Binding Studies on Rabbit‐Muscle Phosphoglucose Isomerase

Abstract

Binding studies utilizing the techniques of gel filtration, rate dialysis and equilibrium dialysis yielded a value of 2.0 binding sites per molecule for either substrate or inhibitors like 6‐phospho‐gluconate and pyridoxal 5′‐phosphate to rabbit muscle phosphoglucose isomerase, thereby establishing that the enzyme has one catalytic site per subunit. For the substrates and 6‐phospho‐gluconate studies were performed at various pH values. For 6‐phosphogluconate, the dissociation constant was found to change dramatically with increasing pH, following a pattern analogous to that deduced from catalytic rate measurements. For the catalytic equilibrium mixture of glucose 6‐phosphate and fructose 6‐phosphate, the Kd showed excellent agreement with the Km values reported earlier. The binding studies are discussed in relation to the current concept of the catalytic mechanism.

Retardation by the soybean Bowman‐Birk inhibitor of elastin hydrolysis catalyzed by leukocyte proteinases

“…Preadsorption of the enzymes was achived by incubating elastin and enzymes in a buffer for 5 rain at 37°C with agitation prior to addition of the inhibitor. As it was noticed earlier for HLE [19,20] and shown by us for HLCG, the absorption is complete in a matter of 5 min.…”

“…It is known, for example, that adsorption of HLE on elastin protects the enzyme from the action of physiological ~I-PI. The residual enzymatic activity equals 25% and does not decrease further on addition the inhibitor [19]. Apparently, the steric hindrance disfavors the association between the inhibitor and the enzyme adsorbed on insoluble substrate.…”

“…Apparently, as in the case of other reversible protein inhibitors of HLE [19,21], BBI, on administration, immediately forms a complex with HLE or HLCG, which is stable in the presence of elastin. Preferable complexation of HLE or HLCG with BBI, rather than with elastin, is suggested by analysis of the rates of these three processes.…”

“…Apparently, the steric hindrance disfavors the association between the inhibitor and the enzyme adsorbed on insoluble substrate. In contrast, low molecular weight protein inhibitors, secretory leukocyte Table 1 Suppression by BBI of elastinolysis catalyzed by leukocyte proteinases proteinase inhibitor and eglin C, inhibit HLE in solution and on the support in a similar way [19,21].…”

Comparative inactivation and inhibition of the anomerase and isomerase activities of phosphoglucose isomerase

“…The latter concentration is still approximately three times the K m. Concurrently, the concentration of the inhibitor in the assay must also be above its K i. found to aIlect ATP inhibition of the isomerase activity (22). The anomerase K m values derived from plots of inhibitor concentration versus reciprocal initial velocity agree well with previously published data (8), but are approximately 40-fold lower than indicated by kinetic data as an isomerase (4,12), by a non-linear optimization scheme as an anomerase (25), and by binding studies (26). This difference may arise from the unavoidable differences in assay conditions for the two activities, eg.…”