Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.
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PMID:Myoglobin of the shark Heterodontus portusjacksoni: isolation and amino acid sequence. 50 2

Myoglobin isolated from skeletal muscle of the platypus contains 153 amino acid residues. The complete amino acid sequence has been determined following cleavage with cyanogen bromide and further digestion of the four fragments with trypsin, chymotrypsin, pepsin and thermolysin. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed 25 differences from human myoglobin and 24 from kangaroo myoglobin. Amino acid sequences in myoglobins are more conserved than sequences in the alpha- and beta-globin chains, and platypus myoglobin shows a similar number of variations in sequence to kangaroo myoglobin when compared with myoglobin of other species. The date of divergence of the platypus from other mammals was estimated at 102 +/- 31 million years, based on the number of amino acid differences between species and allowing for mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha- and beta-chain sequences and a constant rate of mutation of globin chains is not supported.
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PMID:Studies on monotreme proteins. VII. Amino acid sequence of myoglobin from the platypus, Ornithoryhynchus anatinus. 96 22

The beta o-thalassaemia gene of an individual who was a mixed heterozygote for this allele (GAA to TAA in codon 121) and beta(+)-thalassaemia (IVS-1 position 110 G to A) was examined to determine if the beta o-thalassaemia allele directed the synthesis of any detectable protein product. This beta o-thalassaemia allele was of particular interest, because it is the only example of a premature chain termination codon in the third exon of the beta-globin gene that produces thalassaemia. A very small amount of an abnormal protein was found in the red blood cells of the proband and was purified by preparative column chromatography. This abnormal protein was digested with trypsin, the peptides were separated by reverse phase high performance liquid chromatography (HPLC), and the amino acid content of each peptide was determined. All of the soluble beta-globin peptides were found except for T-13, T-14 and T-15 (residues 121-146), indicating the presence of a truncated protein that corresponded to the translation product of the beta 121 Glu----Term mRNA. This truncated globin was estimated to comprise between 0.05% and 0.1% of the total non-alpha-globin protein. These results may explain the phenotype of inclusion body beta-thalassaemia in heterozygotes, which is atypical of heterozygous beta o-thalassaemia.
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PMID:Isolation and characterization of the translation product of a beta-globin gene nonsense mutation (beta 121 GAA----TAA). 220 8

Siamese cats are homozygous for the recessive cs allele of the color (albino) locus. The c locus is shown here by backcross analysis to be linked to the beta-hemoglobin (HBB) locus in the cat at a distance of approximately eight centiMorgans. The HBB locus and, by inference, the c locus were assigned to feline chromosome D1, by analysis of genomic DNAs from a panel of rodent X cat somatic cell hybrids with a molecular clone of the human beta-globin locus. Evolutionary conservation of the synthetic homology of feline chromosome D1 and human chromosome 11 is extensive. Comparison of high resolution G-trypsin-banded preparations of the two chromosomes permitted cytological alignment of the long arm of the conserved chromosomes providing that a minimum of one paracentric inversion is hypothesized. The placement of the albino locus on conserved syntenic groups of several markers (HBB, HRAS, LDHA) in both cat and mouse strongly indicates the conservative placement of the as yet unmapped human albino locus in the homologous syntenic group on human chromosome 11p.
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PMID:Chromosomal mapping of beta-globin and albino loci in the domestic cat. A conserved mammalian chromosome group. 355 63

A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2. 647 77

A 68-year-old female visited our hospital because of low hemoglobin A1c content, which was found by chance at a health checkup. She did not have any symptom or sign except hypertension and low HbA1c. To determine the reason why HbA1c was so low in usual laboratory test, we carried out isoelectrofocusing (IEF) of the hemolysate, Hb instability test, and detection and isolation of the abnormal globin chain by urea CM-cellulose column chromatography. The abnormal beta-globin chain was digested with TPCK-trypsin, and the tryptic peptides were separated by HPLC on a reversed phase column. Finally the determination of amino acid composition and amino acid sequence of the abnormal peptide were performed. The Hb variant was identified as Hb Riyadh (beta 120Lys-->Asn). The detection and analysis of abnormal hemoglobin will be expected to increase in accordance with the increased opportunity of public health checkup.
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PMID:[An abnormal hemoglobin which was found by chance at a health checkup]. 767 38

