Purification and sequence determination of guanylate kinase from pig brain.
A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M(r) of 21,831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.
Zschocke
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Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Schiltz
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Schulz
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014357:Trypsin
D014357:Trypsin; D006162:Guanylate Cyclase; D003488:Cyanogen Bromide