Matrix-Bound Phosphoglucose Isomerase. Formation and Properties of Monomers and Hybrids

Abstract

Dimeric phosphoglucose isomerase from rabbit muscle was immobilized by reaction with cyanogen-bromide-activated Sepharose 4B. The catalytic parameters and stability properties of the free and matrix-bound isomerases were essentially identical. Total monomerisation of the matrix-bound enzyme was achieved with 8 M urea as determined by a study in which one subunit was labelled with i~do['~C]acetate and the other with the 3H-labelled reagent. Although matrixbound monomers were devoid of isomerase activity, they were still capable of binding the substrate. Matrix-bound monomers exhibited the ability to redimerize with soluble isomerase subunits from either rabbit or human yielding catalytically active dimers. Yeast isomerase monomers, in contrast, did not yield active hybrids with the rabbit monomers. Furthermore, soluble subunits, which had been inactivated with pyridoxal 5'-phosphate were also capable of hybridizing with and inducing catalytic activity in the matrix-bound monomers. These studies indicate the prerequisite of dimer formation for catalytic activity but the independent action of the catalytic centers of the dimer.Dissociation of phosphoglucose isomerase into subunits has been achieved in 3 mM sodium dodecylsulfate, 6 M guanidine hydrochloride and 8 M urea [l -31. The demonstration of the dimeric structure of the enzyme, identical polypeptide chains [4] and of two substrate binding sites [5] does not exclude the functional non-equivalence of the two active sites.

Crystal structure of phosphoglucose isomerase from pig muscle and its complex with 5‐phosphoarabinonate

“…Applying a symmetry operation can generate the dimer, and this is prerequisite for viewing either active-site region, each of which is comprised of chains from both monomers. This arrangement agrees with biochemical data showing that the dimer is required for enzymatic activity 7 but is at odds with data suggesting that AMF exhibits PGI enzyme activity and yet appears to exist as a monomer. 3 As expected, the structure of porcine PGI is highly similar to those of PGIs from other mammalian sources.…”

The Use of Biochemical Solid‐Phase Techniques in the Study of Alcohol Dehydrogenase

“…Hybridization experiments on a solid phase might provide important information about subunit interactions in oligomeric proteins, as demonstrated here and in other studies [6,38]. An inherent advantage in the solid-phase approach is that the desired hybrid is obtained directly without the need for any separation steps.…”

“…(EE)cm denotes carboxymethylated dimeric EE; other details are the same as in Table 2 Preparation Activity immobilized subunits but also that matrix-bound S subunits are not inactivated due to the immobilizationper se. A similar approach to check that covalently bound inactive subunits are in fact active upon reassociation with soluble subunits was used in studies of matrix-bound glycogen phosphorylase monomers [4] and immobilized phosphoglucose isomerase subunits [6]. However, in these studies enzyme activity was restored on addition of soluble subunits which were previously inactivated by modification-in their active sites.…”

pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate

“…Acid-dependent sequestration of PGI/AMF PGI/AMF is an oligomeric protein whose enzymatic function is dependent on dimer formation (Bruch et al, 1976) and acidification to pH 5 is associated with loss of enzymatic activity due to disruption of PGI/AMF oligomeric structure (Amraei et al, 2003;Dyson and Noltmann, 1968). We have previously shown that the acid-induced association of PGI/AMF with FN is associated with both changes in the tertiary structure of the protein and disruption of the oligomeric structure of the protein (Amraei et al, 2003).…”