The Isolation and Crystallization of Yeast and Rabbit Liver Triose Phosphate Isomerase and a Comparative Characterization with the Rabbit Muscle Enzyme

Abstract

Triose phosphate isomerase was isolated from brewer's yeast and rabbit liver and was obtained in crystalline form. Chemical, physical and kinetic properties were compared to rabbit muscle triose phosphate isomerase. The molecular weight of all three enzymes is in the range of 56000 to 60000. In dodecyl sulfate or as modified maleylated protein, the enzymes dissociate into two polypeptide chains each having a molecular weight in the range of 24000 to 29000. The rabbit muscle and liver enzymes appear to be indistinguishable in terms of their amino acid composition, electrophoretic mobility, kinetic properties, inhibition sensitivity, pH optimum, molecular weight and N‐terminal amino acid (alanine). The yeast enzyme, on the other hand, was found different in the following respects; it has a several fold lower content of sulfur amino acids, is highly resistant to inactivation through photooxidation as well as sulfhydryl and alkylating agents. Furthermore, it contains different N‐terminal amino acids (valine and alanine) and has kinetic properties differing from those of the rabbit enzymes. The crystalline liver and muscle enzymes could be resolved into three distinct electrophoretic forms in starch and polyacrylamide gels. The possible interpretation of the multiple forms in terms of hybrids or conformers is discussed.

The Preparation and Properties of Triosephosphate Isomerase from Chicken Muscle and a Comparison with that from Rabbit Muscle

“…Using the Yphantis short-column technique of equilibrium ultracentrifugation [23], which is liable to inaccuracy because of difficulties in measuring the relatively broad schlieren lines, Krietsch et al [3] obtained a value of 56800 f 4100 for the molecular weight of their preparation of the rabbit muscle enzyme. They chose a value for 9 of 0.75 ml/g which on correction to our value of 0.741 ml/g yields 54800 f 4100 as the value of the molecular weight to be derived from their results.…”

“…To these must be added the values of 56000-60000 obtained by Krietsch et al [3] and of 44000-49000 obtained by Lee et al [4]. The value of 60000 (unpublished data of Esnouf and Waley quoted by Johnson and Waley [5] was the result of a single determination on a specimen of doubtful purity and the value of 48000 (unpublished data of Esnouf and Jesty quoted by Coulson et al [22]) used a value for the partial specific volume, B, of 0.73 ml/g which on the basis of the value now reported (namely, .ii = 0.741 ml/g) ought to be corrected to 50000, in closer agreement with subsequent results obtained in this laboratory.…”

“…I n recent years interest has centred on the enzyme obtained from rabbit muscle and preparative procedures have been described by Czok and Bucher {I] and Norton et al [2]. These last authors and also Krietsch et al [3] and Lee et al [4] have studied the molecular weight of the enzyme without, however, direct determinations of the partial specific volumes, and considerable evidence has been presented to show that the native enzyme is composed of two subunits [2,3,.…”

Inhibition of glycolytic enzymes by 2-phosphotartronate

“…is known to be inhibited by phosphoenolpyruvate (with a KII/Km ratio of 7-9:1), but is not affected by 2-or 3-phosphoglycerate (Krietsch et al, 1970); 2-phosphotartronate is also without effect (Table 2). 2-Phosphotartronate does not compete with 3-phosphoglycerate in the 3-phosphoglycerate kinase reaction, suggesting that the position of the phosphate group is important in binding.…”

The tryptic peptides of rabbit muscle triose phosphate isomerase

“…is not only the most active, but also one of the smallest of the glycolytic enzymes. The molecular weight of the rabbit muscle enzyme has been estimated from ultracentrifuge data to lie in the range 50000-60000 (McVittie et al, 1972;Krietsch et al, 1970;Norton et al, 1970). Crystallographic data suggested a value of about 53000; moreover the hexagonal crystals had a twofold axis of rotation that related two subunits of molecular weight 26200 (Johnson & Waley, 1967).…”