Properties of crosslinked polymers of glutamic dehydrogenase

Josephs, Robert; Eisenberg, Henry; Reisler, Emil

doi:10.1021/bi00745a006

Cited by 30 publications

(8 citation statements)

References 23 publications

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“…GTP (1 mM) and NADH (1 mM) were added to the reaction mixture because under these conditions the macromolecule is cross-linked in the hexameric form. The extent of cross-linking was verified by electrophoresis on SDS-PAGE gels and substantially confirmed the pattern reported in the literature (Josephs et al, 1973). As the modified protein eluted as a single band on gel filtration chromatography, no further purification was attempted.…”

Section: Methodssupporting

confidence: 84%

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

Strambini¹,

Cioni²,

Puntoni³

1989

Biochemistry

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The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form.

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Section: Methodssupporting

confidence: 84%

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

Strambini¹,

Cioni²,

Puntoni³

1989

Biochemistry

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“…However, in our studies with the porcine OAT, we were not able to find higher aggregates of linear or other form as with glutamate dehydrogenase. Even mild cross-linking of the enzyme at high protein concentrations with glutardialdehyde (Josephs et al, 1973) or suberimidate did not help to obtain appropriate polymers (data not shown). Instead, Tsai et al (1987) proved, for the porcine OAT, that the molecular mass of this enzyme was dependent simply on the rotor speed during sedimentation equilibrium runs.…”

Section: Resultsmentioning

confidence: 99%

High‐resolution Electron Microscopic Studies on the Quaternary Structure of Ornithine Aminotransferase from Pig Kidney

Lünsdorf

Hecht

Tsai

1994

European Journal of Biochemistry

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The ornithine aminotransferase (OAT) from pig kidney has been studied on the basis of highresolution electron microscopy and the morphological appearance of the apoenzyme and holoenzyme have been examined. The quaternary structure of the OAT molecules in the presence of 5 mM pyridoxal 5'-phosphate could be established. The enzyme molecule appears to be built up of two morphological units, called M1. The native holoenzyme, termed morphological unit M2, measures . 10.9 nm in length and 5.8 nm in width and its molecular mass is approximately 168 kDa, based on electron microscopical calculations. Since the enzyme is composed of only one type of 45-kDa subunit, the holoenzyme is a homotetramer. Each M1 is composed of two subunits and, as seen in top-view projection, has an oval to triangular shape. Upon tilting to 40" the triangular shape changes into three distinct centers of mass. This morphological differentiation reflects the inner organization of M1, i.e. the shape of the individual subunit deviates from strictly globular proteins. This observation is compatible with the notion that the 45-kDa subunit consists of one large and one small domain. By tilting to 40", both large domains in M1 represent two of the three centers of mass, while the third center of mass is attributed to the superposition of both small domains. Thus, the four domains of both subunits in M1, in accordance with the triangular top-view projection, are quasi-tetrahedrally arranged. Since the change in shape of M1 upon tilting is only obvious in one of the two halves of the native OAT, it suggests that both morphological units of M2 are oriented asymmetrically relative to one another.

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“…boGDH aggregates over a wide range of conditions and forms long helical fibers extending along the threefold axis of the hexamer [50,51]. Although the aggregated state does not affect catalytic activity [52,53], allosteric regulators do alter the polymeric state of GDH; GTP inhibits this aggregation, whereas ADP promotes it [20,21,54]. GDH aggregation is also affected by some chemical modification agents.…”

Section: Enzyme Polymerizationmentioning

confidence: 99%

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery

1999

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We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.

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Properties of crosslinked polymers of glutamic dehydrogenase

Cited by 30 publications

References 23 publications

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

High‐resolution Electron Microscopic Studies on the Quaternary Structure of Ornithine Aminotransferase from Pig Kidney

The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery

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