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)

The dimeric haemoglobin CTT VIIB (Erythrocruorin) of Chironomus thummi thummi, Diptera, has been sequenced. Either globin or globin with maleic acid blocked lysines were cleaved with trypsin. The separation of peptides and the sequence analysis are given in detail.
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PMID:[Hemoglobins. XXVI. Analysis of the primary structure of the dimeric insect haemoglobin CTT VIIB (Erythrocruorin) from Chironomus thummi thummi, Diptera]. 42 21

Hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis are closely related, congenital disorders of the red blood cell usually associated with defective spectrin self-association and abnormal limited tryptic digestion of the N-terminal of domain of spectrin. Enhanced cleavage by trypsin of spectrin from affected individuals at arginyl residue 45* and lysyl residue 48* frequently yields increased amounts of an alpha 1/74-Kd fragment at the expense of the normal alpha 1/80-Kd parent fragment. Limited tryptic digestion of three unrelated individuals with HPP showed the alpha 1/74 defect. To ascertain the molecular defect responsible for the abnormality, the structure of exon 2 of the alpha-spectrin gene was examined. Genomic DNA from the subjects was amplified by the polymerase chain reaction using primers flanking exon 2. Restriction endonuclease digestion of amplified products showed the loss of the HindIII site at codons 47 and 48 in one allele of subject 1 and abolished the AhaII site at codons 27 and 28 in one allele of subjects 2 and 3. Nucleotide sequence analysis of subcloned amplified DNA from the HPP subjects showed three novel amino acid substitutions. In subject 1 (a black individual), a single base substitution (AAG----AGG) at codon position 48 changes amino acid residue lysine to arginine. In subject 2 (a white individual), a single base substitution (CGT----AGT) at codon 28 changes arginine to serine. In subject 3 (a black individual), a different base substitution at position 28 (CGT----CTT) changes arginine to leucine. These mutations occur at positions of the alpha l domain where other mutations have also been described, indicating that the normal residues at these positions play an important role in spectrin dimer self-association and thus, in membrane stability.
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PMID:Heterogeneity of the molecular basis of hereditary pyropoikilocytosis and hereditary elliptocytosis associated with increased levels of the spectrin alpha I/74-kilodalton tryptic peptide. 187 97

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.
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PMID:Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe). 238 1

The dimeric hemoglobin CTT VI (Erythrocruorin) was isolated from the hemolymph of the larvae of Chironomus thummi thummi. The globin from CTT VI was subjected to trypsin, limited trypsin and cyanogen bromide digestion. For elucidation of the sequence in the C-terminal region, cleavage with Staphylococcus aureus V8 protease was also carried out. The peptides were separated by gel and ion-exchange chromatography. The handling of some large fragments was facilitated by maleylation and subsequent ion-exchange chromatography with conservation of the maleyl groups. The amino acid sequence was determined by automatic Edman degradation. The order of the peptides was provided by overlaps, but in two cases by homology only (for residues 98 and 99, and 109 and 110). The hemoglobin consists of 2 x 147 amino acids with a molecular weight of 32411. The sequence of CTT VI is compared with a monomeric (CTT III) and a dimeric hemoglobin (CTT II beta) and the human alpha-chains. The structural differences are discussed.
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PMID:Hemoglobins, XXXVIII. Amino acid sequence of a dimeric hemoglobin (erythrocruorin), component VI from Chironomus thummi thummi (CTT VI). 722 79

Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid-point potential redox centres. The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.
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PMID:Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli. 959 8