role of Lys108. In RNase Le2 from Lentinus edodes, a homologous enzyme of RNase Rh, Lys108 is replaced by Thr. In this paper, the enzymatic properties of a K108T mutant and its analogous enzyme, K108S, were investigated to determine the effect of Thr and its analog, Ser at the 108th position on enzyme activity. The enzymatic properties of these mutant enzymes were compared with those of other mutant enzymes at this position (K108M, K108A, K108L). The results showed that Thr and Ser could replace Lys108 but resulted in only 2-20% of the activity of the native enzyme depending on the substrates used.
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PMID:Enzymatic activities of several K108 mutants of ribonuclease (RNase) Rh isolated from Rhizopus niveus. 887 21


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