Amino Acid Sequence Studies on Sheep Liver Fructose-bisphosphatase. II The Complete Sequence

Abstract

The cyanogen bromide fragments of S-carboxymethylated fructose-bisphosphatase were purified. The amino acid sequences of the small fragments were determined by the dansyl-Edman method. The large fragments were subjected to proteolytic digestion to give smaller peptides more amenable for purification and sequencing by similar methods. Enzyme digests of the S-carboxymethylated enzyme gave overlap peptides containing the methionine residues.In conjunction with the amino acid sequence of the 60-residue N-terminal fragment previously determined on the S-peptide released by limited proteolysis with subtilisin the complete sequence of 336 residues was deduced. The sequence has been compared with the 335 residue sequence of pig kidney fructose-bisphosphatase and some areas of sequence for rabbit liver enzyme. The strong homology previously noted for the S-peptide sequences is maintained for the complete enzyme with only 34 changes in 336 residues when comparing the pig and sheep enzymes.

Studies on the Regulation of Rat Liver Pyruvate Kinase and Fructose-1,6-Bisphosphatase

“…Mammalian Fru-1,6-P2:ase is a tetrameric enzyme having identical subunits and having a molecular weight of about 140 000, The complete amino acid sequence has been determined for Fru-1,6-P2:ase from pig kidney cortex (48) and sheep liver (29). These sequences show a high degree of homology with 90 % being identical (36).…”

Amino Acid Sequence of the Globin lIB Chain of the Dimeric Haemoglobin of the Bivalve Mollusc Andara trapezia

“…The methods of enzyme digestion with trypsin, chymotrypsin, elastase, pepsin, papain and staphylococcal protease; peptide mapping, amino acid analysis and sequence determination were substantially the same as previously described (Air and Thompson 1969;Fisher and Thompson 1983). Because of inadequate facilities' for quantitative amino acid analysis of all peptides generated, the hydrolysates were first examined by paper ionophoresis at pH 1·8 (Dreyer and Bynum 1967) while saving a small portion for quantitative analysis on the Beckman 121M analyser (0·5-2 nmol) if it later became essential.…”

“…The radioactive peak was recovered after dialysis and freeze-drying. Further purification involved paper ionophoresis in 20% (v/v) formic acid combined with paper chromatography at right angles to the original direction of migration with butanol-pyridine-acetic acid-water (15: 10: 3: 12 v/v) as solvent (Fisher and Thompson 1983).…”

“…The globin was dissolved in 7 M guanidine hydrochloride contammg buffer, treated with dithiothreitol to ensure that oxidation ofthiol groups was avoided and alkylated with [2-'4C]iodoacetic acid as described by Fisher and Thompson (1983). After recovery, samples of the labelled protein were digested separately with trypsin and chymotrypsin and the digests fractionated on a Sephadex G25 column (140 by 2·8 cm) equilibrated with 1% (v/v) ammonia.…”

Fructose‐1,6‐bisphosphatase. Primary structure of the rabbit liver enzyme. ‘Intermediate’ variability of an oligomeric protein

“…A divalent metal ion, such as Mg ~+, Mn 2+, or Zn 2+, is required for activity, and two metal binding sites per subunit have been suggested; AMP is an allosteric inhibitor, and fructose-2,6-bisphosphate a competitive and probably also an allosteric inhibitor [2][3][4][5]. The primary structures have been reported for the enzyme from a wide variety of sources, including mammals [6][7][8][9], plants [10][11][12], yeasts [13,14] and bacteria [15][16][17]. The tertiary structure of the pig apoprotein [18] and its complexes with inhibitors, product, substrate, and substrate analogs [19][20][21] have been determined.…”