Linda A. Fothergill

Purification and properties of enkephalin — The possible endogenous ligand for the morphine receptor☆

Abstract The purification and properties of a peptide of low molecular weight (800–1200) which has been extracted from the pig brain is described. This substance acts as an agonist at opiate receptor sites. It is suggested that the peptide may have a wide neurophysiological role in the brain and possibly in other tissues.

The amino acid sequence of yeast phosphoglycerate mutase.

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N‐terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N‐terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

AMINO ACID SEQUENCES OF ACTIVE-SITE HISTIDINE PEPTIDES FROM RABBIT MUSCLE PHOSPHOGLYCERATE MUTASE

Monophosphogiyc~rate mutase (MPGM) catalyses the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, in a reaction mechanism involving a pl~osphohistidine illter~nediate [ 1 ,I?]. A correlation of the 2.8 _& resolution electron-density map ffom X-ray crystallographic studies ofyeast MPGM with its amino acid sequence shows 2 histidines at the active site [3]. Structural studies of muscle MGP~I are much less complete, although the enzyme has been used extensively in kinetic work, e.g. [4,5]. The muscle and yeast enzymes have been shown to differ in several respects. The muscle enzyme is a dimer with identical subunits of mol. wt 28 000 [6], whereas the yeast enzyme is isolated as a tetramer composed of 4 identical subunits of mol. wt 27 000 [7]. The activity of the muscle enzyme is sensitive to modification of cysteinyl and arginyl residues [8,9], in contrast to the yeast enzyme that has no thiol [lo], and is only moderately sensitive to butanedione [l l]. In addition, a phosphohistidine peptide isolated from chicken breast muscle MGPM has been sequenced [ 121, and shows no homology with any of the 4 histidine sequences of the yeast enzyme (see table 2). We report here the purification and determination of the amino acid sequences of histidine-containing peptides from rabbit muscle MPGM to obtain an estimate of the extent of structural similarity at the active sites of the muscle and yeast enzymes.

Amino acid sequence of an active site fragment from human diphosphoglycerate mutase

Diphosphoglycerate mutase (DPGM, bisphosphoglycerate synthase, EC 2.7 5.4) controls the concentration of 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes [l-4]. The discovery that 2,3-DPG profoundly influences the affinity of haemoglob~ for oxygen [S ,6] has stimulated much interest in this system. It is now apparent that DPGM catalyses three reactions: the conversion of 1,3-diphosphoglycerate to 2,3-DPG; the dephosphorylation of 2,3-DPG; and the interconversion of 3and 2-phosphoglycerates (2-41. These three reactions are also catalysed by the glycolytic enzyme, monophosphoglycerate mutase (MPGM, EC 2.7.5.3), although at different relative rates [2-4]. The two enzymes have other features in common including subunit size [2,7], tryptic peptide elution profiles f8], and a phosphorylated histidine intermediate [29]. A correlation of the amino acid sequence of yeast MPGM with its 2.8 A resolution electron density map shows two histidines (His 8 and His 179) at the active site [lo]. The sequence of a tryptic phosphohistid~e containing peptide from horse DPGM homologous with residues 8-l 5 of yeast MPGM has been determined [9]. We report here the isolation and ammo acid sequence determination of a 32 residue fragment from the N-terminus of human DPGM. A comparison with the yeast MPGM sequence shows them to be highly homologous, and implies a structurally very similar active site, and a common evolutionary origin.

Yeast Phosphoglycerate kinase purified by affinity elution has tightly bound 3-phosphoglycerate

Phosphoglycerate kinase (PGK, ATP3-phospho-Dglycerate-1 -phospho-transferase, EC 2.7.2.3) is the glycolytic enzyme that catalyses the fol

Inactivation of chicken muscle enolase by carbodiimide and glycine methyl ester

wing reaction: 1,3diphosphoglycerate t ADP !!!& 3-phosphoglycerate + ATP [ 11. The enzyme is active as a monomer with a relative molecular mass J4, of 44 000 [ 2,3], Treatment of PGK with the arginine-modifying reagent, phenylglyoxal leads to loss of activity [4&6]; the substrate, 3-phosphoglycerate can protect against inactivation. We report here that PGK purified by affinity elution is not inactivated by phenylglyoxal, and it is shown that this is due to the presence of tightly bound 3-phosphoglycerate.

The Complete Amino-Acid Sequence of Hen Ovalbumin

Enolase (EC 4.2.1.11) catalyses the dehydration of D-2-phosphoglycerate (2-PGA) to 2-phospho- enolpyruvate (PEP) during glycolysis. The enzyme is a dimer with identical subunits (Mr - 45 000) and requires divalent metal ions such as Mg2+ for catalytic function. It has been shown that glycidol phosphate, a substrate analogue, inactivates rabbit muscle enol- ase, and this is interpreted as due to reaction with a carboxyl group at the active site