The Journal of biological chemistry
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Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. ⋯ The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.
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The complete primary structure of the Escherichia coli B/r galactose-binding protein was determined by the automated sequencing of fragments produced by cleavage with cyanogen bromide, o-iodosobenzoic acid, limited trypsin digestion, mild acid hydrolysis, and Staphylococcus aureus strain V8 protease. The protein, which has 309 amino acids, is notable in the extent to which it differs from the L-arabinose-binding protein. ⋯ The galactose-binding protein is the chemoreceptor initiating chemotaxis toward galactose, and it thus becomes the first protein component required for chemotaxis for which the primary structure is known. GM 24602
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The amino acid sequence of the histidine binding protein of Salmonella typhimurium was determined by automated sequence analysis of reduced and S-pyridylethylated histidine binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage at methionine residues by cyanogen bromide, cleavage at tryptophan residues by 2-nitrophenylsulfenyl-3-methyl-3-bromo-3H-indole (BrNps-skatole), limited enzymatic digestion at arginine residues, and enzymatic digestion at Glu-X bonds by the Staphylococcus aureus V8 protease. The sequence of the COOH-terminal residues was determined using bovine carboxypeptidases A and B and amino acid analysis. The histidine binding protein was found to contain 238 amino acid residues and to have a molecular weight of 26,104 calculated from sequence.
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The amino acid sequence of the single-stranded DNA-binding protein encoded by gene 32 of bacteriophage T4 has been determined by manual and automated sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin, chymotrypsin, and staphylococcal protease. Tryptic digestion of citraconylated or succinylated gene 32 protein yields five peptides containing 4, 27, 42, 65, and 163 residues, respectively, which can be separated by Sephadex chromatography. Each of these tryptic peptides was subjected to automated sequencing and, if necessary, more extensive cleavage. ⋯ The "A" region (residues 254 to 301) has been implicated in controlling the helix-destabilizing "activity" of gene 32 protein and in interacting with other T4 DNA replication proteins. The "A" region has a net charge of -10 and, in addition, contains two unusual stretches of 4 serine residues separated by glycine 284. The region between positions 72 and 116 contains 6 of the 8 tyrosine residues in the protein and may be important for DNA binding.
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Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector of ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Therefore it was proposed that the immunodeficiency associated with adenosine deaminase deficiency is mediated through inhibition of ribonucleotide reductase and hence DNA replication. HeLa cells, treated with an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, and deoxyadenosine to mimic the adenosine deaminase-deficient state, were monitored to determine directly the effects on ribonucleotide reductase activity and levels. ⋯ Removal of deoxynucleotides, particularly dATP, from these extracts increased ribonucleotide reductase activity to several-fold higher than control values. The reduced activity of ribonucleotide reductase in the simulated adenosine deaminase-deficient HeLa cells provides direct evidence for the thesis that adenosine deaminase deficiency disease is mediated through elevated levels of dATP which inhibit ribonucleotide reductase. In addition, the cell cycle patterns and ribonucleotide reductase levels suggest that the regulatory substance(s) that controls the level of ribonucleotide reductase is not operative until the late S or G2 phase of the cell cycle.