The basic helix-loop-helix transcription factor USF43 binds to E box motifs on certain promoters and enhancers, as well as the beta-globin locus control region. We have used gel filtration chromatography, velocity centrifugation, and chemical cross-linking methods to investigate the stoichiometry and shape of USF43 in solution and when bound to DNA. USF43 has a very large Stokes' radius (44 A) and a high frictional ratio (1.64), consistent with an asymmetric elongated oligomer. Under a variety of conditions, the only detectable USF43 species in solution and bound to DNA is a dimer. The carboxyl-terminal leucine zipper is absolutely essential for a stable dimer but not for the elongated conformation. We used a protease footprinting assay to demonstrate that, when USF43 binds to DNA, a approximately 15-kDa USF43 domain becomes resistant to cleavage with trypsin. This domain includes sequences that are not expected to interact with the DNA helix, suggesting that trypsin cleavage sites are masked by a conformational change. Our results show that the oligomerization state of USF43 does not change upon binding to DNA, and the helix-loop-helix oligomerization motif of USF43 is not itself sufficient to form a high affinity dimerization interface.
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PMID:The leucine zipper is necessary for stabilizing a dimer of the helix-loop-helix transcription factor USF but not for maintenance of an elongated conformation. 806 30

Here we report the occurrence of five different beta chain hemoglobin variants not previously described in Sweden. The variants were found during quantification of HbA1c using ion exchange high performance liquid chromatography (HPLC) or isoelectrofocusing. Samples were examined either at protein level by separation of globin chains on C8 reversed phase HPLC, digestion with trypsin or lysylendopeptidase and separation of peptides by C18 reversed phase HPLC, or at DNA level by direct nucleotide sequencing of double-stranded DNA fragments amplified from exon 1 + 2 of the beta-globin gene. The variants were: Hb Raleigh [beta 1 (NA1)Val-->Ac-Ala], Hb J-Baltimore [beta 16(A13)Gly-->Asp], Hb Tacoma [beta 30(B12)Arg-->Ser], Hb K-Ibadan [beta 46(CD5)Gly-->Glu], and Hb Fukuyama [beta 77(EF1)His-->Tyr]. Hb Tacoma, Hb K-Ibadan, and Hb Fukuyama were slightly unstable in the isopropanol test, but no signs of hemolysis were found in the patients who all had normal hematological findings.
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PMID:Rare beta chain hemoglobin variants found in Swedish patients during HBA1c analysis. 822 93

A new beta chain variant was accidentally found through the assay of Hb A1c in a diabetic patient. The variant was detected by polyacrylamide gel isoelectrofocusing and electrospray ionization mass spectrometry. For sequence determination, globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liquid chromatography connected to electrospray ionization mass spectrometry. An abnormal betaT-5 peptide was found by reconstructed selected ion monitoring. The collision-induced dissociation spectrum of an ion derived from the abnormal betaT-5 peptide revealed a new substitution, [beta52(D3)Asp-->Gly], named Hb Hokusetsu. The sequence was confirmed with an automatic sequencer using peptides isolated by reversed phase high performance liquid chromatography. Amplification of the beta-globin exon 2 and nucleotide sequencing revealed a GAT-->GGT mutation in codon 52 corresponding to an Asp-->Gly replacement. Electrospray ionization mass spectrometry analysis of the hemolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal stability in the isopropanol test. Oxygen equilibrium studies on the patient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.
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PMID:A new hemoglobin variant found during Hb A1c measurement: Hb Hokusetsu [beta52(D3)Asp-->Gly]. 973 Mar 66

The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3'-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLE(min)) and the maximum amount of PCR products (CONC(max)). The hematoxylin stain showed a significantly prolonged CYCLE(min) (P < .01) and lower CONC(max) (P < .05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLE(min) (P < .01), although the CONC(max) was not significantly different from that of the control (P = .051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONC(max) (P < .05), whereas the CYCLE(min) was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE(min) (P < .01), and the PCR amplification did not reach the "plateau" level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLE(min) nor CONC(max) being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker.
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PMID:Influence of histochemical and immunohistochemical stains on polymerase chain reaction. 1069 71


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